Activities of Enzymes of Carbohydrate Metabolism during Development of Okra [Abelmoschus esculentus (L.) Moench] Seed

Changes in activity levels of important enzymes of carbohydratemetabolism (and ß-amylases, sucrose synthetase, acidinvertase, acid phosphatase, glucose phosphate isomerase, aldolase,phosphofructokinase and pyruvate kinase) during seed developmentwere determined. Changes in the activities of these enzymesand their functional significance in developing seeds are described.A close correlation was found between the stage of maximum carbohydrateoxidation and the accumulation of reserve materials in the developingseeds. ¹Present address: School of Agriculture and Forestry, Universityof Melbourne, Parkvilie 3052, Victoria, Australia. ²Present address: School of Botany, University of Melbourne,Parkville 3052, Victoria, Australia. (Received October 8, 1982; Accepted January 10, 1983)     

Effect of complement depletion on lung fluid balance after thrombin

We examined the effect of complement depletion on lung fluid and protein exchange after thrombin-induced pulmonary thromboembolization. Sheep were prepared with lung lymph fistulas to assess pulmonary transvascular fluid and protein dynamics. Studies were made in three groups: in group I (n = 5) pulmonary thromboembolization (PT) was induced by an iv infusion of thrombin (55.0 +/- 12.9 NIH U/kg); in group II (n = 6) cobra venom factor (CVF) was given ip (94.5 +/- 18.8 U/kg/day) for 2 days to deplete complement, and then thrombin (66.4 +/- 37.0 NIH U/kg) was infused to raise pulmonary vascular resistance to the same level as in group I; in group III (n = 10) left atrial pressure (Pla) was increased by 10-15 Torr in normal animals by inflation of a Foley balloon catheter. In group I, thrombin infusion caused an increase in pulmonary lymph flow (Qlym) with a gradual increase in the lymph-to-plasma protein concentration ratio (L/P). In complement-depleted sheep, thrombin caused a transient increase in Qlym, which was associated with a decrease in L/P. In group I an increase in Pla further increased Qlym but without a change in L/P, indicating an increase in lung vascular permeability to proteins; whereas in the decomplemented-thrombin sheep raising Pla increased Qlym but decreased L/P. Results in the latter group were similar to those obtained in normal animals after left atrial hypertension (group III). Therefore the complement system participates in the increase in lung vascular permeability following thrombin-induced microembolization.     

Pulmonary transvascular flux of transferrin

We compared the pulmonary transvascular fluxes of transferrin and albumin in the intact sheep lung. Anesthetized sheep were prepared with lung lymph fistulas. The vascular blood pool was marked with 99mTc-erythrocytes, autologous transferrin was labeled with 113mIn, and albumin was labeled with 125I. Samples of blood, plasma, lymph, and lung were obtained up to 180 min after tracer infusion. Lymph tissue radioactivities were corrected for the intravascular component and expressed as extravascular-to-plasma concentration ratios. Clearance of transferrin and albumin from the plasma space followed a two-compartment model. The clearance rate constant was 2.1 +/- 0.1 x 10(-3) min for albumin and 2.4 +/- 0.1 x 10(-3) min for transferrin (P less than 0.05). Lymph-to-plasma ratios for albumin and transferrin were not different. However, the extravascular-to-plasma ratio for albumin was greater than transferrin (P less than 0.05). The lymph and lung data were deconvoluted for the plasma input function and fit to a two-compartment model. The results indicate that albumin and transferrin have similar permeabilities across the vascular barrier but have different pulmonary circulation to lymph kinetics because the extravascular volume of distribution of albumin is greater than transferrin.     

Relative regeneration proficiency ofArabidopsis thaliana ecotypes

We have compared regeneration proficiency for cultured explants from different tissues of different ecotypes ofArabidopsis thaliana. Proficiency varies widely with both tissue and ecotype, and is highest when the flux of light during regeneration is low. Analysis of F1 hybrids suggests that high proficiency is dominant to low proficiency.     

The mechanism of photodynamic inactivation ofStaphylococcus aureus by deuteroporphyrin

The mechanism ofStaphylococcus aureus inactivation by deuteroporphyrin (DT) and light was studied with singlet oxygen quenchers or hydroxyl radical scavengers. The light-activated DT (10 /ml) reduced the viability of the culture to less than 1%, whereas methionine, tryptophan, and 1,4-diazabicyclo-2,2,2-octane (DBCO) used as singlet oxygen quenchers provided almost 60% protection. Propylgallate, which is a hydroxyl free radical scavenger, also provided 60% protection. The presence of a singlet oxygen quencher and propylgallate provided almost complete protection from inactivation (96%). Photoinactivation in the absence of culture media (in saline) increased the killing rate and decreased the ability of the singlet oxygen quenchers to protect. In the same conditions damage from hydroxl free radicals was well protected by propyl gallate. The present results indicate thatS. aureus photoinactivation by DT and light is mediated by both singlet oxygen and hydroxyl free radicals.     

On the biological occurrence and regulation of 1-acyl and 1-O-alkyl-diradylglycerols in human neutrophils. Selective destruction of diacyl species using Rhizopus lipase

The occurrence and regulation of 1-ether-linked diradylglycerol in human neutrophils were investigated using a sensitive and practical analytical mass method which distinguishes 1-O-alkyl- (EAG) versus 1-acyl (DAG) diglycerides. After phosphorylation of diglycerides to the corresponding [32P]phosphatidic acids using [gamma-32P]ATP and diglyceride kinase (Preiss, J., Loomis, C. R., Bishop, W. R., Stein, R., Niedel, J. E., and Bell, R. M. (1986) J. Biol. Chem. 261, 8597-8600), lipase from Rhizopus arrhizus selectively degraded the 1-acyl-containing species (DAG), but the ether lipid (EAG) was resistant and was identified and quantified after thin layer chromatography separation. By using this method, unstimulated neutrophils were demonstrated to contain both DAG and EAG (100-180 and 40-95 pmol/10(7) cells, respectively). The chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) caused a rapid (30 s) and transient increase (1.6-fold) in DAG, but no increase in EAG. Opsonized zymosan produced a 6-8-fold sustained increase in DAG peaking at 2 to 3 min, but only a small (1.7-fold) increase in EAG which was not seen until later times (10 min). Thus, under these stimulation conditions, the major diglyceride was DAG. However, in neutrophils "primed" with cytochalasin B or phorbol ester, formyl-methionyl-leucyl-phenylalanine caused a significant increase in EAG. Neutrophils pretreated with cytochalasin B and then stimulated by fMLP showed a rapid (15-60 s) increase (more than 3-fold) in total diglycerides which was sustained beyond 5 min. At the earliest time points (15-30 s), the increase was due almost entirely to DAG (3-fold), but at 1 min and beyond, EAG comprised as much as 40% of the total (up to a 5-fold increase in EAG). Neutrophils pretreated with phorbol ester prior to fMLP stimulation showed a rapid (around 30 s) more than 2-fold increase in both DAG and EAG. Thus, priming conditions (in particular cytochalasin B) may alter either the access of phospholipase(s) C and/or D to membrane phospholipids or may affect their activities, allowing hydrolysis of 1-O-alkyl-containing lipids to generate 1-O-alkyl-containing diglycerides.     

Pulmonary microvascular endothelial cells constitutively release xanthine oxidase.

The generation of oxidants in reperfused ischemic tissues by xanthine oxidase (XO) may contribute to tissue damage. We exposed bovine pulmonary microvascular endothelial (BPMVE) cells to hypoxia and subsequent reoxygenation and examined alterations in intracellular and extracellular XO activities. BPMVE cells incubated 24 h under hypoxic conditions (less than 1% O2) showed a twofold increase in intracellular xanthine dehydrogenase activity and a smaller increase in intracellular XO activity compared to normoxic BPMVE. Both normoxic and hypoxic BPMVE cells constitutively released XO activity into their culture media. Incubation of hypoxic or normoxic BPMVE cells with oxygenated medium (95% O2) stimulated the release of XO activity into the extracellular medium within 5 min. The XO activity could not be detected in the oxygenated medium after 60 min incubation with 95% O2. These results indicate that endothelial cells in culture constitutively release XO and that oxygenation rapidly enhances XO release. The released XO activity may play an important role in generation of oxidants in the extracellular milieu during reperfusion.     

Purification, crystallization, and X-ray diffraction studies of lactotransferrin from buffalo colostrum.

Lactotransferrin is an iron-binding protein. It has been purified from buffalo colostrum. The purified lactotransferrin has been crystallized in 10% ethanol solution. The crystals are orthorhombic and the space group is P2(1)2(1)2(1) with unit cell dimensions a = 161.70 A, b = 155.75 A, c = 113.48 A. The asymmetric unit contains three molecules of the protein with a solvent content of about 59%. The crystals were stable in the X-ray beam and diffract beyond 3.5 A resolution. The native data have been collected and the structure determination is in progress.     

Cell-free translocation of recombinant p47-phox, a component of the neutrophil NADPH oxidase: effects of guanosine 5'-O-(3-thiotriphosphate), diacylglycerol, and an anionic amphiphile.

We reported previously that diacylglycerol (diC8) and GTP gamma S synergize with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to produce high rates of superoxide generation in a cell-free system consisting of neutrophil plasma membrane plus cytosol [Burnham, D. N., Uhlinger, D. J., & Lambeth, J. D. (1990) J. Biol. Chem. 265, 17550-17559]. Here we investigate the effects of these activating factors on the plasma membrane association in an in vitro translated radiolabeled recombinant p47-phox protein. Apparent translocation, assayed by cosedimentation with plasma membranes, required the presence of excess cytosol and an anionic amphiphile, was enhanced by both GTP gamma S and diC8, and was inhibited by high salt, correlating qualitatively with activation; up to 70% cosedimentation was observed with the combination of activators (compared with less than 20% in their absence). Similar results were obtained using heat-inactivated cytosol, wherein another oxidase component, p67-phox, has been inactivated. Unexpectedly, from 50 to 80% of the apparent translocation occurred in the absence of membranes, indicating that protein aggregation accounted for a significant part of the observed translocation. Nevertheless, the percent translocation was increased in all cases by the presence of membranes, indicating some degree of protein-membrane interaction. While a control in vitro translated protein failed to translocate, cosedimentation of p47-phox occurred equally well when red blood cell or neutrophil plasma membranes lacking cytochrome b558 were used. Also, the peptide RGVHFIF, which is contained within the C-terminus of the large subunit of cytochrome b558, failed to inhibit translocation/aggregation of p47-phox, despite its ability to inhibit cell-free activation of the oxidase. The data are consistent with the following: (a) SDS, diC8, and GTP gamma S all act on cytosolic components to alter protein-protein and/or protein-membrane associations, and these changes are necessary (but not sufficient) for activation; (b) these altered associations are likely to function by increasing the local concentration of p47-phox and other components at the plasma membrane; (c) a high background of nonspecific associations in the cell-free activation system is likely to obscure any specific, functionally relevant associations (e.g., with cytochrome b558); and (d) the mechanism of translocation in the cell-free system differs from that seen in intact neutrophils.     

Role of disulfide exchange in alpha 1-protease inhibitor.

The major endogenous inhibitor of neutrophil elastase in the plasma, alpha 1-protease inhibitor (alpha 1-PI), has a single cysteine residue which has been shown to form mixed disulfides with a number of thiols in vitro. Under normal physiological conditions, the plasma concentrations of reduced and oxidized thiols are such that a major fraction of alpha 1-PI in the circulation in vivo is in the form of mixed disulfides [Laurell, C.-B. (1979) in The Chemistry and Physiology of Human Plasma Proteins (Bing, D. H., Ed.) pp 329-341, Pergamon, New York]. We show here that the mixed disulfide between glutathione or cysteine and alpha 1-PI (alpha 1-PI-SSG or alpha 1-PI-SScys) has an intrinsic fluorescence which distinguishes it from the reduced form of alpha 1-PI. By employing the fluorescence difference, we have measured the ratio of alpha 1-PI-SH to mixed disulfide alpha 1-PI in redox buffers of different ratios of reduced to oxidized glutathione (GSH to GSSG) or reduced to oxidized cysteine (cys to cysSScys) and have calculated an equilibrium constant and redox potential of 0.74 +/- 0.08 and 8 +/- 2 mV, respectively, for the alpha 1-PI-SH/alpha 1-PI-SSG couple and of 0.32 +/- 0.02 and 29 +/- 2 mV, respectively, for the alpha 1-PI-SH/alpha 1-PI-SScys couple. We are unable to detect any change in Trp fluorescence in the complex of alpha 1-PI and elastase when the preformed complex is added to the same GSH/GSSG or cys/cysSScys redox buffers.(ABSTRACT TRUNCATED AT 250 WORDS)