Superposition properties of interacting ion channels.

Quantitative analysis of patch clamp data is widely based on stochastic models of single-channel kinetics. Membrane patches often contain more than one active channel of a given type, and it is usually assumed that these behave independently in order to interpret the record and infer individual channel properties. However, recent studies suggest there are significant channel interactions in some systems. We examine a model of dependence in a system of two identical channels, each modeled by a continuous-time Markov chain in which specified transition rates are dependent on the conductance state of the other channel, changing instantaneously when the other channel opens or closes. Each channel then has, e.g., a closed time density that is conditional on the other channel being open or closed, these being identical under independence. We relate the two densities by a convolution function that embodies information about, and serves to quantify, dependence in the closed class. Distributions of observable (superposition) sojourn times are given in terms of these conditional densities. The behavior of two channel systems based on two- and three-state Markov models is examined by simulation. Optimized fitting of simulated data using reasonable parameters values and sample size indicates that both positive and negative cooperativity can be distinguished from independence.     

The effect of ethanol on lateral and rotational mobility of plasma membrane vesicles isolated from cultured mar 18.5 hybridoma cells

Intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py-3-Py) and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) were used to evaluate the effect of ethanol on the rate and range of the lateral mobility and the range of the rotational mobility of bulk bilayer structures of the plasma membrane vesicles (ATCC-PMV) isolated from cultured hybridoma cells (ATCC TIB 216). In a concentration-dependent manner, ethanol increased the excimer to monomer fluorescence intensity ratio (I/I) of Py-3-Py in the ATCC-PMV and decreased the anisotropy (r), limiting anisotropy (r) and order parameter (S) of DPH in the ATCC-PMV. This indicates that ethanol increased both the lateral and rotational mobility of the probes in the ATCC-PMV. Selective quenching of DPH by trinitrophenyl groups was utilized to examine the range of transbilayer asymmetric rotational diffusion of the ATCC-PMV. The anisotropy (r), limiting anisotropy (r ) and order parameter (S) of DPH in the inner monolayer were 0.024, 0.032, and 0.069, respectively, greater than calculated for the outer monolayer of the ATCC-PMV. Selective quenching of DPH by trinitrophenyl groups was also used to examine the transbilayer asymmetric effects of ethanol on the range of the rotational mobility of the ATCC-PMV. Ethanol had a greater increasing effect on the range of the rotational mobility of the outer monolayer as compared to the inner monolayer of the ATCC-PMV. It has been proven that ethanol exhibits a selective rather than nonselective fluidizing effect within the transbilayer domains of the ATCC-PMV.This paper was supported in part by a research grant from the Korea Science and Engineering Foundation (KOSEF 88-1013-01) and from the Korea Research Foundation (1991–1993).     

Regeneration of haploid plants from isolated microspores of asparagus (Asparagus officinalis L.)

High percentages of micro-calli and micro-derived embryos were produced from isolated asparagus microspores at late uninucleate stage on MS liquid medium supplemented with 1.0 mg l^–1 2,4-D and 0.5 mg l^–1 BA. Two types of calli, namely compact callus (CC) and loose callus (LC), were found. Plantlets were regenerated via organogenesis, when these calli were transferred onto MS solid medium supplemented with 1.0 mg l^–1 BA and 0.2 mg l^–1 IBA 6 weeks. Embryos were produced from liquid cultured microspores, or from solid cultured micro-calli. The frequencies of haploid plant production from organogenesis and embryogenesis were compared. Effects of plant growth regulators on callus production, plantlet regeneration, and haploid plant production were tested. The combination of BA 1.0 mg l^–1 and IBA 0.2 mg l^–1 resulted the highest precentage of haploid plant production (7.7% from CC, 4.3% from LC).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA 3-indolybutyric acid - BA 6-binzyladinine - NAA naphtalene acetic acid - MS Murashige and Skoog     

Dopaminergic and adrenergic receptors synergistically stimulate browning in 3T3-L1 white adipocytes

Journal of Physiology and Biochemistry - The browning of white adipose tissue (WAT) has attracted considerable attention in the scientific community as a popular strategy for enhancing energy...     

AREL1 resists the apoptosis induced by TGF-β by inhibiting SMAC in vascular endothelial cells

Abnormal apoptosis of vascular endothelial cells is an important feature of arteriosclerosis (AS). Here, we induced apoptosis in human umbilical vein endothelial cells (HUVECs) using transforming growth factor-β (TGF-β), and investigated the role of antiapoptotic E3 ubiquitin ligase (AREL1) in the apoptosis of vascular endothelial cells. We proved that AREL1 is downregulated in TGF-β treated HUVECs. The overexpression of AREL1 inhibits the activation of Caspase-3 and Caspase-9 and attenuates cell apoptosis induced by TGF-β. According to the result of coimmunoprecipitation, AREL1 interacts with the proapoptotic proteins the second mitochondria-derived activator of caspases (SMAC) in TGF-β treated HUVECs. In addition, miR-320b inhibits the expression of AREL1, and the overexpression of AREL1 attenuates the apoptosis induced by miR-320b mimics in HUVECs. In conclusion, AREL1 is downregulated by miR-320b. AREL1 overexpression inhibits TGF-β induced apoptosis through downregulating SMAC in vascular endothelial cells. Our study explores pathogenesis regulation mechanism and new biological therapeutic targets for vascular disease.     

PCR-mediated screening and labeling of DNA from clones

A simplified and economical protocol for DNA library screening and nonradioactive labeling is described. Bacterial clones are lysed in 1% of Triton X-100 and subjected to polymerase chain reaction in the presence of digoxigenin-11-dUTP to screen and simultaneously to label the DNA inserts. Bacteriallysates are stable in storage at −20°C and can be used repeatedly for PCR-mediated labeling. In this protocol, very low concentrations of dNTP, digoxigenin-dUTP, and primers are used in combination with a reduced reaction volume. This will considerably reduce the expense of screening and labeling bacterial clones and facilitate the exchange of DNA probes among laboratories.     

First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

Larval development of Crangon hakodatei Rathbun (Decapoda: Crangonidae) reared in the laboratory

Complete larval development of Crangon hakodatei Rathbun isdescribed, based on material hatched in the laboratory fromovigerous females. The species has six zoeal stages and onepostlarval stage. The morphological characters of the larvaland postlarval stages are described with illustrative figuresand compared with those of two congeneric species. The zoealstages of C. hakodatei can be distinguished from those of otherCrangon species in the number of segments of the antennule peduncle,the number of setae on the antennal scale and basis of the maxillipeds,and the stages of appearance of pereiopods. The first zoealstage in the seven species of Crangon are compared and an annotatedkey for distinguishing them is also provided.     

1α,25-dihydroxy-24-oxo-16-ene vitamin D3, a metabolite of a synthetic vitamin D3 analog, 1α,25-dihydroxy-16-ene vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells

1α,25(OH)₂-16-ene-D₃, a synthetic analog of the steroid hormone, 1α,25(OH)₂D₃, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1α,25(OH)₂-16-ene-D₃ expresses its biological activities are still not completely understood. Our previous in vitro study in a perfused rat kidney indicated for the first time that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently. 1α,25(OH)₂-24-oxo-16-ene-D₃, an intermediary metabolite of 1α,25(OH)₂-16-ene-D₃ formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1α,25(OH)₂-24-oxo-D₃, the corresponding intermediary metabolite of 1α,25(OH)₂D₃. In a subsequent in vivo study, we also reported that 1α,25(OH)₂-24-oxo-16-ene-D₃ exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1α,25(OH)₂-24-oxo-16-ene-D₃, in generating some of the key biological activities ascribed to its parent, we performed the present in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1α,25(OH)₂-24-oxo-16-ene-D₃ in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1α,25(OH)₂D₃. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1α,25(OH)₂D₃ itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1α,25(OH)₂-24-oxo-16-ene-D₃, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1α,25(OH)₂-16-ene-D₃.