Synthesis of fibronectin in normal and osteoarthritic articular cartilage

The content and the biosynthesis of fibronectin was examined in disease-free articular cartilage and in articular cartilage from osteoarthritic canine joints. Fibronectin content was increased in extracts of cartilage from osteoarthritic joints. Incubation of cartilage in vitro with [³H]phenylalanine and subsequent isolation of [³H]fibronectin from a gelatin affinity column and characterization by SDS-polyacrylamide gel electrophoresis and by immunoprecipitation indicated that disease-free and osteoarthritic cartilage explants synthesized fibronectin. About 50% of the [³H]fibronectin was recovered in the incubation medium. The osteoarthritic cartilage synthesized and accumulated up to 5-fold more [³H]fibronectin than disease-free cartilage.     

Vitamin K epoxidase activity of rough and smooth microsomes from rat liver

The distribution of vitamin K epoxidase activity in rough and smooth microsomes has been studied and compared to the prothrombin precursor and vitamin K-dependent carboxylase activity. All three activities were high in rough microsomes as compared to the low levels found in smooth microsomes. The results are in agreement with the suggestion that there might be a linkage between the vitamin K-dependent carboxylation and epoxidation reaction in vivo.     

Peripheral receptor populations involved in the regulation of gastrointestinal motility and the pharmacological actions of metoclopramide-like drugs

This minireview is concerned with a re-examination of the locus of action and the possible peripheral mechanisms involved in the gastrointestinal (GI) stimulant effects of metoclopramide. Such a re-evaluation is opportune given the increasing use of this drug in the therapy of certain GI tract disorders. To provide an orientation on this subject the location in the GI tract and function of several relevant receptor types have been reviewed. In the past metoclopramide has been reported to enhance contractions of a variety of GI preparations to electrical stimulation, acetylcholine, carbachol and ganglion stimulants, to inhibit responses to alpha 2-adrenoreceptor agonists and 5-hydroxytryptamine, as well as blocking those to dopamine. Also in such preparations metoclopramide facilitates the release of acetylcholine to transmural stimulation. One important question is whether this effect is mediated via a specific prejunctional receptor. In this respect 2 suggestions have been made. Firstly that the drug may act as a preferential, prejunctional muscarinic antagonist thus inhibiting the negative feedback inhibition of acetylcholine release and secondly that metoclopramide may be a prejunctional agonist (partial) at 5-hydroxy-tryptamine receptors. Although the latter possibility appears most tenable at present, the involvement of a specific receptor remains to be confirmed. The important finding that dopamine receptors are probably not involved in the local stimulant effects of metoclopramide has important implications for future research orientated towards the discovery of a new generation of GI drugs lacking the side effects associated with central dopamine receptor blockade. Several compounds (cinitapride, BRL 20627A and cisapride) are now in the early stages of clinical evaluation.     

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.     

Adenosine mediates transforming growth factor-beta 1 release in kidney glomeruli of diabetic rats

Up regulation of the transforming growth factor-beta 1 (TGF-β1) axis has been recognized as a pathogenic event for progression of glomerulosclerosis in diabetic nephropathy. We demonstrate that glomeruli isolated from diabetic rats accumulate up to sixfold more extracellular adenosine than normal rats. Both decreased nucleoside uptake activity by the equilibrative nucleoside transporter 1 and increased AMP hydrolysis contribute to raise extracellular adenosine. Ex vivo assays indicate that activation of the low affinity adenosine A_2B receptor subtype (A_2BAR) mediates TGF-β1 release from glomeruli of diabetic rats, a pathogenic event that could support progression of glomerulopathy when the bioavailability of adenosine is increased.     

Polymyxin B Enhances Formation of a New Pigment,a Peptide–Ferropyrimine Complex,in Serratia marcescens

We previously isolated a Serratia marcescens O5: HI Z-54 strain which produces a new reddish-violet pigment, a peptide- ferropyrimine complex. This study showed that polymyxin B enhances the formation of the pigment about threefold. This occurs because polymyxin B in the medium causes the formation of an iron-polymyxin B complex which imposes a low iron stress on the bacteria and, in turn, enhances pigment production. This shows that polymyxin B is both a membrane-disrupting and ionophoric antibiotic.     

An automated technique for monitoring carbon dioxide respired by insects*

ABSTRACT. A non-dispersive infrared gas analyser equipped with a Luft-type sonic detector and flow-through reference cell was automated to monitor the total volume of carbon dioxide (CO₂) respired by single insects or groups of insects. The infrared analyser was interfaced with an integrator for quantification, a microprocessor to control intermittent air flow through the insect respiration chambers, and a microcomputer for data storage and reduction. This technique has been used to monitor the CO₂ Output of diapausing and non-diapausing mature fifth instar larvae and of developing pupae of the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). The resulting data were accurate, quantitative and reproducible.     

Subcellular distribution of enzymes in the yeast Saccharomycopsis lipolytica, grown on n-hexadecane,with special reference to the ω-hydroxylase

The subcellular localization of the ω-hydroxylase of Saccharomycopsis lipolytica was assessed by the analytical fractionation technique, originally described by de Duve C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F., and hitherto little, if at all, applied to yeast. Protoplasts were separated in six fractions by differential centrifugation. Some of these fractions were further fractioned by density gradient centrifugation. The distribution of ω-hydroxylase and 15 other constituents chosen as possible markers of its subcellular membranes has been established. ω-Hydroxylase resulted in being bound to a membrane that containes also cytochrome P-450 and NADPH-cytochrome c reductase. This membrane clearly differs from five other subcellular entities. (1) Mitochondria were characterized by particulate malate dehydrogenase, particulate Antimycin A-insensitive NADH-cytochrome c reductase, oligomycin-sensitive and K⁺-stimulated ATPase pH 9. (2) Most if not all of the catalase and urate oxidase is peroxisomal. (3) Free ribosomes account for most RNA. (4) Nucleoside diphosphatase is for the first time reported in a yeast and appears to belong to an homogeneous population of small membranes. (5) The soluble compartment contains magnesium pyrophosphatase, alkaline phosphatase, 5′-nucleotidase and part of the NADH-cytochrome c reductase. Latent arylesterase and ATPase pH7 have an unspecific distribution. Alkaline phosphodiesterase I has not been detected.     

Characterization by [3H]Dihydroergocryptine Binding of Alpha-Adrenergic Receptors in Neuroblastoma × Glioma Hybrid Cells

[3H]Dihydroergocryptine ([3H]DHE) was shown to bind to sites in membranes from neuroblastoma X glioma hybrid cells (NG 108-15) that had the characteristics expected of alpha-adrenergic receptors. The binding was saturable with 0.3 pmol [3H]DHE bound per mg of protein and of high affinity, with an apparent dissociation constant (KD) of 1.8 nM. The specificity of the binding site for various ligands was more similar to that of alpha 2 receptors than to that of alpha 1. No specific binding of [3H]WB-4101 was found in the membranes derived from NG 108 cells. This finding also indicated that the [3H]DHE binding site in the cell is the alpha 2 receptor. GTP lowered the affinity of agonists for the [3H]DHE binding site, although the nucleotide hardly affected the affinity of antagonists including [3H]DHE.