Proteinase Inhibitor II Gene Expression Induced by Electrical Stimulation and Control of Photosynthetic Activity in Tomato Plants

Mechanical damage and heat stimulation were used to activateproteinase inhibitor II (Pin2) gene expression in tomato plantsin both treated (local induction) and non-treated tissues (systemicinduction). Both stimuli have been shown to generate electricalsignals, leading to a systemic activation of gene expression.Treatment of tomato leaves with electrical current resultedin the accumulation of Pin2 mRNA in the local and systemic leaves.Additionally, all treatments inducing Pin2 gene activity gaverise to a significant alteration of stomatal aperture. However,heat stimulation provoked a different response in the stomatalparameters than mechanical wounding or electric treatment. Bothmechanical damage and electrical stimulation activated two characteristictime constants in the gas exchange relaxation kinetics. Conversely,heat stimulation resulted in only one major time constant. Theresults clearly show that direct current application to tomatoleaves initiates Pin2 mRNA accumulation locally and systemically.In addition, they suggest the participation of a second slowelectrical/hydraulic component in the wound response mechanismof tomato plants and a possible alternative pathway regulatingheat-induced Pin2 gene expression. (Received February 13, 1995; Accepted April 14, 1995)     

The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C.

The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far.     

Modulation of IL-4 production in murine spleen cells by prostaglandins

Recently, it has been reported that IL-4 production by murine Th2 cell lines is insensitive to inhibition by E-type prostaglandins. In the present study, IL-4 production in vitro by freshly isolated concanavalin A (Con A)-stimulated murine spleen cells was readily suppressed by PGE₂ with an I₅₀ of 2 nM. Comparable suppression by PGE₂ was seen after priming by anti-CD3? antibody instead of Con A or with other changes in the culture conditions. PGE₂ was an effective inhibitor after elimination of Ly2.2⁺ T cells, consistent with a direct effect on Th2 cells. In the absence of added prostaglandins, IL-4 production was enhanced 1.5- to 7.0-fold by 0.2–2.0 μM indomethacin, indicating that endogenous arachidonate metabolites such as PGE₂ and PGI₂ regulate IL-4 production in our usual culture system. The inhibition of Th2 cell secretion by PGE₂in vitro may have physiologic and pharmacologic implications for the regulation of Th2 cell function and IgE production in vivo.     

Conservation of the genes for dissimilatory sulfite reductase from Desulfovibrio vulgaris and Archaeoglobus fulgidus allows their detection by PCR.

The structural genes for dissimilatory sulfite reductase (desulfoviridin) from Desulfovibrio vulgaris Hilden-borough were cloned as a 7.2-kbp SacII DNA fragment. Nucleotide sequencing indicated the presence of a third gene, encoding a protein of only 78 amino acids, immediately downstream from the genes for the alpha and beta subunits (dsvA and dsvB). We designated this protein DsvD and the gene encoding it the dsvD gene. The alpha- and beta-subunit sequences are highly homologous to those of the dissimilatory sulfite reductase from Archaeoglobus fulgidus, a thermophilic archaeal sulfate reducer, which grows optimally at 83 degrees C. A gene with significant homology to dsvD was also found immediately downstream from the dsrAB genes of A. fulgidus. The remarkable conservation of gene arrangement and sequence across domain (bacterial versus archaeal) and physical (mesophilic versus thermophilic) boundaries indicates an essential role for DsvD in dissimilatory sulfite reduction and allowed the construction of conserved deoxyoligonucleotide primers for detection of the dissimilatory sulfite reductase genes in the environment.     

Increases in α-mannosidase activity in the arbuscular mycorrhizal symbiosis of Allium schoenoprasum

 Mycorrhizal and nonmycorrhizal roots of Allium schoenoprasum were tested for activities of α-mannosidase, β-glucosidase and arabinosidase. Mannosidase activity was higher by a factor of two in mycorrhizal than in nonmycorrhizal root extracts. The apparent molecular weight of the enzyme was 152 kDa and its K_M was 1.25 mM in colonized roots and 1.85 mM in uncolonized roots. α-Mannosidase activity was further characterized by an acid pH optimum and Zn²⁺ dependency. No significant differences could be found between mycorrhizal and nonmycorrhizal roots for β-glucosidase and arabinosidase activities. Accepted: 28 August 1995     

Biochemical basis for effects of k-deficiency on assimilate export rate and accumulation of soluble sugars in soybean leaves

The effects of K-deficiency on carbon exchange rates (CER), photosynthate partitioning, export rate, and activities of key enzymes involved in sucrose metabolism were studied in soybean (Glycine max [L.] Merr.) leaves. The different parameters were monitored in mature leaves that had expanded prior to, or during, imposition of a complete K-deficiency (plants received K-free nutrition solution). In general, recently expanded leaves had the highest concentration of K, and imposition of K-stress at any stage of leaf expansion resulted in decreased K concentrations relative to control plants (10 millimolar K). A reduction in CER, relative to control plants, was only observed in leaves that expanded during the K-stress. Stomatal conductance also declined, but this was not the primary cause of the decrease in carbon fixation because internal CO₂ concentration was unaffected by K-stress. Assimilate export rate from K-deficient leaves was reduced but relative export, calculated as a percentage of CER, was similar to control leaves. Over all the data, export rate was correlated positively with both CER and activity of sucrose phosphate synthase in leaf extracts. K-deficient leaves had higher concentrations of sucrose and hexose sugars. Accumulation of hexose sugars was associated with increased activities of acid invertase. Neutral invertase activity was low and unaffected by K-nutrition. It is concluded that decreased rates of assimilate export are associated with decreased activities of sucrose phosphate synthase, a key enzyme involved in sucrose formation, and that accumulation of hexose sugars may occur because of increased hydrolysis of sucrose in K-deficient leaves.     

Exoglucanases fromZea mays L. seedlings: their role inβ-D-glucan hydrolysis and their potential role in extension growth

Exoglucanases of corn seedlings were examined and evaluated in terms of their participation in the hydrolysis of cell-wall β-D-glucan and their possible role in extension growth. An exo-β-1,3-glucanase (EC 3.2.1.58), a component of the protein dissociated from isolated wall by use of high salt solutions, was purified using gel-filtration and ion-exchange chromatography. The purified enzyme hydrolyzed a number of polymeric and oligosaccharide substrates, including those of mixedlinkage, and their direct conversion to monosaccharide was evidence that the enzyme was capable of hydrolyzing both β1–4 and β1–3 linkages. The enzyme was considerably more active toward glucan that had been previously hydrolyzed by a cell-wall endo-β-D-glucanase. Similarly, the capacity of the purified exo-β-D-glucanase to degrade isolated wall was enhanced by more than 60% when the wall had been previously treated with the endoenzyme. The exo-β-D-glucanase did not exhibit growth-promoting properties nor was its activity, measured in vivo, enhanced by auxin. Another glucanase was obtained from the soluble fraction of seedling homogenates. It functioned strictly as a β-glucosidase and did not appear to participate in the hydrolysis of wall β-D-glucan.     

Effects of dibromothymoquinone on mung bean mitochondrial electron transfer and membrane fluidity

The effects of the quinone analog dibromothymoquinone on electron transfer in isolated mung bean mitochondria are described. Both the main, cyanidesensitive and the alternate, cyanide-insensitive pathways are inhibited by dibromothymoquinone but in markedly different fashions. Half-maximal inhibition appeared at 40 μM and 20 μM dibromothymoquinone for the cyanide-sensitive and alternate pathways, respectively. With succinate as the electron donor, dibromothymoquinone inhibited the alternate pathway at a single site; showing a mixed, non-competitive type inhibition. On the succinate, cyanide-sensitive pathway dibromothymoquinone showed two sites of inhibition and neither coincides with the site of inhibition associated with the alternate pathway. With malate as the electron donor, two sites of inhibition by dibromothymoquinone were observed regardless of the pathway measured.Dibromothymoquinone also inhibited the rate of valinomycin-induced swelling of isolated mung bean mitochondria. Steady-state kinetics showed the inhibition to be non-competitive with respect to valinomycin. Additionally dibromothymoquinone was observed to increase the fluorescence polarization associated with the hydrophobic probe 1,6-diphenylhexatriene. The results indicated that dibromothymoquinone decreased the fluidity of the inner mitochondrial membrane and suggested that the inhibition of mitochondrial electron transfer by dibromothymoquinone may be associated with this decrease in membrane fluidity.The relationship of the multisite nature of the inhibition of electron transfer by dibromothymoquinone and the possible role of mobile electron carriers such as ubiquinone on the main and alternate respiratory pathways of higher plants is discussed.     

Sequential conversion of the glutathionyl side chain of slow reacting substance (SRS) to cysteinyl-glycine and cysteine in rat basophilic leukemia cells stimulated with A-23187

In rat basophilic leukemia (RBL-1) cells stimulated with A-23187, the major slow reacting substance (SRS) species contain glutathione, cysteinyl-glycine, or cysteine in their side chains, corresponding or closely related to leukotrienes LTC4, LTD4, and LTE4, respectively. Evidence is presented that most of the SRS produced during the first few minutes of stimulation by the ionophore has a glutathionyl side chain which is sequentially converted to cysteinyl-glycine and cysteine.     

Aryl sulfatase inactivation of slow reacting substance. Evidence for proteolysis as a major mechanism when ordinary commercial preparations of the enzyme are used

Type II B arylsulfatases are known to inactivate slow reacting substance (SRS), but the mechanism is unclear. In the present study, ordinary commercial preparations of Sigma limpet arylsulfatase largely inactivated the glutathionyl and cysteinyl-glycyl forms of SRS, but the cysteinyl form of SRS was largely resistant to the enzyme. Evidence is presented which established that a major mechanism for the inactivation of the glutathionyl and cysteinyl-glycyl SRS types, at least by the particular enzyme preparations we have studied, involves cleavage of the glycine moiety from the sulfur containing side chain. This was confirmed by digestion studies with glutathione itself. In addition, there is some evidence to indicate that the enzyme may destabilize the double bond structure of the SRS molecule, contributing to the overall inactivation.