Thamarapu Srikrishnan

Linear quantitation of Aβ aggregation using Thioflavin T: Reduction in fibril formation by colostrinin

Thioflavin T (ThT) fluorescence is a commonly used method to monitor Abeta protein fibril formation. This method is particularly attractive since ThT fluoresces only when bound to fibrils, the reaction is completed within 1min and ThT does not interfere with aggregation of Abeta fibrils. One of the drawbacks of this method is the lack of a strict quantitative relationship between ThT fluorescence and fibril content. It was observed that, when the same gram molecular weight of Abeta (1-40) is dissolved into varying amounts of base then placed into a constant volume of aqueous buffer, a non-linear fluorescent response is obtained. By maintaining a strict relationship between Abeta content and the volume of base, this anomalous result can be alleviated and a linear dose response curve is obtained at much lower Abeta concentrations than is typically observed. In addition, differences in Abeta batch to batch preparations are alleviated. It was previously reported that colostrinin (CLN), a proline-rich peptide derived from colostrum, reduces fibril content and protects neuroblastoma cells against Abeta peptide-induced toxicity. The newly developed ThT fluorescence protocol was used to quantify Abeta fibril content after treatment with CLN. We also demonstrate that CLN, can solubilize Abeta fibrils in a dose and time-dependent fashion.

Protective effect of colostrinin on neuroblastoma cell survival is due to reduced aggregation of β-amyloid

Colostrinin (CLN), a mixture of proline-rich polypeptides, has shown a stabilizing effect on cognitive function in Alzheimers patients measured by the Alzheimers disease Assessment Scale-cognitive (ADAS-cog) and in Instrumental Activities of Daily Living (ILDL) in recently conducted clinical trials. The aim of this study was to elucidate a possible mode of action of CLN in the treatment of Alzheimers disease. Here, we report that CLN prevents the aggregation of beta-amyloid peptide Abeta (1-40) in vitro. The impact of CLN on the fibril formation was monitored by optical and electron microscopy. The electron micrographs illustrate that, at 25 microM, Abeta (1-40) peptides formed fibrils after 24-48 h of incubation. The presence of 0.25 microM CLN completely abolished the fibril formation. Abeta (1-40) peptides grow into dense fibers when examined at the 20th day. In the presence of CLN, however, the fibrils are much shorter and less dense. Addition of CLN as late as the 17th day can still dissolves the preformed fibrils. These observations were compared to the effect of CLN on the neurotoxic activity of beta-amyloid peptides in the cell culture model (SHSY-5Y). The beta-amyloid peptides were pre-incubated with CLN at various times and used to treat SHSY-5Y neuroblastoma cells for up to 4 days. The cytotoxic effect was monitored by trypan blue exclusion. We demonstrated that 24-48 h treatment was the onset of toxicity of 10-50 microM of beta-amyloid peptides. Pre-incubation of 0.0025-0.25muM of CLN with 25 microM of beta-amyloid peptides leads to near-complete abolition of cytotoxicity. Low doses of CLN (2.5 nM) can attain cytotoxic protection levels similar to those of highest doses (0.25 microM). Thus, the time course for the appearance of beta-amyloid fibrils coincides with that for cytotoxicity, and that the reduction of fibrils of beta-amyloid peptides by CLN is concomitant with the reduction of the cytotoxic effects of beta-amyloid on SHSY-5Y neuroblastoma cells. Our studies suggest that the neuroprotective effects exerted by CLN are related to the reduction of beta-amyloid fibrils.

Chemical synthesis of sulfated oligosaccharides with a β-d-Gal-(1→3)-[β-d-Gal-(1→4)-(α-l-Fuc-(1→3)-β-d-GlcNAc-(1→6)]-α-d-GalNAc sequence

Abstract The syntheses of two sulfated pentasaccharides: β- d -Gal6SO 3 Na-(1→3)-[β- d -Gal-(1→4)-α- l -Fuc-(1→3)-β- d -GlcNAc-(1→6)]-α- d -GalNAc→OMe ( 1 ) and β- d -Gal6SO 3 Na-(1→3)-[β- d -Gal-(1→4)-α- l -Fuc-(1→3)-β- d -GlcNAc6SO 3 Na-(1→6)]-α- d -GalNAc→OMe ( 2 ) by using Lewis X trisaccharides 12 and 16 as glycosyl donors are described. Sulfated oligosaccharides 1 – 2 and intermediate compounds are fully characterized by 2D 1 H– 1 H DQF-COSY and 2D ROESY experiments.

Crystal and molecular structure of methyl O-α-d-mannopyranosyl-(1→2)-α-d-mannopyranoside

Abstract The crystal and molecular structure of a synthetic mannosyl disaccharide, methyl O-α- d -mannopyranosyl-(1→2)-α- d -mannopyranoside, has been determined from X-ray diffractometer data by direct methods by use of the MULTAN programs. The crystals are monoclinic, space group P21 with unit cell dimensions, a 8.086(1), b 9.775(1), c 9.975(2) A, β 104.58(1)°, Z 2, and Dm 1.54 g/cm3. The structure was refined to an R-value of 0.033 for 1359 reflections measured with CuKα radiation. The mannopyranose units have the chair conformations 4C1( d ) with C-5′ and C-2′ deviating from the best plane through the other four atoms of the ring by −0.68 and +0.53 A in the nonreducing group, and C-3 and O-5 deviating from the mean plane through the other four atoms by +0.57 and −0.66 A, respectively, in the “potentially” reducing residue. The ring-to-ring conformation can be described as (φ,ψ) = (−64.5, 105.5°). The conformation across the C-5–C-6 bond is gauche-gauche in both the sugars. The crystal structure is stabilized by a network of intermolecular O-H O hydrogen bonds.

Study of Creatinine and its 5-Alkoxy Analogs: Structure and Conformational Studies in the Solid and Solution States by X-Ray Crystallography, NMR, UV and Mass Spectrometry

Abstract Creatinine (2-amino-1,5- dihydro-1-methyl-4-imidazolone) is a natural by-product of cellular metabolism related to muscular mass. It is excreted in human urine and is necessary for normal kidney function. Urinary secretion of creatinine serves as a bench mark for several clinical measurements. Recently, in our laboratories, during the course of an investigation of the urine of cancer patients for tumor markers, we found some new metabolites of creatinine. These were identified as 5-methoxy and 5-ethoxy creatinine by UV, NMR and Mass spectrometry and their tautomeric structures confirmed by crystal structural investigations of the metabolites. The x-ray crystallographic analysis confirmed the structures of the compound and showed that it exists in the 2-amino form. The spectral characteristics of these new compounds and the generality of the reaction are discussed in this paper. Creatinine, when allowed to react with methanol, ethanol and propanol respectively, in the presence of charcoal and air ...

Crystal structure and conformation of 5-fluorouridine: conformational preferences for 5-fluorinated pyranosides.

Crystals of 5‐fluorouridine (5FUrd) have unit cell dimensions a = 7.716(1), b = 5.861(2), c = 13.041(1)Å, α = γ = 90°, β = 96.70° (1), space group P21, Z = 2, ρobs = 1.56 gm/c.c and ρcalc = 1574 gm/c.c The crystal structure was determined with diffractometric data and refined to a final reliability index of 0.042 for the observed 2205 reflections (I ≥ 3σ). The nucleoside has the anti conformation [χ = 53.1(4)°] with the furanose ring in the favorite C2′–endo conformation. The conformation across the sugar exocyclic bond is g+, with values of 49.1(4) and − 69.3(4)° for Φθc and Φ∞ respectively. The pseudorotational amplitude τm is 34.5 (2) with a phase angle of 171.6(4)°. The crystal structure is stabilized by a network of N–H…O and O–H…O involving the N3 of the uracil base and the sugar O3′ and O2′ as donors and the O2 and O4 of the uracil base and O3′ oxygen as acceptors respectively. Fluorine is neither involved in the hydrogen bonding nor in the stacking interactions. Our studies of several 5‐fluorinated nucleosides show the following preferred conformational features: 1) the most favored anti conformation for the nucleoside [χ varies from − 20 to + 60°] 2) an inverse correlation between the glycosyl bond distance and the χ angle 3) a wide variation of conformations of the sugar ranging froni C2′–endo through C3′–endo to C4′–exo 4) the preferred g+ across the exocyclic C4′–C5′ bond and 5) no role for the fluorine atom in the hydrogen bonding or base stacking interactions.

A convergent synthesis of core 2 branched sialylated and sulfated oligosaccharides.

A convergent pathway for the syntheses of core 2 oligosaccharide analogues 1 and 2, and a natural form sialylated and sulfated hexasaccharide 3 was developed. Construction of pentasaccharides 24, 27 and hexasaccharide 28 was achieved by complete regioselective glycosylation of the 6-OH in the acceptors 5, 7 and 8, respectively, owing to the much higher reactivity of the primary hydroxyl group over the secondary axial hydroxyl group in these structures. Stereoselective sialylation was accomplished using donor 10 with defined configuration established through X-ray crystallographic analysis. Target oligosaccharides 1-3 were then obtained by the systematic deprotection of intermediates 24, 27 and 29. With these target oligosaccharides 1-3 obtained, biological evaluations of these molecules as enzyme substrates was undertaken and selectin binding studies are planned.

Structure and Conformation of 1-β-D-Ribofuranosylpyridin-4-One-3- Carboxamide, A Novel Nucleoside from Human Urine with a Rare Ribose Pucker

Abstract The pyridine nucleoside, 1-β-d-ribofuranosylpyridin-4-one-3- carboxamide (PCR) is one of several novel nucleosides isolated in our laboratory from the urine of chronic myelogenous leukemia patients. Its crystal structure and conformation were studied to complete its characterization. This nucleoside exhibits an anti (XCN = 66.9°) conformation across the glycosidic bond, a rare pucker of the ribose ring, C(4′)exo-C(3′)endo (4T3), and g+ across the C(4′)-C(5′) bond. The amino group of the carboxamide is proximal to and hydrogen bonded intramolecularly to 0(4). Nuclear magnetic resonance studies show that the intramolecular hydrogen bond is present in the solution state also, but the solution conformation of the furanose ring is not the same as that observed in the solid.

Crystal structure of methyl-3, 4-O-ethylidene-β-L- arabinopyranoside

The acid-catalyzed ethylidenation of several methyl pentopyranosides has been studied and syntheses of many cyclic alkyl orthoacetates from pentopyranosides have been reported. In a study of the scope and limitation of the reduction of sugar epoxides with diborane/sodium borohydride, methyl-4-O-acetyl-2,3-anhydro-β-L-lyxopyranoside was reduced, yielding two products: methyl-2,3-anhydro-β-L-lyxopyranoside and methyl-3,4-O-ethylidene-β-L-arabinopyranoside. The latter product was characterized by X-ray diffraction and its structure indicates that the epoxide was not reduced as expected; rather, the epoxide was opened by the hydroxyl group of the reduced acetyl. Crystals of the title compound are monoclinic, space group P21 with the following cell dimensions: a = 5.818(l) Å, b = 10.842(2) Å, c =7.251(1) Å, β = 91.47(1)°, Z = 2. Complete three-dimensional data (2θmax = 150° for Cu Kα) were collected on a CAD-4 diffractometer by the ω−2θ scan method. The structure was solved by the application of direct methods and refined by full-matrix least-square methods to a final reliability index of 0.04 for the observed 1031 reflections (I ≥ 3σ). The ethylidene ring has a twist conformation with a torsion angle of −42.8° about the C4–O bond. The pyranose ring is in a flattened chair conformation with the C2–C3–C4–C5 torsion angle of 33.5°. Of the acetal bond lengths, the C5–O5 length, (1.423 Å) is shorter than the values found in other methyl pyranosides. O2–H is involved in a bifurcated hydrogen bond intramolecularly to O1 and intermolecularly to O4.

Crystal and molecular structure of glutaconic acid

AbstractCrystals of Glutaconic acid, C5O4H6, are triclinic, space group P