The reverse-phase evaporation method [Szoka, F., Jr., and Papahadjopoulos, D. (1978): Proc. Natl. Acad. Sci. USA., 75, 4194-4198] was modified to enable the preparation of liposomes entrapping plasmids without damage to DNA strands due to sonication. In our procedure, reverse-phase micelles were prepared first by sonication and then plasmids were added to form liposomes entrapping plasmids. The efficiency of the liposomes for entrapping of DNA by the present method was higher than that by the original method. By the present method, circular-pCH 110 was entrapped in liposomes without damage, while by the original method, some of the plasmids were cut during the preparation. The expression of the β-galactosidase gene encoded in plasmid pCH 110 was detected in COS-7 cells when the gene was transferred with the liposomes prepared by the present method, but not with the vesicles prepared by the original method.
A pCH 110 plasmid encoding the β-galactosidase gene of E. coli was effectively entrapped in novel positively charged liposomes consisting of N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride, dilauroyl phosphatidylcholine, and dioleoyl phosphatidylethanolamine (1:2:2, molar ratio) by an improved reverse-phase evaporation method. The DNA entrapping efficiency of these liposomes was 5-70 fold higher than that of liposomes containing no positively charged lipid. By using such positively charged liposomes, we could achieve transfection of the gene into COS-1 cells by simply adding the liposomes to the medium covering the cells; and the transfection efficiency exceeded that obtained by the calcium phosphate precipitation method, especially when a small amount of DNA was used.
The effects of 3 doses and 2 durations of iron supplement on iron-deficient rats were investigated. Two groups of 15 rats were given, respectively, a low iron-containing diet (4-5mg Fe/kg diet) and a medium iron-containing diet (15mg Fe/kg diet) for 4 weeks. At that time the first group was anemic and the second group was moderately iron-deficient, as shown by their iron status parameters. The two groups were then each divided into 3 subgroups of 5 rats each that received the low iron-containing diet supplemented with 35, 150, or 250mg of iron in the ferrous sulfate form per kg diet. Changes in their iron status and mean body weight was followed after 8 and 14 days of supplementation. Results showed that, after 14 days of supplementation, a dose of 35ppm was not sufficient to normalize hematological parameters. However, doses of 150 and 250mg Fe/kg diet normalized all hematological parameters after 8 days of diet, and corrected the decrease in body weight gain.
Monoamine oxidase (MAO) activities in rat liver perfusate obtained by perfusion with sucrose buffer and lipid peroxide and superoxide dismutase (SOD) activity levels in liver tissue were investigated after pretreatment of rats with total hepatic ischemia or the perilobular hepatotoxin allyl formate. MAO activities in rat liver perfusate assessed with β-phenylethylamine and 5-hydroxytryptamine as substrates and liver tissue lipid peroxide of these rats were increased, but SOD activity was decreased. The accumulation of lipid peroxides was partly attributed to the decreased activity of SOD as a consequence of membrane disorders. When allyl formate was administered to the rats, amine oxidase activities in plasma were not completely inhibited by MAO inhibitors (clorgyline or deprenyl), and the activity measured with 1μM benzylamine as substrate was elevated. However, the complete inhibition of amine oxidase activities in the perfusates was obtainable and the activity toward 1μM benzylamine was not observed. The Km values of these amine oxidases for β-phenylethylamine and 5-hydroxytryptamine were the same as those of liver mitochondrial MAO. These results suggest that MAO in liver mitochondria was released into the blood as a consequence of membrane disorder but that two or more other distinct amine oxidase activities that were detected in the plasma were not released from the liver.
Lipid hydroperoxides (LHP) were synthesized from linoleic acid and injected into the eyes of albino rabbits. This produced a decline in electroretinographic (ERG) activity, a corresponding loss of key molecules localized in the photoreceptor outer segments, and formation of new compounds related to oxidative damage. During the first 2 days of the experimental period, a rapid and irreversible drop in protein, phospholipid, total polyunsaturated fatty acids, vitamin A, and α-tocopherol was observed. This change was so complete that retinal tissue lacked sufficient substrate to undergo any further oxidative stress. Following this initial event, there was an increase in the level of a compound with properties similar to those of the injected LHP, which suggests a propagation phenomena. Corresponding increases in other products of LHP metabolism and formation of conjugated fluorescent pigments also occurred. In response to the oxidative insult, antioxidant enzymes such as peroxidase and glutathione-S-transferase and lysosomal enzymes in general showed changes, but at different time intervals. Thus, an early rise at 2 days post-injection was seen in peroxidase, whereas the rise in transferase activity occurred at 14 days and remained elevated. Lysosomal enzymes rose sharply at 2 days and stayed above baseline levels over the following 2-week period. The present study has demonstrated the LHP are capable of producing severe damage to the retina and are the cause of decreased ERG activity in this type of degeneration.
The anti-atherosclerotic effect of probucol was tested on weaned male New Zealand white rabbits. The rabbits, weighing 2kg, were first fed regular rabbit food ad libitum; and when their weight reached 3kg, they were placed on one of the following diets for 10 weeks: 1% cholesterol and 1% probucol rabbit food diet, or 1% cholesterol and regular rabbit food diet. At the end of the experiment we found that the presence of probucol in the diet reduced the level of plasma cholesterol and increased the resistance of low density lipoprotein to oxidation, whereas the drug had no significant beneficial effect of reducing the area of the aorta occupied by atherosclerotic plaques. When the data were probed further, it became apparent that the area of atherosclerotic plaques of the probucol/cholesterol-fed rabbit group was bimodal in distribution. In one subgroup of these rabbits, whose body weight reached 3kg between 6 and 8 weeks (fast grower) during the conditioning period, the area of atherosclerotic plaques (91.0±1.8% of total aortic surface) was the same as that of the cholesterol-fed fast growers (81.8±8%). In the other subgroup of rabbits, whose body weight reached 3kg between 10 and 11 weeks (slow grower) during the conditioning period, the area of atherosclerotic plaques (37.6±6.2%) was significantly (p<0.05) less than that of the cholesterol-fed slow growers (66.4±11.1%). In the comparison of the fast and slow growers on the probucol diet, the rate of weight gain was the only significant difference found. These observations suggest that the anti-atherosclerotic effect of probucol depends upon the rate of weight gain and that the effect may be mediated by an unknown mechanism.
We have investigated the effect of oligopeptides on brush border membrane aminopeptidase and cytosol peptidase activities in rat small intestine. Activities of these enzymes were significantly higher in the group fed a diet containing small peptides (SP group) than in the group fed a diet containing a mixture of amino acids (AA group). There were no significant differences in the activities of maltase, sucrase, lactase, and alkaline phosphatase between the two diet groups. The elevation of activity of brush border membrane aminopeptidase was inhibited by cycloheximide, indicating this enzyme to be an adaptive enzyme induced by oligopeptides as intraluminal substrates. The SP group also showed increased absorption of not only Gly-Leu, the dipeptide, but also of Gly and Leu, the free amino acids. The present study indicates that there could be advantages of using oligopeptides rather than amino acids as the nitrogen source in chemically defined diets from the viewpoint of peptidase activities as well as absorption of amino acids.
This is a clinical study covering 55 persons of male sex with ages ranging from 25 to 55 years. The patients were divided up into 3 groups as follows: Group A, operated on for perforating gastroduodenal ulcer where the postoperative period ran a course free of any complications (18 patients); Group B, operated on for gastroduodenal ulcer, complicated by postoperative diffuse purulent peritonitis, with a successful cure as a final outcome (16 patients); Group C, operated on for perforating gastroduodenal ulcer, complicated by postoperative diffuse purulent peritonitis, with lethal outcome (21 patients). In all groups under study assessment was made of the levels of thyrotropic hormone (TTH), triiodothyronine (T3), and thyroxine (T4) at 1, 3, and 5 days after laparotomy in Group A, and after relaparotomy in Groups B and C. In the patients of Group A, the dynamic changes in TTH, T3, and T4 levels disclosed values within normal limits. In Group B, the levels of TTH and T4 were within normal limits, while T3 showed a low value during the first and third day, reaching the lowermost limit of the norm on the fifth day. In the patients of Group C, inhibition of the entire thyrotropin-thyroid axis was observed: TTH, T3, and T4 levels fell abruptly, with the change in T3 being the most significant. These results suggest that the thyroid hormones may be related to these pathological conditions and that these hormones may be a good indicator for the prognosis of peritonitis.
Patients with hepatocellular carcinoma (HCC) frequently also suffer from chronic hepatitis or liver cirrhosis (LC). Preoperative nutritional assessment was made of HCC patients with or without LC and of patients with esophageal varices or cholelithiasis as controls. The patients with HCC and esophageal varices associated with LC were malnourished and were especially lacking in protein and other nutrients. The blood molar ratio of branched-chain amino acids (BCAA) to aromatic amino acids was low in LC patients. The effects of post-operative parenteral nutrition with a BCAA-enriched amino acid solution on the plasma amino acid profile and on protein renewal were evaluated in HCC patients with LC. Although the ratio was unchanged after intravenous hyperalimentation with a 21% BCAA-enriched solution, it rose in the group receiving a 31% BCAA-enriched solution. This group showed a rise in BCAA and a fall in aromatic amino acids in the plasma. Moreover, following hepatectomy, the serum level of prealbumin, a rapid turnover protein, improved significantly after intravenous hyperalimentation with the 31% BCAA-enriched solution. These results suggest that nutritional support with BCAA is a useful measure for the post-operative management of HCC patients with LC.