Decreased uptake and retention of rhodamine 123 by mitochondria in feline sarcoma virus-transformed mink cells

A reduce uptake and retention of the mitochondria-specific membrane potential probe rhodamine 123 by feline sarcoma virus (FeSV)-transformed mink fibroblasts (64F3) has been detected. The decreased accumulation of rhodamine 123 by 64F3 mitochondria is not due to abnormal plasma membrane dye permeability, since after microinjection of the dye these cells are still unable to retain the dye at levels comparable to the untransformed parental cells, CCL 64. Nigericin, an ionophore that mediates an electrically neutral exchange of protons for potassium ions resulting the elimination of the pH gradient across the mitochondrial membrane and a compensatory increase in mitochondrial membrane potential with continued respiration, increases both the dye uptake and the retention time in transformed 64F3 cells. These results suggest that mitochondria in FeSV-transformed mink cells may have an abnormally low mitochondrial membrane potential accompanied by a relatively high pH gradient. Since anioic metabolites such as pyruvate and glutamate are accumulated by mitochondria in proportion to the delta pH across the mitochondrial membrane, the abnormal mitochondria described here may contribute to the abnormal metabolic state of FeSV-transformed cells.     

Computer simulations of mass transport for nonidentical interacting molecules.

We have developed a system for simulating the mass transport properties of interacting nonidentical macromolecules where the association is of the form A + B ? C, C + B ? D. Our simulation programs operate in a minicomputer (PDP 1104) with 16K of core and provide results identical to methods previously usable only with large computers. We use a rectangular approximation for the centrifuge cell which greatly simplifies calculation, although it introduces a few percent error into any attempt at quantitative fitting of actual data. The program as written is directly applicable to gel chromatography, simply by substitution of flow for centrifugal field and dispersion for diffusion. Simulations of centrifuge results have been compared with experimental results for two systems which have been proposed to fit the association pattern described—nitrogenase components and an antigen-antibody interaction. In both cases the results of our simulations suggest that the accepted interpretation of the experimental results may need to be modified. For the antigen-antibody interaction, the presence of multivalent higher order complexes apparently is required to explain the centrifuge results. For nitrogenase, one cannot readily distinguish the case of association to form both 1:1 and 1:2 molar complexes from that of formation of only the 1:1 complex on the basis of the published data. Criteria for making such a discrimination are discussed.     

Photocontrol of the germination of onoclea spores: v. Analysis of germination processes by means of temperature

The physiological nature of photoinduced germination of Onoclea sensibilis L. spores was investigated by temporarily applying a range of temperatures, particularly 40 C, before and after short light treatment. Controls were germinated at 25 C.     

LONGITUDINAL MICROFIBRILLAR ALIGNMENT IN THE WALL OF CYLINDRICAL NITELLA RHIZOIDAL CELLS: OBSERVATION UNDER POLARIZING AND INTERFERENCE MICROSCOPES

Polarizing and interference microscopes were employed to measure overall orientation of microfibrils and dry matter (expressed in optical thickness) in the cell wall of Nitella rhizoids. The microfibrils are aligned predominantly parallel to the cell's long axis (positively birefrengent), except in the apical dome where the arrangement appears to be random. The optical thickness, however, is greater at the very tip and the base region. The wall contains about 50-60% of acid extractable amorphous, noncrystalline substance. This extraction does not make a significant change in the alignment, but the remaining dry matter becomes less at the tip and increases slightly toward the base. The alignment parallel to the direction of cell growth in the rhizoidal cell is different from that of the elongating Nitella internodal cell where the alignment is transverse.     

Perturbance analysis of nuclear determination in Tetrahymena I: background, rationale, and illustrative example, employing temperature responses

A model based on comparative considerations, is presented for the nuclear determination of mating type in Tetrahymena thermophila. The model proposes a system of three binary control elements, each capable of stable persistence in one of two states. A general method is proposed for evaluating the model and for assigning particular mating types to particular compound states. Preliminary assignments of mating types are made from the responses of nuclei to temperature differentials.     

Hyphal anastomosis inPyricularia oryzae cav.

Summary Hyphal anastomosis inPyricularia oryzae occurs naturally in the lower epidermal cells and in the vascular bundles of young lesions on rice. In those cells the invading blast fungus are active. Two hyphal cells lying side by side start an anastomosis by forming two, one from each, very short penetration pegs which are opposite to each other. The cell wall of the pegs and the wall in the vicinity of them are then gradually eliminated and thus form a fusion aperture of 0.2–0.6 m in diameter that is big enough for the migration or exchange of nucleus and cytoplasm between two hyphal cells. The explanation of genetical variation inP. oryzae may be sought on the basis of the ultrastructural evidence of hyphal anastomoses presented in this paper.     

Interaction of methotrexate with polycationic carriers

Binding parameters were measured for the $\text{in vitro}$ interaction of methotrexate with poly-L-lysine and diethylaminoethyl dextran. The complexes were found to have apparent affinity constants of 2590 and 440 M^?1 respectively at 37°, ionic strength 0.02. For hypotonic solutions containing therapeutic concentrations of methotrexate, this results in 87% binding of the drug to poly-L-lysine and 74% to diethylaminoethyl dextran. While the binding decreased to about 50% upon increasing ionic strength to physiologic levels, sufficient drug-carrying capacity was retained at isotonicity to support the potential utility of such complexes as tissue-specific drug carriers.     

Recombinant human acid alpha-glucosidase generated in bacteria: antigenic, but enzymatically inactive.

Genetic deficiency of acid alpha-glucosidase (GAA) results in glycogen storage disease type II. To investigate whether we could generate a functional recombinant human GAA protein for future enzyme replacement therapy, we subcloned the GAA cDNA into the bacterial expression plasmid pMaI and analyzed the recombinant protein produced. This nonglycosylated recombinant human GAA was found to be antigenic by reacting with polyclonal rabbit antibody to human placental GAA using ELISA and Western techniques. However, the protein was not enzymatically active, suggesting that glycosylation may play a role in enzymatic function.     

Purification and characterization of 3-hydroxymethylglutaryl-coenzyme A reductase of Schistosoma mansoni: regulation of parasite enzyme activity differs from mammalian host

The enzyme 3-hydroxymethylglutaryl-CoA (HMG-CoA) reductase plays a critical role in regulating the production of cholesterol, dolichols, and ubiquinones in mammals. The inhibition of this enzyme in Schistosoma mansoni is accompanied by a cessation of egg production by the female parasite and a reduced ability of the parasite to properly glycoslyate their proteins. Furthermore, we recently demonstrated that mevinolin, if given continuously over a period of 10-14 days, is a potent antischistosomal drug. In this paper, we describe the properties of purified HMG-CoA reductase from S. mansoni. Using affinity chromatography, we were able to obtain a 417-fold purification of the enzyme which had Km values similar to the rat enzyme for HMG-CoA and NADPH. The Ki value for mevinolin, a potent and selective inhibitor of the rat reductase (Ki = 0.6 nM), was significantly higher (Ki = 46 nM) for the schistosome enzyme. SDS-PAGE and HPLC of the purified enzyme resulted in the appearance of a single protein, which had a molecular weight (66,000) in the range reported for the rat enzyme. Parasite reductase activity, unlike that of its host, did not display a circadian rhythm. Furthermore, agents which elevate (cholestyramine) or decrease (cholesterol) mammalian reductase activity had no effect on the parasite enzyme. Our results suggest that the mechanism which regulates production of the parasite's enzyme may differ from its mammalian host.     

肾综合征出血热病毒糖蛋白的提纯和鉴定

以感染肾综合征出血热病毒(HFRSV)的Vero E6细胞为材料,用免疫亲和层析结合制备聚丙烯酰胺凝胶电泳(PAGE)从感染细胞中提纯了HFRSV两种糖蛋白。先用免疫亲和层析从感染细胞的粗制抗原中获得含有四种蛋白的混合液,用[~3H]-氨基葡萄糖在感染细胞中标记病毒糖蛋白,观察到[~3H]-氨基葡萄糖只结合入78K和57K的病毒蛋白。再用制备SDS-PAGE从HFRSV混合液中提纯78K和57K两种蛋白。实验证明这两种糖蛋白均具中和抗原决定簇,57K的糖蛋白尚具血凝活性,初步鉴定表明这两种糖蛋白相当于文献报道的HFRSV G_1和G_2。