Carbohydrate-recognition domains of galectin-9 are involved in intermolecular interaction with galectin-9 itself and other members of the galectin family

Galectin-9 (Gal-9) is a tandem-repeat-type member of the galectin family associated with diverse biological processes, such as apoptosis, cell aggregation, and eosinophil chemoattraction. Although the detailed sugar-binding specificity of Gal-9 has been elucidated, molecular mechanisms that underlie these functions remain to be investigated. During the course of our binding study by affinity chromatography and surface plasmon resonance (SPR) analysis, we found that human Gal-9 interacts with immobilized Gal-9 in the protein-protein interaction mode. Interestingly, this intermolecular interaction strongly depended on the activity of the carbohydrate recognition domain (CRD), because the addition of potent saccharide inhibitors abolished the binding. The presence of multimers was also confirmed by Ferguson plot analysis of result of polyacrylamide gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Moreover, this intermolecular interaction was observed between Gal-9 and other galectin members, such as Gal-3 and Gal-8, but not Gal-1. Because such properties have not been reported yet, they may explain an unidentified mechanism underlying the diverse functions of Gal-9.     

DEVELOPMENT, SEXUAL DIMORPHISM, and INDIVIDUAL VARIATION IN THE SKELETON OF THE FINLESS PORPOISE, NEOPHOCAENA PHOCAENOIDES, IN THE COASTAL WATERS OF WESTERN KYUSHU, JAPAN

Development, sexual dimorphism, and individual variation were examined in the skeleton of the finless porpoise in the coastal waters of western Kyushu, Japan. Skulls ceased growing by 4 yr. Postcranial skeletons ceased increasing in size at an age older than 11 yr. The finless porpoise was estimated to attain cranial maturity by 4 yr and physical maturity at 14–23 yr. Sexual dimorphism was not detectable in most of the cranial characters but was detected in more than half of the postcranial characters. Females tended to show larger values of postcranial characters. The shape of the pelvic bone was obviously different between males and females. Thus, a discriminant function was proposed to determine sex using measurements of this bone. Individual variation was greatest in the feeding apparatus such as length of the rostrum, and least in the braincase.     

Fine structure and lectin histochemistry of the apical surface of the free neuromast of Lampetra japonica

The surface coat, ciliary process, and microvilli of the lamprey neuromast were examined with electron microscopy after tannic acid prefixation and lectin histochemistry. The neuromast was found to exist in the form of a dermal mound with a furrow in the middle. On the bottom of the furrow, the hair cell was characterized by a kinocilium and 15–20 stereocilia, arranged along the longitudinal axis of the furrow. Spanning structures were demonstrated between the kinocilium and stereocilia as well as between stereocilia. The surface coat, enhanced by tannic acid prefixation, was particularly rich over the surface of the supporting cell; by contrast, it was thin over the hair cell. Some lectins (PNA, GS-I, SBA, WGA) showed affinity to the surface coat of the supporting cell as well as the hair cell, and the others (RCA-I, MPA, ConA) showed affinity only to the supporting cell. These differences in the structure and affinities of the surface coat suggest an extracellular milieu highly specialized for the hair cell in this particular form of the mechanoreceptor.     

Westerdykella globosa,a proposal for a new combination

The surface ornamentation of ascospores ofPreussia globosa was compared in an isolate from paddy soil in Japan and a culture derived from the holotype. The ascospores of two cultures were characterized by the surface ornamentation of a single, semicircular spiral ridge. This new finding strongly suggested that the fungus should be transferred to the genusWesterdykella. Therefore, the morphological and cultural characters of the fungus were re-examined, and the new combinationWesterdykella globosa is herein proposed.     

Phylogenetic position of Rickettsia tsutsugamushi and the relationship among its antigenic variants by analyses of 16S rRNA gene sequences

Abstract The 16S rRNA gene sequences of Rickettsia tsutsugamushi and Rickettsia sibirica were determined by PCR and DNA sequencing. Phylogenetic analysis revealed that R. sibirica is positioned in a cluster of the genus Rickettsia with a similarity value of 98.1–99.6%, whereas R. tsutsugamushi is located apart from the cluster with a similarity value of 90.2–90.6%. This evidence suggests that R. tsutsugamushi should be excluded taxonomically from the genus Rickettsia . The phylogenetic classification of six antigenic variants in R. tsutsugamushi moderately reflected their antigenic relationship known in closely and distantly related strains.     

Sequence of three members and expression of a new major subfamily of glutelin genes from rice

Three members have been isolated of an additional glutelin gene subfamily, named subfamily B, consisting of about five members per haploid rice genome. Restriction fragment length polymorphism analysis showed major differences between Japonica and Indica lines, indicating the divergence of the subfamily since the split between the two varieties. While corresponding exons of the subfamily B showed 80 to 88% nucleotide sequence homology, those exons were only 60–65% homologous to those of the glutelin A subfamily [15, 19, 24], distinguishing them from the subfamily A. Intron position and derived polypeptide structure, in addition to the nucleotide sequence, confirm the subfamily B members as glutelins. Analysis of RNA from seeds of different stages of development showed that the subfamily B members were expressed at the same time as those of subfamily A, demonstrating coordinated regulation of the two subfamilies.     

Purification and properties of tobacco ferredoxin-dependent glutamate synthase,and isolation of corresponding cDNA clones

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C₁₆) from a tobacco gt11 expression library. A longer Fd-GOGAT cDNA clone (C₃₅) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco gt10 cDNA library using C₁₆ as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C₃₅ was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.Abbreviations bp base pairs - Fd-GOGAT ferredoxin-dependent glutamate synthase - GS glutamine synthetase - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate The authors wish to thank Juan Luis Gómez Pinchetti (Marine Plant Biotechnology Laboratory) for his assistance during the experiments. This study was supported by grants received from SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Tryggers Fund for Scientific Research (K. Haglund), SJFR (Swedish Council for Forestry and Agricultural Research) (M. Björk, M. Pedersén), CITYT Spain (SAB 89-0091 and MAR 91-1237, M. Pedersén) and CICYT Spain (Z. Ramazanov, invited professor of Ministerio de Educatión y Ciencia, Spain). The planning of this cooperation was facilitated by COST-48.     

Oxidation of methionine residues in recombinant human interleukin-1 receptor antagonist: implications of conformational stability on protein oxidation kinetics

Oxidation of methionine residues is involved in several biochemical processes and in degradation of therapeutic proteins. The relationship between conformational stability and methionine oxidation in recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was investigated to document how thermodynamics of unfolding affect methionine oxidation in proteins. Conformational stability of rhIL-1ra was monitored by equilibrium urea denaturation, and thermodynamic parameters of unfolding (DeltaGH2O, m, and Cm) were estimated at different temperatures. Methionine oxidation induced by hydrogen peroxide at varying temperatures was monitored during "coincubation" of rhIL-1ra with peptides mimicking specific regions of the reactive methionine residues in the protein. The coincubation study allowed estimation of oxidation rates in protein and peptide at each temperature from which normalized oxidation rate constants and activation energies were calculated. The rate constants for buried Met-11 in the protein were lower than for methionine in the peptide with an associated increase in activation energy. The rate constants and activation energy of solvent exposed methionines in protein and peptide were similar. The results showed that conformational stability, monitored using the Cm value, has an effect on oxidation rates of buried methionines. The rate constant of buried Met-11 correlated well with the Cm value but not DeltaGH2O. No correlation was observed for the oxidation rates of solvent-exposed methionines with any thermodynamic parameters of unfolding. The findings presented have implications in protein engineering, in design of accelerated stability studies for protein formulation development, and in understanding disease conditions involving protein oxidation.     

Simplified model for Belousov-Zhabotinsky reaction

Noyes' scheme of the elementary processes for the Belousov-Zhabotinsky reaction has been simplified on the basis of reaction kinetic consideration. The simplest possible analogue (model K) has been described by a set of kinetic equations, and it is solved to examine the varieties of new ordered phases, which include temporal rhythm and spatial pattern. Particular attention has been given to the onset of new phases, which is associated with an anomalous enhancement of fluctuations. A stochastic theory of fluctuations, which was developed in our previous work, has been applied to the present case. Theoretical results compare reasonably well with experimental findings, i.e. with (1) spatial periodic structure and (2) limit cycle behaviour.     

Stability enhancement and circular dichroism spectral anomaly as an indication of non-covalent interactions in histidine- and tyrosine-containing ternary copper(II)—amino acid systems

Visible absorption and CD spectral and potentiometric studies on the His- and Tyr-containing ternary copper(II) complexes Cu(A)(L-B), where A refers to L-His, D-His, or L-Tyr and B to Lys, Tyr, Trp, Phe, Ala, Val, Arg, Glu, Asn, Gln, Ser, or Thr, were made to study ligand-ligand interactions in the complexes. While the CD spectral magnitudes in the d—d region are additive in the absence of side chain interactions and can be estimated from the magnitudes for the ternary systems involving DL-A or DL-B, deviation from the additivity was observed for Cu(L-His)(L-B) (B = LysH, Tyr, Trp, or Phe) and Cu(L-Tyr)(L-Trp). From the stability constants determined at 25 °C and I = 0.1 M (KNO₃), the equilibrium constants, K, for the following hypothetical equilibria were calculated to be large (0.14–0.60) for formation of Cu(L-/D-His)(L-B)(B = Tyr or Trp) and Cu(D-His)(L-Phe) with Cu(en)(L-Ala) as standard: $Cu(A)(L?Ala)+Cu(en)(L?b)\stackrel{K}{?}Cu(A)(L?B)+Cu(en)(L?Ala)$ The positive values indicate the stabilization due to the stacking between the imidazole ring of His and the aromatic side chain of L-B. Solvent dependence of the CD spectra for Cu(L-His)(L-LysH) and Cu(L-His) L-Trp) further supported the existence of the intramolecular electrostatic and hydrophobic interactions.