Some properties of human and bovine brain cathepsin B

Cathespin B has been purified 750-fold to apparent homogeneity from human and bovine brain cortex using ammonium sulfate fractionation (30–70%), chromatography on Sephadex G-100, CM-Sephadex C-50, and concanavalin A-Sepharose. Enzyme was assayed fluorometrically at pH 4.0 with pyridoxyl-hemoglobin in the presence of 1 mM DTT and 1 mM EDTA. Properties of the enzyme from the two sources proved to be similar. On disc PAGE the purified preparation produced two bands associated with proteinase activity that are due to existence of two multiple forms of brain cathepsin B with pI 6.1 and 6.8. The enzyme is completely inactivated by thiol-blocking reagents, leupeptin, E-64, and demands thiol compounds for its ultimate activity. Z-Phe-Ala-CHN₂ is a potent inhibitor of the enzyme (K _2nd=1280 M⁻¹s⁻¹) in contrast to Z-Phe-Phe-CHN₂ (K _2nd=264 M⁻¹s⁻¹). pH optimum in the reaction of hydrolysis of Pxy-Hb is 4.0–6.0,K _M(app.) =10⁻⁵ M. Cathepsin B splits azocasein: pH optimum 5.0–6.0,K _M(app.)=2.2·10⁻⁵ M, but inclusion of urea in the incubation medium depresses the azocaseinolytic activity of the enzyme 1.5-fold. It does not split Lys-NNap, Arg-NMec and is not inhibited by bestatin. The specific activity of brain cathepsin B with Z-Arg-Arg-NNapOMe at pH 6.0 is 10-fold higher than with Bz-Arg-NNap, Z-Gly-Gly-Arg-NNap is a poor substrate. With Z-Arg-Arg-NMec and Bz-Phe-Val-Arg-NMec the specific acitivity is 80 and 35%, respectively of that with Z-Phe-Arg-NMec. Special Issue dedicated to Dr. Eugene Kreps.     

Self-Renewal Capacity and Clonal Succession of Haemopoietic Stem Cells In Long-Term Bone Marrow Culture

Abstract. The CFU-s proliferative potential varied greatly during long-term cultivation. Most of the CFU-s in the cultures were represented by cells with low renewal capacity. Pre-CFU-s cells capable of producing multipotential colonies in methylcellulose, which contained CFU-s with a high proliferative potential, were identified in the culture. In cultivation of a mixture of cells of different karyotype their ratio changed rapidly from week to week. the findings were consistent with the hypothesis that haemopoietic stem cells are maintained in the culture by the products of a small number of clones which arise and decline in succession, and that pre-CFU-s, but not the CFU-s themselves, are clonogenic progenitors.     

Relationship between ion requirements for respiration and membrane transport in a marine bacterium.

Intact cells of the marine bacterium Alteromonas haloplanktis 214 oxidized NADH, added to the suspending medium, by a process which was stimulated by Na+ or Li+ but not K+. Toluene-treated cells oxidized NADH at three times the rate of untreated cells by a mechanism activated by Na+ but not by Li+ or K+. In the latter reaction, K+ spared the requirement for Na+. Intact cells of A. haloplanktis oxidized ethanol by a mechanism stimulated by either Na+ or Li+. The uptake of alpha-aminoisobutyric acid by intact cells of A. haloplanktis in the presence of either NADH or ethanol as an oxidizable substrate required Na+, and neither Li+ nor K+ could replace it. The results indicate that exogenous and endogenous NADH and ethanol are oxidized by A. haloplanktis by processes distinguishable from one another by their requirements for alkali metal ions and from the ion requirements for membrane transport. Intact cells of Vibrio natriegens and Photobacterium phosphoreum oxidized NADH, added externally, by an Na+-activated process, and intact cells of Vibrio fischeri oxidized NADH, added externally, by a K+-activated process. Toluene treatment caused the cells of all three organisms to oxidize NADH at much faster rates than untreated cells by mechanisms which were activated by Na+ and spared by K+.     

Effects of physical and chemical treatments on chloroplast manganese. NMR and ESR studies

Measurements were made of the water proton relaxation rate (T^?1₂ = R₂), electron spin resonance (ESR) six-line signal of ‘free’ Mn²⁺, and O₂-evolution activity in thylakoid membranes from pea leaves. The main results are: (1) Aging of thylakoids at 35°C causes a parallel decrease in O₂-evolution activity, in R₂ and in the content of bound Mn, suggesting that R₂ may be related to the loosely bound Mn involved in O₂ evolution. (2) Treatment of thylakoids with tetraphenylboron (TPB) at [TPB] > 2 mM produces a 2-fold increase in R₂, without release of Mn²⁺. The titration curve exhibits three sharp end points. The first end point occurs at a $\text{[}\text{TPB}\text{]}\text{[}\text{chlorophyll}\text{]}$ of 1.25, at which the O₂ evolution is completely inhibited. (3) Treatment of thylakoids with NH₂OH also increases R₂ by nearly 2-fold, either by the reduction of the higher oxidation states of Mn to Mn²⁺ and / or by exposing the Mn to solvent protons. Also, progressive release of bound Mn occurs at [NH₂OH] ≥ 1 mM as shown by an increase increase in the Mn²⁺ ESR signal and a decrease in R₂. (4) Addition of H₂O₂ (0.1–1.0%) to thylakoids causes an enhancement of R₂ similar to that by NH₂OH, but without the release of Mn²⁺. (5) Heat treatment of thylakoids at 40–50°C releases Mn²⁺ and increases R₂. Conversely, pH values of 7 to 4 release Mn²⁺ without changing R₂ while pH values of 7–9 increase R₂ without releasing Mn²⁺. Thus, both high and low pH values as well as the heat treatment cause structural changes enhancing the relaxivity of the bound Mn or of other paramagnetic species.     

Glabrachalcone,a chromenochalcone from Pongamia glabra seeds

Glabrachalcone, a new chromenochalcone has been isolated along with a known chromenochalcone from an ethanolic extract of the seed oil of Pongamia glabra. The structure of glabrachalcone has been established as 2′-hydroxy-2,4,5-trimethoxy-6″,6″-dimethylchromeno(4′,3′:2″,3″)chalcone on the basis of spectral evidence and was confirmed by synthesis.     

Effect of certain inorganic chemicals on growth and spore germination of Alternaria tenuis auct., the fungus causing core rot of mandarin oranges in India

Summary Laboratory studies to determine the tolerance of an isolate ofAlternaria tenuis causing a black core rot of mandarin oranges (Citrus reticulata) have shown that presence of low amounts of manganese, zinc, iron, copper, boron, and molybdenum in a synthetic medium stimulated growth and sporulation of the fungus. Concentrations of 20 ppm or more of copper sulphate, boric acid, and ammonium molybdate, however, caused significant reduction of growth. Conidia of the fungus were very sensitive to even low amounts of copper, zinc, and iron. Copper sulphate was most toxic to spore germination followed, in order of inhibitory effect, by zinc sulphate, ferrous sulphate, and boric acid. Ammonium molybdate and manganese sulphate were stimulatory to spore germination at 5 ppm. There was a positive correlation between percentage of spore germination and average length of germ tubes in different concentrations of various chemicals. The possibility of using copper sulphate, zinc sulphate, and ferrous sulphate as spray materials for trees and as dip for ripe fruits to prevent black core rot are discussed.Assoc. Professor and Research Assistant, respectively. Publication under journal series No. 27 from Experiment Station, U.P. Agricultural University.     

Role of Ca2+ in induction and secretion of dengue virus-induced cytokines

The present study was undertaken to investigate the role of calcium ions (Ca₂₊) in the induction and secretion of the dengue type 2 virus induced cytotoxic factor and the cytotoxin. This was done by using calcium channel blocking drugs such as verapamil, nifedipine or diltiazem hydrochloride. The production of cytotoxic factor was significantly reduced by treatment of dengue type 2 virus infected mice with verapamil. Similarly, a dosedependent inhibition of the secretion of cytotoxic factor was observed, when spleen cells of the virus-primed mice were treatedin vitro with the 3 calcium channel blockers. The production of cytotoxin by macrophages was abrogated by pretreatment with calcium channel blockers but had little effect on its secretion as shown by treatment of macrophages with verapamil at 1 h after the induction to later periods up to 18 h. The findings thus show that in the induction of both the cytokines Ca₂₊ plays a critical role; on the other hand it is required for the secretion of the cytotoxic factor but not for that of the cytotoxin.