Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver

We have used isopycnic density gradient centrifugation to study the distribution of several rat liver microsomal enzymes of cholesterol synthesis and metabolism. All of the enzymes assayed in the pathway from lanosterol to cholesterol (lanosterol 14-demethylase, steroid 14-reductase, steroid 8-isomerase, cytochrome P-450, and cytochrome b5) are distributed in both smooth (SER) and rough endoplasmic reticulum (RER). The major regulatory enzyme in the pathway, hydroxymethylglutaryl-CoA reductase, also was found in both smooth and rough fractions, but we did not observe any associated with either plasma membrane or golgi. Since cholesterol can only be synthesized in the presence of these requisite enzymes, we conclude that the intracellular site of cholesterol biosynthesis is the endoplasmic reticulum. This is consistent with the long-held hypothesis. When the overall pathway was assayed by the conversion of mevalonic acid to non-saponifiable lipids (including cholesterol), the pattern of distribution obtained in density gradients verified its general endoplasmic reticulum localization. The enzyme acyl-CoA-cholesterol acyltransferase which removes free cholesterol from the membrane by esterification, was found only in the rough fraction of endoplasmic reticulum. In addition, when the RER was degranulated by the addition of EDTA, the activity of acyl-CoA-cholesterol acyltransferase not only shifted to the density of SER but was stimulated approximately 3-fold. The localization of these enzymes coupled with the stimulatory effect of degranulation on acyl-CoA-cholesterol acyltransferase activity has led us to speculate that the accumulation of free cholesterol in the RER membrane might be a driving factor in the conversion of RER to SER.     

Nitroguanidines induce bud break and change sterol content in apple

Bud break in apple (Malus domestica Borkh cv. Golden Delicious) was induced by 1-(3,5-dichlorophenyl)-3-nitroguanidine or 1-(-ethylbenzyl)-3-nitroguanidine. The optimum dose was 1000 M. An increase in bud fresh weight, dry weight, and length was more prominent in buds treated with 1-(-ethylbenzyl)-3-nitroguanidine than in those treated with 1-(3,5-dichlorophenyl)-3-nitroguanidine. The sterol compositional changes during bud break induced by 1-(3,5-dichlorophenyl)-3-nitroguanidine were similar to those induced by 1-(-ethylbenzyl)-3-nitroguanidine. -Sitosterol and sitosteryl ester were the predominant sterols. The amounts of these sterols increased immediately after dormancy was broken and then declined. A decrease in the percentage of the sitosterol and sitosteryl ester was accompanied by an increase in campesterol and stigmasterol at the beginning of rapid growth. A decrease in the ratio of free sterols to phospholipids and an increase in the ratio of campesterol + stigmasterol to sitosterol upon breaking dormancy occurred in apple buds induced by 1-(3,5,-dichlorophenyl)-3-nitroguanidine or 1-(-ethylbenzyl)-3-nitroguanidine. 1-(m-Methoxybenzyl)-3-nitroguanidine did not affect breaking of apple bud dormancy and also had no effect on changes in sterol content. The sterols in apple buds were confirmed by gas chromatography-mass spectrometry.     

Genetic and physical mapping of the biglycan gene on the mouse X Chromosome

A human cDNA for biglycan (BGN) has recently been mapped to proximal Xq28. We have mapped the murine locus, Bgn, approximately 50 kb distal to DXPas8, using a combination of genetic mapping in an interspecific backcross of B6CBA-A ^w-J/A-Bpa x Mus spretus and physical mapping using pulsed field gel electrophoresis and analysis of murine yeast artificial chromosomes (YACs) containing both DXPas8 and Bgn. Our mapping studies also appear to exclude Bgn as a candidate gene for the bare patches (Bpa) mutation and for the homologous human disorder X-linked dominant chondrodysplasia punctata (CDPX2).     

Involvement of the PKC family in regulation of early development

Protein kinase C (PKC) isotypes have been implicated in a number of key steps during gametogenesis, fertilization, and early development. The 11‐member family of PKC isotypes, many with different cofactor requirements for activation, can provide for differential activation of the specific kinases. In addition the enrichment of particular PKC isotypes to unique locations within gametes, zygotes, and early embryos likely promotes specific substrate interactions. Evidence exists to indicate involvement of PKC isotypes during sperm capacitation and the acrosome reaction, during resumption of meiosis in the oocytes, regulating the spindle organization in meiosis I and II, at fertilization, in the pronuclei, in the mitotically dividing blastomeres of the embryo, and at the plasma membranes of blastomeres at the time of embryonic compaction. Evidence also exists for crosstalk with other signaling pathways and one or more isotypes of PKC appear to be active at each major developmental transition. Mol. Reprod. Dev. 77: 95–104, 2010. © 2009 Wiley‐Liss, Inc.     

The Bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 Is Reduced by Flavodoxin and Ferredoxin and Is Essential under Mixotrophic,Nitrate-limiting Conditions

Cyanobacteria are able to use solar energy for the production of hydrogen. It is generally accepted that cyanobacterial NiFe-hydrogenases are reduced by NAD(P)H. This is in conflict with thermodynamic considerations, as the midpoint potentials of NAD(P)H do not suffice to support the measured hydrogen production under physiological conditions. We show that flavodoxin and ferredoxin directly reduce the bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 in vitro. A merodiploid ferredoxin-NADP reductase mutant produced correspondingly more photohydrogen. We furthermore found that the hydrogenase receives its electrons via pyruvate:flavodoxin/ferredoxin oxidoreductase (PFOR)-flavodoxin/ferredoxin under fermentative conditions, enabling the cells to gain ATP. These results strongly support that the bidirectional NiFe-hydrogenases in cyanobacteria function as electron sinks for low potential electrons from photosystem I and as a redox balancing device under fermentative conditions. However, the selective advantage of this enzyme is not known. No strong phenotype of mutants lacking the hydrogenase has been found. Because bidirectional hydrogenases are widespread in aquatic nutrient-rich environments that are capable of triggering phytoplankton blooms, we mimicked those conditions by growing cells in the presence of increased amounts of dissolved organic carbon and dissolved organic nitrogen. Under these conditions the hydrogenase was found to be essential. As these conditions close the two most important sinks for reduced flavodoxin/ferredoxin (CO₂-fixation and nitrate reduction), this discovery further substantiates the connection between flavodoxin/ferredoxin and the NiFe-hydrogenase.     

Synthesis,Characterization, and Antibacterial Activity of Structurally Complex 2‐Acylated 2,3,1‐Benzodiazaborines and Related Compounds

A set of 2‐acylated 2,3,1‐benzodiazaborines and some related boron heterocycles were synthesized, characterized, and tested for antibacterial activity against Escherichia coli and Mycobacterium smegmatis. By high‐field solution NMR, the heretofore unknown class of 2‐acyl‐1‐hydroxy‐2,3,1‐diazaborines has been found to be able to exist in several interconvertable structural forms along a continuum comprised of an open hydrazone a , a monomeric B‐hydroxy diazaborine b , and an anhydro dimer c . X‐Ray crystallography of one of the anhydro dimers, 17c , revealed it to have an unprecedented structure featuring a double intramolecular O→B chelation. The crystal structure of another compound, 37 , showed it to be based on a new pentacyclic B heterocycle framework. Nine compounds were found to possess activities against E. coli, and two others were active against M. smegmatis. The finding that these two contain isoniazid covalently embedded in their structures suggests that they might possibly be acting as prodrugs of this well‐known antituberculosis agent in vivo.