Variation in the Frequency and Extent of Hybridization between Leucosceptrum japonicum and L. stellipilum (Lamiaceae) in the Central Japanese Mainland

Variations in the frequency and extent of hybridization among mixed populations located in the same contact zone provide natural laboratories for the study of extrinsic reproductive isolation maintaining species integrity. In this study, we examined the pattern of hybridization between L. japonicum and L. stellipilum among mixed populations in different localities of a contact zone. The genetic structures from three sympatric populations and six mixed populations in the hybrid zone, and five reference populations far from the contact zone, were characterized using 10 neutral nuclear microsatellite markers. Evidence from genetic distance-based clustering analysis, the frequency distribution of admixture proportion values, and the hybrid category assignment approaches indicated that the frequency and extent of hybridization varied considerably among populations in the contact zone between L. japonicum and L. stellipilum. One likely explanation is that variation in exogenous (ecological) selection among populations might contribute to differences in frequency and extent of hybridization. The present study will facilitate future research exploring the evolution of reproductive isolation between L. japonicum and L. stellipilum.     

Changes in Targeting Efficiencies of Proteins to Plant Microbodies Caused by Amino Acid Substitutions in the Carboxy-terminal Tripeptide

It has been demonstrated that the carboxyl terminus of microbodyenzymes functions as a targeting signal to microbodies in higherplants. We have examined an ability of 24 carboxy-terminal aminoacid sequences to facilitate the transport of a cytosolic passengerprotein, ß-glucuroni-dase, into microbodies in greencotyledonary cells of trans-genic Arabidopsis. Immunoelectronmicroscopic analysis revealed that carboxy-terminal tripeptidesequences of the form [C/A/S/P]-[K/R]-[I/L/M] function as amicrobody-targeting signal, although tripeptides with prolineat the first amino acid position and isoleucine at the carboxylterminus show weak targeting efficiencies. All known micro-bodyenzymes that are synthesized in a form similar in size to themature molecule, except catalase, contain one of these tripeptidesequences at their carboxyl terminus. (Received April 14, 1997; Accepted April 8, 1997)     

Optically detected magnetic resonance studies of porcine pancreatic phospholipase A2 binding to a negatively charged substrate analogue

The direct binding of porcine pancreatic phospholipase A2 and its zymogen to 1,2-bis(heptanylcarbamoyl)-rac-glycerol 3-sulfate was studied by optical detection of triplet-state magnetic resonance spectroscopy in zero applied magnetic field. The zero-field splittings of the single Trp3 residue undergo significant changes upon binding of phospholipase A2 to lipid. Shifts in zero-field splittings, characterized mainly by a reduction of the E parameter from 1.215 to 1.144 GHz, point to large changes in the Trp3 local environment which accompany the complexing of phospholipase A2 with lipid. This may be attributed to Stark effects caused by the binding of a charged group near Trp3 in the enzyme-lipid complex. The cofactor, Ca2+, which is strongly bound to the enzyme active site, has an influence on the bonding, as reflected by smaller zero-field splitting shifts. A relatively small change in the Trp environment was observed for the interaction of the zymogen with lipid.     

Investigation of phospholipase-lipid interactions by optical detection of triplet state magnetic resonance spectroscopy

We have investigated the binding of porcine pancreatic phospholipase A2 (PA2) to n-hexadecylphosphocholine (C16PN) micelles using optical detection of triplet state magnetic resonance (ODMR) spectroscopy. The zero field splittings (zfs) of the single Trp3 residue undergo significant changes upon binding of PA2 to C16PN micelles. Zfs titrations of PA2 vs C16PN indicate that the binding stoichiometry is C16PN:PA2 approximately 25. A reduction of the (E) parameter from 1.227 to 1.135 GHz is postulated to result from Stark effects caused by the binding of a polar group (possibly phosphocholine) near Trp3 in the PA2-C16PN micelle complex.     

Separate Intramolecular Targets for Protein Kinase A Control N-Methyl-d-aspartate Receptor Gating and Ca2+ Permeability

Protein kinase A (PKA) enhances synaptic plasticity in the central nervous system by increasing NMDA receptor current amplitude and Ca²⁺ flux in an isoform-dependent yet poorly understood manner. PKA phosphorylates multiple residues on GluN1, GluN2A, and GluN2B subunits in vivo, but the functional significance of this multiplicity is unknown. We examined gating and permeation properties of recombinant NMDA receptor isoforms and of receptors with altered C-terminal domain (CTDs) prior to and after pharmacological inhibition of PKA. We found that PKA inhibition decreased GluN1/GluN2B but not GluN1/GluN2A gating; this effect was due to slower rates for receptor activation and resensitization and was mediated exclusively by the GluN2B CTD. In contrast, PKA inhibition reduced NMDA receptor-relative Ca²⁺ permeability (P_Ca/P_Na) regardless of the GluN2 isoform and required the GluN1 CTD; this effect was due primarily to decreased unitary Ca²⁺ conductance, because neither Na⁺ conductance nor Ca²⁺-dependent block was altered substantially. Finally, we show that both the gating and permeation effects can be reproduced by changing the phosphorylation state of a single residue: GluN2B Ser-1166 and GluN1 Ser-897, respectively. We conclude that PKA effects on NMDA receptor gating and Ca²⁺ permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. This separate control of NMDA receptor properties by PKA may account for the specific effects of PKA on plasticity during synaptic development and may lead to drugs targeted to alter NMDA receptor gating or Ca²⁺ permeability.     

Isolation and characterization of a cDNA encoding mitochondrial chaperonin 10 from Arabidopsis thaliana by functional complementation of an Escherichia coli groES mutant

Chaperonin (Cpn) is one of the molecular chaperones. Cpn10 is a co-factor of Cpn60, which regulates Cpn60-mediated protein folding. It is known that Cpn10 is located in mitochondria and chloroplasts in plant cells. The Escherichia coli homologue of Cpn10 is called GroES. A cDNA for the Cpn10 homologue was isolated from Arabidopsis thaliana by functional complementation of the E. coli groES mutant. The cDNA was 647 bp long and encoded a polypeptide of 98 amino acids. The deduced amino acid sequence showed approximately 50% identity to mammalian mitochondrial Cpn10s and 30% identity to GroES. A Northern blot analysis revealed that the mRNA for the Cpn10 homologue was expressed uniformly in various organs and was markedly induced by heat-shock treatment. The Cpn10 homologue was constitutively expressed in transgenic tobaccos. Immunogold and immunoblot analyses following the subcellular fractionation of leaves from transgenic tobaccos revealed that the Cpn10 homologue was localized in mitochondria and accumulated at a high level in transgenic tobaccos.     

Concordant clines and significant correlation between floral and pollen characters in Asarum heterotropoides var. heterotropoides (Aristolochiaceae)

To examine geographic differentiation in Asarum heterotropoides var. heterotropoides in Hokkaido Is. in the northern Japan, in which two putative cryptic species have been suspected to exist, extensive and detailed morphological research on 794 individuals from 44 populations throughout Hokkaido Is. was performed. Among the characters examined, the angle between and tip shape of the calyx lobes and the supratecta of the pollen grains were significantly correlated and were found in similar geographic clines. Among them, the pollen showed two discrete states in almost distinct distribution. Multidimensional scaling analysis showed that individuals within each of the two pollen types had different trends in flower characters. Consequently, we assumed a cline from south to north on Hokkaido Is. For the causes of the cline, the two hypotheses were proposed, primary geographic differentiation or extensive introgressive hybridization between two distinct geographical species existing in the past.     

An approach to the mechanism of the asymmetrical radical formation in yttrium-90-beta-irradiated D- and L-alanines

Several attempts were made to investigate the mechanism of the asymmetrical radical formation in yttrium-90-beta-irradiated D- and L-alanines which was reported in the preceding paper (1). The experiments demonstrated that the magnitude of the asymmetry was dependent on beta-ray dose, namely the lower the dose the larger the observed difference was, and that no difference could be detected when the alanines were irradiated in aqueous state. The results in the present study seem to that different interaction between the crystal structure of the two enantiomers and polarized beta-rays may be responsible for the observed phenomenon.