Purification and properties of rabbit liver phosphorylase phosphatase.

A procedure for the purification of rabbit liver phosphorylase phosphatase is described. The specific activity of the preparation is 2,100 units/mg of protein, representing a 25,000-fold purification. During the initial steps of the purification a large activation of enzyme activity was observed. The molecular weight of the purified enzyme was estimated by Sephadex G-75 chromatography to be 35,000, and by sucrose density ultracentrifugation to be 34,000 (2.9 S). On Na dodecyl-SO4 polyacrylamide disc gel electrophoresis a single component with a molecular weight of 34,000 was observed. The pH optimum is 6.9 to 7.4, and the Km for rabbit muscle phosphorylase alpha is 2 muM. The same procedure is also applicable to the extensive purification of phosphorylase phosphatase from rabbit muscle.     

Properties of a clonal muscle cell line from rat heart

Two clonal cell lines have been derived from the thoracic aorta of embryonic rats. Both of these cell lines, at some stage of their development possess membranes capable of generating overshooting action potentials spontaneously. Contiguous cells of each of these lines are electrically coupled. Ultrastructural analysis consistently reveals the presence of rows of pinocytotic vesicles, a well-developed rough endoplasmic reticulum, massive tracts of thin filaments oriented parallel to the longitudinal axis of the cell and randomly dispersed intermediate sized filaments. The specific activities of the enzymes myokinase and creatine phosphokinase (CPK) increase 3- to 5-fold after growth has ceased. These two cell lines synthesize a muscle type CPK isoenzyme after the cessation of cell division. It is concluded that these cell lines proliferate as myoblasts and develop into cells which phenotypically resemble smooth muscle. A third clonal cell line, from fetal rat aorta, with properties of both smooth and skeletal muscle, is also described.     

Sites of action of D2O in intact and skinned crayfish muscle fibers

Summary The effect on tension development of replacing 90% of the H₂O of the bathing saline with D₂O was studied on intact single fibers, and on skinned fibers before and after the latter were treated so as to eliminate Ca-accumulation by the sarcoplasmic reticulum (SR). Excitation-contraction coupling (ECC) of intact fibers is not abolished, but is depressed by D₂O so that higher depolarizations are required to elicit a given tension. The reduction in tension at a given level of depolarization is not due to inhibition of the contractile system. The latter showed an enhanced Ca sensitivity; that is, skinned fibers respond to Ca concentrations that are 1–2 orders of magnitude smaller in D₂O than in H₂O saline. When bathed in D₂O saline, intact fibers or skinned fibers with functional SR can still accumulate and release Ca in sufficient quantities to allow repeated induction of maximum tensions. Relaxation is slowed in all three types of preparation, perhaps because of an increased affinity of troponin to Ca in D₂O salines.     

The complete mitochondrial genome of yarrowia lipolytica

We here report the complete nucleotide sequence of the 47.9 kb mitochondrial (mt) genome from the obligate aerobic yeast Yarrowia lipolytica. It encodes, all on the same strand, seven subunits of NADH: ubiquinone oxidoreductase (ND1-6, ND4L), apocytochrome b (COB), three subunits of cytochrome oxidase (COX1, 2, 3), three subunits of ATP synthetase (ATP6, 8 and 9), small and large ribosomal RNAs and an incomplete set of tRNAs. The Y. lipolytica mt genome is very similar to the Hansenula wingei mt genome, as judged from blocks of conserved gene order and from sequence homology. The extra DNA in the Y. lipolytica mt genome consists of 17 group 1 introns and stretches of A+Trich sequence, interspersed with potentially transposable GC clusters. The usual mould mt genetic code is used. Interestingly, there is no tRNA able to read CGN (arginine) codons. CGN codons could not be found in exonic open reading frames, whereas they do occur in intronic open reading frames. However, several of the intronic open reading frames have accumulated mutations and must be regarded as pseudogenes. We propose that this may have been triggered by the presence of untranslatable CGN codons. This sequence is available under EMBL Accession No. AJ307410.     

The automatic pilot of honeybees

Using scanning harmonic radar, we make visible for the first time the complete trajectories of "goal-vector" flights in honeybees. We demonstrate that bees captured at an established feeding station, and released elsewhere, nevertheless embark on the previously learned vector flight that would have taken them directly home from the station, had they not been artificially displaced. Almost all of the bees maintained accurate compensation for lateral wind drift, and many completed the full length of the vector flight before starting to search for their hive. Our results showed that bees tend to disregard landscape cues during these vector flights, at least initially, and rely on the "optic flow" of the ground beneath them, and their sun compass, to judge both direction and distance.     

Human CD34+ HLA-DR- bone marrow cells contain progenitor cells capable of self-renewal, multilineage differentiation, and long-term in vitro hematopoiesis.

Human bone marrow cells expressing CD34 but not HLA-DR were isolated by immunofluorescence flow cytometric cell sorting. These cells contained a hematopoietic cell (CFU-B1) capable of producing, in an in vitro semisolid culture system, blast-cell-containing colonies, which possessed the capacity for self-renewal and commitment to multipotential differentiation. In addition, CD34+ HLA-DR- marrow cells contained primitive megakaryocyte progenitor cells, the burst-forming unit-megakaryocyte (BFU-MK). A subset of CD34+ HLA-DR- marrow cells lacking the expression of CD15 and CD71 was obtained by flow cytometric cell sorting and was capable of sustaining in vitro hematopoiesis in suspension culture for up to 8 weeks in the absence of a preestablished adherent marrow cell layer. The combination of IL-3 + IL-1 alpha and IL-3 + IL-6 sustained proliferation of these cells for 8 weeks, induced maximal cellular expansion, and increased the numbers of assayable progenitor cells. These studies demonstrate that human CD34+ HLA-DR- marrow cells and their subsets contain primitive multipotential hematopoietic cells capable of self-renewal and of differentiation into multiple hematopoietic lineages.     

StructureActivity Relationships of Antimicrobial Gallic Acid Derivatives from Pomegranate and Acacia Fruit Extracts against Potato Bacterial Wilt Pathogen

Bacterial wilts of potato, tomato, pepper, and or eggplant caused by Ralstonia solanacearum are among the most serious plant diseases worldwide. In this study, the issue of developing bactericidal agents from natural sources against R. solanacearum derived from plant extracts was addressed. Extracts prepared from 25 plant species with antiseptic relevance in Egyptian folk medicine were screened for their antimicrobial properties against the potato pathogen R. solancearum by using the disc‐zone inhibition assay and microtitre plate dilution method. Plants exhibiting notable antimicrobial activities against the tested pathogen include extracts from Acacia arabica and Punica granatum. Bioactivity‐guided fractionation of A. arabica and P. granatum resulted in the isolation of bioactive compounds 3,5‐dihydroxy‐4‐methoxybenzoic acid and gallic acid, in addition to epicatechin. All isolates displayed significant antimicrobial activities against R. solanacearum (MIC values 0.5–9 mg/ml), with 3,5‐dihydroxy‐4‐methoxybenzoic acid being the most effective one with a MIC value of 0.47 mg/ml. We further performed a structure–activity relationship (SAR) study for the inhibition of R. solanacearum growth by ten natural, structurally related benzoic acids.