Discovery of a human erythroid differentiation factor (EDF) and its large-scale production

A novel human protein exhibiting erythroid differentiation activity was discovered in the culture fluids of phorbol ester-stimulated human cells. The differentiation assay system involving Friend virus-derived mouse leukemia cells was used. THP-1 cells of myelomonocytic origin were typical producers. 4beta-Phorbol 12-myristate 13-acetate (PMA) was essential for inducible excretion of the erythroid differentiation factor (EDF). The factor was stable toward heat and pH (acidic or alkaline) but lost its activity on pronase treatment, which suggested its proteinous nature. After an optimization of the condition, production of EDF was performed on a 200-L scale for purification of the protein.     

Spin-label studies of the oligomeric structure of band 3 protein in erythrocyte membranes and in reconstituted systems

A spin-labeled fatty acid (16-doxylstearic acid), linked by an ester bond to a maleimide or a nitrene residue, was covalently attached to band 3 of erythrocyte membranes. The electron spin resonance spectrum of the spin-labeled protein was examined at different temperatures in: (a) whole erythrocyte ghosts; (b) ghosts depleted of spectrin and actin; (c) alkaline-treated ghosts; (d) vesicles made with purified band 3 reassociated with dimyristoylphosphatidylcholine. Most spectra are composite with a major component corresponding to a large overall splitting. The determination of the percentage of the immobilized component was carried out by pairwise subtraction. At low temperatures (1–7°C), the highest fraction of immobilized component was found in dimyristoylphosphatidylcholine vesicles (approx. 100%); alkaline-treated membranes had approx. 75% of the immobilized component at the same temperature; whole erythrocyte, spectrin/actin-depleted and spectrin/actin/ankyrin-depleted ghosts gave identical results (approx. 60% of immobilized component). The immobilized fraction decreased in all samples with increasing temperature or addition of a nonsolubilizing concentration of dodecyl octaethylene glycol monoether. In dimyristoylphosphatidylcholine vesicles, however, the modification in the ratio of the two components was obtained only above the lipid transition temperature (23°C). The strong immobilization of the spin-labeled lipid chain at all temperatures suggested trapping of the lipid chain between proteins. At low temperature, in dimyristoylphosphatidylcholine vesicles or in alkaline-treated ghosts, lipid-protein segregation is likely to take place. In whole erythrocyte ghosts, on the other hand, the large contribution of the motionally restricted component at physiological temperature indicates the oligomeric nature of band 3. Partial dissociation of the oligomers occurs as the temperature is increased, but the presence or absence of cytoskeletal proteins has no influence on the state of oligomerization of band 3.     

Effects of an N-methyl-d-aspartate receptor agonist and its antagonist CPP on the levels of dopamine and serotonin metabolites in rat striatum collected in vivo by using a brain dialysis technique

3-((±)-2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) is an antagonist at the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. In the present study, levels of dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA) were measured after intracerebroventricular injection of NMDA, CPP or both in rat striatum using a brain dialysis method. The injection of NMDA produced a significant increase in DOPAC level. HVA level was also increased by NMDA injection. The level of 5-HIAA was not affected by NMDA injection. The injection of CPP had no effect on DOPAC, HVA and 5-HIAA levels. The injection of CPP restrained the increase of DOPAC and HVA levels induced by NMDA injection. The results suggest that intracerebral injection of NMDA may increase dopamine release from rat striatum, but have no effect on serotonin release. Furthermore, CPP inhibits NMDA induced release of dopamine.     

Stochastic model for multipotent hemopoietic progenitor differentiation

In this study, the authors propose a stochastic model for multipotent hemopoietic progenitor differentiation, which assumes that there is a fixed probability (P) that a progenitor with a potential for differentiation along a particular lineage maintains the potential in each cell division in each daughter cell, and this differentiation process of each lineage proceeds independently. To examine the applicability of this model, a sequential micromanipulation of paired progenitors was carried out and followed by cytological examination of the cells contained in the colonies derived from these progenitors; then calculation was made of the ratio of the number of paired colonies containing cell(s) with a particular lineage to the number of paired colonies in which only one colony contained cell(s) with the lineage at the first and second cell division. The ratios were similar at the first and second cell division within each lineage. Furthermore, the frequences of each lineage in multilineage hemopoietic colonies were calculated using the P values obtained from these micromanipulation experiments. The expected frequencies were similar to those in the actual experiments. These results suggested that the stochastic model was applicable to multipotent hemopoietic progenitor differentiation.     

Repression of the K+ Uptake and Cation-Stimulated ATPase Activity Associated with the Plasma Membrane-Enriched Fraction of Cucumber Roots Due to Ca2+ Starvation

The uptake of K⁺ by cucumber plants decreased markedly duringCa²⁺ starvation. A plasma membrane-enriched fraction, judgedfrom the distribution of marker enzymes, was prepared from controland Ca²⁺-starved roots. The Mg²⁺- and K⁺-Mg²⁺-ATPase activitiesassociated with the plasma membrane-enriched fraction of controlroots were maxima at pH 6.5. Various monovalent cations andpotassium salts of monovalent anions stimulated Mg²⁺-ATPaseactivity. Vanadate, DES and DCCD inhibited K⁺- Mg²⁺-ATPase activity.Of the divalent cations and phosphate esters tested, Mg²⁺ andATP were most effective for the stimulation of ATPase by K⁺,whereas Ca²⁺ was ineffective in replacing Mg²⁺. Mg²⁺- and K⁺-Mg²⁺-ATPase activities associated with the plasmamembrane enriched fraction of Ca²⁺-starved roots were much lowerthan those of control roots. K_m values of K⁺-Mg²⁺-ATPase forATP were comparable for control and Ca²⁺-starved roots. The K⁺-stimulated activity of Mg²⁺-ATPase in Ca²⁺-starved rootswas approximately one fourth that of the control, whereas therate of stimulation was only slightly lower in Ca²⁺-starvedroots. (Received May 9, 1984; Accepted September 17, 1984)     

Refolding of lactate dehydrogenase by zeolite beta

We used zeolite beta as an adsorbing matrix to refold recombinant lactate dehydrogenase (LDH) protein collected as an insoluble aggregate from a bacterial expression system. The adsorption isotherm revealed that 1 g of zeolite adsorbed 200 mg of denatured LDH solubilized with a buffer containing 6 M of guanidine hydrochloride. The pH of the buffer had little effect on the adsorption, but this property was abolished by preincubation of the zeolite with polyethylene glycol (PEG) in a weight ratio of 1:10. These data suggest that the adsorption of LDH depends on the hydrophobicity of the zeolite surface, and that the adsorption of PEG to zeolite is sufficient to release LDH from its surface. LDH was thus released by refolding buffer containing PEG and arginine, and soluble LDH was obtained in its active enzymatic form. The addition of arginine dramatically increased the yield of LDH in a dose‐dependent manner. The overall refolding efficiency was optimized to 35%. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Identification of new inhibitors for alternative NADH dehydrogenase (NDH-II)

In bacterial membranes and plant, fungus and protist mitochondria, NADH dehydrogenase (NDH-II) serves as an alternative NADH : quinone reductase, a non-proton-pumping single-subunit enzyme bound to the membrane surface. Because NDH-II is absent in mammalian mitochondria, it is a promising target for new antibiotics. However, inhibitors for NDH-II are rare and unspecific. Taking advantage of the simple organization of the respiratory chain in Gluconobacter oxydans , we carried out screening of natural compounds and identified scopafungin and gramicidin S as inhibitors for G. oxydans NDH-II. Further, we examined their effects on Mycobacterium smegmatis and Plasmodium yoelii NDH-II as model pathogen enzymes.     

High-level expression and purification of apoaequorin.

A fairly rapid and improved method for producing large amounts of highly pure apoaequorin, the apoprotein of aequorin which emits light on binding Ca2+, is described. The method consists of fusing the gene of the outer membrane protein A (ompA) secretion signal peptide of Escherichia coli to the apoaequorin gene and expressing the fused gene in the bacterium. The expressed protein is correctly cleaved in the process of being secreted across the cell membrane into the culture medium. The apoaequorin is subsequently purified by acid precipitation and DEAE-cellulose chromatography, yielding a product of greater than 95% purity. The availability of pure apoaequorin makes possible detailed studies of the physical-chemical properties of this Ca2(+)-binding protein and allows for the preparation of pure aequorin for use in highly specific and sensitive assays for Ca2+.