Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Extramedullary hematopoiesis in a case of benign mixed mammary tumor in a female dog: cytological and histopathological assessment

Backgroud  

Extramedullary hematopoiesis (EMH) is defined as the presence of hematopoietic stem cells such as erythroid and myeloid lineage plus megakaryocytes in extramedullary sites like liver, spleen and lymph nodes and is usually associated with either bone marrow or hematological disorders. Mammary EMH is a rare condition either in human and veterinary medicine and can be associated with benign mixed mammary tumors, similarly to that described in this case.     

The active site of factor IXa is located far above the membrane surface and its conformation is altered upon association with factor VIIIa. A fluorescence study.

The topography of membrane-bound blood coagulation factor IXa (fIXa) and the nature of its interaction with its cofactor, factor VIIIa (fVIIIa), were examined using fluorescent derivatives of fIXa. A fluorescein dye was covalently attached to the active-site histidine of fIXa via a D-Phe-Pro-Arg tripeptide tether to form Fl-A-FPR-fIXa; similarly, a 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) dye was covalently attached via Glu-Gly-Arg to form DEGR-fIXa. When either Fl-A-FPR-fIXa or DEGR-fIXa was titrated with phosphatidylcholine-phosphatidylserine vesicles containing octadecylrhodamine in the presence of Ca2+, fluorescence energy transfer was observed. Assuming a random orientation of dyes, the distance of closest approach between the donor dyes in the active sites of the membrane-bound enzymes and the acceptor dyes at the membrane surface was found to be 89 +/- 3 A for Fl-A-FPR-fIXa and 73 +/- 4 A for DEGR-fIXa. Although the exact distance remains uncertain, it is clear that the active site of fIXa is positioned more than 70 A above the surface, and hence that the elongated fIXa molecule projects approximately perpendicularly from the surface when bound to the membrane. The binding of fVIIIa to membrane-bound Fl-A-FPR-fIXa or DEGR-fIXa did not alter the location of the active site relative to the membrane surface, but did alter both the emission intensity and anisotropy of the fluorescein and dansyl probes and hence their environments. Cofactor stimulation of fIXa activity therefore appears to be mediated, at least in part, by a conformational change in the active site that occurs when fVIIIa binds to the enzyme on the phospholipid surface.     

Expression and characterization of the naturally occurring mutation L394R in human gamma-glutamyl carboxylase

Patients with mutation L394R in gamma-glutamyl carboxylase have a severe bleeding disorder because of decreased biological activities of all vitamin K-dependent coagulation proteins. Vitamin K administration partially corrects this deficiency. To characterize L394R, we purified recombinant mutant L394R and wild-type carboxylase expressed in baculovirus-infected insect cells. By kinetic studies, we analyzed the catalytic activity of mutant L394R and its binding to factor IX's propeptide and vitamin KH(2). Mutant L394R differs from its wild-type counterpart as follows: 1) 110-fold higher K(i) for Boc-mEEV, an active site-specific, competitive inhibitor of FLEEL; 2) 30-fold lower V(max)/K(m) toward the substrate FLEEL in the presence of the propeptide; 3) severely reduced activity toward FLEEL carboxylation in the absence of the propeptide; 4) 7-fold decreased affinity for the propeptide; 5) 9-fold higher K(m) for FIXproGla, a substrate containing the propeptide and the Gla domain of human factor IX; and 6) 5-fold higher K(m) for vitamin KH(2). The primary defect in mutant L394R appears to be in its glutamate-binding site. To a lesser degree, the propeptide and KH(2) binding properties are altered in the L394R mutant. Compared with its wild-type counterpart, the L394R mutant shows an augmented activation of FLEEL carboxylation by the propeptide.     

Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles.

The activation of factor X by factor IXa (fIXa) in the presence of phosphatidylcholine-phosphatidylserine (PCPS) vesicles is markedly accelerated by thrombin-activated factor VIII (fVIIIa). The interaction between highly purified fVIIIa and fIXa in this complex was studied fluorometrically at 25 degrees C by using a derivative of D-phenylalanyl-prolyl-arginyl-fIXa which was modified at the active site with fluorescein-5-maleimide (Fl-M-FPR-fIXa). Titration of Fl-M-FPR-fIXa with fVIIIa at fixed PCPS resulted in a large, saturable increase in anisotropy (delta r = 0.09). The titration data were fit to a model assuming a reversible equilibrium between fVIIIa and fIXa, resulting in an apparent dissociation constant of 2 nM and a stoichiometry of 1 mol of fVIIIa/mol of Fl-M-FPR-fIXa. The initial velocity of factor X activation was measured under identical conditions except that active fIXa and factor X were included, which yielded binding parameters similar to those determined fluorometrically. Thus, the fluorescence method accurately reflects complex formation between fVIIIa and fIXa on the phospholipid surface, and the fVIIIa-fIXa interaction is not influenced by the presence of the substrate, factor X. Addition of fVIII to Fl-M-FPR-fIXa and PCPS produced a small, saturable increase in anisotropy (delta r = 0.03), followed by a larger increase (delta r = 0.07) upon addition of thrombin to activate fVIII. Thus, fVIII binds fIXa, but proteolytic modification of fVIII must occur before the complete fVIIIa-dependent structural change in the active site of fIXa, as reflected in the anisotropy change, occurs     

Effect of Vitamin K-dependent Protein Precursor Propeptide,Vitamin K Hydroquinone,and Glutamate Substrate Binding on the Structure and Function of γ-Glutamyl Carboxylase

The γ-glutamyl carboxylase utilizes four substrates to catalyze carboxylation of certain glutamic acid residues in vitamin K-dependent proteins. How the enzyme brings the substrates together to promote catalysis is an important question in understanding the structure and function of this enzyme. The propeptide is the primary binding site of the vitamin K-dependent proteins to carboxylase. It is also an effector of carboxylase activity. We tested the hypothesis that binding of substrates causes changes to the carboxylase and in turn to the substrate-enzyme interactions. In addition we investigated how the sequences of the propeptides affected the substrate-enzyme interaction. To study these questions we employed fluorescently labeled propeptides to measure affinity for the carboxylase. We also measured the ability of several propeptides to increase carboxylase catalytic activity. Finally we determined the effect of substrates: vitamin K hydroquinone, the pentapeptide FLEEL, and NaHCO₃, on the stability of the propeptide-carboxylase complexes. We found a wide variation in the propeptide affinities for carboxylase. In contrast, the propeptides tested had similar effects on carboxylase catalytic activity. FLEEL and vitamin K hydroquinone both stabilized the propeptide-carboxylase complex. The two together had a greater effect than either alone. We conclude that the effect of propeptide and substrates on carboxylase controls the order of substrate binding in such a way as to ensure efficient, specific carboxylation.     

Studies on the partial characterization of extracted glycosaminoglycans from fish waste and its potentiality in modulating obesity through in-vitro and in-vivo

Glycoconjugate Journal - Glycosaminoglycans (GAGs) are bioactive polysaccharides or glycoconjugates found in the fish waste having significant health impacts. In the present study it has been...     

Determination of disulfide bond assignment of human vitamin K-dependent gamma-glutamyl carboxylase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Vitamin K-dependent gamma-glutamyl carboxylase is a 758 amino acid integral membrane glycoprotein that catalyzes the post-translational conversion of certain protein glutamate residues to gamma-carboxyglutamate. Carboxylase has ten cysteine residues, but their form (sulfhydryl or disulfide) is largely unknown. Pudota et al. in Pudota, B. N., Miyagi, M., Hallgren, K. W., West, K. A., Crabb, J. W., Misono, K. S., and Berkner, K. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13033-13038 reported that Cys-99 and Cys-450 are the carboxylase active site residues. We determined the form of all cysteines in carboxylase using in-gel protease digestion and matrix-assisted laser desorption/ionization mass spectrometry. The spectrum of non-reduced, trypsin-digested carboxylase revealed a peak at m/z 1991.9. Only this peak disappeared in the spectrum of the reduced sample. This peak's m/z is consistent with the mass of peptide 92-100 (Cys-99) disulfide-linked with peptide 446-453 (Cys-450). To confirm its identity, the m/z 1991.9 peak was isolated by a timed ion selector as the precursor ion for further MS analysis. The fragmentation pattern exhibited two groups of triplet ions characteristic of the symmetric and asymmetric cleavage of disulfide-linked tryptic peptides containing Cys-99 and Cys-450. Mutation of either Cys-99 or Cys-450 caused loss of enzymatic activity. We created a carboxylase variant with both C598A and C700A, leaving Cys-450 as the only remaining cysteine residue in the 60-kDa fragment created by limited trypsin digestion. Analysis of this fully active mutant enzyme showed a 30- and the 60-kDa fragment were joined under non-reducing conditions, thus confirming Cys-450 participates in a disulfide bond. Our results indicate that Cys-99 and Cys-450 form the only disulfide bond in carboxylase.     

Yield and quality of forage maize (<i>Zea mays</i> L.) with different levels of subsurface drip irrigation and plant density

The scarcity of water in arid and semiarid regions of the world is a problem that every day increases by climate change. The subsurface drip irrigation (SDI) and changes in population density of plants are alternatives that can be used to make a sustainable use of water. Therefore, the objectives of this study were to determine the combination that allows for an increased corn performance and efficient use of water without losing the quality of forage. Three different irrigation levels were applied through a system of a SDI at three different densities of forage maize plants in an arid region. The results demonstrated that by applying different levels of water, either enough or lack of soil moisture is created, which is directly reflected in crop yield, and its determining variables such as green forage and dry matter yield, and nutritional quality. The irrigation level to a 100% of potential evapotranspiration (PET), at a density of 80000 plants/ha, increased yield of green forage to 57664 kg/ha; crude protein was 8.59%, while the rest of the quality parameters decreased. This study allowed to conclude that the irrigation level was the major factor in the response of the crop.