A Simple Method for Preparation of 18α-Glycyrrhetinic Acid and Its Derivatives

Treatment of 18-glycyrrhizic acid with a methanolic solution of HCl resulted in 1 : 1 mixture of methyl esters of 18- and 18-glycyrrhetinic acids. Benzoylation of the mixture led to methyl esters of 3-benzoyl-18-glycyrrhetinic acid and 3-benzoyl-18-glycyrrhetinic acid, which were separated by chromatography on silica gel. 18-Glycyrrhetinic acid was prepared by alkaline hydrolysis of methyl 3-benzoyl-18-glycyrrhetinate and was further used for the syntheses of 3-keto-18-glycyrrhetinic acid and methyl esters of 18-glycyrrhetinic acid and 3-keto-18-glycyrrhetinic acid.     

Subunit structures of multiple hemoglobins in carp

Three hemoglobin components in carp designated CI, CII, and CIII, were isolated by DEAE-Toyopearl ion-exchange chromatography. Constituent globin chains, ¹, ², ¹ and ², were analyzed by urea-Triton acid polyacrylamide gel electrophoresis and isolated by high performance liquid chromatography with a reversed-phase column. Tryptic peptide mapping indicated that the -globin chains of the three hemoglobin components have slightly different structures. In addition, N-terminal amino acid sequence analysis indicated that the ¹-globin chain has a primary structure different from that of the ²-chain. A series of hybridization experiments between isolated hemoglobins, together with such structural properties of globin chains, suggested that the three hemoglobins have the following compositions: CI (^{1 2} ₂ ¹ ), CII (^{1 2 1 2}), and CIII (^{1 2} ₂ ² ). Hemoglobin CII was a hybrid between the two types each of - and -chain and could be constructed in vitro from two hemoglobin components CI and CIII.Abbreviations a-a amino acid - Hb hemoglobin - HPLC high performance liquid chromatography - P ₅₀ oxygen pressure at half saturation - PAGE polyacrylamide gel electrophoresis - TFA trifluoroacetic acid     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Attenuation of changes in sarcoplasmic reticular gene expression in cardiac hypertrophy by propranolol and verapamil

The prothymosin a kinase (ProTK) is an apparently novel enzyme that is responsible for the phosphorylation of prothymosin (ProT), involved in the proliferation of mammalian cells. The present study investigated the properties of this enzyme. ProTK is more effectively activated by Mn²⁺ than by other divalent cations, and its activity is unaffected by RNA. Its principal substrate in proliferating cells appears to be ProTa. Both in vivo and in vitro, it is unable to phosphorylate the peptides thymosin ₁ and thymosin ₁₁, derived from the amino terminus of ProT, despite the fact that the sites of phosphorylation of ProT are contained within this part of its sequence. In trials in vivo, inhibition of gene expression abolished both phosphorylation of ProT and ProTK activity. ProTK is located in the cytosolic fractions throughout the cell cycle. Its activity, which is dependent on cell proliferation, increases markedly during S phase and begins to decline as the cell enters G2. Studies of the effects of activators and inhibitors of protein kinases involved in signal transduction pathways suggest that ProTK is activated by phosphorylation in a mitogen-initiated pathway that is dependent on PKC; however, PKC does not itself phosphorylate ProTK, which is therefore presumably phosphorylated by another kinase.     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Syntheses of 1-O-benzyl-α-glucoside and 1-O-benzyl-α-maltoside by transglycosylation of α- amylase from soluble starch in aqueous solution

Glycosides, 1-O-benzyl--glucoside (BG) and 1- O-benzyl--maltoside (BM), were synthesized from soluble starch and benzyl alcohol by transglycosylation with an -amylase in a water system. BG was mostly obtained in a reaction mixture of pH 5.0, while BM was synthesized in pH 8.0. The synthesized glycosides had -configuration linkage between sugar and benzyl alcohol. The BG was rapidly hydrolyzed to benzyl alcohol and glucose by -glucosidase. The BM was hydrolyzed to BG and glucose below pH 5.0 by the -amylase used for its synthesis but it was not hydrolyzed above pH 8.0.     

Evaluation of hydrophobicity versus chaperonelike activity of bovine alphaA- and alphaB-crystallin

Calf lens A-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than B-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to A-crystallin than to B-crystallin at room temperature. Bis-ANS binding to both A- and B-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of A-crystallin is lower than that of B-crystallin whereas at higher temperatures, A-crystallin shows significantly higher protection against aggregation of substrate proteins compared to B-crystallin. Therefore, calf lens A-crystallin is more hydrophobic than B-crystallin and chaperone-like activity of -crystallin subunits is not quantitatively related to their hydrophobicity.     

An alpha-L-fucosidase from Thermus sp. with unusually broad specificity

An -L-fucosidase (E.C. 3.2.1.51) exhibiting a wide aglycon specificity expressed in ability of cleaving 1 6-, 1 3-, 1 4-, and 1 2-O-fucosyl bonds in fucosylated oligosaccharides, has been isolated from culture filtrate of Thermus sp. strain Y5. The -L-fucosidase hydrolyzes p-nitrophenyl -L-fucopyranoside with V _max of 12.0 ± 0.1 M/min/mg and K _m = 0.20 ± 0.05 mM and is able to cleave off about 90% of total L-fucose from pronase-treated fractions of fucosyl-containing glycoproteins and about 30% from the native glycoproteins. The purified enzyme is a tetramer with a molecular mass of 240 ± 10 kDa consisting of four identical subunits with a molecular mass of 61.0 ± 0.5 kDa. The N-terminal sequence showed homology to some -L-fucosidases from microbial and plant sources. Hydrolysis of p-nitrophenyl -L-fucopyranoside occurs with retention of the anomeric configuration. Transglycosylating activity of the -L-fucosidase was demonstrated in reactions with such acceptors as alcohols, N-acetylglucosamine and N-acetylgalactosamine while no transglycosylation products were observed in the reaction with p-nitrophenyl -L-fucopyranoside. The enzyme can be classified in glycosyl hydrolase family 29.     

Inhibition of protein synthesis enhances the lytic effects of tumor necrosis factor α and interferon γ in cell lines derived from gynecological malignancies

Summary Few clinical responses have occurred in preliminary studies using the cytokines tumor necrosis factor (TNF) or interferon (IFN) in cancer patients. This may be related to the observation that many malignant cell lines are resistant to lysis by these cytokinesin vitro. Resistance to lysis by TNF or IFN in many cells is controlled by a protein-synthesis-dependent mechanism, such that when protein synthesis is inhibited cells become sensitive to lysis by these cytokines. Because there is some evidence that TNF and IFN act through different lytic mechanisms and are opposed by different resistance mechanisms, we treated a panel of eight cell lines, five derived from human cervical carcinomas (ME-180, MS751, SiHa, HT-3, and C-33A) and three derived from ovarian carcinomas (Caov-3, SK-OV-3, and NIH: OVCAR-3) with both TNF and IFN to determine whether such combination treatment might maximizein vitro cell lysis. Our results showed that pretreatment with IFN followed by exposure to TNF in the presence of protein synthesis inhibitors increased lysis of seven of the eight cell lines above that seen with either TNF or IFN and inhibitors of protein synthesis. Only the cell line C-33A was resistant to lysis by TNF and IFN, when exposed to these agents both alone and in combination with protein synthesis inhibitors. Clinically, combining the cytokines TNF and IFN with protein synthesis inhibitors may maximize thein vivo lytic effects of these cytokines.Supported by American Cancer Society Career Development Award 90-221     

The effect of modification and fragmentation of α-lactalbumin on lactose and lactosamine synthase reactions

In the presence of the modifier protein -lactalbumin, bovine milk galactosyltransferase transfers galactose to glucose forming lactose instead of transferring toN-acetylglucosamine formingN-acetyllactosamine. At low concentrations of -lact-albumin, the lactosamine synthase activity is stimulated by -lactalbumin and decreases when the lactose synthase activity develops along a sigmoidal curve. The observation suggests that different interactions between -lactalbumin and enzyme were responsible for the modulating effect of the -lactalbumin in the lactose and lactosamine synthase reactions.To study the nature of the protein-protein interactions, -lactalbumin was both modified and cleaved chemically. Reduction and alkylation with iodoacetic acid, iodoacetamide or 4-vinylpyridine abolished the ability of the -lactalbumin to induce lactose synthase activity but stimulated lactosamine synthase activity 7-to 12-fold.A peptide fragment corresponding to residues 26–60 of -lactalbumin isolated from a 2-(2-nitrophenylsulphenyl)-3-methyl-3-bromo-indolene (BNPS-skatole) fragmentation of the molecule was active in the lactosamine but not lactose synthase reaction. We concluded that, whereas lactose synthase required -lactalbumin, in the native conformation, lactosamine synthase activity was stimulated by a linear sequence of amino acids in peptide 26–60.Abbreviations MES 4-N-morpholinoethanesulfonic acid - TRIS 2-amino-2-(hydroxymethyl)-1,3-propanediol - UDP-Gal uridinediphosphogalactose - BNPS-skatole 2-(2-nitrophenylsulphenyl)-3-methyl-3-bromo-indolene - EDTA ethylene diamine tetra acetic acid     

Distribution of Fucoidan Hydrolases and Some Glycosidases among Marine Invertebrates

Thirty-three species of marine invertebrates from the Sea of Japan were analyzed for contents of fucoidan hydrolases and some glycosidases. Fucoidan hydrolase activity was assessed by examining the effect of animal tissue extracts on fucoidans from the two brown seaweeds Laminaria cichorioides and Fucus evanescens, which have different structural characteristics. The activity of glycosidases (-glucosidase, -galactosidase, -fucosidase, and -mannosidase) was determined using p-nitrophenyl derivatives of sugars as substrates. It was found that glycosidases and fucoidan hydrolases of different specificities are fairly widely distributed among marine invertebrates. Mollusks and some species of echinoderms and arthropods showed the highest enzymatic activity. This research will enable us to choose organisms for the separation and study of fucoidan hydrolases and glycosidases, which may be useful in determining the structure of fucoidans.     

The sialyl-α2,6-lactosaminyl-structure: Biosynthesis and functional role

Sialylation represents one of the most frequently occurring terminations of the oligosaccharide chains of glycoproteins and glycolipids. Sialic acid is commonly found ,3- or ,6-linked to galactose (Gal), ,6-linked to N-acetylgalactosamine (GalNAc) or ,8-linked to another sialic acid. The biosynthesis of the various linkages is mediated by the different members of the sialyltransferase family. The addition of sialic acid in ,6-linkage to the galactose residue of lactosamine (type 2 chains) is catalyzed by -galactoside ,6-sialyltransferase (ST6Gal.I). Although expressed by a single gene, this enzyme shows a complex pattern of regulation which allows its tissue- and stage-specific modulation. The cognate oligosaccharide structure, NeuAc,6Gal1,4GIcNAc, is widely distributed among tissues and is involved in biological processes such as the regulation of the immune response and the progression of colon cancer. This review summarizes the current knowledge on the biochemistry of ST6Gal.I and on the functional role of the sialyl-,6-lactosaminyl structure.     

Liver and intestinal fatty acid binding proteins in control and TGFβ1 gene targeted deficient mice

The effect of transforming growth factor beta-1 (TGF1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF1-deficient mice. Homozygous TGF1-deficient 129 × CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF1-deficient/immunodeficient C3H mice on a SLID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (1-FABP) which decreased 3-fold in the TGF1-deficient/immunodeficient C3H mice only. Thus, TGF1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.Abbreviations L-FABP liver fatty acid binding protein - I-FABP intestinal fatty acid binding protein - TGF1 transforming growth factor beta-1 - TNF- tumor necrosis factor- - MIP- macrophage inflammatory protein- - PMSF phenylmethyl sulfonyl fluoride - PBS phosphate buffered saline     

New pharmacokinetic and pharmacodynamic tools for interferon-alpha (IFN-α) treatment of human cancer

Interferon (IFN-) has been widely used in the treatment of human solid and haematologic malignancies. Although the antitumour activity of IFN- is well recognised at present, no major advances have been achieved in the last few years. Recent findings have provided new information on the molecular mechanisms of the antitumour activity of the cytokine. In fact, IFN- appears to block cell proliferation, at least in part, through the induction of apoptotic effects. This cytokine can also regulate the progression of tumour cells through the different phases of the cell cycle inducing an increase of the expression of the cyclin-dependent kinase inhibitors p21 and p27. However, it must be considered that IFN- is a physiologic molecule with ubiquitously expressed receptors that is likely to activate survival mechanisms in the cell. We have recently identified an epidermal growth factor (EGF) Ras-dependent protective response to the apoptosis induced by IFN- in epidermoid cancer cells. The identification of tissue- and/or tumour-specific survival pathways and their selective targeting might provide a new approach to improve the efficacy of IFN-–based treatment of human cancer. Moreover, new pegylated species of IFN- are now available with a more favourable pharmacokinetic profile. We will review these achievements, and we will specifically address the topic of IFN-–based molecularly targeted combinatory antitumour approaches.     

Two forms of α-glucosidase from sugar-beet seeds

Two forms of -glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from sugarbeet (Beta vulgaris L.) seeds by a procedure including fractionation with ammonium sulfate and ethanol, carboxymethyl-cellulose column chromatography, and preparative disc gel electrophoresis. The two enzymes were homogeneous by polyacrylamide disc gel electrophoresis. Their molecular weights were 98,000 (I) and 60,000 (II). -Glucosidase I readily hydrolyzed maltose, isomaltose, kojibiose, maltotriose, panose, amylose, soluble starch, amylopectin and glycogen. -Glucosidase II also hydrolyzed maltose, kojibiose and maltotriose but hydrolyzed the other substrates only very weakly or not at all. -Glucosidase I hydrolyzed soluble starch at a faster rate than maltose. It produced isomaltose and panose as the main -glucosyltransfer products from maltose, whereas maltotriose was the main product of -glucosidase II. -Glucosidase I hydrolyzed amylose liberating -glucose. The neutral-sugar content was calculated to be 2.7% for -glucosidase I and 8.8% for -glucosidase II. The main neutral sugar was mannose in -glucosidase I, and glucose in -glucosidase II.     

Purification and partial characterisation of and α-tocopherol-binding protein from rabbit heart cytosol

An -tocopherol-binding protein has been isolated and purified from rabbit heart cytosol. The purified protein had an apparent molecular mass of 14,200, as derived from SDS-PAGE. The content of the protein in rabbit heart was around 11.8 g per g of tissue. The binding of -tocopherol to the purified protein was rapid, reversible, and saturable. Neither nor tocopherol could displace the bound -tocopherol from the protein, suggesting a high specificity for -tocopherol. -Tocopherol-binding protein did not bind oleate. Transfer of -tocopherol from liposomes to mitochodria was stimulated 8-fold in the presence of the binding protein, suggesting that this protein may be involved in the intracellular transport of -tocopherol in the heart.     

Endosperm-specific demethylation and activation of specific alleles of α-tubulin genes of Zea mays L.

We have investigated the methylation status of the -tubulin genes, and the degree of accumulation of their mRNAs in endosperm, embryo and seedling tissues of Zea mays L. We have found that many of the -tubulin genes are differentially demethylated in the endosperm relative to the embryo and seedling. However, only for tub2 and tub4 could a correlation between DNA demethylation and increased RNA accumulation be detected. By analyzing the inbred lines W64A and A69Y and their reciprocal crosses, we have also identified in the endosperm two -tubulin genes, tub3 and tub4, that are differentially demethylated if transmitted by the maternal germline, but that remain hypermethylated when transmitted by the paternal germline.