The role of a cholesta-8,14-dien-3β-ol system in cholesterol biosynthesis

The biosynthesis of cholesterol from squalene and tritiated water is described. Degradation of the cholesterol indicated that C-15 may be involved in cholesterol biosynthesis. In accordance with this view it is shown that in the conversion of [2RS-(3)H(2)]mevalonic acid into cholesterol one of the hydrogen atoms at C-15 is removed. A mechanism for the removal of the 14alpha-methyl group in steroid biosynthesis that involves the labilization of a C-15 hydrogen atom is outlined. In accordance with the requirement of this scheme it is shown that 4,4'-dimethyl-cholesta-8,14-dien-3beta-ol is converted into cholesterol.     

The pathway of biosynthesis of nicotinamide–adenine dinucleotide in rat mammary gland

1. The pathway of NAD synthesis in mammary gland was examined by measuring the activities of some of the key enzymes in each of the tryptophan, nicotinic acid and nicotinamide pathways. 2. In the tryptophan pathway, 3-hydroxyanthranilate oxidase and quinolinate transphosphoribosylase activities were investigated. Neither of these enzymes was found in mammary gland. 3. In the nicotinic acid pathway, nicotinate mononucleotide pyrophosphorylase, NAD synthetase, nicotinamide deamidase and NMN deamidase were investigated. Both NAD synthetase and nicotinate mononucleotide pyrophosphorylase were present but were very inactive. Nicotinamide deamidase, if present, had a very low activity and NMN deamidase was absent. 4. In the nicotinamide pathway both enzymes, NMN pyrophosphorylase and NMN adenylyltransferase, were present and showed very high activity. The activity of the pyrophosphorylase in mammary gland is by far the highest yet found in any tissue. 5. The apparent K(m) values for the substrates of these enzymes in mammary gland were determined. 6. On the basis of these investigations it is proposed that the main, and probably only, pathway of synthesis of NAD in mammary tissue is from nicotinamide via NMN.     

The role of β-alanine in the biosynthesis of nitrate byAspergillus flavus

d-Serine (0.05m) inhibited nitrification byAspergillus flavus in media containing either peptone, aspartate,a-alanine or -alanine as the sole nitrogen source. A similar inhibition was observed in an aspartate + peptone medium, but nitrate was formed in a -alanine + peptone medium in the presence of the inhibitor. Exceptionally high yields of nitrate were obtained in the -alanine + peptone medium. In replacement cultures,d-serine inhibited nitrification of aspartate but not of -alanine. Manometric studies indicated that aspartate was decarboxylated byA. flavus and that the reaction was inhibited byd-serine. In view of these results, it is suggested that aspartate is a precursor and -alanine is an intermediate in nitrification by this fungus.     

The central role of the brain aldosterone–“ouabain” pathway in salt-sensitive hypertension

Na⁺-transport regulating mechanisms classically considered to reflect renal control of sodium homeostasis and BP, i.e. aldosterone–mineralocorticoid receptors (MR)—epithelial sodium channels (ENaC)—Na⁺/K⁺-ATPase have now been demonstrated to also be present in the central nervous system. This pathway is being regulated independently of the peripheral/renal pathway and contributes to regulation of cerebrospinal fluid [Na⁺] by the choroid plexus, of brain tissue [Na⁺] by the ependyma and to neuronal responses to e.g. Na⁺ or angiotensin II. Increases in CSF [Na⁺] by central infusion of Na⁺-rich aCSF or by high salt intake in Dahl S or SHR cause sympatho-excitation and hypertension. These responses appear to depend on activation of a CNS cascade starting with aldosterone–MR–ENaC–“ouabain,” the latter lowering neuronal membrane potential leading to enhanced angiotensin II release in e.g. the PVN. Specific CNS blockade of any of the steps in this cascade from aldosterone synthase blockade to AT₁-receptor blockade prevents the sympathetic hyperactivity and hypertension on high salt intake, irrespective of the presence of a “salt-sensitive kidney.” We propose that in salt-sensitive hypertension an increase in CSF [Na⁺] causes a local increase in aldosterone biosynthesis which activates an aldosterone dependent neuromodulatory pathway which enhances activity of angiotensinergic sympatho-excitatory pathways leading to hypertension.     

The reversibility of the Δ8-cholestenol–Δ7-cholestenol isomerase reaction in cholesterol biosynthesis

The incubation of Delta(7)-cholestenol with a 10000g supernatant or 105000g microsomes in the presence of tritiated water is studied. The reisolated Delta(7)-cholestenol contained up to 0.67g.atom of tritium/mole. This result can best be explained by assuming the reversibility of the reaction Delta(8)-cholestenol right harpoon over left harpoon Delta(7)-cholestenol.     

The stereochemistry of hydrogen transfer from reduced nicotinamide–adenine dinucleotide phosphate in the reduction of ethylenic linkages during cholesterol biosynthesis

It is shown that during the saturation of steroid carbon-carbon double bonds at Delta(24,25) and Delta(14,15) the ;hydride ion' originates from the 4B side of the NADPH.     

The role of bone sialoprotein in the tendon–bone insertion

Tendons/ligaments insert into bone via a transitional structure, the enthesis, which is susceptible to injury and difficult to repair. Fibrocartilaginous entheses contain fibrocartilage in their transitional zone, part of which is mineralized. Mineral-associated proteins within this zone have not been adequately characterized. Members of the Small Integrin Binding Ligand N-linked Glycoprotein (SIBLING) family are acidic phosphoproteins expressed in mineralized tissues. Here we show that two SIBLING proteins, bone sialoprotein (BSP) and osteopontin (OPN), are present in the mouse enthesis. Histological analyses indicate that the calcified zone of the quadriceps tendon enthesis is longer in Bsp^−/− mice, however no difference is apparent in the supraspinatus tendon enthesis. In an analysis of mineral content within the calcified zone, micro-CT and Raman spectroscopy reveal that the mineral content in the calcified fibrocartilage of the quadriceps tendon enthesis are similar between wild type and Bsp^−/− mice. Mechanical testing of the patellar tendon shows that while the tendons fail under similar loads, the Bsp^−/− patellar tendon is 7.5% larger in cross sectional area than wild type tendons, resulting in a 16.5% reduction in failure stress. However, Picrosirius Red staining shows no difference in collagen organization. Data collected here indicate that BSP is present in the calcified fibrocartilage of murine entheses and suggest that BSP plays a regulatory role in this structure, influencing the growth of the calcified fibrocartilage in addition to the weakening of the tendon mechanical properties. Based on the phenotype of the Bsp^−/− mouse enthesis, and the known in vitro functional properties of the protein, BSP may be a useful therapeutic molecule in the reattachment of tendons and ligaments to bone.     

Studies on the mechanism and regulation of C-4 demethylation in cholesterol biosynthesis. The role of adenosine 3′:5′-cyclic monophosphate

1. An assay for demethylation has been developed based on the release of tritium from 4,4-dimethyl[3alpha-(3)H]cholest-7-en-3beta-ol (II). 2. The maximum release of (3)H from 3alpha-(3)H-labelled compound (II) in a rat liver microsomal preparation occurs in the presence of NADPH and NAD(+) under aerobic conditions. 3. Incubation of 3alpha-(3)H-labelled compound (II) with NADPH under aerobic conditions leads to the formation of a 3alpha-(3)H-labelled C-4 carboxylic acid. This compound undergoes dehydrogenation on subsequent anaerobic incubation with NAD(+). 4. The (3)H released from the steroid was located in [4-(3)H]nicotinamide and the medium. Incubation with synthetic [4-(3)H(2)]NADH gave a similar result. 5. In the presence of glutamate dehydrogenase and alpha-oxoglutarate part of the (3)H released from the steroid was transferred to glutamate. 6. A series of 3-oxo steroids were reduced equally well by [4-(3)H(2)]NADH and [4-(3)H(2)]NADPH. The reduction of 5alpha-cholest-7-en-3-one was shown to use the 4B H atom from the nucleotide. 7. 3':5'-Cyclic AMP was shown to be a competitive inhibitor of the 3beta-hydroxy dehydrogenase enzyme in the demethylation reaction.     

Characterization of the GDP-D-mannose biosynthesis pathway in Coxiella burnetii: the initial steps for GDP-β-D-virenose biosynthesis

Coxiella burnetii, the etiologic agent of human Q fever, is a gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars β-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-β-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is critical to fully understand the biosynthesic pathway of GDP-β-D-virenose and LPS structure in C. burnetii.     

The role of β-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower

The β-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus L.) developing seeds, a cDNA coding for HaKAS III (EF514400) was isolated, cloned and sequenced. Its protein sequence is as much as 72% identical to other KAS III-like ones such as those from Perilla frutescens, Jatropha curcas, Ricinus communis or Cuphea hookeriana. Phylogenetic study of the HaKAS III homologous proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons. Heterologous expression of HaKAS III in Escherichia coli altered their fatty acid content and composition implying an interaction of HaKAS III with the bacterial FAS complex. Testing purified HaKAS III recombinant protein by adding to a reconstituted E. coli FAS system lacking condensation activity revealed a novel substrate specificity. In contrast to all hitherto characterized plant KAS IIIs, the activities of which are limited to the first cycles of intraplastidial fatty acid biosynthesis yielding C6 chains, HaKAS III participates in at least four cycles resulting in C10 chains.     

The molecular dissection of the chaperone–usher pathway

Secretion systems are specialized in transport of proteins, DNA or nutrients across the cell envelope of bacteria and enable them to communicate with their environment. The chaperone–usher (CU) pathway is used for assembly and secretion of a large family of long adhesive protein polymers, termed pili, and is widespread among Gram-negative pathogens [1]. Moreover, recent evidence has indicated that CU secretion systems are also involved in sporulation  and . In this review we focus on the structural biology of the paradigmatic type 1 and P pili CU systems encoded by uropathogenic Escherichia coli (UPEC), where recent progress has provided unprecedented insights into pilus assembly and secretion mechanism. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

The introduction of the C-22–C-23 ethylenic linkage in ergosterol biosynthesis

Methods for the chemical synthesis of [23-(3)H(2)]lanosterol, [23,25-(3)H(3)]24-methyldihydrolanosterol and [24,28-(3)H(2)]24-methyldihydrolanosterol are described. It is shown that, in the biosynthesis of ergosterol from [26,27-(14)C(2),23-(3)H(2)]lanosterol by the whole cells of Saccharomyces cerevisiae, one of the original C-23 hydrogen atoms is lost and the other is retained at C-23 of ergosterol. It is also shown that 24-methyldihydrolanosterol is converted into ergosterol in good yield and without prior conversion into a 24-methylene derivative. On the basis of these results possible pathways for the formation of the ergosterol side chain from a 24-methylene side chain are discussed.     

The role of p38α mitogen-activated protein kinase gene in the HELLP syndrome

Mitogen-activated protein kinase (MAPK) p38α was shown to be implicated in the organogenesis of the placenta, and such placental alteration is crucial for the development of hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. We aimed to analyze for the first time human placental expression of MAPK p38α in pregnancies complicated by HELLP. The placental expression of MAPK p38α was investigated by semiquantitative polymerase chain reaction using cDNA extracted from placental tissue of 15 pregnancies with HELLP syndrome and 15 gestational age-matched controls. Seven patients with HELLP also had intrauterine fetal growth restriction (IUGR). In placenta from pregnancy complicated by HELLP, the expression of MAPK p38α is significantly decreased compared to the group with normal pregnancy (p < 0.001), while no difference was found between the HELLP and HELLP with IUGR subpopulations. Our study shows for the first time that MAPK p38α is expressed in the human placenta. Pregnancies with placental dysfunction and hypertensive complications are characterized by a significantly decreased expression of MAPK p38α. Our observations suggest that p38 MAPK signaling may be essential in placental angiogenesis and functioning.