Chloroplast DNA phylogeography of Fagus crenata (Fagaceae) in Japan

 CpDNA variation in Japanese beech, Fagus crenata (Fagaceae), was studied in 45 populations distributed throughout the species' range. Two cpDNA regions were sequenced: the non-coding region between the trnL (UAA) 5′exon and trnF (GAA), and the trnK region (including matK). Thirteen distinct cpDNA haplotypes were recognized and each haplotype was found to be geographically structured. Two major clades (I and II+III) were revealed in phylogenetic analyses among the haplotypes using F. sylvatica as an outgroup. The haplotypes of Clade I were distributed mainly along the Japan Sea side of the Japanese Archipelago, while those of Clade II+III occurred chiefly along the Pacific Ocean side. Consequently, the distribution of the two major cpDNA clades suggests that there were two migration routes in the history of F. crenata; one along the Japan Sea and the other along the Pacific Ocean side of the Japanese Islands. Received March 19, 2001 Accepted November 22, 2001     

Development of host-dependent high-grade tumor-specific immunity through a novel mechanism triggered by the Lyt-2+ tumor-specific T cell clone (K7L) that induces temporal growth of L1210 leukemia-K7L-variant

When a murine leukemia L1210-specific Lyt-2+ T cell clone, K7L, was injected i.p. into CD2F1 mice together with L1210, the normal growth of L1210 in the peritoneal cavity of the mice at the early stage (days 0 to 5) was strongly inhibited, but L1210 grew progressively at the middle-stage (days 5 to 10), and then was rejected at the late stage (days 10 to 20). The mice thus survived for long times (more than 60 days), whereas the normal control injected with L1210 alone died within 14 days. The L1210 that grew at the middle stage in mice initially inoculated with L1210 together with K7L was a K7L-insensitive (K7L-) variant. All of eight tumor clones established from L1210-K7L- by limiting dilution was insensitive to the antitumor activity of K7L, and this property of tumor clones was stable after repeated in vitro passage. The initial depression of the L1210 growth by K7L followed by growth and rejection of the variant L1210-K7L- by the host T cell activity was then found to prepare a strong, long-lasting (more than 3 mo) immunity to protect mice against the high-dose (10(7) cells per mouse) challenge of original L1210. Corresponding to this result, definite tumor (L1210)-specific cytotoxic T lymphocyte (CTL) activity against both variant and original L1210 targets was developed by antigen (L1210) restimulation in the culture of spleen cells from these mice, but was not increased to a detectable level before L1210-K7L- variant started to grow. It was suggested that the 1210-K7L- variant and the original L1210 should have the common tumor-specific antigen that was independent of the K7L-reactive antigen, and that original L1210, whose growth was retarded by K7L, primed the host with the common antigen to be enormously boosted by the subsequently growing L1210-K7L- variant.     

pH dependence of individual tryptophan N-1 hydrogen exchange rates in lysozyme and its chemically modified derivatives

Nuclear magnetic resonance analyses have been made of the individual hydrogen-deuterium exchange rates of tryptophan indole N-1 hydrogens in native lysozyme and its chemically modified derivatives including lysozyme with an ester cross-linkage between Glu-35 and Trp-108, lysozyme with an internal amide cross-linking between the epsilon-amino group of Lys-13 and the alpha-carboxyl group of Leu-129, and lysozyme with the beta-aspartyl sequence at Asp-101. The pH dependence curves of the exchange rates for Trp-63 and Trp-108 are different from those expected for tryptophan. The pH dependence curve for Trp-108 exchange exhibits the effects from molecular aggregation at pH above 5 and from a transition between the two conformational fluctuations at around pH 4. The exchange rates for tryptophan residues in native lysozyme and modified derivatives are not correlated with the thermodynamic or kinetic parameters in protein denaturation, suggesting that the fluctuations responsible for the exchange are not global ones. The exchange rates for tryptophan residues remote from the modification site are perturbed. Such tryptophan residues are found to be involved in a small but distinct conformational change due to the modification. Therefore, the perturbations of the N-1 hydrogen exchange rates are related to the minor change in local conformation or in conformational strain induced by the chemical modification.     

A novel proenkephalin processing carboxypeptidase and its activation by cyclic AMP dependent protein kinase

Carboxypeptidase B-like enzymes cleaving Met-enkephalin-Arg from synaptosomes of the rat striatum purified using a DEAE-cellulose column and Met-Arg-CH-Sepharose 4B affinity column proved to be different from enkephalin-convertase, lysosomal carboxypeptidase B-like enzyme, pancreas carboxypeptidase B and carboxypeptidase Y, in effects of inhibitors and activators, pH optimum (7.5-8.5) and molecular size (50,000). This enzyme, named "Processin CP-E" was activated by cAMP dependent protein kinase, and the Vmax was increased from 4.3 to 13.3 microM/min/mg protein, while the Km (28.2 microM) was unchanged.     

400 MHz two-dimensional nuclear Overhauser spectroscopy on anesthetic interaction with lipid bilayer

Interaction between a volatile anesthetic, methoxyflurane, and dipalmitoylphosphatidylcholine (DPPC) vesicle membrane was analyzed by nuclear Overhauser effect (NOE) difference spectroscopy and two-dimensional nuclear Overhauser spectroscopy (NOESY). The NOE difference spectra were obtained by selectively irradiating methoxy protons (hydrophobic end) of the anesthetic: a negative nuclear Overhauser effect of -2.94% was observed with the choline methyl protons of DPPC. The NOESY spectra revealed a cross-peak between the anesthetic methoxy protons and the choline methyl protons. A dipole-dipole interaction exists between the hydrophobic end of the anesthetic and the hydrophilic head group of DPPC. No other cross-peaks were observed. The anesthetic orients itself at the membrane/water interface by interacting with the hydrophilic surface of the DPPC membrane, leaving the hydrophilic end of the anesthetic molecule in the aqueous phase. The preferred residence site of dipolar volatile anesthetics is the membrane/water interface.     

Differential affinity of charged local anesthetics to solid-gel and liquid-crystalline states of dimyristoylphosphatidic acid vesicle membranes

Cationic local anesthetics decreased the transition temperature of the anionic phospholipid (dimyristoylphosphatidic acid, DMPA) vesicles. The counterion concentration changes the electrical double layer effect, and affects the magnitude of temperature depression caused by anesthetics. From the counterion effect on the transition-temperature depression, the partition coefficients of cationic local anesthetics to liquid-crystalline and solid-gel DMPA membranes were separately estimated. The differences in the partition coefficients between solid-gel and liquid-crystalline membranes correlated to the nerve blocking potencies. There are at least two states in the nerve membranes: resting state at higher temperature and excited state at lower temperature. We speculate that the resting state corresponds to the liquid-crystalline state, and the excited state to the solid-gel state. The difference in the partition coefficients to the resting and excited states is the cause of local anesthesia.     

A dicarba analog of beta-atrial natriuretic peptide (beta-ANP) inhibits guanosine 3',5'-cyclic monophosphate production induced by alpha-ANP in cultured rat vascular smooth muscle cells

The synthesis and biological properties are described of [Asu7,23']-beta-ANP-(7-28) (Asu, L-alpha-aminosuberic acid), a dicarba analog of beta-atrial natriuretic peptide (beta-ANP, an antiparallel dimer of human alpha-ANP with the chains linked by 7-23' and 7'-23 disulfide bonds). This Asu-analog (referred to as analog III) displaced 125I-alpha-ANP specifically bound to cultured rat vascular smooth muscle cells (VSMC) with an apparent Ki of 2.1 x 10(-8) M, but did not stimulate formation of intracellular cGMP at 10(-8) -10(-5) M. Analog III inhibited the alpha-ANP-stimulated cGMP production in VSMC competitively with a pA2 value of 7.45 and behaved as an antagonist of alpha-ANP in rat aorta smooth muscle relaxation. In addition, beta-ANP was also shown to inhibit the alpha-ANP-induced cGMP production in a dose-dependent manner. The mechanism of action of beta-ANP is also discussed.