Synthesis and Biological Evaluation of 4-Carbamoyl-2-β-D-Ribofuranosyl-Pyridine

Abstract

D-Allo/D-altro 2-(2,4:3, 5-di-O-benzylidenepentitol-1-y1)-4-(4,4-dimethyloxazolin-2-y1)pyridine was synthesized from 2-lithio-4-(4,4-dimethyloxazolin-2-y1)pyridine and 2, 4:3,5-di-O-benzylidenealdehydo-D-ribose. After mesylation and subsequent treatment of the adduct with CF₃COOH/H₂O and then ammonia, 4-carbamoyl-2-D-ribofuranosylpyridine was formed. The α- and β-anomers were separated by semipreparative hplc on a LICHROSORB 10 DIOL column. The β-anomer had no antiviral activity, but it had modest cytostatic activity against tumor cells.     

Purification of Human α2-Antiplasmin with Chicken IgY Specific to Its Carboxy-Terminal Peptide

ABSTRACT

α₂-antiplasmin, a plasma glycoprotein of the serpin superfamily, is the primary physiological inhibitor of plasmin, the key enzyme in fibrin degradation. Previous purification methods utilize lengthy multistep protocols with low yields or use monoclonal antibodies that are expensive or difficult to make. With a relatively small investment, a chicken was immunized with keyhole limpet hemocyanin-conjugated to α₂-antiplasmin C-terminal 26 residue synthetic peptide and the peptide-specific antibody (IgY) was isolated from the egg yolks of hens using the peptide affinity column. Based on the interaction between this IgY and α₂-antiplasmin, pure α₂-antiplasmin was isolated from human plasma in two steps: (a) citrated plasma was precipitated with 15% PEG-8000 to remove the bulk of plasma proteins while retaining the majority of α₂-antiplasmin activity; and (b) the α₂-antiplasmin was affinity-purified from the supernatant using the IgY column. Yields were typically 48% and the purity and authenticity of the α₂-antiplasmin were verified by gel electrophoresis, Western Blot analysis, N-terminal sequence, and amino acid analysis.     

Separation and biological properties of Phaseolus vulgaris isolectins

Two isolectins (L₄E₀-PHA and L₀E₄-PHA) from red kidney beans (Phaseolus vulgaris) were isolated by affinity chromatography on immobilized thyroglobulin and by chromatography on hydroxyapatite. The L₄E₀-PHA siolectin was not retarded on the affinity column and was eluted from the hydroxyapatite column with a 0.1 M Phosphate buffer. This isolectin had no erythroagglutinating activity but had high lymphoagglutinating and lymphocyte stimulating activities. The L₀E₄-PHA isolectin was adsorbed on the affinity column and was eluted from the hydroxyapatite column with a 0.25 M Phosphate buffer. This isolectin has a high erythroagglutinating activity, a very low lymphoagglutinating activity and no lymphocyte stimulating activity. These two isolectins are shown however to be closely related with respect to their oligomeric structure and reactivity towards anti-PHA antibodies. The lack of mitogenic activity of the L₀E₄-PHA isolectin suggests that in the other isolectins, the E monomer is not responsible for their mitogenic activity and that the membrane glycoproteins, which contain the E monomer — specific oligosaccharide, are not involved in the process inducing mitosis.     

Synthesis and carbonic anhydrase inhibitory properties of novel coumarin derivatives

A newly series of water-soluble 1-alkyl-3-(4-methyl-7, 8-dihydroxy-2H-chromen-2-one) benzimidazolium chloride salts (3a-j) were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase (hCA) I and II were evaluated. hCA I and II from human erythrocytes were purified by a simple one step procedure by using Sepharose 4B-L-tyrosine-sulphanilamide affinity column. The result showed that all the synthesized compounds were inhibited the CA isoenzymes activity. Among them, 3g and 3j were found to be most active (IC₅₀ = 22.09 µM and 20.33 µM) for hCA I and hCA II, respectively.     

白花鬼针草中多烯炔类成分的分离及其生物活性研究

为了解白花鬼针草(Bidens pilosa var.radiata)的化学成分,采用多种色谱技术从其提取物中分离多烯炔类成分,并对其生物活性进行研究.结果表明,从白花鬼针草乙酸乙酯提取部位中分离鉴定出4个多烯炔类化合物,分别为5-acetoxy-2-phenyl-ethinyl-thiophene(1)、1-phen...     

Oligomycins A and C,major secondary metabolites isolated from the newly isolated strain <Emphasis Type="Italic">Streptomyces diastaticus</Emphasis>

During the screening program for fungicides, one actinomycete strain ECO 00047 was isolated with the potential activity against fungus. According to the morphology and analysis of the nucleotide sequence of the 16S rRNA gene (1500 bp) this isolate was identified as Streptomyces diastaticus. The active compounds were separated by silica gel column chromatography, Sephadex LH-20 gel filtration and then purified by flash chromatography on C₁₈ (20–45 μm). The chemical structure of the bioactive compounds I and II were elucidated, based on the spectroscopic data of MS, IR, UV, ¹H-NMR, ¹³C-NMR and X-ray single crystal diffraction analysis. Compounds I and II were identical with oligomycins A and C, the macrolide antibiotics which have been known to be produced by Streptomyces diastatochromogenes, S. libani and S. avermitilis. The two compounds exhibited a strong activity against Aspergillus niger, Alternaria alternata, Botrytis cinerea and Phytophthora capsici but no activity toward bacteria. Although the two above antibiotics were known, their isolation has so far not been reported from S. diastaticus.     

Antifouling diketopiperazines produced by a deep-sea bacterium,Streptomyces fungicidicus

Abstract

Modern antifouling coatings use heavy metals and toxic organic molecules to prevent biofouling, the undesirable growth of marine organisms on man-made substrata. In an ongoing survey of deep-sea microorganisms aimed at finding low toxic antifouling metabolites, an actinomycete bacterium was isolated from the Pacific sediment at the depth of about 5000 m. The bacterium was closely related to Streptomyces fungicidicus (99% similarity) according to 16S ribosomal RNA sequence information. The spent culture medium of this bacterium inhibited barnacle larval attachment. Bioassay-guided fractionation was employed to isolate antifouling compounds. The ethyl acetate extract was fractionated by using an open silica gel column. Active fractions were further purified on a HPLC C₁₈ column. Five diketopiperazines, cyclo-(L-Leu-L-Pro), cyclo-(L-Phe-L-Pro), cyclo-(L-Val-L-Pro), cyclo-(L-Trp-L-Pro), and cyclo-(L-Leu-L-Val) were isolated for the first time from a deep sea bacterium, and the structures of the compounds were elucidated by nuclear magnetic resonance spectroscopy and mass spectrometry. The pure diketopiperazines were tested for antilarval activity using the barnacle Balanus amphitrite. Effective concentrations that inhibited 50% larval attachment (EC₅₀) after 24 h ranged from 0.10 – 0.27 mM. The data suggest that diketopiperazines and other compounds from deep-sea bacteria may be used as novel antifoulants.     

Use of Receptor Affinity Chromatography in Purification of the Growth Hormone-Like Factor Produced by Plerocercoids of the Tapeworm Spirometra Mansonoides

Abstract

The plerocercoid stage of the tapeworm Spirometra mansonoides produces a functional analog of human growth hormone (hGH). Among the similarities between plerocercoid growth factor (PGF) and hGH is competition for the same receptors on rabbit liver membranes. To take advantage of this characteristic in a purification scheme for PGF, rabbit liver microsomes were solubilized in Triton X-100 and the hGH receptors were purified over an hGH affinity column. The purified receptors from six rabbit livers were coupled to Affi-Gel-10 to create a receptor affinity column which was used to purify PGF. Chromatography of crude PGF over the receptor column resulted in a 1044 fold increase in specific activity. SDS-PAGE in the presence of 2-mercaptoethanol showed that the affinity-purified PGF contained three protein bands with apparent M_rs of 27.5 K, 22 K, and 16.7 K. Injections of the partially-purified PGF into hypophysectomized rats produced a dose-dependent growth response and 400 ng eq of PGF each day for 10 days stimulated a growth response not significantly different from that produced by 250 μg of bovine GH each day. Receptor affinity chromatography was an effective method to purify small amounts of PGF in a single step with negligible loss of biological activity.     

Purification and characterization of an extracellular β-xylosidase from a newly isolated Fusarium verticillioides

An extracellular β-xylosidase from a newly isolated Fusarium verticillioides (NRRL 26518) was purified to homogeneity from the culture supernatant by concentration by ultrafiltration using a 10,000 cut-off membrane, ammonium sulfate precipitation, DEAE Bio-Gel A agarose column chromatography and SP-Sephadex C-50 column chromatography. The purified β-xylosidase (specific activity, 57 U/mg protein) had a molecular weight (mol. wt.) of 94,500 and an isoelectric point at pH 7.8. The optimum temperature and pH for action of the enzyme were 65°C and 4.5, respectively. It hydrolyzes xylobiose and higher xylooligosaccharides but is inactive against xylan. The purified β-xylosidase had a K _m value of 0.85 mM (p-nitrophenol-β-D-xyloside, pH 4.5, 50°C) and was competitively inhibited by xylose with a K _i value of 6 mM. It did not require any metal ion for activity and stability. Journal of Industrial Microbiology & Biotechnology (2001) 27, 241–245. Received 20 May 2001/ Accepted in revised form 06 July 2001     

A Rapid,High-Yield Method of Producing M 0-[12]a14 Iodoinsujlin

Abstract

We have developed a rapid method for producing homogeneous mono-[¹²]A₁₄ iodoinsulin with high specific activity and yield. After iodination by the lactoperoxidase method, the labeled peptides were applied to a C₁₈ Porasil^R prs-column, washed with aqueous buffer to eliminate the free [¹²]-iodide and placed “in-line” with a C-18 HPLC column; mono-[12]A₁₄ iodoinsulin was then eluted isocratically with 29% aceto-nitrile in 16 minutes. The labeled hormone was extremely stable, and proved suitable for various biological studies.     

Characterization of Substances that Restore Impaired Cell Division of UV-Irradiated E. coli B

Substances which restore impaired cell division in UV-irradiated E. coli B were surveyed among various bacteria. The active substance was found only in several genera of Gram-negative bacteria, i.e., Escherichia, Enterobacter, Salmonella and some species of Pseudomonas. The activity in the dialyzed cell extract of E. coli B/r was observed in the presence of β-NAD and was enhanced by Mg^{2 +} and Mn²⁺. The active substance was very labile, but the activity was protected by 1 mM dithiothreitol in the process of purification. The activity of a fraction recovered through DEAE-cellulose column chromatography was stimulated by the presence of membrane fraction. Upon treatment with lipid-degrading enzymes and proteases, the division-stimulating activity was lost or reduced. It appears that the inactivation by lipase and phospholipase A₂ was due to the formation of lysophospholipids and that a proteinous substance participated in the recovery of impaired cell division of UV-irradiated E. coli B.     

A Novel Phospholipase C Inhibitor,S-PLI Produced by Streptomyces Sp. Strain No. A-6288

Abstract

S-PLI, an inhibitor of phospholipase C (PLC) produced by Strepromyces sp. strain No. 6288, was purified from the culture filtrate by salting-out with solid ammonium sulfate, column chromatography on CM-cellulose and gel filtration on Sephadex G-75. The molecular weight of S-PLI was estimated to be 65,000 by SDS-polyacrylamide gel electrophoresis. The inhibitor was found to be a glycoprotein with a composition of 609 amino acids and 19 glucose residues having an isoelectric point at 7.8. S-PLI was stable from pH 3 to 10 at 37°C and up to 40° at pH 6.0. The inhibitory activity showed pH-and temperature-dependence with a maximum around pH 7.0 at 50°C. S-PLI inhibited phospholipase C in a competitive manner (K_i value; 9.5 × 10-⁶ mM), but did not inhibit S-Hemolysin, phospholipase A₂, phospholipase B, phospholipase D and phosphatases. S-PLI is the first reported example of a glycoproteinaceous inhibitor of microbial origin which is able to specifically inhibit phospholipase C.     

Zirconia-Cu(I) stabilized copper oxide mesoporous nano-catalyst: Synthesis and DNA reactivity of 1,2,4-oxadiazole-quinolinepeptidomimetics-based metal(II) complexes

Abstract

Contemporary research reveals an undemanding protocol for the catalytic synthesis of 1,2,4-oxadiazole-quinolinepeptide in the incidence of a cost-effective and reusable mesoporous ZrO₂-supported Cu₂O (Cu₂ZrO₃) catalyst. This paper depicts a unique system for peptide bond synthesis staying away from toxic solvents and reactants. The catalyst was reused for four cycles without noteworthy loss in the activity, and the catalyst was genuinely heterogeneous. The method followed a simple workup procedure, and no column chromatography was needed. Further, the synthesized 1,2,4-oxadiazole-quinolinepeptide ligand (L), and its complexes of type, [FeLCl₂] and [CuL]Cl₂ were synthesized and characterized by spectral and analytical techniques. An octahedral geometry has been projected for Fe(II) complexes, while the Cu(II) complex exhibits a square planar geometry. The binding properties of the complexes with CT-DNA were studied by absorption spectral analysis, followed by viscosity measurement and thermal denaturation studies. The photo-induced cleavage studies revealed that the complexes possess photonuclease activity against pUC19 DNA under UV–visible irradiation.     

Antioxidant and α-Amylase Inhibitory Compounds from Aerial Parts of Varthemia iphionoides Boiss

Various extracts of aerial parts of Varthemia (Varthemia iphionoides Boiss) were investigated for radical-scavenging activity, antioxidative activity, and porcine pancreas α-amylase inhibitory activity. The ethanol and water extracts showed a pronounced 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, with inhibition of about 90% at a concentration of 100 μg/ml, and α-amylase inhibitory activity of about 70% at a concentration of 200 μg/ml by the 2-chloro-4-nitrophenyl α-maltotrioside (CNP-G₃) degradation method. The ethanol extract was purified by column chromatography to give seven 3-methoxyflavones (1–7) and eudesmane sesquiterpene, selina-4,11(13)-dien-3-on-12-oic acid (8). The structures of these compounds were established by NMR, MS, and UV spectroscopy. Of 3-methoxyflavones, 5,7,4′-trihydroxy-3,6-dimethoxyflavone (1), 5,7,4′-trihydroxy-3,3′-dimethoxyflavone (2), and 5,4′-dihydroxy-3,7,3′-trimethoxyflavone (3,7,3′-tri-O-methyl-quercetin) (7) exhibited pronounced radical-scavenging activity. The antioxidative activity in the linoleic acid system was considerable in compounds 1, 2, and 5,4′-dihydroxy-3,6,7-trimethoxyflavone (4). Compounds 1, 2, 4, 5 (5,7,4′-trihydroxy-3-methoxyflavone), and 6 (5,4′-dihydroxy-3,7-dimethoxyflavone) showed markedly high inhibitory activity against porcine pancreas α-amylase. Eudesmane sesquiterpene did not show any activity.     

Extracellular Lytic Enzymes of Myxococcus virescens II. Purification of Three Bacteriolytic Enzymes and Determination of their Molecular Weights and Isoelectric Points

The bacteriolytic enzymes produced by Myxococcus virescens and previously concentrated and separated from most of the non-bacteriolytic proteins have been further separated and purified. The bacteriolytic enzyme solution was concentrated by lyo-philization. When applied to a Sephadex G-100 column, three peaks of bacteriolytic activity were eluted. Polyacrylamide gel electrophoresis showed that all the three enzyme fractions were contaminated with at least four non-bacteriolytic proteins. In the first enzyme fraction the bacteriolytic enzymes could be freed from the contaminating proteolytic activity by adsorption on a hydroxylapatite column. The bacteriolytic enzymes could then be adsorbed on a CM-cellulose column. The remaining contaminating proteins passed the column un-adsorbed while the bacteriolytic enzymes could be eluted with a gradient of 0.02–0.10 M ammonium hydrogen carbonate solution. The second enzyme fraction was adsorbed on a CM-cellulose column and then eluted with 0.03–0.15 M NH4 HCO₃. After rechromatography on a new column under the same conditions, all of the contaminating proteins had disappeared. For purification of the third enzyme fraction chro-matography on one single CM-cellulose column was sufficient. The elution of the adsorbed enzymes was performed with a gradient of 0.15–0.30 M NH₄HCO₃. The recovery of activity for each of the ion-exchange chromatography separations was at least 90%. The purity of the enzymes was tested by polyacrylamid gel electrophoresis. Each of the purified enzymes gave only one coloured band which coincided with the enzyme activity assayed in sliced gels. The molecular weights of the enzymes were determined by electrophoresis on acryl-amide gels containing sodiumdodecylsulphate. The molecular weights determined in this way (about 40,000, 30,000 and 20,000, respectively) were about 10,000 daltons higher than those obtained by gel chromatography on Sephadex G-100. This discrepancy seems to depend on interactions between the enzymes and the dextran molecules probably caused by the strongly basic nature of the enzymes or by formation of enzyme-substrate complexes.     

Interaction between Wheat-Germ Agglutinin and acid phosphatase present in dry de-embryonated wheat grains

Abstract

Different hydrolases were found to be present in the crude extract of dry de-embryonated wheat grains, including acid phosphatase. When the crude extract was chromatographed on a Wheat-Germ Agglutinin-Sepharose affinity column, an aliquot of acid phosphatase bound to the column and could be eluted specifically by N-acetyl-D-glucosamine solution. Some properties of the WGA-binding acid phosphatase (pH optimum, heat stability, Triton X-100 sensitivity, inhibition by some ions, molecular weight and K_m) were studied. WGA appeared to have a stabilizing effect on the enzyme, while it was ineffective on the K_m.     

Bleomycin A2 and B2 Purification by Flash Chromatography for Chemical and Biochemical Studies

Abstract

The clinically used formulation of the anticancer antibiotic, Blenoxane, is a mixture of bleomycin congeners. A new approach to separating the major A₂ and B₂ congeners has been developed utilizing the flash chromatography technique. A 5–6-inch column of fine mesh silica gel with a solvent system of 1% ammonium formate:methanol (2:3) was used. Low air pressure was applied to the column to increase the flow rate such that separation was complete in ～20 min. Reverse phase size exclusion gravity chromatography with Sephadex G-15 column bedding was an effective, rapid procedure for removal of the 1% ammonium formate, the lowest percentage practical for separating the bleomycins. This separation approach does not damage the antibiotics, as demonstrated by NMR spectroscopy, thin layer chromatography, and DNA cleaving activity. Although not as useful for detection of trace amounts of the drug in biological systems as some of the known HPLC methods, this method is excellent for separating large quantities of the drug (8–32 mg) in order to obtain congeners pure enough for synthetic, biochemical, and biophysical studies.     

A breakthrough column study for removal of malachite green using coco-peat

Abstract

A continuous adsorption study in a fixed bed column using coco-peat (CP) as an adsorbent was carried out for the removal of toxic malachite green (MG) from contaminated water. Fixed bed column studies were carried out to check field application viability. Various parameters like particle size, pH, concentration, dose and interference were exercised to optimize dye removal. Data obtained from breakthrough column studies were evaluated using Thomas and BDST model. Thomas rate constants K_t (0.22?ml min^?1 mg^?1) and adsorption capacity q_o (181.04?mg g^?1) were estimated and found to favor efficiency of CP. Thomas model was tested with several parameters like flow rate, concentration, and bed depth. Upon increase in input dye concentration, flow rate and bed height, adsorption coefficients increased. According to BDST model, maximum dye uptake of 468.26?mg/l was obtained with an input dye concentration of 5?mg/l. HYBRID and MPSD error functions were tested and found that Thomas model fits best. Dilute hydrochloric acid was found best for desorption. Real wastewater from textile industry was analyzed and confirmed the prospect of large-scale industrial application. In conclusion, coco-peat can be used as a promising bio-sorbent in column bed for scavenging of MG from contaminated water.     

Studies on Proteolytic Enzyme of the Cricket,Gryllulus taiwanemma Ohmachi

A protease was isolated from the alimentary canal of crickets. This cricket protease was purified by ammonium sulfate, rivanol and acetone fractionations, and DEAE-cellulose and CM-cellulose (Ca-form) column chromatographies. The optimum temperature was 50°C and the optimum pH was 8.0. For preservation, the enzyme was most stable at pH 3.0. Aluminum had the best stabilizing action with no drops of enzyme activity after 24 hours of dialysis. The cricket protease was specific for the synthetic substrate, α-benzoyl-l-arginine amide. Cricket protease had a K_m. 10³ of 2.8 which is similar to that of trypsin, 1.2. The Ea was 3,770 while that of trypsin was 14,960 using α-benzoyl-l-arginine amide as the substrate. Although cricket protease has the same substrate specificity and similar optimum pH and pH-stability as trypsin, it differed in metal requirements to obtain activity. Certain metals are essential for cricket protease activity.