Effects of canavanine on IAA- and fusicoccin-stimulated cell enlargement, proton extrusion and potassium uptake in maize coleoptiles

In maize coleoptiles (Zea mays F₁ XL 640A, cv. Dekalb) canavanine and cycloheximide strongly and simultaneously inhibit cell elongation, H⁺ extrusion and K⁺ uptake, induced by IAA. They inhibit also, although to a much lesser degree, the same phenomena induced by fusicoccin. Cycloheximide severely depresses the incorporation of leucine into proteins, while canavanine leaves leucine incorporation almost unchanged. The data confirm that elongation, H⁺ extrusion and K⁺ uptake can be regarded as correlated processes; they also support the view that normal protein synthesis is essential for IAA-induced growth, while this requirement is only partial in growth stimulated by fusicoccin.     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.     

Studies on trypsin inhibitors in the tubers of cocoyam (Xanthosoma sagittifolium)

Trypsin (EC 3.4.21.4) inhibitors have been isolated and purified by gelfiltration and ion exchange chromatography from the tubers of cocoyam (Xanthosoma sagittifolium). Three isoinhibitors were obtained in an electrophoretically homogeneous state. Their molecular weights estimated by molecular sieve chromatography were found to be 19 950, 17 780 and 23 390, respectively. They showed varied trypsin inhibitory activity which was lost on boiling for 40 min. They were found to have a maximum activity at pH 7.5–8.0.     

Studies on the lipid metabolism of the metacercariae of Clinostomum complanatum (Trematoda: Digenea)

Metacercariae of Clinostomum complanatum possess considerable amounts of lipids. Fractionation of the lipids shows triglycerides and phospholipids as the major components whereas cholesterol and free fatty acids are minor components. Furthermore phospholipid fractions by thin layer chromatography reveal lecithin and cephalin as the major polar lipids whereas lysolecithin and lysocephalin are present in small fractions. The specific activity of lipase (E.C. 3.1.1.3) is 150.8 μg free fatty acids liberated/mg protein/h. Epinephrine, testosterone, insulin, sodium fluoride and iodoacetate stimulated, but 2-propanol inhibited, the lipase activity.     

Schistosoma mansoni: thiol proteinase properties of adult worm "hemoglobinase".

This report presents evidence that the “hemoglobinase” from adult Schistosoma mansoni, first described by Timms and Bueding and later by Senft and his collaborators, belongs to the class of thiol proteinases. Proteolytic activity is stimulated by SH-containing compounds and inhibited by N-ethylmaleimide as well as other inhibitors of thiol proteinases. The enzyme can be partially purified by affinity chromatography using a Sepharose-linked organomercurial ligand. In addition to its activity on globin and hemoglobin, the enzyme can also be assayed with Azocoll, a general protease substrate, and by the activation of inactive trypsinogen to active trypsin. Extraction of the enzyme is enhanced by the addition of the nonionic detergent Triton X-100.     

Effect of ay-9944 on sterol biosynthesis in Chlorella sorokiniana

When Chlorella sorokiniana was grown in the presence of 4 ppm AY-9944 total sterol production was unaltered in comparison to control cultures. However, inhibition of sterol biosynthesis was shown by the accumulation of a number of sterols which were considered to be intermediates in sterol biosynthesis. The sterols which were found in treated cultures were identified as cyclolaudenol, 4α,14α-dimethyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 4α,14α-dimethyl -5α-ergosta-8,25-dien-3β-ol, 14α-methyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 24-methylpollinastanol, 14α-methyl-5α-ergost-8-en-3β-ol, 5α-ergost -8(14)-enol, 5α-ergost-8-enol, 5α-ergosta-8(14),22-dienol, 5α-ergosta-8,22-dienol, 5α-ergosta-8,14-dienol, and 5α-ergosta-7,22-dienol, in addition to the normally occurring sterols which are ergosterol, 5α-ergost-7-enol, and ergosta-5,7-dienol.The occurrence of these sterols in the treated culture indicates that AY-9944 is an effective inhibitor of the Δ⁸ → Δ⁷ isomerase and Δ¹⁴-reductase, and also inhibits introduction of the Δ²²-double bond. The occurrence of 14α-dimethyl-5α-ergosta-8,25-dien-3β-ol and 14α-methyl-9β,19-cyclo-5α-ergost -25-en-3β-ol is reported for the first time in living organisms. The presence of 25-methylene sterols suggests that they, and not 24-methylene derivatives, are intermediates in the biosynthesis of sterols in C. sorokiniana.     

Phenyl-substituted aminomethylene-bisphosphonates inhibit human P5C reductase and show antiproliferative activity against proline-hyperproducing tumour cells

In certain cancers, such as breast, prostate and some lung and skin cancers, the gene for the enzyme catalysing the second and last step in proline synthesis, δ¹-pyrroline-5-carboxylate (P5C) reductase, has been found upregulated. This leads to a higher proline content that exacerbates the effects of the so-called proline-P5C cycle, with tumour cells effectively using this method to increase cell survival. If a method of reducing or inhibiting P5C reductase could be discovered, it would provide new means of treating cancer. To address this point, the effect of some phenyl-substituted derivatives of aminomethylene-bisphosphonic acid, previously found to interfere with the catalytic activity of plant and bacterial P5C reductases, was evaluated in vitro on the human isoform 1 (PYCR1), expressed in E. coli and affinity purified. The 3.5-dibromophenyl- and 3.5-dichlorophenyl-derivatives showed a remarkable effectiveness, with IC₅₀ values lower than 1 µM and a mechanism of competitive type against both P5C and NADPH. The actual occurrence in vivo of enzyme inhibition was assessed on myelogenous erythroleukemic K562 and epithelial breast cancer MDA-MB-231 cell lines, whose growth was progressively impaired by concentrations of the dibromo derivative ranging from 10⁻⁶ to 10⁻⁴M. Interestingly, growth inhibition was not relieved by the exogenous supply of proline, suggesting that the effect relies on the interference with the proline-P5C cycle, and not on proline starvation.     

Making and working with hydrogen sulfide: The chemistry and generation of hydrogen sulfide in vitro and its measurement in vivo: A review

Hydrogen sulfide is rapidly emerging as an important vasoactive mediator formed in health and disease. Its biological action is centered on its reactivity with heme-proteins and its ability to activate K_ATP channels. Hydrogen sulfide is a signalling molecule of the inflammatory and nervous systems, and in particular the cardiovascular system where it regulates vascular tone, cardiac work, and exerts cardioprotection.This has led to an explosion of papers in which the role of hydrogen sulfide generated in vitro has been used to stimulate biological responses, and where a variety of methods have been used to measure the concentration of this compound in biological fluids. Understanding the chemistry and the inherent problems in the analytical techniques used to measure hydrogen sulfide concentrations is critical to our expanding knowledge on the biology of hydrogen sulfide. In this brief review we will cover the chemistry of hydrogen sulfide, including sources of hydrogen sulfide, its speciation at physiological pH, the susceptibility of sulfide to aerobic oxidation, and the methods used to measure hydrogen sulfide concentrations in solution, including biological fluids. We also give a brief overview of knockout animals and inhibition of the enzymes involved in the formation of hydrogen sulfide in vivo.