Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a

We have synthesized several ganglio-oligosaccharide structures using glycosyltransferases from Campylobacter jejuni. The enzymes, alpha-(2-->3/8)-sialyltransferase (Cst-II), beta-(1-->4)-N-acetylgalactosaminyltransferase (CgtA), and beta-(1-->3)-galactosyltransferase (CgtB), were produced in large-scale fermentation from Escherichia coli and further characterized based on their acceptor specificities. 2-Azidoethyl-glycosides corresponding to the oligosaccharides of GD3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GM2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GD2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), and GM1 (beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) were synthesized in high yields (gram-scale). In addition, a mammalian alpha-(2-->3)-sialyltransferase (ST3Gal I) was used to sialylate GM1 and generate GD1a (alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) oligosaccharide. We also cloned and expressed a rat UDP-N-acetylglucosamine-4'epimerase (GalNAcE) in E. coli AD202 cells for cost saving in situ conversion of less expensive UDP-GlcNAc to UDP-GalNAc.     

4-Substituted boro-proline dipeptides: Synthesis, characterization, and dipeptidyl peptidase IV, 8, and 9 activities

The boroProline-based dipeptidyl boronic acids were among the first DPP-IV inhibitors identified, and remain the most potent known. We introduced various substitutions at the 4-position of the boroProline ring regioselectively and stereoselectively, and incorporated these aminoboronic acids into a series of 4-substituted boroPro-based dipeptides. Among these dipeptidyl boronic acids, Arg-(4S)-boroHyp (4q) was the most potent inhibitor of DPP-IV, DPP8 and DPP9, while (4S)-Hyp-(4R)-boroHyp (4o) exhibited the most selectivity for DPP-IV over DPP8 and DPP9.     

Synthesis and characterization of 6-O-beta-lactosyl-alpha,beta-lactoses, 1-O-(6-O-beta-lactosyl-beta-lactosyl)-(R,S)-glycerols, and 4,6-di-O-beta-D-galactopyranosyl-alpha,beta-D-glucoses.

1,2,3,2',3',4',6'-Hepta-O-acetyl-beta-lactose (4) was coupled with 2,3,6,2',3',4',6'-hepta-O-acetyl-alpha-lactosyl bromide (7) in the presence of Hg(CN)2 to afford 1,2,3,2',3',4',6'-hepta-O-acetyl-6-O-(2,3,6,2',3',4',6'-hepta-O-acetyl-b eta- lactosyl)-beta-lactose (11) which, upon O-deacetylation, gave 6-O-beta-lactosyl-alpha,beta-lactoses (64% from 4). In contrast, the reaction of 7 with benzyl 2,3,2',3',4',6'-hexa-O-acetyl-beta-lactoside in the presence of Hg(CN)2 produced 3,6,2',3',4',6'-hexa-O-acetyl-1,2-O- (2,3,2',3',4',6'-hexa-O-acetyl-1-O-benzyl-beta-lactos-6-yl orthoacetyl)-alpha-lactose (63%) and 3,6,2',3',4',6'-hexa-O-acetyl-1,2-O-(1- cyanoethylidene)-alpha-lactose (27%). The glycosidation of 4 using 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide in the presence of Hg(CN)2 afforded, after deprotection, 4,6-di-O-beta-D-galactopyranosyl-alpha,beta-D-glucoses (66%). The reaction of 11 with 1,2-di-O-benzyl-(R,S)-glycerols and trimethylsilyl trifluoromethanesulfonate yielded, after deprotection, 1-O-(6-O-beta-lactosyl-beta-lactosyl)-(R,S)-glycerols (18%). Under the same coupling conditions 11 reacted with 2-O-benzylglycerol to form 3-O-acetyl-2-O-benzyl-1-O-[2',3',4',6'-hexa-O-acetyl-6-O-(2,3,6,2',3',4' ,6'- hepta-O-acetyl-beta-lactosyl)-beta-lactosyl]-(R,S)-glycerols (16%).     

Synthesis,Chemical Characterization,DNA Binding,Antioxidant, Antibacterial,and Antifungal Activities of Ferrocence Incorporated Selenoureas

Ferrocene‐incorporated selenoureas 1‐(4‐methoxybenzoyl)‐3‐(4‐ferrocenylphenyl)selenourea (P4Me), 1‐(3‐methoxybenzoyl)‐3‐(4‐ferrocenylphenyl)selenourea (P3Me), and 1‐(2‐methoxybenzoyl)‐3‐(4‐ferrocenylphenyl)selenourea (P2Me) were synthesized and characterized by nuclear magnetic resonance, Fourier transform infrared spectroscopy, atomic absorption spectroscopy, CHNS, and single‐crystal X‐ray diffraction. DNA interaction of the compounds was investigated with cyclic voltammetry, UV–visible spectroscopy, and viscometry, which is a prerequisite for anticancer agents. Drug‐DNA binding constant was found to vary in the sequence: K_P4Me (4.9000 × 10⁴ M^?1) > K_P2Me (2.318 × 10⁴ M^?1) > K_P3Me (1.296 × 10⁴ M^?1). Antioxidant (1,1‐diphenyl‐2‐picrylhydrazyl), antifungal (against Faussarium solani and Helmentosporium sativum), and antibacterial (against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis) activities have also been reported in addition.     

Synthesis of spacer-equipped di-, tri-, and the tetrasaccharide fragments of the deacetylated O-PS1 of Citrobacter gillenii O9a,9b, and a related pentasaccharide

The title rhamnooligosaccharides [alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe, alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe, alpha-D-Rhap4NAc-(1-->2)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe, and alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->2)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe] were synthesized in a stepwise fashion from 5-methoxycarbonylpentyl 4-azido-4,6-dideoxy-2-O-benzyl-alpha-D-mannopyranoside and orthogonally protected 1-thioglycoside glycosyl donors. The amorphous, final products were fully characterized as corresponding per-O-acetyl derivatives.     

Rat liver ATP-sulfurylase: purification, kinetic characterization, and interaction with arsenate, selenate, phosphate, and other inorganic oxyanions

ATP-sulfurylase (ATP:sulfate adenylyltransferase; EC 2.7.7.4), the first enzyme of the two-step sulfate activation sequence, was purified extensively from rat liver cytosol. The enzyme has a native molecular mass of 122 +/- 12 kDa and appears to be composed of identical 62 +/- 6-kDa subunits. At 30 degrees C and pH 8.0 (50 mM Tris-Cl buffer containing 5 mM excess Mg2+), the best preparations have "forward reaction" specific activities of about 20 and 2 units X mg protein-1 with MoO4(2-) and SO4(2-), respectively. The reverse (ATP synthesis) specific activity is about the same as the forward molybdolysis activity. The kinetic constants under the above conditions are as follows: KmA = 0.21 mM, Kia = 0.87 mM, KmB = 0.18 mM, KmQ = 0.65 microM, Kiq = 0.11 microM, and KmP = 5.0 microM where A = MgATP, B = SO4(2-), Q = APS, and P = total PPi at 5 mM Mg2+. PPi is a mixed-type inhibitor with respect to MgATP and SO4(2-). SeO4(2-) is an alternative inorganic substrate with a Vmax about 20% that of SO4(2-). The product, APSe, is unstable. But in the presence of a sufficient excess of APS kinase, APSe is completely converted to PAPSe. The rate constant for nonenzymatic PAPSe hydrolysis was determined from measurements of the final steady-state reaction rate in the presence of limiting initial SeO4(2-) and a large excess of MgATP, ATP sulfurylase, APS kinase, and the other coupling enzymes and their cosubstrates. The results yielded a k of 2.4 +/- 0.5 X 10(-3) sec-1 (t1/2 ca. 5 min). Phosphate is an effective buffer for enzyme purification and storage but inhibits catalytic activity, particularly at low substrate concentrations. In the presence of buffer levels of Pi, the MgATP reciprocal plot of the SO4(2-)-dependent reaction is concave-up. Inorganic monovalent oxyanions are dead end inhibitors competitive with SO4(2-) and apparently uncompetitive with respect to MgATP. The relative potencies are in the order ClO3- greater than ClO4- greater than FSO3- greater than NO3-. Thiosulfate is also competitive with SO4(2-) but noncompetitive with respect to MgATP. Several divalent oxyanions (MoO4(2-), WO4(2-), CrO4(2-), and HAsO4(2-] promote the enzyme-catalyzed cleavage of MgATP to AMP and MgPPi. The ratio Vmaxf/KmA ranged from 0.7 to 200 for various reactive inorganic substrates. The cumulative results suggest the random binding of MgATP and the inorganic substrate but the ordered release of MgPPi before APS.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression, purification and characterization of the sulfite reductase hemo-subunit, SiR-HP, from Acidithiobacillus ferrooxidans

Sulfite reductase (SiR) is a large and soluble enzyme which catalyzes the transfer of six electrons from NADPH to sulfite to produce sulfide. The sulfite reductase flavoprotein (SiR-FP) contains both FAD and FMN, and the sulfite reductase hemoprotein (SiR-HP) contains an iron-sulfur cluster coupled to a siroheme. The enzyme is arranged so that the redox cofactors in the FAD-FMN-Fe(4)S(4)-Heme sequence make an electron pathway between NADPH and sulfite. Here we report the cloning, expression, and characterization of the SiR-HP of the sulfite reductase from Acidithiobacillus ferrooxidans. The purified SiR-HP contained a [Fe(4)S(4)] cluster. Site-directed mutagenesis results revealed that Cys427, Cys433, Cys472 and Cys476 were in ligating with the [Fe(4)S(4)] cluster of the protein.     

Ruthenium(II) polypyridyl complexes: synthesis and studies of DNA binding, photocleavage, cytotoxicity, apoptosis, cellular uptake, and antioxidant activity

Two ruthenium (II) complexes [Ru(dmb)2(APIP)](ClO4)2 (APIP=2-(2-aminophenyl)imidazo[4,5-f?][1,10]phenanthroline, dmb=4,4'-dimethyl-2,2'-bipyridine; 1) and [Ru(dmb)2(HAPIP)](ClO4)2 (HAPIP=2-(2-hydroxyl-4-aminophenyl)imidazo[4,5-f?][1,10]phenanthroline; 2) were synthesized and characterized. DNA binding was investigated by electronic absorption titration, luminescence spectra, thermal denaturation, viscosity measurements, and photocleavage. The DNA binding constants for complexes 1 and 2 were 4.20 (±0.14)×10(4) and 5.45 (±0.15)×10(4) M(-1). The results suggest that these complexes partially intercalate between the base pairs. The cytotoxicity of complexes 1 and 2 was evaluated by MTT assay. Cellular uptake was observed under fluorescence microscopy; complexes 1 and 2 can enter into the cytoplasm and accumulate in the nuclei. Apoptosis and the antioxidant activity against hydroxyl radicals (?OH) were also explored.     

Total synthesis of (+)-macrosphelides A,C, E,F, and G based on enzymatic function

The total synthesis of (+)-macrosphelide A (1) (18.5% overall yield in 11 steps), (+)-macrosphelide C (2) (25% overall yield in 9 steps), (+)-macrosphelide E (3) (23.9% overall yield in 11 steps), (+)-macrosphelide F (4) (20% overall yield in 9 steps), and (+)-macrosphelide G (5) (22% overall yield in 9 steps) was achieved from a chemoenzymatic reaction product (4R,5S)-4-benzyloxy-5-hydroxy-2(E)-hexenoate 10.     

Facile, alternative route to lubeluzole, its enantiomer, and the racemate

Lubeluzole [(S)-9] has been synthesized by a convergent synthesis, alkylation of N-methyl-N-piperidin-4-yl-1,3-benzothiazol-2-amine (4) with (+)-(R)-1-chloro-3-(3,4-difluorophenoxy)propan-2-ol [(+)-(R)-8] being the key step. Alcohol (+)-(R)-8 was obtained from commercially available (R)-epichlorohydrin [(R)-6], while the thiazole derivative 4 was easily obtained starting from N-protected piperidin-4-one (1) in a three-step procedure. The same method was used in order to obtain both the (R)-stereoisomer of lubeluzole [(R)-9] and its racemate [(RS)-9]. Overall yields ranged from 20% to 35%. The enantiomeric excess values for (S)-9 and (R)-9 were 97% and 94% respectively, as analyzed by chiral HPLC.     

Bacteriochlorophyll c monomers,dimers, and higher aggregates in dichloromethane,chloroform, and carbon tetrachloride

Bacteriochlorophyll c in vivo is a mixture of at least 5 homologs, all of which form aggregates in CH₂Cl₂, CHCl₃ and CCl₄. Three homologs exist mainly in the 2-R-(1-hydroxyethyl) configuration, whereas the other two homologs, 4-isobutyl-5-ethyl and 4-isobutyl-5-methyl farnesyl bacteriochlorophyll c, exist mainly in the 2-S-(1-hydroxyethyl) configuration (Smith KM, Craig GW, Kehres LA and Pfennig N (1983) J. Chromatograph. 281: 209–223). In CCl₄ the S-homologs form an aggregate of 2–3 molecules whose absorption (747 nm maximum) and circular dichroism spectra resemble those of the chlorosome. In CH₂Cl₂, CHCl₃ and CCl₄ the 4-n-propyl homolog (R-configuration) forms dimers absorbing at ca. 680 nm and higher aggregates absorbing at 705–710 nm. In CCl₄ the dimerization constant is approx. 10 µM^–1 (1000 times that for chlorophyll a). The difference between the types of aggregates formed by the 4-n-propyl and 4-isobutyl homologs is attributed to the difference between the R- and S-configurations of the 2-(1-hydroxyethyl) groups in each chlorophyll.Abbreviations BChl bacteriochlorophyll - CD circular dichroism - Chl chlorophyll - DNS data not shown - EEF 4-ethyl-5-ethyl farnesyl - iBM/EF 4-isobutyl-5-methyl/ethyl farnesyl - MEF 4-methyl-5-ethyl farnesyl - PEP 4-n-propyl-5-ethyl farnesyl     

Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics

The yeast Saccharomyces cerevisiae possesses two genes that encode phosphatidylinositol (PtdIns) 4-kinases, STT4 and PIK1. Both gene products phosphorylate PtdIns at the D-4 position of the inositol ring to generate PtdIns(4)P, which plays an essential role in yeast viability because deletion of either STT4 or PIK1 is lethal. Furthermore, although both enzymes have the same biochemical activity, increased expression of either kinase cannot compensate for the loss of the other, suggesting that these kinases regulate distinct intracellular functions, each of which is required for yeast cell growth. By the construction of temperature-conditional single and double mutants, we have found that Stt4p activity is required for the maintenance of vacuole morphology, cell wall integrity, and actin cytoskeleton organization. In contrast, Pik1p is essential for normal secretion, Golgi and vacuole membrane dynamics, and endocytosis. Strikingly, pik1(ts) cells exhibit a rapid defect in secretion of Golgi-modified secretory pathway cargos, Hsp150p and invertase, whereas stt4(ts) cells exhibit no detectable secretory defects. Both single mutants reduce PtdIns(4)P by approximately 50%; however, stt4(ts)/pik1(ts) double mutant cells produce more than 10-fold less PtdIns(4)P as well as PtdIns(4,5)P(2). The aberrant Golgi morphology found in pik1(ts) mutants is strikingly similar to that found in cells lacking the function of Arf1p, a small GTPase that is known to regulate multiple membrane trafficking events throughout the cell. Consistent with this observation, arf1 mutants exhibit reduced PtdIns(4)P levels. In contrast, diminished levels of PtdIns(4)P observed in stt4(ts) cells at restrictive temperature result in a dramatic change in vacuole size compared with pik1(ts) cells and persistent actin delocalization. Based on these results, we propose that Stt4p and Pik1p act as the major, if not the only, PtdIns 4-kinases in yeast and produce distinct pools of PtdIns(4)P and PtdIns(4,5)P(2) that act on different intracellular membranes to recruit or activate as yet uncharacterized effector proteins.     

Biologically relevant tetraazamacrocyclic complexes of manganese: synthetic, spectral, antimicrobial, antifertility and antiinflammatory approach

The testicular sperm density, sperm morphology, sperm motility, density of cauda epididymis spermatozoa and fertility in mating trials and biochemical parameters of reproductive organs have been examined and discussed for [Mn(N(4)MacL(n))(NO(3))(2)] (where N(4)MacL(n) represents the tetraazamacrocyclic ligand molecule with n=1-4) type of complexes. An attempt also has been made to correlate the structural aspects of the compounds with their anti-inflammatory activity. The 14-18 membered tetraamide macrocyclic ligands N(4)L(1)-N(4)L(4) used during these investigations have been prepared by the condensation of 1,2-diaminoethane or 1,3-diaminopropane with malonic or succinic acid in the presence of condensing reagents, dicyclohexylcarbodiimide and 4-dimethylaminopyridine. On reduction, these macrocyclic ligands give a new series of tetraazamacrocyles [N(4)MacL(n)] and their complexes with manganese(II) nitrate. The ligands and their complexes were characterized by elemental analyses, molecular weight determinations, infrared, electronic, mass and X-ray structural analyses. An octahedral geometry for these complexes has been confirmed by spectral studies. On the basis of the chemical composition, the representation of the complexes as [Mn(N(4)MacL(n))(NO(3))(2)] has been established. The ligands and their complexes have been screened in vitro against a number of pathogenic fungi and bacteria to assess their growth inhibiting potential.     

Anti-estradiol-17beta single-chain Fv fragments: Generation, characterization, gene randomization, and optimized phage display

A single-chain Fv fragment (scFv) against estradiol-17beta (E(2)) was generated to begin the construction of a library of various mutated anti-steroid antibodies with an improved affinity and/or specificity. A hybridoma clone secreting a specific anti-E(2) antibody (Ab#E4-4) was established by the cell fusion using splenocytes from a mouse immunized with an immunogenic E(2)-carrier conjugate. DNA fragments encoding the variable heavy and light domains (V(H) and V(L)) of the Ab#E4-4 were cloned and combined to give the scFv gene fragment encoding the sequence 5'-V(H)-(GGGGS)(3)-V(L)-3'. Compared to the Ab#E4-4 Fab fragment, soluble scFv (scFv#E4-4) protein showed a similar affinity to E(2) (K(a)=8.6x10(7)M(-1)) and a similar cross-reaction profile. To further study the fundamentals for creating a comprehensive library of mutated scFvs, the scFvV(H) and V(L) genes were amplified using error-prone PCR conditions and the frequency and pattern of incorporated mutations were investigated. For this, regular Taq polymerase was used in the presence of unequal concentrations of dNTPs. At 1.0mM MnCl(2), the error frequency reached to 8.5% and 11% for the V(H) and V(L) respectively, although a significant transition/transversion bias was observed. ScFv#E4-4 and the mutated polyclonal scFvs were then displayed on filamentous phage under various packaging conditions. Cultivation of the transformed bacteria was more suitable at 25 degrees C than at higher temperatures for the packaging of scFv-bearing phagemid particles. Based on these experimental conditions, an scFv-displaying phage library, each scFv member in which has mutated complementarity-determining region (CDR) H2, H3, L1, and L3, was constructed. A soluble scFv clone (scFv#m1-e7) with a mutated amino acid (I-->V) in CDR L1, isolated from this library, showed threefold higher affinity (K(a)=2.6 x 10(8)M(-1)) than that of scFv#4-4.     

Glycosphingolipids in insects. Chemical structures of ceramide tetra-, penta-, hexa-, and heptasaccharides from Calliphora vicina pupae (Insecta: Diptera)

Four neutal fraction glycosphingolipids, designated components 4-7, were purified from the pupae of Calliphora vicina and isolated by the use of high performance liquid chromatography. Their chemical structures were determined to be: GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer and Gal(alpha 1-3)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; and GlcNAC(beta 1-3)Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer. By the use of specific exoglycosidases, it was possible to assign anomeric configurations to all the sugar residues present. Analysis of the ceramide moiety by electron-impact mass spectrometry revealed the dominant fatty acid and sphingoid to be arachidic acid (C20:0) and tetradecasphing-4-enine, respectively.     

Three-centre, two-electron bonds in complexes of Mn, Ni, Co and Cd with 3-(4-benzonitrile)pyrazolyl scorpionates

A series of complexes of the form [M(Bp^3(4Bz))(Tp^3(4Bz))] containing a mixture of novel dihydrobis(3-(4-benzonitrile)pyrazolyl)borato (Bp^3(4Bz)) and hydrotris(3-(4-benzonitrile)pyrazolyl)borato (Tp^3(4Bz)) ligands have been synthesised and structurally characterised. The ligands, both containing 3-(4-benzonitrile)pyrazole arms, form isostructural complexes with a variety of transition metals although different crystalline solvates have been obtained depending upon the crystallisation conditions, namely [M(Bp^3(4Bz))(Tp^3(4Bz))] (M = Ni, Co), [Ni((Bp^3(4Bz))(Tp^3(4Bz))]·2MeOH and [M(Bp^3(4Bz))(Tp^3(4Bz))]·MeOH (M = Mn, Cd). In all cases the metal atoms are five-coordinate with an additional agostic B-H interaction from the bis(pyrazolyl)borate ligand.     

Thymosin beta 4 and its N-terminal tetrapeptide, AcSDKP, inhibit proliferation, and induce dysplastic, non-apoptotic nuclei and degranulation of mast cells

Thymosin beta4 (Tbeta4), a 5 kDa polypeptide, is a member of the beta-thymosin family. It acts as the principal intracellular G-actin sequestering peptide and exhibits extracellular functions in angiogenesis and wound healing. The N-terminus of Tbeta4 contains a bioactive tetrapeptide, acSDKP, a negative regulator of hematopoietic stem-cell proliferation. Here, we show that both peptides inhibit mast-cell proliferation over the concentration range of 10(-6) to 10(-17) M with the maximum effect of both at 10(-14) M. Both Tbeta4 and acSDKP caused dysplastic mast-cell nuclei that were confirmed by DAPI fluorescent staining. Flow-cytometric analysis of ploidy revealed that the dysplastic nuclei were not multinucleated, but fragmented nuclei in G2 growth arrest. We could further demonstrate that 10(-8) or 10(-14) M Tbeta4 or acSDKP induce mast-cell degranulation. A concentration of 10(-8) M Tbeta4 or acSDKP caused 57 or 89% degranulation, respectively. A number of tryptic fragments of Tbeta4 were assayed beside intact Tbeta4 and the tetrapeptide, and found to be inactive.     

Preparation and calculated conformations of the 2'-, 3'-, 4'-, and 6'-deoxy, 3'-O-methyl, 4'-epi, and 4'- and 6'-deoxy-fluoro derivatives of methyl 4-O-alpha-D-galactopyranosyl-beta-D-galactopyranoside (methyl beta-D-galabioside)

The glycosyl chlorides of the 3-O-methyl (6) and 4-deoxy-4-fluoro (8) O-benzylated derivatives of D-galactopyranose and 2,3,4,6-tetra-O-benzyl-D-glucopyranose were condensed with methyl 2,3,6-tri-O-benzoyl-beta-D-galactopyranoside to give, after deprotection, the 3'-O-methyl (23), 4'-deoxy-4'-fluoro (25), and 4'-epi (27) derivatives, respectively, of methyl beta-D-galabioside (1). The glycosyl fluorides of 2,3,4-tri-O-benzyl-D-fucopyranose and the 3-deoxy (12) and 4-deoxy (16) O-benzylated derivatives of D-galactopyranose were condensed with methyl 2,3,6-tri-O-benzyl-beta-D-galactopyranoside (21), to give, after deprotection, the 6'-deoxy (31), 3'-deoxy (34), and 4'-deoxy (37) derivatives of 1, respectively. The 2'-deoxy (41) derivative of 1 was prepared by N-iodosuccinimide-induced condensation of 3,4,6-tri-O-acetyl-D-galactal and 21 followed by deprotection. Treatment of methyl 2,3,6-tri-O-benzoyl-4-O-(2,3-di-O-benzoyl-alpha-D-galactopyranosyl)-beta -D- galactopyranoside with Et2NSF3 (DAST), followed by deprotection, provided the 6'-deoxy-6'-fluoro (46) derivative of 1. Molecular mechanics calculations yielded conformations for 23, 25, 27, 31, 34, 37, 41, and 46 with small deviations from the calculated conformation for 1 (phi H/psi H: -40 degrees/-6 degrees).     

Effect of fibroblast growth factors 1, 2, 4, 5, 6, 7, 8, 9, and 10 on avian chondrocyte proliferation.

It has been demonstrated that fibroblast growth factor receptors are key regulators of endochondral bone growth. However, it has not been determined what fibroblast growth factor ligand(s) (FGFs) are important in this process. This study sought to determine whether FGFs 1, 2, 4, 5, 6, 7, 8, 9, and 10 were capable of stimulating avian chondrocyte proliferation in vitro. We have found that FGFs 2, 4, and 9 strongly stimulate avian chondrocyte proliferation while FGFs 6 and 8 stimulate proliferation to a lesser extent. RT-PCR indicates that FGF-2 and FGF-4 are expressed in the postnatal avian epiphyseal growth plate (EGP) while FGF-8 and FGF-9 are not. Thus, FGF-2 and FGF-4 stimulate chondrocyte proliferation and are both present in the EGP. This suggests that FGF-2 and FGF-4 may be important ligands, in vivo, for the regulation of endochondral bone growth. These observations coupled with our observation that multiple avian FGF receptors (Cek1, Cek2, Cek3, and FREK) are expressed in proliferative chondrocytes highlights the complexity of FGF signaling pathways in postnatal endochondral bone growth.     

Synthesis, antimalarial, antileishmanial, antimicrobial, cytotoxicity, and methemoglobin (MetHB) formation activities of new 8-quinolinamines

We report the synthesis, in vitro antiprotozoal (against Plasmodium and Leishmania), antimicrobial, cytotoxicity (Vero and MetHb-producing properties), and in vivo antimalarial activities of two series of 8-quinolinamines. N1-{4-[2-(tert-Butyl)-6-methoxy-8-quinolylamino]pentyl}-(2S/2R)-2-aminosubstitutedamides (21-33) and N1-[4-(4-ethyl-6-methoxy-5-pentyloxy-8-quinolylamino)pentyl]-(2S/2R)-2-aminosubstitutedamides (51-63) were synthesized in six steps from 6-methoxy-8-nitroquinoline and 4-methoxy-2-nitro-5-pentyloxyaniline, respectively. Several analogs displayed promising antimalarial activity in vitro against Plasmodium falciparum D6 (chloroquine-sensitive) and W2 (chloroquine-resistant) clones with high selectivity indices versus mammalian cells. The most promising analogs (21-24) also displayed potent antimalarial activity in vivo in a Plasmodium berghei-infected mouse model. Most interestingly, many analogs exhibited promising in vitro antileishmanial activity against Leishmania donovani promastigotes, and antimicrobial activities against a panel of pathogenic bacteria and fungi. Several analogs, notably 21-24, 26-32, and 60, showed less MetHb formation compared to primaquine indicating the potential of these compounds in 8-quinolinamine-based antimalarial drug development.