Fractions IIb Research Articles

New Insights into the Umami and Sweet Taste of Oolong Tea: Formation of Enhancer N-(1-carboxyethyl)-6-(hydroxymethyl) pyridinium-3-ol (Alapyridaine) in Roasting Via Maillard Reaction.

Roasting is pivotal for enhancing the flavor of Wuyi rock tea (WRT). A study investigated a novel compound that enhances the umami taste of WRT. Metabolomics of Shuixian tea (SXT) and Rougui tea (RGT) under light roasting (LR), medium roasting (MR), and heavy roasting (HR) revealed significant differences in nonvolatiles compounds. Compared LR reducing sugars and amino acids notably decreased in MR and HR, with l-alanine declining by 69%. Taste-guided fractionation identified fraction II-B as having high umami and sweet intensities. A surprising taste enhancer, N-(1-carboxyethyl)-6-(hydroxymethyl) pyridinium-3-ol (alapyridaine), was discovered and identified. It formed via the Maillard reaction, positively correlated with roasting in SXT and RGT. Alapyridaine levels were highest in SXT among the five oolong teas. Roasting tea with glucose increased alapyridaine levels, while EGCG inhibited its formation. HR-WRT exhibited enhanced umami and sweet taste, highlighting alapyridaine's impact on WRT's flavor profile. The formation of alapyridaine during the roasting process provides new insights into the umami and sweet perception of oolong tea.

α-Synuclein and Parkin Contribute to the Assembly of Ubiquitin Lysine 63-linked Multiubiquitin Chains

Mutations in alpha-synuclein, Parkin, and UCH-L1 cause heritable forms of Parkinson disease. Unlike alpha-synuclein, for which no precise biochemical function has been elucidated, Parkin functions as a ubiquitin E3 ligase, and UCH-L1 is a deubiquitinating enzyme. The E3 ligase activity of Parkin in Parkinson disease is poorly understood and is further obscured by the fact that multiubiquitin chains can be formed through distinct types of linkages that regulate diverse cellular processes. For instance, ubiquitin lysine 48-linked multiubiquitin chains target substrates to the proteasome, whereas ubiquitin lysine 63-linked chains control ribosome function, protein sorting and trafficking, and endocytosis of membrane proteins. It is notable in this regard that ubiquitin lysine 63-linked chains promote the degradation of membrane proteins by the lysosome. Because both Parkin and alpha-synuclein can regulate the activity of the dopamine transporter, we investigated whether they influenced ubiquitin lysine 63-linked chain assembly. These studies revealed novel biochemical activities for both Parkin and alpha-synuclein. We determined that Parkin functions with UbcH13/Uev1a, a dimeric ubiquitin-conjugating enzyme, to assemble ubiquitin lysine 63-linked chains. Our results and the results of others indicate that Parkin can promote both lysine 48- and lysine 63-linked ubiquitin chains. alpha-Synuclein also stimulated the assembly of lysine 63-linked ubiquitin chains. Because UCH-L1, a ubiquitin hydrolase, was recently reported to form lysine 63-linked conjugates, it is evident that three proteins that are genetically linked to Parkinson disease can contribute to lysine 63 multiubiquitin chain formation.

Isolation and Identification of Antiradical and Anti-photooxidant Component of Aloe vera chinensis

A potent antiradical and antiphotooxidant compound from Aloe vera chinensis was investigated. The results indicated that the methanolic extracts produced stronger antiradical and antiphotoaxidant activity also gave higher yields of extract than other organic solvents. Silica gel column separated methanolic extracts into five fractions. Of the five fractions, fraction II possessed significant antiradical activity (70.2 % inhibition on free radical DPPH) and its showed significant antiphotooxidant activity (60.1 % inhibition on hydroperoxide formation). Subsequently, fraction II was separated into eight subfractions by silica gel column. The sub fraction II-b exhibited stronger antiradical and antiphotooxidant activity than others and showed a single spot in thin layer chromatography Silica gel 60 F 254 plates. On the basis of mass spectrophotometry in combination with reversed phased, high performance chromatography, this compound has been identified as 1,8 dihydroxy-3- hydroxymethylanthraquinone (Aloe emodin).

Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

The catalytic subunit (alpha) of mitochondrial DNA polymerase (pol gamma) shares conserved DNA polymerase and 3'-5' exonuclease active site motifs with Escherichia coli DNA polymerase I and bacteriophage T7 DNA polymerase. A major difference between the prokaryotic and mitochondrial proteins is the size and sequence of the region between the exonuclease and DNA polymerase domains, referred to as the spacer in pol gamma-alpha. Four gamma-specific conserved sequence elements are located within the spacer region of the catalytic subunit in eukaryotic species from yeast to humans. To elucidate the functional roles of the spacer region, we pursued deletion and site-directed mutagenesis of Drosophila pol gamma. Mutant proteins were expressed from baculovirus constructs in insect cells, purified to near homogeneity, and analyzed biochemically. We find that mutations in three of the four conserved sequence elements within the spacer alter enzyme activity, processivity, and/or DNA binding affinity. In addition, several mutations affect differentially DNA polymerase and exonuclease activity and/or functional interactions with mitochondrial single-stranded DNA-binding protein. Based on these results and crystallographic evidence showing that the template-primer binds in a cleft between the exonuclease and DNA polymerase domains in family A DNA polymerases, we propose that conserved sequences within the spacer of pol gamma may position the substrate with respect to the enzyme catalytic domains.

Effect of Leiurus quinquestriatus hebreus venom on calcium and deoxyglucose uptake in cultured cardiac cells

The effects of scorpion venom Leiurus quinquestriatus hebreus were studied on cardiac cells grown in culture. The venom (30 μg/ml) increased significantly ( P < 0.05) Ca 2+ uptake into intact cardiocytes and to sarcoplasmic reticulum of skinned cells. [ 3H]Deoxyglucose uptake was also increased significantly ( P < 0.05) in venom treated cardiocytes. It was found that fractions I and III of the venom, separated by gel filtration and ion exchange chromatography, are responsible for the increased Ca 2+ uptake by the sarcoplasmic reticulum, whereas fraction IIb, III and IV are responsible for the accelerated rate of uptake of 45Ca and [ 3H]deoxyglucose by intact cells. Ca channel blockers prevented these effects and similar results were obtained by propranolol. Thus, it is concluded that the venom exerts its effect through activation of β-adrenoceptors which causes the opening of L-type Ca channels.

Studies on Wakan-Yakus (traditional herbal drugs) : Especially on the effects of Gaiyoh (Artemisiae folium) on blood coagulation

Wakan-Yakus (traditional herbal drugs) such as Akyoh (Glutinum), Gaiyoh (Artemisiae folium), Sanshishi (Gardeniae fructus), Kizutsu (Aurantii fructus), and Taisoh (Zizyphi fructus) were studied in relation to their effects on blood coagulation-fibrinolysis. (1) All of the water extracts of the Wakan-Yakus prolonged aPTT and PT. The potency of the effectiveness on aPTT was in the order of Gaiyoh (Artemisiae folium)> Kizutsu (Aurantii fructus)> Sanshishi (gardeniae fructus)> Taisoh (Zizyphi fructus )> Akyoh (Glutium). (2) Gaiyoh (Aremisiae folium)> Kizutsu (Aurantii fructus) Akyoh (glutinum) Taisoh (Zizyphi fructus) showed the antifibrinolytic effects in this order. On the other hand, Sanshishi showed the accelerating effect on fibrinolysis. (3) The inhibition modes of both thrombin and plasmin by Gaiyoh (Artemisiae folium) were shown to be competitive on Lineweaver-Burk plot. (4) Gayoh (Artemisiae folium) was gel-filtrated on Sephadex G-25 column (1.5×90cm) equilibrated with distilled water at room temperature. Five fractions were obtained, and in the first to fourth fraction, strong anticoagulant effects on aPTT and PT were observed. We pooled first and second to make fraction I, and make fraction II from peak 3. The recovery rate was 4.2 % by weight, and 36.7 % by inhibition activity, and specific activity on the basis of inhibition to aPTT was 34.8 % U/mg in the case with fraction II. Fraction I was found to be the same characteristically on blood coagulation. Fraction II was further purified by Sephadex LH-20 column (1.5 × 80 cm) at room temperature. Three fractions (Fraction IIa, IIb, IIc) were obtained, and the strong inhibitory effects was observed on both aPTT and PT in each fraction. The first fraction (fraction IIa) showed the strong inhibitory effect on aPTT, and the heightened specific activity with 17.6 % as the recovery rate. Fraction IIb showed strong inhibitory effect on fibrinolysis. Fraction IIb and IIc were found to be similar with fraction IIa characteristically on blood coagulation-fibrinolysis, but could separate the substance for coagulation and fibrinolysis each other on the basis of weight/volume. (5) by ex vivo study using rabbits, aPTTs were prolonged gradually after infusion (60 mg of fraction IIa). White blood cells and platelets were decreased gradually.

An improved method of preparing rat brain synaptic membranes. Eliminatio of a contaminating membrane containing 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity

Synaptosomes were prepared from rat cortex by subjecting a washed crude mitochondrial pellet to centrifugation first on discontinuous Ficoll-isotonic sucrose gradients and then on discontinuous sucrose gradients. The synaptosome fraction, collected from the 7.5–14% Ficoll band (II), was further separated into two additional fractions, designated IIA and IIB, which band at the 0.32–1.05 M and at the 1.05–1.6 M sucrose interfaces, respectively. Electron microscopic analysis showed that fraction IIB contained synaptosomes and extra terminal mitochondria and was essentially free of membrane fragments. Further characterization showed that IIB contained 69% of the protein and 83% of the lactic dehydrogenase activity of fraction II and had a specific activity of a 2′,3′-cyclic nucleotide 3′-phosphohydrolase approximately 1% of that obtained with myelin. Fraction IIA had approximately 50% the specific activity of the 2′,3′-cyclic nucleotide 3′-phosphohydrolase found in myelin. Synaptic plasma membranes were prepared by lysing fraction IIB in 1 mM sodium phosphate, 0.1 mM EDTA at pH 8.5 and subjecting this preparation to centrifugation on a discontinuous sucrose density gradient. Enzymatic analysis indicated that membranes banding at the 0.6–0.8 M sucrose interface had highgh specific activities of plasma membrane enzymes (e.g. acetylcholinesterase, ATPase, 5′-nucleotidase). The specific activity of the (Na + + K +)-ATPase in the purified membrane preparation was 8-fold higher than that in the original homogenate. Specific activities of various marker enzymes indicated that the composition of these membrane preparations for the most part was synaptic plasma membranes, approximately 7% mitochondrial outer membranes and 3% a membrane containing 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity. The polypeptide compositions of three possible contaminating membranes and of synaptic membranes were compared by electrophoresis in 6–20% gradient polyacrylamide gels in the presence of sodium dodecyl sulfate. Whereas mitochondrial and myelin membranes had distinct compositions, the compositions of the microsomal and synaptosomal plasma membranes were similar. Synaptic plasma membranes contained at least 27 polypeptides; the three major polypeptdes had molecular weights of 103,000; 54,000; and 50,000. The major polypeptides of soluble synaptosomal proteins had molecular weights of 54,000 and 42,000.

Fractionation and Characterization of Surface Antigens from Group a Neisseria Meningitidis

Group A meningococcal surface components were first subjected to fractionation with a mixture of chloroform-methanol. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the aqueous phase containing 30 to 40% of the original material revealed only two polypeptide components and a slowly migrating carbohydrate component. The soluble fraction of the interphase was found to contain most of the bacterial surface proteins and the chloroform-methanol phase essentially all of the lipid components. The components of the aqueous phase were further fractionated by use of the hydrophobic affinity column, 4-phenylbutylamino-Sepharose and gradient elution with NaCl to yield fractions I and II. Fraction II was further separated into a minor and a major component (IIb) with Sepharose G-200. Fraction I contained the group A polysaccharide in ionic linkages with a minor polypeptide component (6%). It elicited bactericidal antibodies in rabbits and protected mice against homologous challenge, whereas the polysaccharide alone was non-immunogenic in these animals. Fraction IIb was a polysaccharide-polypeptide complex with unknown linkages; it induced a low concentration of rabbit antibodies that were bactericidal to group A and C meningococci. Mice vaccinated with fraction IIb were most resistant to homologous challenge and the resistance was also extended to challenges with group B and C cells. Fractions I and IIb appeared to be useful alternatives to the currently employed group-specific polysaccharide vaccines for the protection against drug-resistant meningococci. A simplified procedure for the preparation of group-specific polysaccharide was presented.

Structure of the Carbohydrate Units of IgA1 Immunoglobulin: I. COMPOSITION, GLYCOPEPTIDE ISOLATION, AND STRUCTURE OF THE ASPARAGINE-LINKED OLIGOSACCHARIDE UNITS

Abstract The carbohydrate composition of an IgA (α1 subtype) myeloma protein has been determined. The carbohydrate, present only on the heavy chain, was found to consist of 3 moles of sialic acid, 9 mole of galactose, 5.4 moles of mannose, 0.8 moles of fucose, 8.8 moles of N-acetylglucosamine, and 5 moles of N-acetylgalactosamine per mole of heavy chain. Four major glycopeptide-containing fractions were isolated following pronase degradation of the protein. Glycopeptide I consisted of galactose, N-acetylgalactosamine, threonine, serine, and proline in the molar ratio 4:5:4:5:9. Each of the N-acetylgalactosamine residues was found to be involved in an O-glycosidic linkage, demonstrating that there are five O-glycosidically linked oligosaccharide units per heavy chain. The structure of this glycopeptide is presented in the following paper (Baenziger, J., and Kornfeld, S. (1974) J. Biol. Chem. 249, 7270–7281. The three other glycopeptide fractions contained 1 to 2 residues of sialic acid, 0 to 0.7 residue of fucose, 2 residues of galactose, 3 residues of mannose, 4 to 5 residues of N-acetylglucosamine, and 1 residue of asparagine. Glycopeptide IIA was a homogeneous glycopeptide with the following structure: Glycopeptide fractions IIB and IIC did not appear to be homogeneous, but instead each contained 2 glycopeptides. In both cases, one of the glycopeptides was probably the disialyl form of IIA containing a residue of sialic acid linked α2,6 to the terminal galactose. The other glycopeptide had 2 rather than 3 nonreducing termini arising from the core mannose. Both of these terminated in sialic acid and had the same sequence as that found for the sialic acid-containing terminus of IIA. These termini arose from positions 3 and 6 of the core mannose. The core of this glycopeptide differed from that of IIA in having a residue of fucose which was most likely linked α1,6 to the N-acetylglucosamine involved in the N-glycosidic linkage to the peptide.

Addition of histones to histone-depleted nuclei: effect on template activity toward DNA and RNA polymerases.

The ability of histones to block the accessibility of DNA in chromatin to DNA and RNA polymerases was measured by addition of lysine-rich or arginine-rich histones to nuclei selectively depleted of these histones. By this procedure nuclei were obtained in which all of the original lysine-rich histone in the chromatin was replaced by arginine-rich histone. Conversely in other nuclei, additional lysine-rich histone replaced some of the endogenous arginine-rich histone. Lysine-rich histone was much more effective than arginine-rich histone in blocking accessibility to DNA polymerase. Both classes of histone inhibited template activity toward RNA polymerase to a similar extent. In addition to lysine-rich histone and total arginine-rich histone, phosphorylated lysine-rich histone, two fragments of lysine-rich histone produced by cleavage with N-bromosuccinamide, and fractions IIB and IV of arginine-rich histone were added to histone-depleted nuclei. With both DNA and RNA polymerases as probes, no differences in inhibition of template activity were found when native lysine-rich histone was compared to phosphorylated lysine-rich histone. Similarly, fractions IIB and IV were indistinguishable from total arginine-rich histone. On a molar basis, the carboxyl fragment of lysine-rich histone was as effective as intact lysine-rich histone only when the amino fragment was added to it. By itself, the amino portion of lysine-rich histone was without inhibitory effect in the RNA polymerase assay and resulted in only slight inhibition of template activity toward DNA polymerase.

Enzymic hydrolysis of the potassium chloride soluble fraction of carrageenan: properties of "lambda carrageenases" from Pseudomonas carrageenovora.

An enzyme complex which hydrolyzed the KCl soluble carrageenan extracted from the red alga Chondrus crispus has been isolated from the cell-free medium of a culture of Pseudomonas carrageenovora grown on this polysaccharide. Three hydrolases could be separated. Fraction I, which caused a rapid decrease in the specific viscosity of the polysaccharide preparation with only minimal release of reducing sugar, could be distinguished from fraction II chromatographically on Sephadex G-100 and electrophoretically on agarose gel. Fraction IIa caused release of reducing sugar at pH 6.2, which activity was depressed at pH 7.5. Fraction IIb exhibited viscometric activity only at both pH 6.2 and pH 7.5. Fraction IIa had a sharp pH optimum at pH 6.2 and a temperature optimum at 28°. All hydrolases were inactivated by freezing, by dialysis against distilled water, by heating at 35° for 30 min, and by Hg2+ and 0.0001 mM EDTA. When fraction II (a and b) isolated after chromatographic resolution on Sephadex G-100 was incubated at pH 6.2 with KCl soluble carrageenan from C. crispus, products which had Rgal values of 0.74 and 0.17 were detected, were sulfated, and contained no 3,6-anhydrogalactose.

The dissociation of chicken erythrocyte deoxyribonuleoprotein and some properties of its partial nucleoproteins.

Histones were completely dissociated from their native complex with DNA in 2.0m-sodium chloride. Histone fractions IIb, V and I were dissociated in 1.2m-sodium chloride, fractions V and I in 0.7m-sodium chloride and fraction I in 0.45m-sodium chloride. Repeated extraction of partial dRNP (deoxyribonucleoprotein) preparations with sodium chloride of the same concentration as that from which they were prepared resulted in release of histones that previously had remained associated with the DNA of the complex. Gradual removal of histones from dRNP was paralleled by an improvement in solubility, a decrease in wavelength of the u.v.-absorption minimum, and a fall in sedimentation coefficient of the remaining partial dRNP. X-ray diffraction patterns of partial dRNP preparations showed that removal of histone fractions I and V from dRNP did not destroy the super-coil structure of the dRNP, but further removal of histones did. Infrared spectra of partial dRNP preparations showed that in native dRNP histone fraction I was present in the form of extended, isolated polypeptide chains, and that the other histone fractions probably contain a helical component that lies roughly parallel to the polynucleotide chains in the double helix and an extended polypeptide component that is more nearly parallel to the DNA helix axis. An analysis of the sedimentation of partial dRNP preparations on sucrose gradients showed that native dRNP consists of DNA molecules each complexed with histone fractions of all types.

The stepwise removal of histones from chicken erythrocyte nucleoprotein.

1. A fractionation of chicken erythrocyte histones was achieved simultaneously with their extraction from saline-washed nuclei by stepwise titrations to progressively lower pH values. 2. Different acids and dilute buffer solutions of comparable pH behaved similarly in stepwise extractions of histones. 3. The histone preparations so obtained were characterized by their amino acid composition and behaviour on zone electrophoresis in starch gels. 4. The fractionation by titration was quite sharp at appropriate pH ranges, and the histone fraction that is apparently unique to avian erythrocytes was obtained without contamination by other histone fractions. 5. Histones prepared by stepwise titration were fractionated further by cation-exchange and exclusion chromatography. The chromatographic behaviour and amino acid composition of the components permitted comparison with histones prepared by other methods. 6. Histone fraction IIb was resolved into its subfractions IIb(1) and IIb(2) by exclusion chromatography on Bio-Gel P-60. 7. Histone fractions III and IV, previously reported to be absent from chicken erythrocyte nuclei, were found in extracts made at pH1.

The immunological pattern of the water-soluble proteins and glycoproteins of the media layer of normal and atherosclerotic human aortas

The water-extractable protein and glycoprotein constituents of normal (NM) and sclerotic (SM) media have been fractionated by means of continuous flow paper electrophoresis and Sephadex G-200 column chromatography. Rabbit antisera against the five SM and NM fractions isolated were obtained and employed in a series of immunological studies with especial emphasis on Immunoelectrophoresis; the “Partigen” test was also used for the quantitative estimation of the serum albumin content of the fractions. It was found that: Each of the five fractions shows a characteristic immunoelectrophoretic pattern both in the NM and SM series. All ten fractions contain constituents immunologically identical with serum albumin. It would appear, however, that except in the case of IIb the SM fractions contain substantially larger amounts of these constituents than the corresponding NM fractions. A number of other qualitative and/or quantitative differences between NM and SM series are present the most important being in the α1 area of fraction IV and in the γ area of fraction IIb.

The identification of metabolites of retinal and retinoic acid in rat bile

1. 1. The present work was conducted to determine whether retinoic acid is normally formed from retinal in the rat and what the metabolic products of retinoic acid are. Within 6 h after the intraportal injection of 3 mg of [6,7- 14C 2]retinal to rats with cannulated bile ducts, a minimum of 6% of the injected dose appeared in the bile as free or conjugated acid. 2. 2. In similar experiments with 6,7- 14C 2]retinoic acid (Fraction IIA) was demonstrated both in the liver and the intestine at 4.5 h, and in the bile which was collected for 17 h. Non-acidic esters of retinoic acid accounted for at least 60% of the radioactivity in Fraction I of bile, whereas retinoyl-β-glucuronide accounted for at least 45% of the radioactivity in Fraction III of bile. Furthermore, another acidic metabolite of retinoic acid (Fraction IIB) accounted for about 10% of the biliary radioactivity. 3. 3. We concluded that retinoic acid is normal, but not necessarily an obligatory, metabolite of retinal in the rat, and that retinoic acid is rapidly converted in the liver to its β-glucuronide and to other conjugates which are excreted in the bile. Presumably little or no cleavage of the carbon chain of retinoic acid occurs during these processes.