Purification and properties of d-galactose-binding lectins from some erythrina species: Comparison of properties of lectins from E. indica,E. arborescens,E. suberosa, and E. lithosperma

The lectins of the seeds of four species of the genus Erythrina, namely E. indica, E. arborescens, E. lithosperma, and E. suberosa were isolated by affinity chromatography on acid-treated ECD-Sepharose 6B. The lectins were found homogeneous in polyacrylamide gel electrophoresis and immunochemical tests. In SDS-gel electrophoresis, E. indica and E. lithosperma lectins each gave two bands with subunit molecular weights of 30,000 and 33,000 in the case of the former and 26,000 and 28,000 in the case of the latter. E. arborescens and E. suberosa gave single bands corresponding to polypetide chain molecular weight of 28,000. The lectins were found to be glycoproteins with their neutral sugar contents ranging from 4–9%. In carbohydrate specificity all the lectins were d-galactose specific. Their close similarity was also demonstrated by their homologous cross-reaction against the antiserum to E. indica lectin. In hemagglutinating activity toward human erythrocytes, E. indica and E. suberosa lectins showed higher activity toward the O group and E. arborescens toward the B group. The results show the similarity of the lectins derived from different species of the same genus in respect of immunochemical properties and carbohydrate specificity. In studies on E. indica lectin, the protein was found homogeneous by electrophoretic, immunochemical, and sedimentation experiments. Its molecular weight of 68,000 determined from sedimentation and diffusion data indicated that the molecule was a dimer of two noncovalently bound unequal subunits whose SDS-gel electrophoretic molecular weights are noted above. The lectin was devoid of cysteine and methionine and contained valine as its N-terminal amino acid. It had 9% neutral sugars and 1.5% glucosamine. Equilibrium dialysis studies with lactose showed that the values of the association constant K at different temperatures were of similar orders of magnitude to other lectins and the dimeric molecule possessed two noninteracting binding sites.     

Sulfamethizole attenuates poloxamer 407-induced atherosclerotic neointima formation via inhibition of mTOR in C57BL/6 mice

Mammalian target of Rapamycin C1 (mTORC1) inhibition limits plaque progression in atherosclerosis. The present study evaluated the protective effect of sulfamethizole on poloxamer 407-induced atherosclerotic neointima formation in C57BL/6 mice via mTOR inhibition. Poloxamer 407 (P-407) (0.5 g/kg body weight) was administered intraperitoneally to male C57BL/6 mice every third day for 148 days to induce chronic hyperlipidemia. From Day 121 to 148, animals were additionally administered Sulfamethizole (5, 10, and 50 mg/kg, p.o.), Rapamycin (0.5 mg/kg, positive control), or vehicle (1 ml/kg). Plasma lipid levels were measured on Days 120 and 148. Upon sacrifice, histological studies were performed, and aortic tissue interleukin (IL)-6, tumor necrosis factor-α (TNF-α), and mTOR levels were evaluated. A molecular docking study was carried out to mimic the interaction of sulfamethizole with mTOR protein. Chronic P-407 administration significantly (p < 0.001) elevated plasma lipid levels, compared with those of the normal control group. Chronic hyperlipidemia resulted in increased tunica intima thickness, collagen deposition, and IL-6, TNF-α, and mTOR levels. Treatment with Sulfamethizole attenuated these parameters significantly in a dose-dependent manner. Molecular docking studies showed a significant interaction of Sulfamethizole with mTOR. In conclusion, this study suggests that sulfamethizole significantly limits poloxamer 407-induced atherosclerotic neointima formation in C57BL/6 mice via mTOR inhibition.     

Hypocholesterolemic effect of anhydrous milk fat ghee is mediated by increasing the secretion of biliary lipids

The anhydrous milk fat ghee is one of the important sources of fat in the Indian diet. Our earlier studies showed that rats fed diets containing greater than 2.5 wt% of ghee had lower levels of serum cholesterol compared with rats fed diets containing groundnut oil. To evaluate the mechanism of the hypocholesterolemic effect of ghee, male Wistar rats were fed a diet containing 2.5 or 5.0 wt% ghee for a period of 8 weeks. The diets were made isocaloric with groundnut oil. Both native and ghee heated at 120 degrees C containing oxidized lipids were included in the diet. The ghee in the diet did not affect the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity in the liver microsomes, but it significantly increased biliary excretion of cholesterol, bile acids, uronic acid, and phospholipids. The rats fed ghee had lower levels of cholesterol esters in the serum as well as in the intestinal mucosa. Both native and oxidized ghee influenced cholesterol metabolism. These results indicate that supplementation of diets with ghee lipids would increase the excretion of bile constituents and lower serum cholesterol levels.     

Protein kinase C plays an essential role in sildenafil-induced cardioprotection in rabbits

Sildenafil citrate (Viagra) is the most widely used pharmacological drug for treating erectile dysfunction in men. It has potent cardioprotective effects against ischemia-reperfusion injury via nitric oxide and opening of mitochondrial ATP-sensitive K(+) channels. We further investigated the role of protein kinase C (PKC)-dependent signaling pathway in sildenafil-induced cardioprotection. Rabbits were treated (orally) with sildenafil citrate (1.4 mg/kg) 30 min before index ischemia for 30 min and reperfusion for 3 h. The PKC inhibitor chelerythrine (5 mg/kg i.v.) was given 5 min before sildenafil. Infarct size (% of risk area) reduced from 33.65 +/- 2.17 in the vehicle (saline) group to 15.07 +/- 0.63 in sildenafil-treated groups, a 45% reduction compared with vehicle (mean +/- SE, P < 0.05). Chelerythrine abolished sildenafil-induced protection, as demonstrated by increase in infarct size to 31.14 +/- 2.4 (P < 0.05). Chelerythrine alone had an infarct size of 33.5 +/- 2.5, which was not significantly different compared with DMSO-treated group (36.8 +/- 1.7, P > 0.05). Western blot analysis demonstrated translocation of PKC-alpha, -, and -delta isoforms from cytosol to membrane after treatment with sildenafil. However, no change in the PKC-beta and -epsilon isoforms was observed. These data provide direct evidence of an essential role of PKC, and potentially PKC-alpha, -, and -delta, in sildenafil-induced cardioprotection in the rabbit heart.     

Singlet oxygen: a potential culprit in myocardial injury?

The purpose of this study was to explore the role of singlet oxygen in cardiovascular injury. To accomplish this objective, we investigated the effect of singlet oxygen [generated from photoactivation of rose-bengal] on the calcium transport and Ca²⁺-ATPase activity of cardiac sarcoplasmic reticulum and compared these results with those obtained by superoxide radical, hydrogen peroxide and hydroxyl radical. Isolated cardiac SR exposed to rose bengal (10 nM) irradiated at (560 nm) produced a significant inhibition of Ca ²⁺ uptake; from 2.27 ± 0.05 to 0.62 ± 0.05 µmol Ca⁺/mg.min (mean ± SE) (P < 0.01) and Ca²⁺-ATPase activity from 2.08 ± 0.05 µmol Pi/min. mg to 0.28 ± 0.04 µmol Pi/min. mg (mean ± SE) (P < 0.01). The inhibition of calcium uptake and Ca²⁺-ATPase activity by rose bengal derived activatedoxygen (singlet oxygen) was dependent on the duration of exposure and intensity of light. The singlet oxygen scavengers ascorbic acid and histidine significantly protected SR Ca²⁺-ATPase against rose bengal derived activated oxygen species but superoxide dismutase and catalase did not attenuate the inhibition. SDS-polyacrylamide gel electrophoresis of SR exposed to photoactivated rose bengal up to 14 min, demonstrated complete loss of Ca²⁺-ATPase monomer band which was significantly protected by histidine. Irradiation of rose bengal also caused an 18% loss of total sulfhydryl groups of SR. On the other hand, superoxide (generated from xanthine oxidase action on xanthine) and hydroxyl radical (0.5 mM H₂O₂ + Fe²⁺ -EDTA) as well as H₂O₂ (12 mM) were without any effect on the 97,000 dalton Ca²⁺-ATPase band ofsarcoplasmic reticulum. The results suggest that oxidative damage of cardiac sarcoplasmic reticulum may be mediated by singlet oxygen. This may represent an important mechanism by which the oxidative injury to the myocardium induces both a loss of tension development and arrhythmogenesis.     

Phospholipid fatty acid modification of rat liver microsomes affects acylcoenzyme A:cholesterol acyltransferase activity

The effect of phospholipid fatty acyl composition on the activity of acylcoenzyme A:cholesterol acyltransferase was investigated in rat liver microsomes. Specific phosphatidylcholine replacements were produced by incubating the microsomes with liposomes and bovine liver phospholipid-exchange protein. Although the fatty acid composition of the microsomes was modified appreciably, there was no change in the microsomal phospholipid or cholesterol content. As compared to microsomes enriched for 2 h with dioleoylphosphatidylcholine, those enriched with dipalmitoylphosphatidylcholine exhibited 30-45% less acyl-CoA:cholesterol acyltransferase activity. Enrichment with 1-palmitoyl-2-linoleoylphosphatidylcholine increased acyl-CoA:cholesterol acyltransferase activity by 20%. By contrast, dilinoleoylphosphatidylcholine abolished microsomal acyl-CoA:cholesterol acyltransferase activity almost completely. Addition of cofactors that stimulated microsomal lipid peroxidation inhibited acyl-CoA:cholesterol acyltransferase activity by only 10%, however, and did not increase the inhibition produced by submaximal amounts of dilinoleoylphosphatidylcholine. Certain of the phosphatidylcholine replacements produced changes in palmitoyl-CoA hydrolase, NADPH-dependent lipid peroxidase, glucose-6-phosphatase and UDPglucuronyl transferase activities, but they did not closely correlate with the alterations in acyl-CoA:cholesterol acyltransferase activity. Electron spin resonance measurements with the 5-nitroxystearate probe indicated that microsomal lipid ordering was reduced to a roughly similar extent by dioleoyl- or by dilinoleoylphosphatidylcholine enrichment. Since these enrichments produce widely different effects on acyl-CoA:cholesterol acyltransferase activity, changes in bulk membrane lipid fluidity cannot be the only factor responsible for phospholipid fatty acid compositional effect on acyl-CoA:cholesterol acyltransferase. The present results are more consistent with a modulation resulting from either changes in the lipid microenvironment of acyl-CoA:cholesterol acyltransferase or a direct interaction between specific phosphatidylcholine fatty acyl groups and acyl-CoA:cholesterol acyltransferase.     

Ischemic preconditioning in isolated perfused mouse heart: Reduction in infarct size without improvement of post-ischemic ventricular function

Genetically engineered mice provide an excellent tool to study the role of a particular gene in biological systems and will be increasingly used as models to understand the signal transduction mechanisms involved in ischemic preconditioning (IP). However, the phenomenon of IP has not been well characterized in this species. We therefore attempted to examine whether IP could protect isolated mouse heart against global ischemia/reperfusion (GI/R) injury. Thirty adult mice hearts were perfused at constant pressure of 55 mmHg in Langendorff mode. Following 20 min equilibration, the hearts were randomized into three groups (n = 10/each): (1) Control Group; (2) IP2.5 Group: IP with two cycles of 2.5 min GI + 2.5 min R; (3) IP5 Group: IP with 5 min GI + 5 min R. All hearts were then subjected to 20 min of GI and 30 min R (37°C). Ventricular developed force was measured by a force transducer attached to the apex. Leakage of CK and LDH was measured in coronary efflux. Infarct size was determined by tetrazolium staining. Following sustained GI/R, infarct size was significantly reduced in IP2.5 (13.8 ± 2.3%), but not in IP5 (20.1 ± 4.0%), when compared with non-preconditioned control (23.6 ± 3.8%) hearts. CK and LDH release was also reduced in both IP2.5 and IP5 groups. No significant improvement in post-ischemic ventricular contractile function was observed in either IP groups. We conclude that IP with repetitive cycles of brief GI/R is able to reduce myocardial infarct size and intracellular enzyme leakage caused by a sustained GI/R in the isolated perfused mouse heart. This anti-necrosis cardioprotection induced by IP was not associated with the amelioration of post-ischemic ventricular dysfunction.     

The anhydrous milk fat, ghee, lowers serum prostaglandins and secretion of leukotrienes by rat peritoneal macrophages

Ghee, the anhydrous milk fat, is one of the most important sources of dietary fat in India. Male Wistar rats were fed diets containing 2.5, 5.0 and 10 wt% ghee for a period of 8 weeks. The diets were made isocaloric with groundnut oil. The results showed that serum thromboxane levels decreased by 27-35%, and 6-keto-prostaglandin F1alpha by 23-37% when ghee was incorporated at level of 10% in the diet. Prostaglandin E2 levels in serum and secretion of leukotrienes B4, C4 and D4 by peritoneal macrophages activated with calcium ionophore decreased when increased amounts of ghee from 2.5 to 10% were included in the diet. Arachidonic acid levels in macrophage phospholipids decreased when incremental amounts of ghee were fed to rats. These studies indicate that ghee in the diet not only lowers the prostaglandin levels in serum but also decreases the secretion of leukotrienes by macrophages.     

Tissue distribution of cholinesterases and anticholinesterases in native and transgenic tomato plants

Acetylcholinesterase, a major component of the central and peripheral nervous systems, is ubiquitous among multicellular animals, where its main function is to terminate synaptic transmission by hydrolyzing the neurotransmitter, acetylcholine. However, previous reports describe cholinesterase activities in several plant species and we present data for its presence in tomato plants. Ectopic expression of a recombinant form of the human enzyme and the expression pattern of the transgene and the accumulation of its product in transgenic tomato plants are described. Levels of acetylcholinesterase activity in different tissues are closely effected by and can be separated from -tomatine, an anticholinesterase steroidal glycoalkaloid. The recombinant enzyme can also be separated from the endogenous cholinesterase activity by its subcellular localization and distinct biochemical properties. Our results provide evidence for the co-existence in tomato plants of both acetylcholinesterase activity and a steroidal glycoalkaloid with anticholinesterase activity and suggest spatial mutual exclusivity of these antagonistic activities. Potential functions, including roles in plant-pathogen interactions are discussed.     

Preincubation of Mesorhizobium ciceri with flavonoids improves its nodule occupancy

Strains ofM. ciceri, symbionts of chickpea (Cicer arietinum) were incubated with the flavonoids naringenin, daidzein and quercetin which have earlier been reported as inducers and inhibitors ofnodABC-lacZ fusion ofM. ciceri. Preincubation ofM. ciceri with naringenin and daidzein (100 nmol/L) for 1 d improved the competitive ability of the inoculated strain while preincubation with quercetin decreased the nodule occupancy of inoculated strain under sterile conditions. Under nonsterile conditions induced strains of Rcd 301 and HT-6 formed by 23 and 18% more nodules, respectively, than untreated control. Quercetin-treated strains showed by 13–20% fewer nodules than untreated controls. Therefore, it is possible to regulate the competitive ability of inoculated strains by flavonoid treatment.