Fromβ-lactams toα- andβ-amino acid derived peptides

Summary The potential of-lactams as intermediates for the access to- and-amino acid-derived peptides is shortly reviewed, with major focus on the technologies developed in our group. The two general strategies lie, on one side, in the oxidative ring expansion of 3-hydroxy-lactams toN-carboxy-amino acid anhydrides or Leuch's anhydrides and subsequent coupling with-amino acid esters and, on the other side, in the nucleophilic ring opening ofN-Boc--lactams. Both approaches have been successfully applied to the synthesis of,-diamino acid,-amino--hydroxy acid, polyhydroxylated-amino acid,,-disubstituted-amino acid,-amino acid,-amino--hydroxy acid and,-disubstituted-amino acid derived peptides. Because of the mild reaction conditions needed for the above transformations and the highly stereoselective procedures employed for the construction of the starting-lactam ring, the whole process allows the production of optically pure final products.     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Studies of α- and βL-Crystallin Complex Formation in Solution at 60°C

Studies of molecular mechanisms of chaperone-like activity of -crystallin became an active field of research over last years. However, fine interactions between -crystallin and the damaged protein and their complex organization remain largely uncovered. Complexation between - and _L-crystallins was studied during thermal denaturation of _L-crystallin at 60°C using small-angle X-ray scattering (SAXS), light scattering, gel-permeation chromatography, and electrophoresis. A mixed solution of - and _L-crystallins at concentrations about 10 mg/ml incubated at 60°C was found to contain their soluble complexes with a mean radius of gyration 14 nm, mean molecular mass 4 MDa and maximal size over 40 nm. In pure _L-crystallin solution, no complexes were observed at 60°C. In SAXS studies, transitions in the -crystallin quaternary structure at 60°C were shown to occur and result in doubling of the molecular weight. This suggests that during the temperature-induced denaturation of _L-crystallin it binds with modified -crystallin or, alternatively, _L-crystallin complexation and -crystallin modifications are concurrent. Estimates of the -_L-crystallin complex size and relative contents of - and -_L-crystallins in the complex suggest that several -crystallin molecules are involved in complex formation.     

Liver and intestinal fatty acid binding proteins in control and TGFβ1 gene targeted deficient mice

The effect of transforming growth factor beta-1 (TGF1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF1-deficient mice. Homozygous TGF1-deficient 129 × CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF1-deficient/immunodeficient C3H mice on a SLID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (1-FABP) which decreased 3-fold in the TGF1-deficient/immunodeficient C3H mice only. Thus, TGF1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.Abbreviations L-FABP liver fatty acid binding protein - I-FABP intestinal fatty acid binding protein - TGF1 transforming growth factor beta-1 - TNF- tumor necrosis factor- - MIP- macrophage inflammatory protein- - PMSF phenylmethyl sulfonyl fluoride - PBS phosphate buffered saline     

A Simple Method for Preparation of 18α-Glycyrrhetinic Acid and Its Derivatives

Treatment of 18-glycyrrhizic acid with a methanolic solution of HCl resulted in 1 : 1 mixture of methyl esters of 18- and 18-glycyrrhetinic acids. Benzoylation of the mixture led to methyl esters of 3-benzoyl-18-glycyrrhetinic acid and 3-benzoyl-18-glycyrrhetinic acid, which were separated by chromatography on silica gel. 18-Glycyrrhetinic acid was prepared by alkaline hydrolysis of methyl 3-benzoyl-18-glycyrrhetinate and was further used for the syntheses of 3-keto-18-glycyrrhetinic acid and methyl esters of 18-glycyrrhetinic acid and 3-keto-18-glycyrrhetinic acid.     

Neurotoxic Potential of Three Structural Analogs of β-N-oxalyl-α,β-Diaminopropanoic Acid (β-ODAP)

Lathyrism is a non-progressive motor neuron disease produced by consumption of the excitatory amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (-ODAP). To learn more about the mechanisms underlying Lathyrism three structural analogs of -ODAP were synthesized. Carboxymethyl-,-diaminopropanoic acid (CMDAP) evoked inward currents which were antagonized by APV (30 M), but not by CNQX (10 M). N-acetyl-,-diaminopropanoic acid (ADAP) evoked no detectable ionic currents but potentiated N-methyl-D-aspartate (NMDA)-activated currents. The potentiation of NMDA currents by ADAP was blocked by 7-chlorokynurenic acid. Carboxymethylcysteine (CMC) did not activate any detectable ionic currents. None of the three -ODAP analogs produced visible symptoms of toxicity in day old chicks when administered for 2–3 consecutive days. Ligand binding studies demonstrated that all the three compounds were effective to in displacing [³H]glutamate. The maximum inhibition was 92% for CMDAP, 61% for ADAP, 65% for CMC and 99% for -ODAP. These data indicate that analogs of -ODAP may interact with glutamate receptors without producing neurotoxicity.     

The Structural Basis for the Susceptibility of Gangliosides to Enzymatic Degradation

The conformational properties of GM2, GalNac-4(Neu5Ac-3) Gal-4Glc-1Cer have been compared to those of 6-GM2, in which the linkage between the GalNAc and Gal was altered from GalNac-4Gal- to GalNac-6Gal-, and to those of GD1a, Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-3)Gal-4Glc-1Cer, and GalNAc-GD1a.Our results revealed that unlike the compact and rigid oligosaccharide head group found in GM2, where the Neu5Ac and the GalNAc residues interact, the sugar chain of 6-GM2 is in an open spatial arrangement, with the Neu5Ac no longer interacting with GalNAc, freely accessible to external interactions.The structure of GD1a can be regarded as that of GM2 with an extension of the terminal Neu5Ac-3Gal-disaccharide. The inner portion of GD1a is that of GM2 comprising the very rigid GalNAc-[Neu5Ac-]Gal trisaccharide. The terminal Neu5Ac-Gal linkage is flexible and fluctuates between two limiting conformations. In GalNAc-GD1a the outer sialic acid gains conformational rigidity due to the presence of the outer GalNAc in position 4 of galactose. This ganglioside has two core GalNAc-[Neu5Ac-]Gal trisaccharide linked in tandem.     

Locus-control-region-coupled betaS Antilles- and alpha2-hemoglobin genes select for high alpha2-hemoglobin expression in adult transgenic mice

Two transgenic lines of mice were produced which contained the ^S _Antilles- and ₂-hemoglobin genes trandemly coupled to the micro locus control region (LCR). The LCR^S _Antilles2-hemoglobin transgenic mice expressed high levels of ₂-hemoglobin while ^S _Antilles-hemoglobin expression was virtually undetectable. Abundant ₂-hemoglobin protein was observed in the blood of transgenic mice, while ^S _Antilles-hemoglobin chains could not be detected. Transgenic red blood cells had substantially decreased sensitivity to osmotic lysis. Attempts to produce homozygotes containing the transgene were unsuccessful. The phenotype of these mice closely resembles that of -thalassemic mice. The LCR^S _Antilles2 transgenic mice demonstrate that if the LCR is coupled to the ^S _Antilles- and ₂-hemoglobin genes in tandem, only the distal ₂-hemoglobin gene is selected for expression to significant levels in adult mice. These results support a reciprocally competitive model for LCR-hemoglobin developmental switching.     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat

A -amylase (EC 3.2.1.2) was identified in the outer pericarp (P) of developing seeds of wheat (Triticum aestivum L.) and compared with the well known -amylase which is synthesized during seed development in the starchy endosperm (E). The enzyme P already exists in the tissues before anthesis and vanishes at the time when E starts to accumulate. The isoelectric-focusing patterns of P and E are very similar. The relative molecular weight (M_r) of P is slightly higher than that of E (66 and 64.5 kDa, respectively). Both P and E exhibit common epitopes in addition to epitopes specific for each of them. The two enzymes were identified in small amounts in the green tissues of the developing seeds (inner pericarp and testa). No antigenic difference was detected between P and the -amylases of roots and leaves.Abbreviations P pericarp -amylase - E endosperm -amylase - IS1 anti--amylase immune serum - IS2 anti- and anti- amylase immune serum - IS3 anti- amylase immune serum - IEF isoelectric focusing - IgG immunoglobulin G The authors thank Dr. P. Ziegler (Universität Bayreuth, FRG) for stimulating discussion and for useful suggestions during the writing of the text. The authors thank Miss C. Mayer for her skillful technical assistance.     

Tail-to-tail orientation of the Atlantic salmon alpha- and beta-globin genes

We report the cloning of a cDNA and two corresponding -globin genes of the Atlantic salmon (Salmo salar L.) as well as two genes for -globins. Nucleotide sequence analysis of the cDNA shows that the predicted -globin peptide comprises 148 amino acids with a calculated molecular mass of 16,127 Da and an overall amino acid similarity of 40–50% to higher vertebrates and 60–90% to fish sequences. The study of the genomic organization of - and -globin genes shows that, as is the case in Xenopus, the salmon genes are adjacent. Two sets of linked - and -globin genes were isolated and restriction-enzyme polymorphisms indicate that they belong to two distinct loci, possibly as a result of the salmon tetraploidy. In each locus the - and -globin genes are oriented 3 to 3 relative to each other with the RNA coding sequences located on opposite DNA strands. This is the first evidence for this type of arrangement found for globin genes. Moreover, while the linkage found in salmon and Xenopus supports the hypothesis of an initial tandem duplication of a globin ancestor gene, our results raise the question of the actual original orientation of the duplicated genes. Correspondence to: F. Gannon     

Subunit Interacting Surfaces of Human Hemoglobin in Solution: Localization of the α–β Subunit Interacting Surfaces on the α-Chain by a Comprehensive Synthetic Strategy

By using synthetic overlapping peptides encompassing the entire -chain of adult human hemoglobin (HbA), we have mapped on the -chain the regions responsible for its binding to the -chain in solution. These binding surfaces were, in general, in good agreement with those expected from the crystal structure (peptides 81–95, 101–115, 111–125, and 131–141). However, we observed some significant differences in the levels of binding found here in solution and those expected from the crystal structure. Peptide 31–45, which in the crystal had the highest number of contact residues of all the -chain peptides, did not bind the -chain in solution. Similarly, peptide 91–105, with seven contact residues in the crystal, showed low binding with the -chain in solution. On the other hand, peptides 41–55 and 121–135 possessed much higher binding activity in solution than would be expected from their contribution to subunit association in the crystal. In fact, peptide 121–135 had the highest binding activity of the -chain peptides. These studies and our previous findings, which localized on the -chain the regions that bind to the -chain in solution, have shown that the regions of subunit association in solution are close to, but not identical with, those in the crystal. The approach should be quite useful for mapping subunit association in oligomeric proteins and could even be applied to proteins that are isolated only in traces or whose three-dimensional structure is not yet known.     

Comparison of the apoptotic processes induced by the oxysterols 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

Oxysterols have been shown to induce apoptosis in a variety of cell lines. The mechanism of oxysterol-induced apoptosis is mainly known at the post-mitochondrial level. The aim of the present study was to compare the pathway of apoptosis induced by the oxysterols 7-hydroxycholesterol (7-OH) and cholesterol-5,6-epoxide (-epoxide) in U937 cells. To this end, we employed a range of inhibitors of apoptosis; a broad-spectrum caspase inhibitor, a specific caspase-3 inhibitor and an inhibitor of cytochromec release and the antioxidants; trolox, ebselen and resveratrol. The three inhibitors of apoptosis prevented cell death induced by 7-OH; however, in -epoxide-treated cells, the inhibitor of cytochromec release did not protect against apoptosis. The cellular antioxidant glutathione was depleted in 7-OH-treated cells but not in cells incubated with -epoxide. Trolox, a water-soluble synthetic analogue of -tocopherol, prevented 7-OH-induced apoptosis but did not protect against cell death induced by -epoxide. Ebselen and resveratrol did not protect U937 cells against apoptosis induced by either 7-OH or -epoxide. Our results suggest that differences occur in the pathways of apoptosis induced by 7-OH and -epoxide in U937 cells.     

Interferons up-regulate with different potency HLA class I antigen expression in M14 human melanoma cell line. Possible interaction with glucocorticoid hormones

The relative potency of interferon (IFN), interferon (IFN), and interferon (IFN) in inducing the expression of HLA class I antigens, as well as their capacity to counteract the inhibition induced by glucocorticoid hormones on HLA class I antigen expression, were analysed in the human melanoma cell line M14, both at membrane and at mRNA level. The data obtained indicate that (a) IFN enhance with different potency (IFN>IFN>IFN) the expression of HLA class I antigens in M14 cells, (b) prednisone inhibits HLA class I antigen expresion, (c) glucocorticoid hormones, when associated with IFN or IFN, inhibit the HLA class I enhancement induced by IFN alone, and ffinally, (c) the association between 1 M prednisone or 1 M deflazacort and IFN seems to potentiate the enhancing capacity of IFN on the expression of HLA class I molecules at the mRNA level. These findings, if confirmed, might indicate that IFN and glucocorticoid hormones are not mutually exclusive in the management of human melanoma.     

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

Structure and conformation in solution of the parallel-stranded hybrid α-d(CGCAATTCGC)·β-d(GCGTTAAGCG) by high-resolution 2D NMR

Summary The unnatural duplex oligonucleotide -d(CGCAATTCGC)·-d(GCGTTAAGCG) was analyzed by high-resolution NMR methods. All of the exchangeable imino and nonexchangeable protons of the duplex were assigned. Detection of all 10 of the exchangeable imino protons confirms that a parallel, unsymmetrical duplex is formed. The thermal stability of the parallel duplex is similar to the analogous antiparallel - duplex. The right handedness of the helix is confirmed by inter-residue [H8/H6-H1] and [H8/H6-H2] NOEs to the 5-neighbor in the -strand and [H8/H6-H1] NOEs to the 3-neighbor in the -strand. Intra-residue and inter-residue distances between base protons and deoxyribose protons in both strands were determined using the isolated spin-pair approximation for NOESY cross peaks acquired with mixing times 50 ms or less. The NOE data are consistent with a B-form geometry adopted by the / hybrid decamer.     

Endogenous interferon α/β produced by Kupffer cells inhibits interleukin-1, tumor necrosis factor α production and interleukin-2-induced activation of nonparenchymal liver cells

Summary We have previously reported liver-specific interferon (IFN) / production by murine Kupffer cells that was not observed with other tissue macrophages incubated in the absence of stimulators such as IFN or lipopolysaccharide (LPS). Consequently, while interleukin-2 (IL-2) alone induced pronounced lymphokine-activated killer (LAK) activity from splenocytes, combination of anti-IFN/ antibody with IL-2 was required to generate significant LAK activity from nonparenchymal liver cells. This endogenous IFN/ production by Kupffer cells was not induced by LPS because (a) addition of polymyxin B did not abolish the positive effects of anti-IFN/ antibody on nonparenchymal liver cells, and (b) similar results were obtained when comparing the responses of LPS-responsive C3HeB/FeJ and LPS-hyporesponsive C3H/HeJ mice. The possibility of hepatotropic infection was also ruled out in that anti-IFN/ antibody enhanced hepatic but not splenic LAK cell induction in vitro in both conventional and germfree C3H/HeN mice. IFN/ played an autoregulatory role by down-regulating the production of IL-1 and tumor necrosis factor by Kupffer cells. However, the augmenting effect of anti-IFN/ antibody on LAK induction from non-parenchymal liver cells was not mediated through an increase in the level of either IL-1 or TNF, as specific antisera against either cytokine did not abrogate this positive effect. Finally, flow-cytometry analysis showed that IFN/ significantly diminished the expression of IL-2 receptor chain, indicating an inhibition of LAK cell generation at a relatively early stage of induction.This work is supported by NIH grant RO1-28 835 and by Medical Research Funds from the Veterans Administration     

Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.