An improved, simple and efficient method for preparation of monomethoxypolyethylene glycol (PEG) activated with p-nitrophenylchloroformate (PNP-PEG) and its use as a potent modifier of protein under mild conditions are described. Modification of bovine serum albumin with PNP-PEG was compared with that done with PEG activated with N, N′-carbonyldiimidazole or cyanuric chloride. The reaction of PEG, activated with either p-nitrophenylchloroformate or cyanuric chloride, with bovine serum albumin at 4°C reached a plateau within 1h, whereas protein modification using PEG activated with N, N′-carbonyldiimidazole was rather slow and gave a low yield. The remaining activity of L-asparaginase modified with PNP-PEG was much higher than that of the enzyme modified to the same degree with PEG activated with cyanuric chloride. At a 20 molar excess of PNP-PEG having a molecular weight of 5, 000, 55% of the free amino acid groups were modified at 4°C for 2h, and the modified enzyme still had 33% residual enzyme activity. Immunochemical studies showed that the highly modified enzyme (67% modification with 18% residual enzyme activity) had lost its immunogenicity and had become much less sensitive to protease digestion.
Naturally occurring plant phenols, which included ellagic acid (EA), tannic acid (TA), caffeic acid (CA), and ferulic acid (FA), were tested for their superoxide anion radical (SOR)-scavenging activities. SOR was produced by interaction of tumor promoters, i.e. phorbol-12-myristate-13-acetate (PMA) or mezerein, in vitro with murine peritoneal macrophages. The levels of SOR were assessed microscopically by counting the number of formazan-positive (F+) cells/250 cells produced due to the reduction of nitroblue-tetrazolium. PMA at a concentration of 0.1μg/1.85×106cells/0.5ml induced maximum formation of SOR both in resident and thioglycollate-elicited cells, whereas mezerein induced the maximum formation of SOR at 10μg/1.85×106cells/0.5ml. All the tested polyphenols were able to inhibit the formation of SOR induced by the tumor promoters to a variable degree. The maximum inhibition by EA of SOR formation induced by PMA or mezerein was at a concentration of 250μM or 125μM, respectively. The polyphenols CA, TA, and FA were more effective in inhibition of SOR stimulation at a concentration of 120 and 60μM with promoters PMA and mezerein, respectively. However, ferulic acid was found to be the most effective inhibitor of SOR formation.
The effect of eugenol on lipoxygenase-catalyzed lipid peroxidation was studied in aqueous solution and in microsomal lipid liposomes. Eugenol did not inhibit lipoxygenase-catalyzed linoleic acid hydroperoxide formation in aqueous medium as assessed by oxygen uptake and linoleic acid hydroperoxide formation. However, using the same criteria and formation of thiobarbituric acid-reactive substances, we found that eugenol inhibited lipid oxygenation when the reaction was carried out in linoleic acid-containing liposomes. These results suggest that the peroxyl and carbon-centered radical intermediates generated by lipoxygenase in a membrane environment are readily accessible to eugenol for quenching and concomitant inhibition of lipid peroxidation.
In this study using everted sacs of rat jejunum, we investigated the difference in absorption of two types of soybean peptides (small peptide: SP, and large peptide: LP). We investigated the influence of peptide length on absorption and intact transport of hydrolyzed soybean peptides. The everted sacs were incubated for 5 or 10min in bicarbonate-saline buffer containing a 1% (w/v) concentration of each soybean peptide. After the incubation, the amounts of free amino acids and peptides that were transported to the serosal fluids and the mucosa were measured with an automatic amino acid analyzer both before and after the hydrolysis of the serosal fluids and mucosa. The results indicated that SP was absorbed more rapidly than LP and that the more rapid absorption of SP was due to the greater intact transport of SP. When the everted sacs were made from jejunum that had been injured by intraperitoneal injection of cyclophosphamide (300mg/kg), no significant differences were noted between the absorption of SP and that of LP. We also ascertained that an aminopeptidase inhibitor (bestatin) decreased the aminopeptidase activities of the rat jejunum. In the presence of 1×10-4M bestatin in the incubation buffer, no significant differences were noted between the absorption of SP and that of LP. We conclude that SP is absorbed more rapidly than LP in normal rat jejunum due to greater intact transport.
The modifying effects of naturally occurring plant products such as neem leaf and turmeric extracts on 4-nitroquinoline-1-oxide (4NQO)-induced oral carcinoma were investigated in male Wistar strain rats. All the animals painted with 4NQO on their cheek mucosa for 20 weeks developed oral neoplasms identified histologically as squamous cell carcinoma. The oral tumor tissue showed a decrease in lipid peroxidation with concomitant changes in antioxidant activity. In groups of animals painted with 4NQO and simultaneous oral administration of neem leaf and turmeric extract, respectively, no tumors were observed; and the lipid peroxidation and antioxidant levels showed a pattern similar to that of untreated controls. Thus, the anticarcinogenic effect of neem leaf and turmeric extract appears to be related to their antioxidant function. We envisage that measurement of lipid peroxidation and antioxidant status with histological changes could be used to biomonitor tumor progression and evaluate the therapeutic response.
We measured plasma levels of lipoprotein (a) [Lp(a)] in 117 individuals. A highly significant relationship between Lp(a) and increasing age was found in hypertensive patients but not in normals. These patients also showed marked variation in both total cholesterol and low-density lipoprotein (LDL)-cholesterol levels. In diabetic subjects, despite the significant elevation in TG and very low-density lipoprotein-cholesterol, the increase in Lp(a) concentration was not significant, probably due to a more rapid clearance of triglyceride rich particle apo (a) of Lp(a) compared to the slower apo (a) catabolism in the LDL density range.
The effect of sodium orthovanadate treatment on the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, and the level of lipid peroxide and glutathione of liver, kidney, heart, intestine and testes was studied in streptozotocin-induced diabetic rats. Superoxide dismutase and catalase activities were lowered in liver, kidney, intestine, and testes of the diabetic rats, whereas catalase activity in heart and glutathione peroxidase activity in kidney were elevated. Glutathione peroxidase in liver, glutathione S-transferase in liver and intestine, and glutathione content in liver and testes were found to be decreased in the diabetic animals. The levels of lipid peroxide were found to be increased in all the tissues except the heart, where there was no change. Treatment of diabetic rats with sodium orthovanadate restored the altered activities of catalase, superoxide dismutase, glutathione peroxidase, glutathione S-transferase and the level of lipid peroxide and glutathione to their normal level. The present findings suggest that vanadate can effectively normalize the diabetesinduced alterations to tissue defense systems.