Kaempferol triosides from Silphium perfoliatum

Two apiose-containing kaempferol triosides, together with nine known flavonoids were isolated from the leaves of Silphium perfoliatum L. Their structures were elucidated by acid hydrolysis and spectroscopic methods including UV, LSI MS, FAB MS, CI MS, ¹H, ¹³C and 2D-NMR, DEPT, HMQC and HMBC experiments. The two new compounds were identified as kaempferol 3-O-β- -apiofuranoside 7-O-α- -rhamnosyl-(1′→6)-O-β- -galactopyranoside and kaempferol 3-O-β- -apiofuranoside 7-O-α- -rhamnosyl-(1→ 6)-O-β- (2-O-E-caffeoylgalactopyranoside).     

Mutual separation of hinge-glycopeptide isomers bearing five N-acetylgalactosamine residues from normal human serum immunoglobulin A1 by capillary electrophoresis

Immunoglobulin A1 (IgA1) from normal human serum is known to have O-linked sugar chains, sialylated Galβ1,3GalNAc, in the hinge portion. In order to reduce the microheterogenity of the sugar chain, the hinge glycopeptide prepared from IgA1 was sequentially treated with neuraminidase and β-galactosidase. The asialo-, agalacto-hinge glycopeptide (HGP-SG) composed of a 33-mer peptide (HP33) and N-acetylgalactosamine (GalNAc) residues was obtained. The HGP-SG was separated into three major peaks, A, B and C, by high-performance liquid chromatography (HPLC). Each glycopeptide fraction was further separated by capillary electrophoresis (CE). Peaks A, B and C with HPLC abundantly contained HP33 bearing five and six N-acetylgalactosamine residues (HGP33-5,6GN), HGP33-4,5GN and HGP33-3,4GN, respectively. Among these glycopeptide peaks, only the HGP33-5GN peak was partly split into two peaks based on the CE analysis – HGP33-5GN-α and -β. The glycopeptide, HGP25-5GN shortened by the thermolysin digest of HGP33-SG was also well separated into the α and β forms by CE analysis. No differences in their mass and peptide portion were observed between HGP25-5GN-α and -β. Therefore, the obtained result might indicate that HGP25-5GN-α was an isomer of HGP25-5GN-β differing in its stereospecific structure of the peptide portion and/or the attachment site of the GalNAc residue.     

Norleucine, a natural occurrence in a novel ergot alkaloid γ-ergokryptinine

Summary. A novel natural peptide ergot alkaloid γ-ergokryptinine containing norleucine has been isolated from ergot sclerotia of the field-growing parasitic fungus Claviceps purpurea CCM 8059. Its structure was deduced from the NMR and mass spectral data. The final structural proof was provided by the crystal structure determination, which is the first X-ray structure of a natural Nle-containing secondary metabolite. The conformations of three ergopeptinines: γ-ergokryptinine, ergoladinine, and α-ergokryptinine were compared.     

Intracellular Screening of a Peptide Library to Derive a Potent Peptide Inhibitor of α-Synuclein Aggregation

Aggregation of α-synuclein (α-syn) into toxic fibrils is a pathogenic hallmark of Parkinson disease (PD). Studies have focused largely on residues 71–82, yet most early-onset mutations are located between residues 46 and 53. A semirationally designed 209,952-member library based entirely on this region was constructed, containing all wild-type residues and changes associated with early-onset PD. Intracellular cell survival screening and growth competition isolated a 10-residue peptide antagonist that potently inhibits α-syn aggregation and associated toxicity at a 1:1 stoichiometry. This was verified using continuous growth measurements and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity studies. Atomic force microscopy and circular dichroism on the same samples showed a random-coil structure and no oligomers. A new region of α-syn for inhibitor targeting has been highlighted, together with the approach of using a semirational design and intracellular screening. The peptides can then be used as candidates for modification in drugs capable of slowing or even preventing the onset of PD.     

Inhibition of adipose differentiation by 9α, 11β-prostaglandin F2α

Prostaglandin F2α (PGF2α) is a potent adipose differentiation inhibitor for the adipogenic cell line 1246 and for adipocyte precursors in primary culture with an ED50 of 3×10⁻⁸ M. In this paper, we examined the effect of several prostaglandins which have structural similarities with PGF2α on the differentiation of 1246 cells and of adipocyte precursors in primary culture. The results show that only 9α,11β-PGF2α is as potent as PGF2α to inhibit differentiation of adipocyte precursors in primary culture and of the adipogenic cell line 1246. In the presence of 9α,11β-PGF2α, the cells remained fibroblast-like, typical of undifferentiated adipocyte precursors. Triglyceride accumulation and increase of specific activity for glycerol-3-phosphate dehydrogenase were inhibited. In addition, mRNA expression of early markers of differentiation such as lipoprotein lipase (LPL) and fatty acid binding protein (FAB) was decreased. The isomer 9β,11α-PGF2α and other PGF2α derivatives were inactive. These results provide new information on the biological activity of 9α,11β-PGF2α as an inhibitor of adipose differentiation and about the structural characteristics of prostaglandins required for maintenance of a high adipose differentiation inhibitory effect.     

Differences in oligosaccharide pattern of a sample of polar bear colostrum and mid-lactation milk

Although the concentrations of carbohydrate in the colostrum and in the mid-lactation milk of polar bear (Ursus maritimus) were similar, the oligosaccharide patterns differed. The colostrum sample contained Neu5Ac(α2-3)Gal(β1-4)Glc (3′-N-acetylneuraminyllactose), GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)Glc (A-tetrasaccharide), Fuc(α1-2)Gal(β1-4)Glc (2′-fucosyllactose) and Gal(β1-4)Glc (lactose). The mid-lactation milk contained Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)[Fuc(α1-3)]Glc (B-pentasaccharide), GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)[Fuc(α1-3)]Glc (A-pentasaccharide), Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)Glc (B-tetrasaccharide), A-tetrasaccharide, Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]Glc (3-fucosylisoglobotriose), Gal(α1-3)Gal(β1-4)Glc (isoglobotriose) and lactose. The dominant saccharides in the colostrum were 3′-N-Acetylneuraminyllactose and lactose, whereas isoglobotriose was the dominant saccharide in the mid-lactation milk in which lactose was only a minor component. Isoglobotriose, which had previously been found to be a dominant saccharide in mature milk from the Ezo brown bear, the Japanese black bear and the polar bear, was not found in the polar bear colostrum.     

Assignment of the βB1 Crystallin Gene (CRYBB1) to Human Chromosome 22 and Mouse Chromosome 5

By using primers complementary to the rat βB1 crystallin gene sequence, we amplified exons 5 and 6 of the orthologous human gene (CRYBB1). The amplified human segments displayed greater than 88% sequence homology to the corresponding rat and bovine sequences.CRYBB1was assigned to the group 5 region in 22q11.2–q12.1 by hybridizing the exon 6 PCR product to somatic cell hybrids containing defined portions of human chromosome 22. The exon 5 and exon 6 PCR products ofCRYBB1were used to localize, by interspecific backcross mapping, the mouse gene (Crybb1) to the central portion of chromosome 5. Three other β crystallin genes (βB2(−1), βB3, and βA4) have previously been mapped to the same regions in human and mouse. We demonstrate that the βB1 and βA4 crystallin genes are very closely linked in the two species. These assignments complete the mapping and identification of the human and mouse homologues of the major β crystallins genes that are expressed in the bovine lens.     

Identification of tubulin isoforms in different tissues of Ascaris suum using anti-tubulin monoclonal antibodies

Three monoclonal antibodies specific to α- and β-tubulin were used to examine the expression of tubulin isofoms in the intestine, reproductive tract and body wall muscle of A. suum. The tubulins were found to be different in their isoelectric points, number of isoforms and peptide maps with Western blot analysis of one-dimensional polyacrylamide gel confirming the presence of α-, β₁- and β₂- tubulin. Commercial cross-reactive anti-α and anti-β MAbs 356 and 357 recognized tubulin from A. suum tissues as well as from pig brain, whereas anti-A. suum β-tubulin specific MAb P3D6 recognized tubulin from the A. suum tissues only. Two-dimensional gel analysis showed different isoform patterns in different A. suum tissues with anti-A. suum β-tubulin MAb P3D6 and cross-reactive β-tubulin MAb 357 recognizing 2–4 β- tubulin isoforms and anti-α-tubulin MAb 356 recognizing 1–6 α-tubulin isoforms. Different peptide maps of tubulin were observed in the three tissues, when subjected to limited proteolysis followed by SDS-PAGE. The data indicate that different tubulins are found in different tissues of adult A. suum.     

Small Peptide Inhibitor of JNK3 Protects Dopaminergic Neurons from MPTP Induced Injury via Inhibiting the ASK1-JNK3 Signaling Pathway

Introduction and Aims

The ASK1-JNK3 signaling pathway plays a pivotal role in the pathogenesis of Parkinson''s disease (PD). The specific binding of β-arrestin2 to JNK3 is essential for activation of the ASK1-JNK3 cascade, representing a potential therapeutic target for preventing dopaminergic neuronal death in PD. The aim of this study was to identify a novel strategy for the prevention of dopaminergic neuronal death in PD.

Methods

Based on the specific binding of β-arrestin2 to JNK3, a 21-amino-acid fusion peptide, termed JNK3-N-Tat, was synthesized. We evaluated the ability of this peptide to inhibit the binding of β-arrestin2 to its target domain in JNK3 in vitro and in vivo.

Results

The JNK3-N-Tat peptide inhibited activation of the ASK1-JNK3 cascade by disrupting the interaction between β-arrestin2 and JNK3. JNK3-N-Tat exerted beneficial effects through pathways downstream of JNK3 and improved mitochondrial function, resulting in attenuated MPP+/MPTP-induced damage. JNK3-N-Tat protected mesencephalic dopaminergic neurons against MPTP-induced toxicity.

Conclusions

JNK3-N-Tat, a JNK3-inhibitory peptide, protects dopaminergic neurons against MPP+/MPTP-induced injury by inhibiting the ASK1-JNK3 signaling pathway.     

Determination of lycopene, α-carotene and β-carotene in serum by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry with selected-ion monitoring

A selected-ion monitoring (SIM) determination of serum lycopene, α-carotene and β-carotene by an atmospheric pressure chemical ionization mass spectrometry (APCI–MS) was developed. A large amount of serum cholesterols disturbed the SIM determination of carotenoids by contaminating the segment of interface with the LC–MS. Therefore, separation of carotenoids from the cholesterols was performed using a mixed solution of methanol and acetonitrile (70:30) as the mobile phase on a C₁₈ column of mightsil ODS-5 (75 mm×4.6 mm I.D.). The SIM determination was carried out by introducing only the peak portions of carotenoids and I.S. (squalene) by means of an auto switching valve. In the positive mode of APCI–MS, lycopene, α-carotene and β-carotene were monitored at m/z 537 and I.S. was monitored at m/z 411. This method was linear for all analytes in the range of 15–150 ng for lycopene, 7–70 ng for α-carotene and 25–50 ng for β-carotene. The detection limit of LC–APCI–MS-SIM for carotenoids was about 3 ng per 1 ml of serum (S/N=3). The repeatabilities, expressed as C.V.s, were 10%, 8.4% and 5.3% for lycopene, α-carotene and β-carotene, respectively. The intermediate precisions, expressed as C.V.s, were 11. 2%, 8.8% and 6.5% for lycopene, α-carotene and β-carotene, respectively.     

The two enantiospecific dichlorprop/α-ketoglutarate-dioxygenases from Delftia acidovorans MC1 – protein and sequence data of RdpA and SdpA

Two α-ketoglutarate-dependent dioxygenases carrying enantiospecific activity for the etherolytic cleavage of racemic phenoxypropionate herbicides [(RS)-2-(2,4-dichlorophenoxy)propionate and (RS)-2-(4-chloro-2-methylphenoxy)propionate] from Delftia acidovorans MC1 were characterized with respect to protein and sequence data. The (S)-phenoxypropionate/α-ketoglutarate-dioxygenase (SdpA) appeared as a monomeric enzyme with a molecular weight of 32 kDa in the presence of SDS. N-terminal sequences revealed relationship to α-ketoglutarate-dependent taurine dioxygenase (TauD) and to 2,4-dichlorophenoxyacetate/α-ketoglutarate-dioxygenase (TfdA). The (R)-phenoxypropionate/α-ketoglutarate-dioxygenase (RdpA) referred to 36 kDa in the presence of SDS and to 108 kDa under native conditions. Internal sequences of fragments obtained after digestion made evident relationship to TfdA and TauD. Two-dimensional electrophoretic separation resulted in the resolution of up to 3 individual spots with almost identical molecular weights but different isoelectric points with both RdpA and SdpA. The structural differences of these isoenzyme forms are not yet clear.     

Expression of Biologically Active β Subunit of Bovine Follicle-Stimulating Hormone in the Methylotrophic YeastPichia pastoris

Follicle-stimulating hormone (FSH), a pituitary gonadotropin, is a heterodimer composed of an α subunit, which is common to all the glycoprotein hormones, noncovalently associated with the hormone-specific β subunit. The objective of the present study is to develop a recombinant DNA expression system for the β subunit of FSH that can be applied to study structure–function relationships while producing large quantities of the hormone subunit for immunocontraceptive, clinical, and veterinary purposes. We report here the expression of biologically active bovine FSHβ (bFSHβ) in the methylotrophic yeastPichia pastoris.ThePichia-expressed FSHβ (pFSHβ) was secreted into the culture medium and was found to be immunologically very similar to pituitary-derived ovine FSHβ. Replacement of the cognate signal peptide with the yeast α mating factor signal peptide increased the level of expression from 230 ng/ml (cognate signal peptide) to 4 μg/ml (α mating factor signal peptide) of the culture supernatant. pFSHβ^His.tag(pFSHβ with six histidine residues at the C terminus) was purified to apparent homogeneity using one-step nickel affinity chromatography. The molecular weight of purified pFSHβ^His.tagwas approximately 22,000, which was slightly higher than that of the pituitary-derived ovine FSHβ. pFSHβ^His.tagcould assemble with the α subunit to yield a heterodimer capable of binding to the FSH receptors and also elicit biological response. These data show that pFSHβ^His.tagis properly folded and biologically active.     

Construction and use of two α-human atrial natriuretic peptide-fragment affinity chromatography columns in the isolation of C- and N-terminal epitope-specific antibodies for use in a prototype α-hANP biosensor

Two α-human atrial natriuretic peptide (α-hANP) based affinity chromatography columns were produced by covalently immobilizing the C- and N-terminal epitopes of α-hANP. The stationary phase was made from a controlled-pore-glass bead solid support, which was silanized and treated with sulphosuccinimidyl 4-(maleimidomethyl)cyclohexyl carboxylate before the individual fragments were immobilized by substitution at their thiol groups. These columns were used to isolate α-hANP-specific antibodies from a goat anti-α-hANP serum, which were then further sorted according to their epitope specifity. These C- and N-terminal epitope-specific antibodies were in turn used as components in the construction of an α-hANP biosensor based on an enzyme-linked immunosorbent assay (ELISA) sandwich principle. Initial in vitro testing of the sensor using a physiological α-hANP solution showed a reproducible response to the peptide. There is to date no other equally fast, sensitive and precise method available to detect this peptide. This α-hANP sensor may prove to be an invaluable aid in human medicine as a monitor of patient status during transplant surgery, for example, an area inaccessible to radioimmunoassay and normal ELISA techniques.     

Enzymatic Sialylation of N-Linked Oligosaccharides Using an α-(2,3)-Specific trans-Sialidase from Trypanosoma cruzi: Structural Identification Using a Three-Dimensional Elution Mapping Technique

α-(2,3)-Sialylated biantennary and triantennary oligosaccharides were enzymatically prepared from pyridyl-2-amino-oligosaccharides with terminal Gal residues, using an α-(2,3)-specific trans-sialidase from Trypanosoma cruzi (Lee, K. B., and Lee, Y. C. (1994) Anal. Biochem. 216, 358-364). From the pyridyl-2-amino-derivatives of neutral and α-(2,6)-monosialylated biantennary oligosaccharides from human fibrinogen, 5 different sialyl biantennary oligosaccharides were obtained. From two different asialo-triantennary oligosaccharides from fetuin, 35 sialyl oligosaccharides were obtained. The trans-sialidase transferred sialic acids effectively and indiscriminately to different galactosyl residues in the different positions on the substrates. Since the starting materials are neutral oligosaccharide of established structure, and the only α-(2,3)-sialyl residues are added to the nonreducing Gal terminal residues, the structures of these oligosaccharides could be identified unambiguously by using the three-dimensional mapping technique (Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y., and Tomiya, N. (1995) Anal. Biochem. 226, 139-146.) in combinations with strategic digestion with β-galactosidase, β-N-hexosaminidase, and sialidase L.     

β-N-Acetylhexosaminidase-catalysed synthesis of non-reducing oligosaccharides

A large panel of fungal β-N-acetylhexosaminidases was tested for the regioselectivity of the β-GlcNAc transfer onto galacto-type acceptors ( -galactose, lactose, 2-acetamido-2-deoxy- -galactopyranose). A unique, non-reducing disaccharide β- -GlcpNAc-(1→1)-β- -Galp and trisaccharides β- -GlcpNAc-(1→4)-β- -GlcpNAc-(1→1)-β- -Galp, β- -Galp-(1→4)-β- -Glcp-(1→1)-β- -GlcpNAc and β- -Galp-(1→4)-α- -Glcp-(1→1)-β- -GlcpNAc were synthesised under the catalysis of the β-N-acetylhexosaminidase from the Aspergillus flavofurcatis CCF 3061 with -galactose and lactose as acceptors. The use of 2-acetamido-2-deoxy- -galactopyranose as an acceptor with the β-N-acetylhexosaminidases from A. flavofurcatis CCF 3061, A. oryzae CCF 1066 and A. tamarii CCF 1665 afforded only β- -GlcpNAc-(1→6)- -GalpNAc.     

A large-scale synthesis of somatostatin for clinical use by a novel alternating solution/solid-phase procedure

A large-scale synthesis of somatostatin was developed. A stepwise C→N approach in solution was used, employing N(α)-t-butoxycarbonyl amino acid active esters. The scheme of semipermanent protection utilized 2-(methylsulfonyl)-ethoxycarbonyl for the -amino group of lysine; acetamidomethyl for the β-thiol groups of cysteine; the orange-colored 2-[4-(phenylazo)-phenylsulfonyl]-ethoxy group for the C-terminal carboxy group of cysteine. All condensations and N(α)-deprotections were carried out in homogeneous solution, while isolation and purification of peptides carrying the colored group was achieved by precipitation and washing of the solid products. Thus, the “alternating solution/solid-phase peptide synthesis” combines advantages of both the classical solution synthesis and the Merrifield solid-phase technique. The overall yield was 5%, or 16 g of somatostatin from 100 g of the novel amino acid derivative, N(α)-t-butoxycarbonyl-S-acetamidomethyl- -cysteine 2-[4-(phenylazo)-phenylsulfonyl]-ethyl ester. An improved method for the preparation of S-acetamidomethyl- -cysteine, free of thiazolidine carboxylic acid, is described.     

Synthesis of 2,3-di-O-glycosyl derivatives of methyl α- and β- -glucopyranoside

The syntheses are described of 2,3-di-O-glycosyl derivatives of methyl α- and β- -glucopyranoside having α- -manno-, β- -galacto-, α- -rhamno-, α- -fuco-, and β- -fuco-pyranosyl substitutents at O-2 and O-3. The syntheses involved glycoslation of methyl 4,6-O-(benzylidene-α- (24) and β- -glucopyranoside (21), and substituted derivatives of 21 bearing 2-O-(2,3,4,6-tetra-O-benzyl-α- -mannopyranosyl)-, -(2,3,4,6-tetra-O-acetyl-β- -galactopyranosyl)-, -(2,3,4-tri-O-benzyol-α- -rhamnopyranosyl)-, and-(2,3,4-tri-O-benzoyl-β- -fucopyranosyl) groups.     

Comparison of 252californium plasma desorption and fast atom bombardment mass spectrometry for analysis of small peptides

The data obtained with 252Cf plasma desorption (PD) and fast atom bombardment mass spectrometry of eight tri-, tetra- and pentapeptides were compared. Good spectra were obtained with 1-10 nmol of peptide. In both techniques molecular weight information was obtained. The PD mass spectra are often dominated by the cationized molecular ions in contrast to the fast atom bombardment (FAB) mass spectra, where cationization is rarely observed. Amino acid content is reflected in the immonium ions equally well in both techniques. The fragmentation patterns observed with the two techniques are almost identical. However, practical sequencing of peptides based on either FAB or PD mass spectrometry of underivatized peptides alone is difficult. This is due to the unpredictable and sometimes absent cleavage yield at certain peptide bonds. Another difficulty is the many simultaneous fragmentation pathways. However, for many peptides enough information is present to allow sequence determination for at least a major part of the molecule.