The Separation and Characterization of Carbohydrate Rich Component from Ovomucin in Chicken Eggs

We investigated the effects of near-infrared irradiation on the photoconversion of Chenopodium album water-soluble chlorophyll-binding protein (CaWSCP) in the presence of sodium hydrosulfite and found a further photoconversion from CP742 to CP763, a novel form of CaWSCP. Interestingly, one-third of the absorption peak at 668 nm was recovered in CP763, but re-irradiation under oxidative conditions eliminated the photo convertibility of CaWSCP.     

Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy

The nitrogen-15 chemical shift of the N1 (tau)-nitrogen of 15N-labeled histidine and the half-height line widths of proton-coupled resonances of the delta- and omega,omega'-nitrogens of 15N-labeled arginine and of the alpha-nitrogens of 15N-labeled alanine and proline were measured in intact mycelia of Neurospora crassa to obtain to estimates of intracellular pH. For intracellular 15N-labeled histidine, the N1 (tau)-nitrogen chemical shift was 200.2 ppm. In vitro measurements showed that the chemical shift was slightly affected by the presence of phosphate, with which the basic amino acids may be associated in vivo. These considerations indicate a pH of 5.7-6.0 for the environment of intracellular histidine. The half-height line widths of the delta- and omega,omega'-nitrogens of [15N]arginine were 15 and 26 Hz, respectively. In vitro studies showed that these line widths also are influenced by the presence of phosphate, and, after suitable allowance for this, the line widths indicate pH 6.1-6.5 for intracellular arginine. The half-height line widths for intracellular alanine and proline were 17 and 12 Hz, respectively, which are consistent with an intracellular pH of 7.1-7.2. Pools of histidine and arginine are found principally in the vacuole of Neurospora, most likely in association with polyphosphates. Proline and alanine are cytoplasmic. The results reported here are consistent with these localizations and indicate that the vacuolar pH is 6.1 +/- 0.4 while the cytoplasmic pH is 7.15 +/- 0.10. Comparisons of these estimates with those obtained by other techniques and their implications for vacuolar function are discussed.     

Characterization of calcium binding properties of lithostathine

The pancreas secretes primarily two types of metabolically important proteins: digestive enzymes and hormones. Lithostathine (LIT) is the only protein excreted from the pancreas that has no known digestive or hormonal activity. Human lithostathine is a 144-amino acid glycoprotein synthesized by the exocrine pancreas that has been implicated in various physiological functions, including inhibition of pancreatic stone formation. To better understand the physiological function of LIT, we expressed the recombinant LIT protein in Escherichia coli and measured its calcium binding properties by equilibrium dialysis and electron paramagnetic resonance (EPR) spectroscopy. Equilibrium dialysis with (45)Ca(2+) showed that LIT binds Ca(2+) with 1:1 stoichiometry. EPR studies using the divalent vanadyl (VO(2+)) ion as a paramagnetic substitute for Ca(2+) also showed that VO(2+) binds to LIT with a metal:protein binding stoichiometry of 1:1 and that VO(2+) competes with Ca(2+) in binding to LIT. Mutations of a cluster of acidic residues on the molecular surface (E30A, D31A, E33A, D37A, D72A, and D73A) resulted in almost complete loss (95-100%) of binding of Ca(2+) and VO(2+), showing that these residues are critical for calcium binding by LIT.     

Voxel Based Analysis of Surgical Revascularization for Moyamoya Disease: Pre- and Postoperative SPECT Studies

Moyamoya disease (MMD) is a chronic, progressive, cerebrovascular occlusive disease that causes abnormal enlargement of collateral pathways (moyamoya vessels) in the region of the basal ganglia and thalamus. Cerebral revascularization procedures remain the preferred treatment for patients with MMD, improving the compromised cerebral blood flow (CBF). However, voxel based analysis (VBA) of revascularization surgery for MMD based on data from pre- and postoperative data has not been established. The latest algorithm called as Diffeomorphic Anatomical Registration Through Exponentiated Lie Algebra (DARTEL) has been introduced for VBA as the function of statistical parametric mapping (SPM8), and improved registration has been achieved by SPM8 with DARTEL. In this study, VBA was conducted to evaluate pre- and postoperative single photon emission computed tomography (SPECT) images for MMD by SPM8 with DARTEL algorithm, and the results were compared with those from SPM8 without DARTEL (a conventional method). Thirty-two patients with MMD who underwent superficial temporal artery-middle cerebral artery (STA-MCA) bypass surgery as the first surgery were included and all patients underwent pre- and postoperative 3D T1-weighted imaging and SPECT. Pre- and postoperative SPECT images were registered to 3D T1-weighted images, then VBA was conducted. Postoperative SPECT showed more statistically increased CBF areas in the bypassed side cerebral hemisphere by using SPM8 with DARTEL (58,989 voxels; P<0.001), and increased ratio of CBF after operation was less than 15%. Meanwhile, postoperative SPECT showed less CBF increased areas by SPM8 without DARTEL. In conclusion, VBA was conducted for patients with MMD, and SPM8 with DARTEL revealed that postoperative SPECT showed statistically significant CBF increases over a relatively large area and with at most 15% increase ratio.     

Synthesis in vitro of glycoprotein in regenerating rat liver

The metabolism of glucosamine in regenerating rat liver was studied in liver slices. [1-¹⁴C]Glucosamine was incorporated into acid-soluble fraction, rapidly converted to UDP-N-acetylhexosamine and transferred to acid-insoluble fraction. Electrophoretic analysis revealed that most of the radioactive macromolecules released from the slices to the incubation medium were plasma glycoproteins.The incorporation of [1-¹⁴c]glucosamine into UDP-N-acetylhexosamine significantly increased from 6 h to 48 h after partial hepatectomy. On the contrary, the incorporation into acid-insoluble fractions of slice and medium decreased to about 50% of the control values. The rate of transfer of N-acetylhexosamine from UDP-N-acetylhexosamine to acid-insoluble fractions also decreased at 12 h and 48 h respectively. This indicates that the transfer of N-acetylhexosamine to glycoproteins decreases during 48 h of liver regeneration.The enhancement of [1-¹⁴C]glucosamine incorporation into UDP-N-acetylhexosamine is due to an accumulation of the label in the larger pool of this compound. Evidently, some control mechanism may operate on the transfer of N-acetylhexosamine from UDP-N-acetylhexosamine to glycoproteins in regenerating rat liver.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

Intracellular protease (IPLB) of Streptococcus cremoris was extracted from the cells, which were cultivated in liquid media, by momentarily disrupting between two disks by high pressure.

The hydrolyzing modes of α_s-, crude k-, β-, and whole casein by IPLB of Str. cremoris or rennet were observed through the released amounts of tyrosine, sialic acid, NPN, and calcium insensitive substance. Relative specific turbidity of casein solution and dissymmetry coefficient of casein were measured. Particle weight and UV absorption spectrum of each high molecular hydrolyzate of whole casein were also determined.

Among four kinds of casein fractions, α_s- or crude k-casein was most easily hydrolyzed by IPLB of Str. cremoris or rennet. Relative specific turbidity of crude k-casein solution was remarkably, but those of α_s-, β-, and whole casein slightly increased by the action of IPLB of Str. cremoris or of rennet. Changes of dissymmetry coefficients were negligibly induced by these two enzymes. Absorption spectrum of IPLB-Str. cremoris-casein showed some conformational change.

It was recognized that intracellular protease (IPLB) of L. bulgaricus, L. helveticus or Str. lactis, all together, more easily hydrolyzed α_s-casein than crude k-, β-, and whole casein. By the actions of three IPLBs, relative specific turbidity of crude k-casein solution remarkably but those of α_s-, β-, and whole casein slightly increased, and dissymmetry coefficients of these casein fractions changed negligibly.

Particle weight of whole casein hydrolyzed by each IPLB for five days was larger than that of control casein. UV absorption of each whole casein hydrolyzed by a IPLB increased at the wave length range of 280~250 mμ.