Zymographic demonstration of lactate and malate dehydrogenases isoenzymes in the rodent salivary glands

Summary Analysis of lactate and malate dehydrogenase zymograms of rodent salivary glands showed species and organ specific patterns.Lactate dehydrogenase isoenzyme patterns occupied the middle positions in relation to those of skeletal and heart muscle. Activities of the major salivary glands were in the order submaxillary gland>parotid>sublingual gland. Zymogram of the mouse and rat showed LDH₄ and LDH₅ high activity patterns, while that of the rabbit was the fast moving active one. Hamster salivary gland exhibited a neutral type of the former and the latter.Malate dehydrogenase isoenzyme exhibited very similar patterns for the mouse, rat and hamster. Malate dehydrogenase zymogram of rabbit showed 3 active bands, which was different from the other rodents.     

Elucidation of the mechanism enhancing the avidity of lectin with oligosaccharides on the solid phase surface

The mechanism underlying molecular recognition of lectins waselucidated by a novel solid phase binding assay system basedon surface plasmon resonance. When the apparent affinities ofinteractions between chitooligosaccharides and wheat germ agglutininwere compared between lectin-immobilized and oligosaccharide-immobilizedassay systems, the affinity constants (Ka) calculated for theformer system were in good agreement with the previously reportedvalues measured in solution. On the other hand, in the lattersystem, the calculated Ka could be more than 10,000 times higherthan the values in solution at lower lee tin concentrations.To elucidate the reason for this, we systematically investigatedthe effects of the oligosaccha-ride immobilized density andthe lectin valence on the apparent affinity in the oligosaccharide-immobilizedassay system. Both the apparent association (k_ass) and dissociationrate constants (k_diss) showed a tendency to decrease as theoligosaccharide density increased. This effect was most remarkablefor the interaction possessing an extremely fast intrinsic K_ass.Oligomerization of lectin enhanced the avidity due to a significantreduction in k_diss. These phenomena could be explained by consideringthe nonhomogeneous conditions under which binding occurred.The reaction in a nonhomogeneous state is limited by the masstransport effect, and the effect of rebinding becomes so largethat it cannot be disregarded. These findings are the firstto demonstrate the importance of the mass transport effect inmodulating the affinity of lectin for oligosaccharides on asolid phase surface. avidity clustering effect lectin mass transport surface plasmon resonance     

Oxygen concentration-dependent induction of a 140-kDa protein in magnetic bacterium Magnetospirillum magnetotacticum MS-1

Abstract The magnetic bacterium Magnetospirillum magnetotacticum prefers a microaerobic habitat and should be able to sense oxygen. Therefore, the bacterium was cultured under atmospheres containing 0–5% O₂ and analyzed for oxygen-dependent changes in the levels of its protein components by sodium dodecyl sulfate-polyccrylamide gel electrophoresis (SDS-PAGE). The analysis revealed a marked anaerobic induction of a 140-kDa protein, which was suppressed when M. magnetotacticum was switched from microaerobic (<1% O₂) to aerobic (>1% O₂) growth conditions. Although its function remains to be determined, the 140-kDa protein may serve as a useful tool to gain insight into the physiology of the organism.     

l-Acylimidazoles with Broad-spectrum Fungicidal Activity

The membrane lipids of six higher plants that differ in salt tolerance were analyzed and compared. The root lipids increased in a ratio of glycolipid/phospholipid with increasing salt- tolerance. A similar increase in the ratio was observed with increasing external salinity when halophytic orach and salt-sensitive cucumber were exposed to varying salinity, although the latter plant was limited to only a little increase. Measurements of ion-transport rates with artificial lipid membranes revealed that the root lipids from a salt-resistant plant formed a more permeable membrane than those from a salt-sensitive species. It was found that the membrane permeability was related to the glycolipid/phospholipid ratio in the membrane lipids, where the glycolipids were stimulative and the phospholipids were repressive for ion-flow. These different effects of the two lipid classes may be attributed to their molecular species and head groups.     

Py-GC/MS analysis of sediments from Lake Biwa,Japan: characterization and sources of humic acids

Humic substances extracted and purified from bottom sediments of northern Lake Biwa, Japan, in November 2012 and 2013 were characterized using elemental analysis, Fourier-transform infrared spectroscopy, hydrogen-1 nuclear magnetic resonance (¹H NMR) analysis, and pyrolysis gas chromatography/mass spectrometry (Py-GC/MS). The infrared (IR) bands in the spectra of humic acids showed the presence of amide linkages and polysaccharides. Results of ¹H NMR analysis showed that the humic acids contained approximately twice the number of aliphatic protons as those in the Japanese soil standards used for comparison. Results of the Py-GC/MS analysis, which evaluates pyrolysis temperature dependency of the amount of pyrolysis products, showed that the generation of pyrolysis products in humic acids also differed from that in Japanese soil standards but was similar to that of phytoplankton in Lake Biwa. This analysis method is the first to provide extensive information about the chemical structure of humic substances; conventional Py-GC/MS provides limited information for a single temperature. Data suggest that humic acids in lake sediments are related to chemical characteristics of phytoplankton. Results shed new light on the origins of humic substances in deep-water-lake sediments and provide insights into material recycling in such sediments.     

Gliadain, a gibberellin-inducible cysteine proteinase occurring in germinating seeds of wheat, Triticum aestivum L., specifically digests gliadin and is regulated by intrinsic cystatins

We cloned a new cysteine proteinase of wheat seed origin, which hydrolyzed the storage protein gliadin almost specifically, and was named gliadain. Gliadain mRNA was expressed 1 day after the start of seed imbibition, and showed a gradual increase thereafter. Gliadain expression was suppressed when uniconazol, a gibberellin synthesis inhibitor, was added to germinating seeds. Histochemical detection with anti-gliadain serum indicated that gliadain was present in the aleurone layer and also that its expression intensity increased in sites nearer the embryo. The enzymological characteristics of gliadain were investigated using recombinant glutathione S-transferase (GST)-progliadain fusion protein produced in Escherichia coli. The GST-progliadain almost specifically digested gliadin into low molecular mass peptides. These results indicate that gliadain is produced via gibberellin-mediated gene activation in aleurone cells and secreted into the endosperm to digest its storage proteins. Enzymologically, the GST-progliadain hydrolyzed benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin (Z-Phe-Arg-NH(2)-Mec) at K(m) = 9.5 microm, which is equivalent to the K(m) value for hydrolysis of this substrate by cathepsin L. Hydrolysis was inhibited by two wheat cystatins, WC1 and WC4, with IC(50) values of 1.7 x 10(-8) and 5.0 x 10(-8) m, respectively. These values are comparable with those found for GST-progliadain inhibition by E-64 and egg-white cystatin, and are consistent with the possibility that, in germinating wheat seeds, gliadain is under the control of intrinsic cystatins.     

Genetic diversity and origin of Potamogeton anguillanus (Potamogetonaceae) in Lake Biwa,Japan

We analyzed the genetic variation in Potamogeton anguillanus Koidz. and its putative parents, P. malaianus Miq. and P. perfoliatus L., at five allozyme loci of four enzymes to test the hypothesis of a hybrid origin for P. anguillanus, collected in Lake Biwa, Japan. Alleles diagnostic for either P. malaianus or P. perfoliatus were present at four loci. Of 13 single locus phenotypes (SLPs) of P. anguillanus, eight were phenotypes that were expected in F(1) hybrids between P. malaianus and P. perfoliatus. Two SLPs were different from those expected in F(1) hybrids but could have resulted from segregation of parental alleles in later generation hybrids. Each of the remaining three SLPs possessed one allele unique to P. anguillanus. Allozyme analyses thus supported the view that P. anguillanus was derived from hybridization between P. malaianus and P. perfoliatus. It seems likely that the genetic diversity of P. anguillanus found previously originated through multiple hybridizations and sexual processes in P. anguillanus. Other processes such as intragenic recombination, mutation, or hybridization with another lineage are also discussed with reference to the origin of unique alleles.     

Inhibitory effect of hyperglycemia on insulin-induced Akt/protein kinase B activation in skeletal muscle

To determine the molecular mechanism underlying hyperglycemia-induced insulin resistance in skeletal muscles, postreceptor insulin-signaling events were assessed in skeletal muscles of neonatally streptozotocin-treated diabetic rats. In isolated soleus muscle of the diabetic rats, insulin-stimulated 2-deoxyglucose uptake, glucose oxidation, and lactate release were all significantly decreased compared with normal rats. Similarly, insulin-induced phosphorylation and activation of Akt/protein kinase B (PKB) and GLUT-4 translocation were severely impaired. However, the upstream signal, including phosphorylation of the insulin receptor (IR) and insulin receptor substrate (IRS)-1 and -2 and activity of phosphatidylinositol (PI) 3-kinase associated with IRS-1/2, was enhanced. The amelioration of hyperglycemia by T-1095, a Na(+)-glucose transporter inhibitor, normalized the reduced insulin sensitivity in the soleus muscle and the impaired insulin-stimulated Akt/PKB phosphorylation and activity. In addition, the enhanced PI 3-kinase activation and phosphorylation of IR and IRS-1 and -2 were reduced to normal levels. These results suggest that sustained hyperglycemia impairs the insulin-signaling steps between PI 3-kinase and Akt/PKB, and that impaired Akt/PKB activity underlies hyperglycemia-induced insulin resistance in skeletal muscle.     

Laryngeal endocrine cells: topographic distribution and adaptation to chronic hypercapnic hypoxia

The morphology, topographic distribution, effects of denervation, and exposure to hypercapnic hypoxia of endocrine cells were examined in rat larynx. The endocrine cells, which were immunoreactive for protein gene product 9.5 (PGP 9.5) and calcitonin gene-related peptide (CGRP), were observed within the epithelial layer of the laryngeal cavity and in the laryngeal gland, while solitary endocrine cells with apical and/or basal cytoplasmic processes appeared near the glottis. After denervation of the left cervical vagosympathetic trunk and the superior laryngeal nerve, the number of mucosal endocrine cells in the denervated side was not significantly different from that in the intact side. After exposure to hypercapnic hypoxia for 3 months, the number of endocrine cells with PGP 9.5 and CGRP was markedly increased. In conclusion, the secretion of laryngeal endocrine cells may be stimulated by CO2 rather than O2. Furthermore, the endocrine cells and the sensory and autonomic nervous system may regulate each other by an axon reflex mechanism. Endocrine cells appear to play a very important role in the local regulation of the laryngeal mucosa.