One molecule of diphtheria toxin fragment a introduced into a cell can kill the cell

Erythrocyte ghosts containing a known number of molecules of purified fragment A of diphtheria toxin with a constant amount of FITC-BSA as a fluorescence marker were prepared by dialyzing a mixture of erythrocytes and these substances against hypotonic solution. These substances were then introduced into diphtheria toxin-resistant mouse L cells by virus-mediated cell fusion of the cells with the ghosts, and mononuclear recipients that had fused with only one erythrocyte ghost were separated in a fluorescence-activated cell sorter (FACS) on the basis of their cell size and fluorescence intensity. After separation, the viability of cells containing known numbers of fragment A was examined by measuring colony-forming ability. The results demonstrated that a single molecule of fragment A was sufficient to kill a cell.This fact was confirmed by introduction into cells of fragment A from an immunologically related mutant toxin, CRM 176 (fragment A-176); this has a completely functional fragment B region, but in cell extracts, the enzymic activity of its fragment A is about 10 fold less than that of wild toxin. The cytotoxicity of CRM 176 is about two hundredths of that of the wild-type (Uchida, Pappenheimer and Greany, 1973). As expected, about 100–200 fold excess of fragment A-176 was needed to kill the cells.     

13C and 1H NMR studies of helix-coil transition of poly(β-benzyl-L-aspartate) and poly(γ-benzyl-L-glutamate): Behavior in nonprotonating solvent mixtures,and origin of solvent-induced chemical shift

From the results of ¹³C-nmr measurement of poly(β-benzyl-L -aspartate) and its model compounds in dimethyl sulphoxide/deuterated chloroform mixtures, it was found that the side chain of poly(β-benzyl-L -aspartate) is solvated by dimethyl sulphoxide in the region more than dimethyl sulphoxide 20% (v/v), where the backbone maintains the α-helix. The chemical shift differences in the benzyl group carbons of poly(γ-benzyl-L -glutamate) (trifluoroacetic acid/deuterated chloroform) accompanied by the helix-coil transition, originate from the interaction between the ester group of the side chain and trifluoroacetic acid. The chemical shift difference in the ester carbon is similar. On the other hand, the chemical shift differences of the side-chain carbons in the alkyl portion (C^β, C^γ) originate not only from the interaction between the ester group of the side chain and trifluoroacetic acid, but also from some other unknown factors. The chemical shift differences of the side-chain carbons of poly(β-benzyl-L -aspartate) originate from the interaction between the ester group of the side chain and trifluoroacetic acid.     

Production of itaconic acid by Aspergillus terreus immobilized in polyacrylamide gels

Summary Living Aspergillus terreus cells were entrapped in polyacrylamide gels and employed in both replacement batch and continuous column reactors to produce itaconic acid from glucose.With the replacement batch reactor, maximum itaconic acid productivity was observed under the following conditions: pH 2.50, temperature at 35°C, addition of NH₄H₂PO₄ and MgSO₄·7H₂O. Using the continuous reactor, the maximum itaconic acid yield was 60 mg/h/40 g of gel. The biocatalyst activity or half-life was about 10 days.     

Methylamine facilitates demonstration of specific uptake of diphtheria toxin by CHO cell and toxin-resistant CHO cell mutants

We have measured the specific uptake of ¹²⁵I-labelled diphtheria toxin in the presence of methylamine by a number of cell lines with different sensitivities to diphtheria toxin. The results show a strong correlation between the toxin sensitivities of the cell lines and the amount of specific uptake. The specific association of labelled toxin with cells was clearly demonstrated even with CHO cells, a cell line with relatively low sensitivity. Thus, CHO cell mutants that are resistant to diphtheria toxin could be classified as toxin-binding or non-binding cells by this method.     

Biosynthesis of poly(3-hydroxyalkanoate) from amino acids

It was found that an optically active copolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), denoted as P(3HB-co-3HV), is synthesized by Alcaligenes eutrophus H16 from several amino acids under various fermentation conditions. The optimum condition for the biosynthesis from one amino acid, threonine, was investigated and its biosynthetic pathway was discussed on the basis of the relation between the fermentation condition and the co-monomer composition of the produced polyesters.     

Callus regeneration from cotyledon protoplasts ofChamaecyparis obtusa (Hinoki cypress)

Summary Protoplasts were isolated from cotyledons of 1- to 1.5-mo.-old seedlings ofChamaecyparis obtusa using 1% driselase or 0.25% pectolyase Y-23 in combination with 1% cellulase RS in 0.6M mannitol solution. Cell division and colony formation were induced efficiently in liquid Murashige and Skoog’s (MS) medium containing 0.6M mannitol and 10 to 30 μM 2,4-dichlorophenoxyacetic acid or 1 μM of naphthaleneacegic acid at the cell density of 1 to 2×10³ ml. Continued callus proliferations was observed by transferring tissue to fresh medium of the same composition as the induction medium without mannitol. Campbell and Durzan’s medium and ammonium nitrate-free MS medium were less effective than MS medium. High concentration of benzyladenine (1 or 10 μM) was inhibitory to cell division.     

Application of immobilized lipase to regio-specific interesterification of triglyceride in organic solvent

Summary Lipase from Rhizopus delemar was immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers or by binding to various types of porous silica beads. The immobilized lipase preparations thus obtained were examined for their activity in converting olive oil to an interesterified fat (cacao butter-like fat), whose oleic acid moieties at 1- and 3-positions were replaced with stearic acid moieties, in the reaction solvent n-hexane. Although all of the immobilized preparations exhibited some activity, lipase adsorbed on Celite and then entrapped with a hydrophobic photo-crosslinkable resin prepolymer showed the highest activity, about 75% of that of lipase simply adsorbed onto Celite. Entrapment markedly enhanced the operational stability of lipase.Dedicated to Professor H. Holzer, Freiburg University, on his 60th birthday (June 13, 1981)     

Seasonal and areal features of the lagoonal environment in Lake Nakanoumi,a shallow coastal lagoon in Japan

Lake Nakanoumi is a shallow coastal lagoon connected with the Japan Sea by a narrow channel. Over the past decade, land reclamation resulted in a 33% reduction of the lagoon's surface area. The remaining water basin of Lake Nakanoumi is scheduled to be artificially freshened to supply irrigation water for the newly reclaimed lands. This paper deals with the seasonal and areal features of the lagoonal environment prior to the beginning of the artificial desalinization.     

Simultaneous determination of triazolo-benzophenone [2′,5-dichloro-2-(3-glycylaminomethyl-5-methyl-4H-1,2,4-triazol-4-yl)-benzophenone] and its major blood metabolite, triazolam, in monkey plasma by electron-capture gas—liquid chromatography

A gas—liquid chromatographic method for the simultaneous determination of triazolobenzophenone [2′,5-dichloro-2-(3-glycylaminomethyl-5-methyl-4H-1,2,4-triazol-4-yl)-benzophenone, TB] and its major blood metabolite, triazolam, 8-chloro-6-(o-chlorophenyl)-1-methyl-4H-s-triazolo[4,3-a][1,4]benzodiazepine (TZ), in monkey plasma was developed. Decomposition of TB was observed during gas—liquid chromatography. In alkaline medium, TB in plasma was submitted to ring closure reaction to yield triazolo-aminoquinoline, [4-amino-7-chloro-5-(2-chlorophenyl)-1-methyl-4H-s-triazolo[4,3-a]quinoline (TAQ), while TZ remained unaffected, and TAQ and TZ in the benzene extract were assayed by gas—liquid chromatography using an electron-capture detector. The concentration ranges studied were from 5 to 40 ng of TB per 0.5 ml of plasma and from 2 to 20 ng of TZ per 0.5 ml of plasma. This method could be applied to the determination of the plasma levels of TB and TZ in monkeys following intravenous administration of a single 0.2 mg/kg dose of TB.