An electrokinetics-induced stagnation flow was created inside a microscale cross-channel. Compared to hydrodynamic-induced microfluidics, this flow system can be readily assembled and the operation is very simple due to a low pressure drop. Through image analysis, a fairly homogeneous, two-dimensional elongational flow was observed. The initial conformation of DNA molecules and residence time inside the flow field play important roles in determining the extent of DNA stretching. A coarse-grain molecular simulation agrees reasonably well with experimental observations.

Ag+ (0.5-10 microM) is known to produce a transient contraction of intact frog skeletal muscle fibers followed by complete inhibition of excitation-contraction (E-C) coupling. We have carried out physiological and biochemical experiments to investigate the basis of this effect. Dihydropyridine (DHP) Ca2+ channel blockers, which inhibit the voltage sensor of the Ca2+ channel, completely inhibit Ag+ contractions. Removal of extracellular Ca2+, or blockade of Ca2+ entry with cadmium, does not inhibit Ag+ contractions. Activation of the Ca2+ channel's voltage sensor with the Ca2+ channel agonists Bay K 8644 or with perchlorate, potentiates the Ag(+)-induced contraction. Ag+ binds to the partially purified rabbit skeletal muscle Ca2+ channel and inhibits DHP binding (IC50 = 1.1 microM) and sulfhydryl (SH) reactivity (IC50 = 0.11 microM) over the concentration range where it inhibits E-C coupling. Oxidation of free SH groups by H2O2 or their reaction with DTNB prevents Ag+ contractions, while DTT reduction of oxidized SH groups restores Ag+ contractions. These results suggest that Ag+ binds to critical SH groups on the DHP receptor Ca2+ channel, resulting in modification of the channel's voltage sensor and the failure of E-C coupling.

The P-type ATPases (e.g., Na+-K+-ATPase and Ca2+-ATPase) occur widely in living cells of fungi, Protozoa, plants, and animals. These ion pumps show a high degree of divergence in their primary structures but share a limited number of common amino acid residues for their ATP-catalytic function. Particularly, the amino acid sequences for the phosphorylation site (DKTGTLT) and the binding site for ATP (and its analogs; GDGVND) are conserved throughout evolution. Using two degenerate oligonucleotides corresponding to these regions, we applied a polymerase chain reaction (PCR) technique to the search for P-type ATPase isoforms, which will provide a clue to the evolutionary mechanisms of ion pumps in Tetrahymena thermophila. A total of 12 distinct P-type ATPase genes were identified. Sequence comparisons revealed that seven of them can be compiled into a multigene family, which is similar to animal Na+-K+- and H+-K+-ATPase genes. One of them is close to the sarco(endo)plasmic reticulum Ca2+-ATPase gene, and the other four share a significant homology with the gene encoding Plasmodium ATPase-1 whose function is unknown. A Northern blot analysis and reverse transcriptase-PCR demonstrated that all identified genes are expressed, but the expression levels vary widely under different culture conditions. A Southern blot analysis after pulse-field gel electrophoresis showed that all of these genes exist in T. thermophila macronuclei. The Na+-K+- and H+-K+-ATPase gene family has a high multiplicity (at least 10 different genes detected on genomic Southern blot analysis) and is distributed on four different macronuclear chromosomes. On the basis of a calculation with the amino acid sequences of the cloned cytoplasmic loop region (between the phosphorylation and the gamma-[4-(N-2-chloroethyl-N-methylamino)]-benzylamido ATP sites), the genes with >80% identity form a cognate linkage group within the same macronuclei chromosome, whereas the genes with <70% identity are separated in different chromosomes. The phylogenetic analysis showed that this multigene family is the result of a series of gene duplications.

The 2‐bromoacetophenone acetal (I) is converted to the 2‐lithio derivative and then coupled with the p‐benzoquinone monoacetal (II), yielding the spiro‐fused methylenisobenzofuran (IV) after release of methanol.

The spirobenzoisofurancyclohexadienones (IV) and their acetals (VII) are prepared as shown in the reaction scheme.