Estimating glycoprotein carbohydrate chain structures by lectin reactivities in polyacrylamide gels

Complex mixtures of cellular glycoproteins contain a myriad of different carbohydrate chains that cannot be easily analyzed without rigorous purification of each individual glycoprotein. We have analyzed the carbohydrate chains in complex mixtures of cellular glycoproteins by separation using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and interacting the gels with several 125I-labeled lectins. By use of in situ chemical modifications of the glycoproteins after their electrophoretic separation together with the known carbohydrate-binding specifities of several lectins, it has been possible to estimate glycoprotein carbohydrate chain structures. As an example we have examined the cellular glycoproteins of a ovary-colonizing metastatic variant of B16 melanoma and report the types of carbohydrate chains that are found on various melanoma glycoproteins.     

Stimulation of the estrogen synthesizing system of the immature rat ovary by exogenous and endogenous gonadotropins

Immature rat ovaries increase their secretion of estradiol (E₂) when stimulated by gonadotropins but only after a lag period of several hours. Once established, estrogen secretion can be maintained, or increased, by the continued presence of gonadotropin. A combination of ovine FSH+LH given at 2 hr intervals stimulated the estrogen synthesizing system ($\text{ESS}$) of the ovary and serum E₂ showed a pronounced rise between 16 and 20 hrs after the initial injection. When given every 2 hrs for 5 doses (0–8 hrs) serum E₂ was undetectable. However, it was increased if 20 IU PMS was injected at the time of the last dose of FSH+ LH. Endogenous FSH&LH, increased by hourly injections of LH-releasing hormone for a period of 8 hrs, stimulated the $\text{ESS}$; serum E₂ increased at the expected time when this treatment was followed by an injection of PMS.Anti-PMS antiserum given 12 hrs after PMS, prevented the expected rise in serum E₂ at 24 hrs. However, FSH, LH or a combination of the two given every 2 hrs beginning at the time of the anti-PMS produced an increase in E₂ secretion; the combination was more effective than either hormone alone.These results are consistent with the interpretation that a combined FSH-LH action is responsible for induction of the $\text{ESS}$ in the immature rat ovary. The combination of hormones is also very effective in maintaining estrogen secretion but some function appears possible with FSH or LH alone.     

On the estimation of insect population by time unit collecting

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Contributions from the Entomological Laboratory, Kyoto University, No. 230.     

Crystal Structure of a Symmetric Football-Shaped GroEL:GroES2-ATP14 Complex Determined at 3.8 Å Reveals Rearrangement between Two GroEL Rings

The chaperonin GroEL is an essential chaperone that assists in protein folding with the aid of GroES and ATP. GroEL forms a double-ring structure, and both rings can bind GroES in the presence of ATP. Recent progress on the GroEL mechanism has revealed the importance of a symmetric 1:2 GroEL:GroES₂ complex (the “football”-shaped complex) as a critical intermediate during the functional GroEL cycle. We determined the crystal structure of the football GroEL:GroES₂-ATP₁₄ complex from Escherichia coli at 3.8 Å, using a GroEL mutant that is extremely defective in ATP hydrolysis. The overall structure of the football complex resembled the GroES-bound GroEL ring of the asymmetric 1:1 GroEL:GroES complex (the “bullet” complex). However, the two GroES-bound GroEL rings form a modified interface by an ~ 7° rotation about the 7-fold axis. As a result, the inter-ring contacts between the two GroEL rings in the football complex differed from those in the bullet complex. The differences provide a structural basis for the apparently impaired inter-ring negative cooperativity observed in several biochemical analyses.     

Purification and characterisation of β-N-acetylhexosaminidase from the butter clam, saxidomus purpuratus

A β-N-acetylhexosaminidase [EC 3.2.1.30] has been purified ～98-fold from an extract of the digestive organs of Saxidomus purpuratus by using ammonium sulfate fractionation, and chromatography on Toyopearl HW-50, CM-cellulose, and Sepharose 4B. The purified enzyme, the molecular weight of which was estimated to be ～66,000 by gel filtration, was composed of two sub-units of molecular weight 30,000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The purified enzyme had a pH optimum of 3.8 and an optimum temperature of 55°, and its activity was enhanced ～2-fold in the presence of 0.1m sodium chloride. The Michaelis constants toward p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucoside and -galactoside were 1.2 × 10^?4 and 1.3 × 10^?4m, respectively.     

Growth,net photosynthesis and leaf nutrient status of <Emphasis Type="Italic">Fagus crenata</Emphasis> seedlings grown in brown forest soil acidified with H<Subscript>2</Subscript>SO<Subscript>4</Subscript> or HNO<Subscript>3</Subscript> solution

To obtain basic information for evaluating critical loads of acid deposition for protecting Japanese beech forests, growth, net photosynthesis and leaf nutrient status of Fagus crenata seedlings grown for two growing seasons in brown forest soil acidified with H₂SO₄ or HNO₃ solution were investigated. The whole-plant dry mass of the seedlings grown in the soil acidified by the addition of H₂SO₄ or HNO₃ solution was significantly less than that of the seedlings grown in the control soil not supplemented with H⁺ as H₂SO₄ or HNO₃ solution. However, the degrees of reduction in the whole-plant dry mass and net photosynthetic rate of the seedlings grown in the soil acidified by the addition of H⁺ as H₂SO₄ solution at 100 mg l^–1 on the basis of air-dried soil volume (S-100 treatment) were greater than those of the seedlings grown in the soil acidified by the addition of H⁺ as HNO₃ solution at 100 mg l^–1 (N-100 treatment). The concentrations of Al and Mn in the leaves of the seedlings grown in the S-100 treatment were significantly higher than those in the N-100 treatment. A positive correlation was obtained between the molar ratio of (Ca+Mg+K)/(Al+Mn) in the soil solution and the relative whole-plant dry mass of the seedlings grown in the acidified soils to that of the seedlings grown in the control soil. Based on the results, we concluded that the negative effects of soil acidification due to sulfate deposition are greater than those of soil acidification due to nitrate deposition on growth, net photosynthesis and leaf nutrient status of F. crenata, and that the molar ratio of (Ca+Mg+K)/(Al+Mn) in soil solution is a suitable soil parameter for evaluating critical loads of acid deposition in efforts to protect F. crenata forests in Japan.     

Taurine inhibits K+-Cl- cotransporter KCC2 to regulate embryonic Cl- homeostasis via with-no-lysine (WNK) protein kinase signaling pathway

GABA inhibits mature neurons and conversely excites immature neurons due to lower K(+)-Cl(-) cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl(-) homeostasis, which is required for normal brain development.