Influence of phosphatidylcholine/cholesterol molar ratios in liposomes on cholesterol reactivity with cholesterol:oxygen oxidoreductase]

The reactivity of sonicated phosphatidylcholine-cholesterol liposomes with cholesterol : oxygene oxydoreductase, an enzyme which catalyses the oxidation of the 3 beta hydroxyl group of cholesterol to a ketone group, is compared with that of ternary system phosphatidylcholine-cholesterol-Thesit. Regardless to the phosphatidylcholines nature and the phosphatidylcholine/cholesterol molar ratio (R), the enzymatic oxidation rate of liposomal cholesterol is slower than when the reaction is developed in the present of Thesit, a surfactif agent which destroyes the lamellar particles. This is true whether Thesit is added during preparation of dispersions or during incubation with cholesterol oxydase. The enzymatic oxydation rate of cholesterol of ternary systems phosphatidylcholine-cholesterol-Thesit is independent of the (R) value and the phosphatidylcholine fatty acid unsaturation, whereas that of phosphatidylcholine-cholesterol dispersions depends on these two parameters. The reaction rate increases in the order: dipalmitoylphosphatidylcholine to yolk egg phosphatidylcholines, and dioleylphosphatidylcholine. The optimal conditions for cholesterol oxidation were found to be R = 0.5. This result is not affected by the phosphatidylcholines nature. In order to explain these data, various hypotheses are considered. In particular, the weak liposomal cholesterol reactivity with cholesterol oxidase could result from an inhibitory effect on the enzyme-substrate combination due to the polar phosphorylcholine groups.     

Comparison of the acid denaturation of several hemoglobins which differ in amino acid sequence

There are pronounced differences in kinetic and thermodynamic stability between human and horse hemoglobins. Since the amino acid sequences of the α, β dimers of horse and human hemoglobins differ in 61 locations, it is difficult to account for them in terms of specific direct or indirect effects of the sequence differences. Rhesus hemoglobin differs from human in only 12 locations and its stability resembles that of human more closely than does horse, although pronounced differences remain. The stabilities of rhesus ferrihemoglobin and deoxyhemoglobin (Hb⁺ and Hb °) are intermediate between those of the corresponding high-spin forms of horse and human hemoglobin; but there are only small or negligible differences between the low-spin forms (carbonylhemoglobin and oxyhemoglobin) of the two species. The equilibrium isotherm between native and acid unfolded forms of rhesus Hb⁺ resembles that of horse more than that of human, but it is slightly more stable and slightly less cooperative. The effects of octanol on the rates of unfolding of rhesus ferrihemoglobin are only slightly smaller than with human. There is no effect of octanol on the unfolding rate of any of the CO hemoglobins. Unlike the equilibria of horse and human, octanol is also without effect on the unfolding equilibrium of rhesus ferrihemoglobin, and thus qualifies as a true catalyst of the initial stage of the acid unfolding reaction of the monkey ferriprotein. Differences in stability are tentatively attributed to a limited number of the 12 differences between the two proteins.     

Characterization and physico-chemical properties of human transcortin]

The molecular weight of human transcortin, calculated from the sedimentation coefficient, was found to be 49,500, thus slightly lower than previously reported values. After purification, human transcortin trended to polymerize rapidly, with participation of both non covalent bonds and one disulfide bridge per dimer. The physicochemical parameters, the amino-acid and carbohydrate composition were determined; its stability was studied under different conditions. Preliminary structural studies showed that the N-terminal sequence of the polypeptide chain was: Met-Asp-Pro-Asn-Ala-Ala-Tyr-Val and that the C-terminal amino acid was leucine.     

Changes in amino acid transport in the rat pancreas in response to fasting and feeding

Transport of amino acids (in vitro) in the rat pancreas is affected by the nutritional state of the animal. A fast of 24 h (young animals) or 48 h (adult animals) reduces the rate of amino acid uptake in the isolated rat pancreas in vitro. In contrast, refeeding of animals after a fast shows an increase in transport in young as well as adult animals.The effects of refeeding after a fast are mimicked to a significant extent by injection of mixtures of pancreozymin and carbamylcholine. Addition of these agents in vitro has no effect.The incorporation of amino acids into the total proteins of the rat pancreas follows the pattern of amino acid uptake. Even at high external levels of glycine (5 mM), incorporation increases although the glycine level in the cell is in excess of 25 mM. Reduction of glycine uptake by ouabain by 75% results in a substantial (44%) diminution of amino acid incorporation into proteins. The data suggest that inhibition of amino acid incorporation under the various metabolic conditions examined is due largely to a decreased availability of amino acids.     

Ataxia telangiectasia: the effects of chemical mutagens and x-rays on sister chromatid exchanges in blood lymphocytes

It is now possible to examine in detail exchanges between sister chromatids (SCEs) and to attempt to investigate the relationships of such exchanges to aberration formation and DNA-repair mechanisms. The frequency of SCEs is dramatically increased by chemical mutagens and may reflect the level of DNA damage. Lymphocytes from patients with ataxia telangiectasis (AT) show high levels of spontaneous chromosome damage and are hypersentive to ionising radiations and it was of interest to examine the levels of SCE induced in these cells by various mutagens. The frequencies of SCE after treatment with X=rays or three chemical mutagens were equivalent to those in normal cells. The effects of fluorodeoxyuridine and deoxycytidine on SCE frequencies were also tested.     

Effect of algal inoculation on the yield and vitamin C content of two varieties of tomato

Summary Two varieties of tomato (Pusa Rubi and Selection 120) positively responded to algal inoculation in terms of the yield of fruits and shoots, but there was no significant effect on the vitamin C content of the fruits. A combined application of urea and algae was more effective than the application of urea alone.     

Measurement of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in mammalian kidney

Until recently measurement of 25-OH-D3-1 alpha-hydroxylase activity in mammalian kidney has not been possible due to the presence of a protein which inhibits the enzyme by reducing available substrate. However, utilization of sufficient unlabeled 25-OH-D3 (80 nmol/ml renal homogenate) to overcome the effect of the inhibitor while maintaining optimal concentration for 1-hydroxylation has made quantitation of enzyme activity possible. We have modified this existing technique in order to increase the sensitivity and to permit detailed study of 1 alpha-hydroxylate regulation in mouse kidney. The modifications that we have incorporated include (i) simplifying the purification scheme for obtaining measurable 1,25-(OH)2D3 by reducing to one the necessary number of high-performance liquid chromatography steps and (ii) quantifying 1,25-(OH)2D3 by radioligand assay. The sensitivity of the assay is 10 pg, which, corrected for fractionation and recovery (50-60%), allows the measurement of 0.5 fmol 1,25-(OH)2D3 produced per milligram kidney per minute. Moreover, reliability and precision of the assay have been confirmed by demonstrating that samples from carefully matched, identically treated mice have reproducible enzyme activity (interassay coefficient of variation = 9.1%, n = 5) and show appropriate dilution characteristics. We have also demonstrated appropriate modulation of enzyme activity by known effectors of 1-hydroxylation. Kidneys from D-deficient mice exhibit significantly higher enzyme activity (15.28 +/- 1.17, n = 21) than do normal mouse kidneys (5.14 +/- 0.26, n = 33). In contrast, enzyme activity is suppressed significantly in kidneys obtained from calcium-loaded (1.20 +/- 0.04, n = 5) and parathyroidectomized animals (2.94 +/- 0.29, n = 5). Our assay now permits the indepth study of 1 alpha-hydroxylase regulation in mammalian (mouse) kidneys.