Interruption of signal transduction between G protein and PKC-ε underlies the impaired myocardial response to ischemic preconditioning in postinfarct remodeled hearts

We have recently shown that the protective mechanism of ischemic preconditioning (PC) is impaired in the myocardium that survived infarction and underwent postinfarct ventricular remodeling. In this study, we examined the hypothesis that failure of PC to activate PKC- underlies the refractoriness of the remodeling heart to PC. Circumflex coronary arteries were ligated in rabbits to induce infarction and subsequent ventricular remodeling, and only sham operations were performed in controls. Hearts were isolated before (i.e. 4 days later) or after (i.e. 2 weeks later) remodeling of the left ventricle and used for isolated buffer-perfused heart experiments. Myocardial infarction was induced in isolated hearts by 30 min global ischemia/2 h reperfusion, and its size was measured by tetrazolium staining. Using separate groups of hearts, tissue biopsies were taken before and after PC, and PKC translocation was assessed by Western blotting. Areas infarcted in vivo by coronary ligation (CL) were excluded from subsequent infarct size/PKC analyses. In the hearts 4 days after CL, PC with 2 cycles of 5 min ischemia/5 min reperfusion induced PKC- translocation from cytosol to particulate fractions and limited infarct size to 40% of control value. In the hearts remodeled 2 weeks after CL, PC failed to induce PKC- translocation and infarct size limitation. In this group, PKC activity and hemodynamic responses to adenosine were similar to those in sham-operated controls. When remodeling after CL was prevented by valsartan infusion (10 mg/kg/day), an angiotensin II type 1 (AT₁) receptor blocker, PC could induce both infarct limitation and PKC- translocation. The present results suggest that persistent activation of AT₁ receptors during remodeling disturbed the PC signaling between G proteins and PKC-, which underlies the refractoriness of the remodeled myocardium to PC.     

Denitrification of nitrate by the fungus Cylindrocarpon tonkinense

The denitrifying fungus Cylindrocarpon tonkinense was thought to be able to denitrify only nitrite (NO2-) but not nitrate (NO3-) to form nitrous oxide (N2O). Here we found, however, that C. tonkinense can denitrify NO3- under certain conditions. Presence of ammonium (NH3+) in addition to NO3- and the use of a fermentable sugar as an electron donor were key conditions for inducing the denitrifying activity. Such induction accompanied a remarkable increase in the intracellular level of the enzyme activities related to NO3- metabolism. These activities contained assimilatory type NADPH (or NADH)-dependent NO3- reductase (aNar), dissimilatory nitrite reductase (dNir), and nitric oxide reductase (P450nor), but did not contain ubiquinol-dependent, dissimilatory NO3- reductase (dNar). The denitrification was inhibited by tungstate, an inhibitor of Nar. These results demonstrated occurrence of a novel type of denitrification in C. tonkinense, in which assimilatory type Nar is possibly involved.     

Properties of rice cooked with commercial water-soluble soybean polysaccharides extracted under weakly acidic conditions from soybean cotyledons

It has been found that commercial water-soluble soybean polysaccharides (SSPS) can make cooked rice and noodles non-sticky and prevent rice grains and noodles from adhering to each other. We studied in detail the phenomenon of rice cooked with SSPS. We assumed that the phenomenon resulted from the interaction between SSPS and starch during cooking, and studied the effects of SSPS on the gelatinizing behavior of rice starch by using a Rapid-Visco-Analyzer. The addition of SSPS reduced the viscosity of the gelatinized starch. This lower final viscosity of the rice starch was more distinct from than that of potato starch. These results imply that the properties of SSPS in forming a non-sticky condition might result from a decrease in the viscosity of the gelatinized starch.     

Inhibitory effects of brown algal phlorotannins on secretory phospholipase A2s, lipoxygenases and cyclooxygenases

The inhibitory effects of brown algal phlorotannins on secretory phospholipase A₂s (sPLA₂s), lipoxygenases (LOXs) and cyclooxygenases (COXs) were determined with an in vitro assay. Oligomers of phloroglucinol; eckol (a trimer), phlorofucofuroeckol A (a pentamer), dieckol (a hexamer) and 8,8-bieckol (a hexamer) isolated from the brown alga Eisenia bicyclis had pronounced inhibitory effects on sPLA₂ from porcine pancreas and bee venom (IC₅₀ 100–200 M). The phlorotannins inhibited LOX activity more effectively than the well-known LOX inhibitors; resveratrol and epigallocatechin gallate. 8,8-Bieckol, the strongest LOX inhibitor in this study, inhibited soybean LOX and 5-LOX with IC₅₀ values of 38 and 24 M, respectively. Negligible or very weak effects of the phlorotannins on COX-1 and COX-2 were found, except for an inhibitory effect of dieckol on COX-1 (74.7%) and of eckol on COX-2 (43.2%) at 100 M.     

Histochemistry of glycoconjugates in the nasolabial skin of the Japanese serow (Capricornis crispus, Artiodactyla, Mammalia) with special reference to glandular structures

The histochemistry of glycoconjugates in the nasolabial skin of the Japanese serow ( Capricornis crispus ) was studied by light microscopic histochemical methods, particularly lectin histochemistry. The eccrine glands present exhibited neutral and acidic glycoconjugates with different saccharide residues (alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine, alpha-D-galactose and N-acetyl-neuraminic acid) especially in the cells of the secretory acini, the free surface of the collecting duct cells also showed distinct positive reactions with most of the histochemical methods. The thick epidermis of the nasolabial skin contained smaller amounts of glycoproteins. The results obtained are discussed with regard to possible functions of the glandular secretions. This substance mixture may particularly improve water retention on the skin surface, and protect against physical damage as well as microbial contamination. There seem to be no basic differences of muzzle functions between wild and domesticated bovine species.     

Synthesis of chemotactic peptide analogs with a photoaffinity cross-linker as new probes for analysis of receptor-ligand interaction

Formylpeptide receptors are well-characterized receptors which participate in host defense responses of neutrophils. We designed and synthesized chemotactic peptide analog with p-benzoylphenylalanine (Bpa) and biotin to probe structural and mechanistic aspects of peptide-receptor interaction. These peptides possess biological activities which were dependent upon spacer residue length of and Bpa position. The covalent photoaffinity label was detected by Streptavidine-blot, which was inhibited by the parent peptide.     

The role of Grg4 in tectal laminar formation

Mature chick optic tecta consist of 16 laminae and receive retinal fiber projections in a precise retinotopic manner. Retinal axons arborize in laminae a-f of the SGFS, but do not cross the border between lamina f and g. In order to elucidate molecular mechanisms of tectal laminar formation, we first looked at the migration of tectal postmitotic cells. We found that the migration pattern of postmitotic cells changes around E5 and that late migratory cells intervened laminae that were formed by early migratory cells. The coincident appearance of Grg4 expression in the tectal ventricular layer and the change in migration pattern suggested an important role for Grg4. Clonal misexpression of Grg4 resulted in cells migrating to laminae h-j of the SGFS. Massive misexpression of Grg4 resulted in disruption of laminae that were formed by early migratory cells, in particular lamina g of the SGFS. Application of Grg4 morpholino antisense oligonucleotide or the misexpression of a dominant-negative form of Grg4 exerted the opposite effect. We concluded that Grg4 may direct tectal postmitotic cells to follow a late migratory pathway.     

Noninvasive measurement of chloride concentration in rat olfactory receptor cells with use of a fluorescent dye

Inwardly directed Ca(2+)-dependent chloride currents are thought to prolong and boost the odorant-induced transient receptor currents in olfactory cilia. Cl(-) inward current, of course, requires a sufficiently high intracellular Cl(-) concentration ([Cl(-)](i)). In previous measurements using a fluorescent Cl(-) probe, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), [Cl(-)](i) of newt olfactory cells was estimated to be only 40 mM. This low value led us to reexamine the [Cl(-)](i) by an improved procedure. When isolated rat olfactory neurons were bathed in Tyrode's solution (150 mM Cl(-)) at room temperature, the [Cl(-)] was 81.5 +/- 13.5 mM (mean +/- SE) in the tip of the dendrite (olfactory knob) and 81.8 +/- 10.2 mM (mean +/- SE) in the soma. The corresponding Cl(-) equilibrium potentials were -15.4 and -15.3 mV, respectively. Therefore, at resting potentials in the range of -90 to -50 mV, Cl(-) currents are predicted to be inward and capable of contributing to the depolarization induced by odorants. Yet, if the cell was depolarized beyond -15 mV, somal Cl(-) currents would be outward and facilitate repolarization during excitation. The measured [Cl(-)] in soma and knob are of interest, because in the cilia the chloride content may be expected to equilibrate with that of the knob in the resting state. They provide a starting point for the decrease in ciliary [Cl(-)] predicted to occur during transduction.     

Ontogenetic Changes of Photosynthetic and Dark Respiration Rates in Relation to Nitrogen Content in Individual Leaves of Field Crops

Ontogenetic changes of rates of photon-saturated photosynthesis (P _sat) and dark respiration (R _D) of individual leaves were examined in relation to nitrogen content (Nc) in rice, winter wheat, maize, soybean, field bean, tomato, potato, and beet. P _sat was positively correlated with Nc as follows: P _sat = CfNc + P _sat0, where Cf and P _sat0 are coefficients. The value of Cf was high in maize, medium in rice and soybean, and low in field bean, potato, tomato, and beet, of which difference was not explained by ribulose-1,5-bisphoshate carboxylase/oxygenase (RuBPCO) content. R _D was explained by P _sat and/or Nc, however, two models must be applied according to plant species. R _D related linearly with P _sat and Nc in maize, field bean, and potato as follows: R _D = a P _sat + b, or R _D = aNc + b, where a, a, b and b are coefficients. In other species, the R _D/P _sat ratio increased exponentially with the decrease of Nc as follows: R _D/P _sat = a exp(b Nc), where a and b are coefficients. Therefore, R _D in these crops was expressed as follows: In(R _D) = ln(a P _sat) + b Nc, indicating that R _D in these crops was regulated by both P _sat and Nc.     

Construction of humanized anti-ganglioside monoclonal antibodies with potent immune effector functions

 Gangliosides GD3, GD2 and GM2, which are the major gangliosides expressed on most human cancers of neuroectodermal and epithelial origin, have been focused on as effective targets for passive immunotherapy with monoclonal antibodies. We previously developed a chimeric anti-GD3 mAb, KM871, and a humanized anti-GM2 mAb, KM8969, which specifically bound to the respective antigen with high affinity and showed potent immune effector functions. Humanization of anti-ganglioside antibody is expected to enhance its use for human cancer therapy. In the present study, we generated a chimeric anti-GD2 mAb, KM1138, and further developed the humanized form of anti-GD2 and anti-GD3 mAbs by the complementarity-determining regions grafting method. The resultant humanized anti-GD2 mAb, KM8138, and anti-GD3 mAb, KM8871, showed binding affinity and specificity similar to those of their chimeric counterparts. In addition, both humanized mAbs had functional potency comparable to the chimeric mAbs in mediating the immune effector functions, consisting of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. The production of these humanized anti-ganglioside mAbs, with potent effector functions and low immunogenicity, precedes the evaluation of the therapeutic value of anti-ganglioside mAbs in passive immunotherapy and the target validation for ganglioside-based vaccine therapy. Received: 30 November 2000 / Accepted: 30 January 2001