A heterogeneous electrophysiological profile of bone marrow-derived mast cells

Electrophysiological properties of mouse bone marrow-derived mast cells (BMMC) were studied under the whole-cell clamp configuration. About one third of the cells were quiescent, but others expressed either inward or outward currents. Inwardly rectifying (IR) currents were predominant in 14% of the cells, and outwardly rectifying (OR) currents in 24%. The rest (22%) of the cells exhibited both inward and outward currents. The IR currents were eliminated by 1 mm Ba²⁺, and were partially inhibited by 100 μm quinidine. The reversal potential was dependent on extracellular K⁺, thereby indicating that K⁺ mediated the IR currents. The negative conductance region was seen at potentials positive to E _K. The OR currents did not apparently depend on the extracellular K⁺ concentration, but were reduced by lowering the extracellular Cl^? concentration. The OR currents were partially blocked by 1 mm Ba²⁺, and were further blocked by a Cl^? channel blocker, 4,4′-diisothiocyano-2, 2′-stilbenedisulfonate (DIDS). In addition, the reversal potential of the OR currents was positively shifted by decreasing the ratio of external and internal Cl^? concentrations, suggesting that Cl^? was a major ion carrier. In cells exhibiting IR currents, the membrane potential varied among cells and tended to depolarize by elevating the external K⁺ concentration. In cells with OR currents, the resting potential was hyperpolarized in association with an increase in conductance. These results suggest that BMMC have a heterogeneous electrophysiological profile that may underlie a variety of ion channels expressed in different phenotypes of mast cells. Activities of both the inwardly rectifying K⁺ channel and the outwardly rectifying Cl^? channel seem to contribute to the regulation of the membrane potential.     

Histological Observations on Initiation and Morphogenesis in Immature and Mature Embryo Derived Callus of Barley (Hordeum vulgare L.)

Callus was induced from immature and mature embryos of barley(cv. Haruna Nijo) on Murashige and Skoog medium containing 2mg l^-1 2,4-D and 5 mg l^-1 picloram, respectively. Paraffin sections(10 µm thick) were prepared for histology during callusinitiation and plant regeneration. Meristems were regeneratedfrom nodular compact callus (NC) derived from scutellar epidermisin immature embryos, whereas they were regenerated from NC derivedfrom epidermal cells of leaf or coleoptile bases in mature embryos.Regardless of the explant source, regeneration was predominantlythrough organogenesis, although regeneration through somaticembryogenesis infrequently occurred. Thus, the callus inducedfrom immature and mature embryos of barley was regarded as 'nodularcompact' rather than 'embryogenic'.Copyright 1995, 1999 AcademicPress Barley, callus, Hordeum vulgare, histology, immature embryo, mature embryo, regeneration     

Non-local interactions between light induced processes inCalliphora photoreceptors

Summary The prolonged depolarizing afterpotential (PDA) is a phenomenon which is tightly linked to visual pigment conversion. In order to determine whether processes underlying PDA induction and depression can spread in space, the PDA was recorded intracellularly in white-eyedCalliphora R1-6 photoreceptors and used to examine interactions between processes induced by activating statistically different photopigment molecules (Figs. 3–6). It was found that a PDA induced by converting some fraction of rhodopsin (R) molecules forward into the metarhodopsin (M) state can be completely depressed by equal or smaller amounts of pigment conversion, backward from metarhodopsin to rhodopsin even when largely different sets of pigment molecules were shifted in the respective directions, in agreement with previous experiments conducted on the barnacle. The characteristics of the afterpotentials obtained following the cessation of strong blue and green light stimuli which did not cause a net pigment conversion was examined (Figs. 7, 8). It was found that these afterpotentials, obtained when nonet R to M conversion took place, could not be depressed by an opposite net large M to R pigment conversion. Accordingly we propose to restrict the term PDA to an afterpotential which can be depressed by a net M to R pigment conversion. It is concluded: (a) that some processes underlying PDA induction and depression inCalliphora must interact at a distance which extends at least to the nearest neighboring pigment molecule, and (b) that inCalliphora photoreceptors net pigment conversion is required in order to induce and depress a PDA.Abbreviations R rhodopsin - M metarhodopsin - R to M rhodopsin to metarhodopsin pigment conversion - M to R metarhodopsin to rhodopsin pigment conversion - PDA prolonged depolarizing afterpotential - ERG electroretinogram - M potential metarhodopsin potential - ERP early receptor potential     

Purification and properties of ATPase from an alkalophilic Bacillus

ATPase was purified from an alkalophilic Bacillus. The enzyme has a molecular weight of 410,000 and consists of five types of subunits of molecular weights of 60,000 (α), 58,000 (β), 34,000 (γ), 14,000 (δ), and 11,000 (?). The subunit structure is suggested to be α₃β₃γδ?. The enzyme is activated by Mg²⁺ and Ca²⁺. The pH optima of the enzyme with 0.1 and 2.0 mm Mg²⁺ are 9 and 6, and those with 1 and 10 mm Ca²⁺ are 8–9 and 7, respectively. Ca²⁺-ATPase hydrolyzes only ATP, whereas Mg²⁺-ATPase hydrolyzes GTP and, to a lesser extent, ATP. The values of V and K_m of the enzyme with ATP in the presence of 10 mm Ca²⁺ or 0.6 mm Mg²⁺ at pH 7.2 are 17 or 0.5 units/mg protein and 1.2 or 0.3 mm, respectively. The enzyme with Mg²⁺ is appreciably activated by HCO^?₃. Relationship of the ATPase to the active transport system in the bacterium is suggested.     

Coenzyme B12-dependent diol dehydrase: Chemical modification with 2,3-butanedione and phenylglyoxal

The apoenzyme of diol dehydrase was inactivated by two arginine-specific reagents, 2,3-butanedione and phenylglyoxal, in borate buffer. In both cases, the inactivation followed pseudo-first-order kinetics. Kinetic data show that the incorporation of a single reagent molecule per active site of the enzyme is necessary for the complete inactivation. The modification with 2,3-butanedione was reversed by dilution of the reagent and borate concentrations (65% activity recovered). 1,2-Propanediol (substrate) partially protected the enzyme against inactivation. The holoenzyme was almost insensitive to 2,3-butanedione and phenylglyoxal, indicating that the essential arginine residue is prevented from the attack of these reagents either by direct blockage with the bound coenzyme or by an indirect conformational change caused by coenzyme binding. The inactivation of diol dehydrase by 2,3-butanedione did not result in dissociation of the enzyme into subunits. From these results, we concluded that the essential arginine residue is located at or in close proximity to the active site of diol dehydrase.     

Macromolecule synthesis of Escherichia coli BB at a lower or transient growth state.

Macromolecule synthesis in Escherichia coli BB at lower growth rates was investigated. The results indicate that a deviation in ribonucleic acid (RNA) content per cell at a lower growth rate from the exponential relationship to a specific growth rate is entirely attributable to the presence of nonviable cells, in which the RNA content is lower than in viable cells. Based on this fact, a mathematical expression of macromolecule contents versus specific growth rate was devised. Moreover, continuous changes in macromolecule content during unbalanced growth from late-logarithmic phase to stationary phase were measured. Although growth rates changed continuously, the data on deoxyribonucleic acid (DNA) or RNA content versus the specific growth rate calculated from the increments in cell number satisfactorily fitted the exponential lines obtained under balanced growth at a higher growth rate. However, no such relationship was observed in the plot of DNA or RNA content versus the specific growth rate calculated from the increments in optical density.     

Macromolecule synthesis of Escherichia coli BB at a lower or transient growth state.

Macromolecule synthesis in Escherichia coli BB at lower growth rates was investigated. The results indicate that a deviation in ribonucleic acid (RNA) content per cell at a lower growth rate from the exponential relationship to a specific growth rate is entirely attributable to the presence of nonviable cells, in which the RNA content is lower than in viable cells. Based on this fact, a mathematical expression of macromolecule contents versus specific growth rate was devised. Moreover, continuous changes in macromolecule content during unbalanced growth from late-logarithmic phase to stationary phase were measured. Although growth rates changed continuously, the data on deoxyribonucleic acid (DNA) or RNA content versus the specific growth rate calculated from the increments in cell number satisfactorily fitted the exponential lines obtained under balanced growth at a higher growth rate. However, no such relationship was observed in the plot of DNA or RNA content versus the specific growth rate calculated from the increments in optical density.     

Cultivation of mosquito cell lines in serum-free media and their effects on dengue virus replication

Summary Seven mosquito cell lines from five species (Aedes aegypti, Ae. albopictus, Ae. pseudoscutellaris, Culex tarsalis, andToxorhynchites amboinensis) were adapted to three kinds of serum-free media (SEM), which were composed of equal volumes of tryptose phosphate broth and of either Leibovitz (L15) medium, Eagle’s minimum essential medium, or Medium 199 with Hanks’ salts. Population growth rates of the cells cultivated in the SMFs were generally slower than those of original cell cultures maintained in conventional media containing bovine sera. A karyological study showed a significant shift to heteroploidy in two of the four cell lines examined. Four SMF-adapted sublines were compared with parental cultures for replication of dengue viruses.Ae. aegypti RML-12,Ae. albopictus C6/36,Ae. pseudoscutellaris AP-61, andTx. amboinensis TRA-171 demonstrated different levels of alteration in virus replication ranging from lower titers (as inAe. albopictus C6/36) to comparable or higher titers (as inAe. aegypti RML-12) when they were simultaneously inoculated with four dengue serotypes. Use of trade names and commercial sources is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.     

Gray and Red Fragments of the Egg of the Ascidian Ciona savignyi: Preferential Development of Muscle Cells from Gray Fragments

The egg of the ascidian Ciona savignyi is pinkish red with brownish myoplasm that contains the putative determinants responsible for differentiation of muscle cells. When dechorionated unfertilized eggs were centrifuged at moderate speed, eggs were divided into centripetal, small gray fragments and centrifugal, large red fragments. The former contained the female pronucleus and clear cytoplasm, while most of the latter was filled with yolk granules. An antibody raised against the myoplasm of C. intestinalis eggs extensively stained the cortical region of gray fragments, while the antibody stained only small regions of the red fragments. After insemination, both fragments cleaved and gave rise to partial embryos. When development of muscle and epidermal cells in the partial embryos was examined with specific antibodies, muscle development was conspicuous in gray partial embryos, while epidermal differentiation was extensive in red partial embryos. Furthermore, when expression of markers of differentiation was examined in cleavage-arrested gray and red fragments, the number of arrested gray fragments exhibiting the muscle marker was about three-fold greater than in controls. These results suggest that putative muscle determinants are concentrated into gray fragments.     

Expression of the GABAB receptor in Xenopus oocytes and inhibition of the response by activation of protein kinase C.

The functional GABAB receptor was expressed in Xenopus oocytes by injecting mRNA obtained from the cerebellum of the rat. Application of GABA in the presence of bicuculline induced a hyperpolarization under current-clamp conditions and an outward current under voltage-clamp conditions. Baclofen mimicked the effect of GABA in the presence of bicuculline, and the effect of baclofen was antagonized by phaclofen. The GABA-induced outward current was slightly inhibited by treatment with GDP-beta-S and was completely inhibited by treatment with GTP-gamma-S. The activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA), but not 4 alpha-phorbol-12,13-didecanoate, suppressed the GABAB receptor-mediated hyperpolarization, and the effect of TPA was antagonized by sphingosine. Thus, activation of protein kinase C inhibits the expressed GABAB receptor-mediated response.