Iodination of proteins,glycoproteins, and peptides using a solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen)

A new, commercially available oxidizing agent, 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) was compared with chloramine-T and solid-phase lactoperoxidase in the radioiodination of proteins, glycoproteins, and peptides. A method for performing low-level iodinations is described and was used to determine maximum ¹²⁵I incorporation. Iodinated proteins were purified on analytical gel filtration columns and peptides by reverse-phase high-performance liquid chromatography. Both methods were designed to analyze the tracers for the presence of aggregate and breakdown products caused by the iodination. All tracers prepared were tested in antibody dilution and dose-response curves in their respective radioimmunoassays. Results indicate that Iodogen can be used for a wide range of proteins and peptides, can permit theoretical iodine incorporation with minimal oxidation damage, and can produce tracer stable for up to 3 months.     

Concerning the structure of 7α,15β-dichloro-5α-cholest-8(14)-en-3β-ol,a novel inhibitor of cholesterol biosynthesis

Treatment of 3β-p-bromobenzoyloxy-14α, 15α-epoxy-5α-cholest-7-ene with gaseous HCI in chloroform at ?25°C gave 3β-p-bromobenzoyloxy-7α, 15β-dichloro-5α-cholest-8(14)-ene in 93% yield. The structure of the latter compound was unequivocally established by the results of X-ray crystallographic analysis.     

Synthesis of a Stereoisomeric Mixture of 3-Hydroxy-α-damascone

The relationships among the spinnability, the rheological properties, the spun fiber strength, and the inhibition of lysinoalanine (LAL) formation with the addition of reducing agents were studied to get safe, edible, fibrous soy protein, having excellent spun fiber strength, when a dope of 20% protein concentration was used as a normal dope. It was found that cysteine and 2-mercaptoethanol were effective in reducing LAL formation and the dopes prepared with the addition of these agents showed almost the same spun fiber strength as that of the normal dope prepared without agents. Especially, cysteine was the most effective agent for inhibiting LAL formation to get fibrous protein for meat analogues. The most suitable concentration for inhibiting LAL formation was 2% cysteine in the total protein. The dopes with the addition of 2-mercaptoethanol and Na₂SO₃ had lower viscoelastic values and their frequency dependence of G' was higher than that of the normal dope. However, the dopes with the addition of other reducing agents (NaHSO₃, glycine, reduced glutathione) had higher viscoelastic values and lower frequency dependence of G'. The viscoelastic values of the dopes with the addition of 2-mercaptoethanol, Na₂SO₃, and that of the normal dope were decreased with the lapse of time, but the dopes prepared by the addition of other agents had constant viscoelastic values.     

Opposing actions of the progesterone metabolites, 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αHP) on mitosis,apoptosis, and expression of Bcl-2, Bax and p21 in human breast cell lines

Previous studies have shown that breast tissues and breast cell lines convert progesterone (P) to 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αHP) and that 3αHP suppresses, whereas 5αP promotes, cell proliferation and detachment. The objectives of the current studies were to determine if the 5αP- and 3αHP-induced changes in cell numbers are due to altered rates of mitosis and/or apoptosis, and if 3αHP and 5αP act on tumorigenic and non-tumorigenic cells, regardless of estrogen (E) and P receptor status. The studies were conducted on tumorigenic (MCF-7, MDA-MB-231, T47D) and non-tumorigenic (MCF-10A) human breast cell lines, employing several methods to assess the effects of the hormones on cell proliferation, mitosis, apoptosis and expression of Bcl-2, Bax and p21. In all four cell lines, 5αP increased, whereas 3αHP decreased cell numbers, [³H]thymidine uptake and mitotic index. Apoptosis was stimulated by 3αHP and suppressed by 5αP. 5αP resulted in increases in Bcl-2/Bax ratio, indicating decreased apoptosis; 3αHP resulted in decreases in Bcl-2/Bax ratio, indicating increased apoptosis. The effects of either 3αHP or 5αP on cell numbers, [³H]thymidine uptake, mitosis, apoptosis, and Bcl-2/Bax ratio, were abrogated when cells were treated simultaneously with both hormones. The expression of p21 was increased by 3αHP, and was unaffected by 5αP. The results provide the first evidence that 5αP stimulates mitosis and suppresses apoptosis, whereas 3αHP inhibits mitosis and stimulates apoptosis. The opposing effects of 5αP and 3αHP were observed in all four breast cell lines examined and the data suggest that all breast cancers (estrogen-responsive and unresponsive) might be suppressed by blocking 5αP formation and/or increasing 3αHP. The findings further support the hypothesis that progesterone metabolites are key regulatory hormones and that changes in their relative concentrations in the breast microenvironment determine whether breast tissues remain normal or become cancerous.     

Synthesis of a Spacer-Linked Derivative of Heptopyranosyl(α1-3)heptopyranose Expressed in Lipooligosaccharide and Lipopolysaccharide

Glycosylation of penta-O-acetyl heptopyranosyl trichloroacetimidate with the 3-OH acceptor, methyl 2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-α-D-manno-oct-7-enopyranoside, gave the desired α1-3-linked disaccharide in a 94% yield. The oct-enopyranoside moiety of the disaccharide was converted to the heptoside by oxidative cleavage with osmium tetroxide/NaIO₄ and subsequent reduction with NaBH₄. The resulting α1-3-linked heptose disaccharide was converted to a tricholoroacetaimidate derivative containing a benzoyl group at C-2. This donor was glycosylated with 2-(carbobenzoxyamino)-1-ethanol to give an α spacer-linked disaccharide derivative in a 90% yield. Zemplén deacylation of the derivative and subsequent hydrogenolysis gave a 2-aminoethyl glycoside of heptopyranosyl(α1-3)heptopyranose.     

Synthesis of a spacer-linked derivative of heptopyranosyl(α1-3)heptopyranose expressed in lipooligosaccharide and lipopolysaccharide

Glycosylation of penta-O-acetyl heptopyranosyl trichloroacetimidate with the 3-OH acceptor, methyl 2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-α-D-manno-oct-7-enopyranoside, gave the desired α1-3-linked disaccharide in a 94% yield. The oct-enopyranoside moiety of the disaccharide was converted to the heptoside by oxidative cleavage with osmium tetroxide/NaIO(4) and subsequent reduction with NaBH(4). The resulting α1-3-linked heptose disaccharide was converted to a tricholoroacetaimidate derivative containing a benzoyl group at C-2. This donor was glycosylated with 2-(carbobenzoxyamino)-1-ethanol to give an α spacer-linked disaccharide derivative in a 90% yield. Zemplén deacylation of the derivative and subsequent hydrogenolysis gave a 2-aminoethyl glycoside of heptopyranosyl(α1-3)heptopyranose.     

A novel structural tree for wrap-proteins,a subclass of (α+β)-proteins

In this paper, a novel structural subclass of (α+β)-proteins is presented. A characteristic feature of these proteins and domains is that they consist of strongly twisted and coiled β-sheets wrapped around one or two α-helices, so they are referred to here as wrap-proteins. It is shown that overall folds of the wrap-proteins can be obtained by stepwise addition of α-helices and/or β-strands to the strongly twisted and coiled β-hairpin taken as the starting structure in modeling. As a result of modeling, a structural tree for the wrap-proteins was constructed that includes 201 folds of which 49 occur in known nonhomologous proteins.     

Synthesis of 1-(4-Keto-2, 3-O-isopropylidene-α-L-rhamnopyranosyl) uracil

Abstract

Synthesis of 1-(2, 3, 4-tri-0-acetyl-α-L-rhamnopyranosyl) uracil (3), 1-(α-L-rhamnopyranosyl) uracil (4), 1-(2, 3-0-isopropylidene-α-L-rhamnosyl) uracil (5), and 1-(2, 3-0-isopropylidene-4-keto-α-L-rhamnopyranosyl) uracil (6) are reported. Oxidation of (5) to (6) was effected using pyridinium chlorochromate in presence of molecular sieves.     

Isolation and structural analyses of positional isomers of 61,6m-di-O-α-d-mannopyranosyl-cyclomaltooctaose (m=2–5) and 6-O-α-(n-O-α-d-mannopyranosyl)-α-d-mannopyranosyl-cyclomaltooctaose (n=2, 3, 4, and 6)

Eight positional isomers of 6¹,6^m-di-O-α-d-mannopyranosyl-cyclomaltooctaose (γCD) (m=2–5) and 6-O-α-(n-O-α-d-mannopyranosyl)-d-mannopyranosyl-γCD (n=2, 3, 4, and 6) in a mixture of products from γCD and d-mannose by condensation reaction of α-mannosidase from jack bean were isolated by HPLC. The structures of four isomers of 6-O-α-(n-O-α-d-mannopyranosyl)-d-mannopyranosyl-γCD were elucidated by NMR spectroscopy. On the other hand, four positional isomers of 6¹,6^m-di-O-α-d-mannopyranosyl-γCD were determined by LC–MS analysis of degree of polymerization of the branched oligosaccharides produced by enzymatic degradation with bacterial saccharifying α-amylase (BSA), and combination of BSA and glucoamylase. Similarly cyclomaltodextrin glucanotransferase also digested these isomers.     

Synthesis of 2-(p-trifluoroacetamidophenyl)ethylO-α-l-fucopyranosyl-(1–3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl) (1–3)-O-β-d-galactopyranosyl-(1–4)-β-d-glucopyranoside,a tetrasaccharide fragment of a tumour-associated glycosphingolipid

The tetrasaccharide 2-(p-trifluoroacetamidophenyl)ethylO-α-l-fucopyranosyl-(1–3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1–3)-O-β-d-galactopyranosyl-(1–4)-β-d-glucopyranoside was synthesized from thioglycoside intermediates. The key step was a methyl triflate promoted glycosidation of a lactose-derived 3′,4′-diol with a disaccharide thioglycoside to give a β(1–3)-linked tetrasaccharide derivative in 67% yield.     

Localization of a Class II Phosphatidylinositol 3-Kinase,PI3KC2α, to Clathrin-Coated Vesicles

We have analysed phosphatidylinositol 3-kinase activity associated with subcellular fractions prepared from rat brains. Phosphatidylinositol 3-kinase activity is not markedly enriched with synaptic vesicle purification; whilst the activity associated with the most pure fractions is inhibited at low concentrations of wortmannin (IC₅₀ ∼ 4–5 nM). In contrast, clathrin-coated vesicle (CCV) fractions showed increased enzyme activity compared to light membrane fractions from which they are purified. In addition to a wortmannin-sensitive activity, we also detected an activity that could only be inhibited at higher concentrations of wortmannin (IC₅₀ ∼ 400 nM), characteristic of certain class II enzymes (including phosphatidylinositol 3-kinase C2α) to be highly enriched in CCV fractions. Immunoblotting with an antibody raised against phosphatidylinositol 3-kinase C2α, confirmed that this enzyme is highly enriched in CCVs and displays an enrichment profile during the purification that mirrors enrichment of the low nanomolar wortmannin-insensitive activity. If the CCV purification protocol is adapted to favour nerve terminally derived vesicles, we find reduced levels of the C2α enzyme in the CCV fractions, suggesting that the enzyme may principally reside on vesicles associated with the cell body.     

Conformation of (1→3)-α-D-Glucan Tribenzoate

The molecular conformation of (1→3)-α-D-glucan tribenzoate (TBG) was studied by X-ray diffraction measurements coupled with a conformational analysis. Although the fiber pattern obtained was of very low crystallinity, the presence of a meridional reflection at the 5th layer line indicated that the TBG molecule took a five-fold helical conformation with a 19.63 A fiber repeat. A conformational analysis on the five-fold helix, which was done by calculating van der Waals’ repulsion energy between non-bonded atoms comprising the TBG chain, suggested that the most preferable energy-based conformation was –5/1, a left-handed five-fold helix.     

The microbial production of 3aα-H-4α- (3′-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-indan-1-one-δ-lactone from cholesterol

Summary A mutant strain of Rhodococcus australis CSIR-236.457 which accumulates 3a-H-4-(3-propionic acid)-5-hydroxy-7a-methylhexahydro-indan-1-one--lactone from cholesterol, stigmasterol and -sitosterol was studied. The product is produced in a 60% molar yield in a dilute black strap molasses medium containing 6–12g/l cholesterol after a 72 hour fermentation period.     

(S)-α-methyl,α-amino acids: a new stereocontrolled synthesis

A new and convenient stereocontrolled synthesis of the optically pure (S)-α-methyl,α-amino acids 6(a–d) that exploits the chiral synthon 1,4-N,N-[(S)-1-phenylethyl]-piperazine-2,5-dione (1) is described. The (S)-1-phenylethyl group, bonded to each of the N-atoms of the 2,5-diketopiperazine, acts as a chiral inductor in the first alkylation, while the steric hindrance appears to be the determining factor of stereocontrol in third and forth alkylation.     

Unusual oxidative transformations of a steroidal 16α,17α,22-triol

A number of unexpected reactions were observed during attempts to invert configuration at C16 in 16α,17α,22-triol 3a. The PDC oxidation of 3a produced the D-seco-aldehyde 4a. Analogous compound 4b was obtained by Swern oxidation of the 16α,17α-dihydroxy-22-O-TES-ether 3b in addition to the desired 16-ketone 7. The unprotected triol 3a yielded pentacyclic products 5 and 6 under similar conditions. The Mitsunobu reaction of the triol 3a afforded 16-ketone 8 with inverted configuration of the side chain. During heating of a solution of 3a in THF with NaH at reflux autoxidation to the 16-ketone cyclic hemiketal 5, identical to one of the Swern oxidation products, took place.     

Affinity labeling of 3α, 20β-hydroxysteroid dehydrogenase with a nucleoside analog

Incubation of 3α, 20β-hydroxysteroid dehydrogenase (3α, 20β-HSD; E.C.1.1.1.53) with the nucleoside 5'-p-fluorosulfonylbenzoyladenosine (FSA) caused a time-dependent and irreversible loss in enzyme activity. Both 3α- and 20β-hydroxysteroid oxidoreductase activities decreased at equal rates by a first order kinetic process (in 0.05m phosphate buffer at pH 6.0 and 25°C, t$\text{1}\text{2}$ = 170 min). Incubation of 3α, 20β-HSD was quenched by addition of 2-mercaptoethanol which instantaneously reacts with the fluorosulfonyl group of FSA. The cofactor NADH protected 3α, 20β-HSD against inactivation by FSA, in a concentration-dependent manner. However, progesterone did not protect 3α, 20β-HSD against inactivation by FSA. Evidently, FSA causes inactivation of the enzyme by irreversibly binding to the NADH-binding region at the active site of 3α, 20β-HSD. Both 3α- and 20β-hydroxysteroid oxidoreductase activities disappeared at equal rates under a variety of enzyme-inactivating conditions. These results suggest that both 3α- and 20β-activites occur at the same active site of 3α, 20β-HSD.     

重组人肿瘤坏死因子α衍生物3a(rh-TNFαD3a)与化疗联合治疗恶性肿瘤的研究

研究重组人肿瘤坏死因子α衍生物 3a(rh TNFαD3a)与化疗药物联合应用治疗晚期复治乳腺癌、非霍奇金氏淋巴瘤 ,评价其临床疗效及临床安全性。研究表明试验组和对照组疗效比较存在显著性差异 (P <0 .0 1) ,不良反应较轻可耐受 ;rh TNFαD3a和化疗药物联合应用是治疗晚期复治乳腺癌、非霍奇金氏淋巴瘤较为理想的治疗方法     

Synthesis and Antileishmanial Activity of 3-(α-Azolylbenzyl)indoles

The goal of the present study was to evaluate several azolyl-substituted indoles as new antileishmanial agents. Ten 3- (α -azolylbenzyl)indoles have been synthesized using Friedel-Crafts acylation as a key-step. All the target compounds were found to display high levels of activity when tested against Leishmania mexicana promastigotes in vitro. The most active compounds, showing an IC 50 <1 μM, were 5-bromo-1-ethyl-3-[(2,4-dichlorophenyl)(1 H -imidazol-1-yl)methyl]-1 H -indole 15 and its triazole analogue 17. Four representative compounds 15, 17, 22 and, 23 were also tested against intracellular amastigotes of L. mexicana using ketoconazole and meglumine antimoniate as reference compounds, the results of which are discussed.