Use of Fourier transform 1H NMR in the identification of vitamin D2 metabolites

The use of Fourier transform 1H NMR to characterize vitamin D2 metabolites is described. A 300-MHz spectrometer capable of generating a 6-microseconds pulse and a sweep width of 4000 Hz was used. High-resolution spectra were obtained on 5 micrograms of material using standard 5-mm NMR tubes fitted with glass inserts and isotopically enriched chloroform-d solvent. The data acquisition time under these conditions was 4 h. Application of this technique to a variety of both synthetic and naturally occurring vitamin D2 metabolites, in addition to synthetic delta 22-1,25-dihydroxyvitamin D3, resulted in the reassignment of the chemical shifts for the C-21 and C-28 methyl groups of vitamin D2. The C-21 methyl group resonance is now assigned to the doublet appearing at delta 1.01, whereas the C-28 signal corresponds to the doublet at delta 0.90. An examination of the spectrum of 24 (R),25-dihydroxyvitamin D2 also led to the reassignment of the side-chain methyl group resonances. This technique is an additional means of identifying microgram quantities of vitamin D metabolites.     

Influence of high density lipoprotein (HDL), prepared from human blood, on prostanoid formation, serum and tissue lipids and development of arteriosclerosis in cholesterol rich fed rabbits

The i.v. administration of high density lipoprotein (HDL) into cholesterol fed rabbits decreased statistically significantly the serum level of total cholesterol and of low density lipoprotein cholesterol after a feeding period of 8 weeks. These diminished levels of cholesterol were associated with a statistically significant reduction in the levels of cholesterol esters in kidneys and platelets but not in hepatic tissue or in aorta. Macroscopically detectable arteriosclerosis was not statistically significantly diminished. The formation of prostanoids by the aorta remained unchanged. The atherogenic role of immunologic factors acting against the heterologous HDL may have compensated for the antiatherogenic HDL action on plasma and tissue lipids.     

Effect of the chemical surface structure of functionalized polystyrene on the kinetic properties of immobilized yeast pyruvate decarboxylase]

The influence of the loading density of different functional groups and the length of suitable spacer structures on the kinetic properties of yeast pyruvate decarboxylase are investigated on identical polystyrene matrices. At constant concentrations of the fixed protein both the specific activity and the storage stability of the immobilized enzymes increase with increasing concentrations of the protein binding (C = O)-groups. pH-Optimum and K'M-value prove to be functions of the NH3+-content of the supports. Using four spacer resins with an equal content of spacer groups it could be shown that the optimum time of coupling as well as the maximum catalytical activity, storage stability and thermostability depend on the length of the spacer structures. On the other hand, the mobility of an ESR-marker fixed via the same spacers to the resin is not affected by the different spacer structures.     

Developmental changes in rat kidney 1,25-dihydroxyvitamin D receptor

Kidney 1,25-dihydroxyvitamin D receptor (VDR) was examined in both young and aged male Fischer 344 rats. Cytosols prepared by direct homogenization of the kidney indicated no significant difference in the amount of unoccupied VDR in young (149 +/- 8 fmol/mg) and aged (155 +/- 8 fmol/mg) rats. Binding of kidney VDR to DNA-cellulose, however, was significantly different for the two groups. The assay indicated that about 44% and 24% of the VDR prepared from young and aged rats, respectively, were bound to calf thymus DNA. Elution profiles from DNA-cellulose chromatography displayed the presence of two peaks from young kidneys, while a single broad peak was evident from aged rats. Immunoblot analysis confirmed the existence of two receptor bands at 52K and 50K. The presence of the 50K band was greatly diminished or absent in aged samples. The 50K receptor form was observed to elute from DNA-cellulose at a higher salt concentration than the 52K-form. Similarly, prepared receptor extracts from intestinal tissue produced only a single band at 52K. These results demonstrate for the first time that the rat kidney possesses two forms of the receptor which have different affinities for DNA.     

Fatty acid biosynthesis in liver and adipose tissue from dogs

Production of CO2, fatty acids and glycerol from glucose and acetate was measured in slices of liver and adipose tissue taken from mature dogs. Acetate was the predominant carbon source for de novo fatty acid synthesis in both tissues. Fatty acid synthesis occurred at greater rates in adipose tissue than in liver. Glucose provided carbon for glycerol synthesis production in adipose tissue. Results support the concept that adipose tissue, and not liver, is the principal anatomical site for fatty acid synthesis in dogs.     

Recovery of mitochondrial DNA from blood leukocytes using detergent lysis

Mitochondrial DNA (mtDNA) was isolated from leukocytes contained in whole blood of cattle. Leukocyte membranes except the nuclear envelope were solubilized in a buffer that contained 1% Triton X-100. After sedimentation of cell nuclei, mtDNA was purified from the cell lysate by organic solvent extraction and ethanol precipitation. Approximately 5 µg of mtDNA was recovered from 400 ml of whole blood, a quantity sufficient for routine DNA cloning procedures or for detailed restriction mapping studies. mtDNA isolated with this method is a suitable substrate for several DNA-modifying enzymes. Thus, preparation of mtDNA from blood by detergent lysis provides a noninvasive alternative to tissue biopsy for characterization of mitochondrial genotypes in studies of evolutionary genetics and population dynamics.This work was supported in part by the Iowa State University Biotechnology Council and by the Holstein Association.Journal Paper No. J-13683 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2736.     

Inhibition by potential metabolic inhibitors of in vitro adipose tissue lipogenesis

To study the pathway of lactate utilization as a carbon source for fatty acid synthesis, the effect of (-)-hydroxycitrate, agaric acid, sodium oxamate, 2-n-butyl malonate and alpha-cyano-4-hydroxycinnamate on the rate of in vitro conversion of lactate, acetate and glucose to fatty acids was measured in bovine and rat adipose tissues. Sodium oxamate and hydroxycitrate caused less fatty acid to be synthesized from lactate in bovine adipose tissue. Hydroxycitrate depressed fatty acid synthesis from glucose in rat adipose tissue. alpha-Cyano-4-hydroxycinnamate was an effective inhibitor of lipogenesis from all substrates and may act as a specific inhibitor in adipose tissue. Although the inhibitors were absorbed poorly into adipocytes, the results indicate that conversion of lactate to fatty acids probably occurs by way of the citrate cleavage pathway.     

Lipogenesis in liver and adipose tissue of the domestic cat (Felis domestica)

1. Production of fatty acids, CO2 and glycerol from glucose and acetate was determined in slices of liver and adipose tissue taken from mature cats. 2. Acetate was the predominant carbon source for de novo fatty acid synthesis in both liver and adipose tissue. 3. Fatty acid synthesis occurred at much greater rates in adipose tissue than in liver. 4. Relative lipogenic capacity (ratio of production of long-chain fatty acids and glycerol to oxidation of substrates) was 20 times greater in adipose tissue than in liver with both substrates.     

Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2

The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis.