Study of the conformational behaviour of trehalose mycolates by FT-IR spectroscopy.

Mycolic acids are fundamental cell wall components, found in the outer membrane barrier (mycomembrane) of Mycobacterium related genera, that have shown antigenic, murine innate immunity inducting and inflammatory activity triggering action. The mycolic acid derivatives, such as the lipid extractable trehalose monomycolates (TMM) and dimycolates (TDM), have been extensively investigated by several biochemical and biological methods and, more recently, we have performed the first neutron scattering measurements on these molecules in order to characterize their dynamical behavior as well as their rigidity properties. In the present paper, we show the first systematic FT-IR study on TMM, TDM and glucose monomycolate (GMM). It includes the analysis of individual lipids but also mixtures of TMM/TDM (ratio of 1:1) or TMM/GMM (ratio of 1:2). The present work is aimed to the first characterization of the vibrational behavior of mycolates and their mixtures enabling us to elucidate the molecular mechanisms responsible for the capability of mycolic acids to affect the flexibility and permeability properties of the mycomembrane. As a whole, the present FT-IR findings provide information that have relevant biological implications, allowing to demonstrate that the membrane fluidity is not only linked to the chain length, but also to the specific conformational behavior adopted by mycolates, which in the mixtures is strongly affected by their mutual interactions. In addition, the capability of trehalose to drive the mycolate conformational behavior and then the chain order and packing is emphasized; due to the TDM relevant evidences shown by our data, this trehalose effect could be related to the TDM toxicity and inflammation action.

Nodule-inducing activity of synthetic Sinorhizobium meliloti nodulation factors and related lipo-chitooligosaccharides on alfalfa. Importance of the acyl chain structure.

Sinorhizobium meliloti nodulation factors (NFs) elicit a number of symbiotic responses in alfalfa (Medicago sativa) roots. Using a semiquantitative nodulation assay, we have shown that chemically synthesized NFs trigger nodule formation in the same range of concentrations (down to 10(-10) M) as natural NFs. The absence of O-sulfate or O-acetate substitutions resulted in a decrease in morphogenic activity of more than 100-fold and approximately 10-fold, respectively. To address the question of the influence of the structure of the N-acyl chain, we synthesized a series of sulfated tetrameric lipo-chitooligosaccharides (LCOs) having fatty acids of different lengths and with unsaturations either conjugated to the carbonyl group (2E) or located in the middle of the chain (9Z). A nonacylated, sulfated chitin tetramer was unable to elicit nodule formation. Acylation with short (C8) chains rendered the LCO active at 10(-7) M. The optimal chain length was C16, with the C16-LCO being more than 10-fold more active than the C12- and C18-LCOs. Unsaturations were important, and the diunsaturated 2E,9Z LCO was more active than the monounsaturated LCOs. We discuss different hypotheses for the role of the acyl chain in NF perception.

Up-regulation of laminin B2 gene expression in dorsal root ganglion neurons and nonneuronal cells during sciatic nerve regeneration.

A recent study in our laboratory showed that B2 laminin gene is expressed by all L4 and L5 dorsal root ganglion (DRG) neurons as well as by satellite and Schwann cells. Because the laminin B2 subunit has a domain that supports neurite extension in culture, the present study was undertaken to test the hypothesis that laminin B2 gene expression would increase during sciatic nerve regeneration. In situ hybridization was used to examine B2 laminin gene expression in L4 and L5 DRGs 28 days after creating and bridging a 10-mm sciatic nerve gap with an impermeable silicone tube. Overall there was a nearly threefold increase in DRG B2 laminin chain mRNA at this timepoint, a time when axons are known to show vigorous regrowth. Both neurons and nonneuronal cells contributed to this increase. These data suggest that an up-regulation of B2 laminin gene expression by DRG neurons and nonneuronal cells may play a role in peripheral nerve regeneration.

Selectively 13C-enriched DNA: dynamics of the C1'-H1' vector in d(CGCAAATTTGCG)2.

In order to examine the internal dynamic processes of the dodecamer d(CGCAAATTTGCG)2, the 13C-enriched oligonucleotide has been synthesized. The three central thymines were selectively 13C-labeled at the C1' position and their spin-lattice relaxation parameters R(CZ), R(CX,Y), R(HZ-->CZ), R(2HZCZ), R(2HZCX,Y) and R(HZC) were measured. Density functions were computed for two models of internal motions. Comparisons of the experimental data were made with spin-lattice relaxation rates rather than with the density functions, whose values were altered by accumulation of the uncertainties of each relaxation rate measurement. The spin-lattice relaxation rates were computed with respect to the motions of the sugar around the C1'-N1 bond. A two-state jump model between the anti- and syn-conformations with P(anti)/P(syn) = 91/9 or a restricted rotation model with delta chi = 28 degrees and an internal diffusion coefficient of 30 x 10(7) s-1 gave a good fit with the experimental data. Twist, tilt or roll base motions have little effect on 13C1' NMR relaxation. Simulation of spin-relaxation rates with the data obtained at several temperatures between 7 and 32 degrees C, where the dodecamer is double stranded, shows that the internal motion amplitude is independent of the temperature within this range, as expected for internal motion. Using the strong correlation which exists in a B-DNA structure between the chi and delta angle, we suggest that the change in the glycosidic angle value should be indicative of a sugar puckering between the C1'-exo and C2'-endo conformations.

Stereocontrolled synthesis of C-glycosides: further studies on the organolithium reagents derived from 2-deoxy-D-glucose and D-glucose.

The addition of alpha- and beta-2-deoxy-D-glucopyranosyl lithium reagents i and ii to prochiral aldehydes is a syn-selective process, synthetically useful only with the alpha-lithio reagent i (syn:anti selectivity of approximately 10:1 with saturated aldehydes). This has been demonstrated by using propionaldehyde and converting the syn-isomers of both series to an easily identified acyclic meso-chain (alpha-series) or a C2 symmetric acyclic chain (beta-series). The preparation of alpha- and beta-D-glucopyranosyl dilithio reagents 26 and 27 is possible but a notable decrease in efficiency and facial selectivity is observed in coupling reactions with model aldehydes.

The use of 2-deoxy-2-trichloroacetamido-D-glucopyranose derivatives in syntheses of oligosaccharides.

3,4,6-Tri-O-acetyl-2-deoxy-2-trichloroacetamido-alpha-D-glucopyran osyl trichloroacetimidate and its O-benzylated analogue were tested as glycosyl donors in the reaction with a set of sugar acceptors unsubstituted on O-3 and O-4, typically encountered in the synthesis of oligosaccharides. Glycosides were obtained in good to excellent yields with only a slight excess (1.1-1.2 equiv) of the donor, and with a high degree of 1,2-trans stereoselectivity. The corresponding 2-(trichloromethyl)oxazolinium ion was postulated to be the major reactive intermediate. The N-trichloroacetyl groups in the disaccharide products were easily transformed into N-acetyl under neutral conditions by reduction with tributylstannane.

Selectively 13C-enriched DNA: 13C and 1H assignments of a triple helix by two-dimensional relayed HMQC experiments.

We present NMR studies of an intramolecular triple helix, the three strands of which have been linked by a hexaethylene glycol chain. To overcome the generally encountered difficulties of assignment in the homo-pyrimidine strands, the carbon Cl' of the pyrimidines were selectively 13C-enriched. Assignments of the aromatic and sugar protons were obtained from NOESY-HMQC and TOCSY-HMQC spectra. We show that the recognition of a DNA duplex by a third strand via triplex formation is easily carried out in solution by observing the changes of the 1Hl'-13Cl' connectivities as a function of pH. Furthermore, the conformation of the sugars has been found to be C2'-endo, on the basis of the coupling constant values directly measured in an HSQC spectrum.

Synthesis of sulfated and phosphorylated glycopeptides from the carbohydrate-protein linkage region of proteoglycans.

The synthesis of the tetrasaccharide dipeptide beta-D-GlcpA-(1-->3)-beta-D-Galp4SO3Na-(1-->3)-beta-D-Galp-( 1-->4)-beta-D-Xylp- (1-->O)-L-Ser-Gly was achieved by coupling a suitably protected tetrasaccharide trichloroacetimidate, built up from the nonreducing end by the stepwise addition of monosaccharide units, to the protected dipeptide Z-L-Ser-Gly-OBn. Sulfation at O-4 of the second D-Gal unit and complete deprotection afforded the target molecule in high yield. Its phosphorylated analogue beta-D-GlcpA-(1-->3)-beta-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-beta- D-Xylp2PO3Na2 - (1-->O)-L-Ser-Gly was synthesized by coupling a protected trisaccharide trichloroacetimidate to the 2,3-O-isopropylidene derivative of Z-(D-Xyl-)L-Ser-Gly-OBn. Hydrolysis of the O-isopropylidene group, regioselective acetylation at O-3 of the O-Xyl unit, and phosphorylation at O-2 followed by complete deprotection gave the phosphorylated tetrasaccharide dipeptide in high yield. These structures are found in the carbohydrate-protein linkage region of several proteoglycans.

Leukocyte common antigen-related receptor-linked tyrosine phosphatase. Regulation of mRNA expression.

Receptor-linked tyrosine phosphatases regulate cell growth by dephosphorylating proteins involved in tyrosine kinase signal transduction. Within this gene family, the leukocyte common antigen-related (LAR) gene is of particular interest with respect to the nervous system because it has sequence similarity to the neural cell adhesion molecule N-CAM and is located in a chromosomal region (1p32-33) frequently deleted in neuroectodermal tumors. However, immunostaining has detected LAR in non-neural tissues, but not in the central nervous system, peripheral neurons, or adrenal medulla. In this study, rat brain cDNA library LAR clones corresponding to cytoplasmic and 3'-untranslated regions of human LAR were identified. Using probes derived from these clones, high stringency Northern blots revealed approximately 8 kilobase and variable length tissue- and cell-specific LAR transcripts in cortex, brainstem, cerebellum, spinal cord, peripheral tissues, and cultured neural, glial, and pheochromocytoma cells. In situ hybridization showed expression by brain and dorsal root ganglion neurons. LAR expression was developmentally regulated in a region-dependent manner. Changes in LAR expression were also found during nerve growth factor-induced PC12 pheochromocytoma cell differentiation and with contact-mediated inhibition of fibroblast growth. These observations and studies demonstrating neurotrophins functioning via tyrosine kinase receptors suggest that LAR represents an additional mechanism regulating neural development.

Total synthesis of the carbohydrate-protein linkage region common to several mammalian proteoglycans.

A stereocontrolled synthesis of beta-D-GlcpA-(1--> 3)-beta-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Xylp-(1 --> O)-L-Ser-Gly, the common glycopeptide sequence of the carbohydrate-protein linkage region of most mammalian proteoglycans, was achieved by use of O-[2-(trimethylsilyl)ethyl 2,3,4-tri-O-benzoyl-beta-D-glucopyranosyluronate] -(1-->3)-O-(2,4,6-tri-O-benzoyl-beta-D-galactopyranosyl)-(1-->3)-O-(2,4, 6-tri -O-benzoyl-beta-D-galactopyranosyl)-(1-->4)-2,3-di-O-benzoyl-alpha, beta-D- xylopyranosyl trichloroacetimidate as the key intermediate. Condensation of this glycosyl donor with suitably protected L-seryl-glycine dipeptide segments, and peptide chain elongation, allowed the construction in high yield of complex structures of this linkage region.

Diabetic neuropathies.

Diabetic neuropathy is a common complication of diabetes that may be associated both with considerable morbidity (painful polyneuropathy, neuropathic ulceration) and mortality (autonomic neuropathy). The epidemiology and natural history of diabetic neuropathy is clouded with uncertainty, largely caused by confusion in the definition and measurement of this disorder. We have reviewed various clinical manifestations associated with somatic and autonomic neuropathy, and we herein discuss current views related to the management of the various abnormalities. Although unproven, the best evidence suggests that near-normal control of blood glucose in the early years after diabetes onset may help delay the development of clinically significant nerve impairment. Intensive therapy to achieve normalization of blood glucose also may lead to reversibility of early diabetic neuropathy, but again, this is unproven. Our ability to manage successfully the many different manifestations of diabetic neuropathy depends ultimately on our success in uncovering the pathogenic processes underlying this disorder. The recent resurgence of interest in the vascular hypothesis, for example, has opened up new avenues of investigation for therapeutic intervention. Paralleling our increased understanding of the pathogenesis of diabetic neuropathy, refinements must be made in our ability to measure quantitatively the different types of defects that occur in this disorder. These tests must be validated and standardized to allow comparability between studies and more meaningful interpretation of study results.

Synthesis of C-glycopyranosyl compounds by a palladium-catalyzed coupling reaction of 1-tributylstannyl-D-glucals with organic halides.

1-Tributylstannyl-D-glucals, prepared from the corresponding 1-phenylsulfonyl-D-glucals, were coupled efficiently to various organic halides in the presence of a palladium(0) catalyst. This mild reaction is specially useful for the preparation of 1-C-aryl-D-glucals and compatible with unprotected hydroxy groups or hindered aromatic bromides. It has been shown that the resulting 1-C-aryl(alkyl)-D-glucals are suited for further synthetic manipulation of the enol ether group, including stereoselective hydrogenation, hydroboration-oxidation, or epoxidation. All compounds formed resulted from the attack of the alpha-face of the glucal derivatives by the reagent. The reaction, extended to 1,3-, 1,4-di-, and 1,3,5-tri-bromobenzenes, leads to the corresponding symmetrical di-(tri)-C-glucosylbenzenes. Finally, a sequential di-C-glucosylation of 1,3-dibromobenzene with two different 1-stannylated glucals was obtained.

Growth factor expression in normal and diabetic rats during peripheral nerve regeneration through silicone tubes.

The silicone tube model of regeneration has proved to be an invaluable tool for experimental studies aimed at understanding expression of growth factors during normal and abnormal metabolic states of regeneration. Since the morphological parameters of nerve growth and myelination are well-defined and easily identified in this model, the expression of both diffusible and intracellular-acting growth factors can be readily correlated with the occurrence of these cellular events. These studies facilitate the study of the cellular and molecular events that accompany regeneration. Further, because the sciatic nerve can be traced up to its corresponding neurons, growth factor gene expression can also be studied by in situ hybridization and Northern blotting techniques. This is particularly important in defining the cell source of extracellularly released growth factors. Finally, and most importantly, the regeneration process in the normal or diseased metabolic state (such as diabetes) can be manipulated via the administration of adjuncts to the tube that either promote or inhibit regeneration. Further studies in this regard, and in the identification of growth factors involved and their role during regeneration should shed some light on the pathogenesis and possible means of mitigating or reversing diabetic neuropathy.

Papulacandins and chaetiacandin: a stereoselective route to their basic skeleton by a palladium-mediated arylation of 4,6-O-benzylidene-3-O-tert- butyldimethylsilyl-l-tributyl-stannyl-D-glucal.

Palladium(0)-catalysed coupling of 1,5-anhydro-4,6-O-benzylidene-3-O-tert- butyldimethylsilyl-2-deoxy-1-tributylstannyl-D-arabino-hex-1-eni tol (7) with 3,5-dibenzyloxy-2-bromobenzyl alcohol gave 1,1(2)-anhydro-4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-2-deoxy-1-( 4,6- dibenzyloxy-2-hydroxymethyl-phenyl)-alpha-D-arabino-hexopyranos e (13). The same reaction buffered by sodium carbonate provided 1,5-anhydro-4,6- O-benzylidene-3-O-tert-butyldimethylsilyl-2-deoxy-1-(4,6-dibenzyloxy+ ++-2- hydroxymethyl-phenyl)-D-arabino-hex-1-enitol (11). Stereoselective oxidative spiroacetalisation of 11 provided 1,1(2)- anhydro-4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-1-(4,6-dibenzyl oxy-2- hydroxymethyl-phenyl)-alpha-D-glucopyranose (15), the basic tricyclic structure of papulacandins. In a model study, 15 was converted in three steps into 1,1(2)-anhydro-1-(4,6-dihydroxy-2-hydroxymethylphenyl)-3-O- octadecanoyl-alpha-D-glucopyranose (24), a structural analogue of papulacandin D. Moreover, stereoselective hydroboration-oxidation of 11 furnished 2-(4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-beta-D- glucopyranosyl)-3,5-dibenzyloxy-1-hydroxymethylbenzene (26), the structural skeleton of the chaetiacandin 3.

Synthesis of glycopeptides from the carbohydrate-protein linkage region of proteoglycans.

2,3,4,6-Tetra-O-benzoyl-alpha-D-galactoyranosyl trichloroacetimidate was condensed with benzyl 2,3-O-isopropylidene-beta-D-xylopyranoside to give the corresponding beta-(1----4)-linked disaccharide derivative, which was transformed into 2,3-di-O-benzoyl-4-O-(2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl)- alpha-D-xylopyranosyl trichloroacetimidate. This glycosyl donor was condensed with a set of selectively C,N-protected L-seryl-glycine dipeptide units. Selective deblocking at the C- or N-termini of the glycosylated or non-glycosylated dipeptide segments, and coupling using the mixed-anhydride procedure allowed the construction in high yield of partially or fully glycosylated oligopeptides from the carbohydrate-protein linkage region of proteoglycan.

Increased expression of pp60c-src protein-tyrosine kinase during peripheral nerve regeneration.

Since little is known about the intracellular changes that take place in response to Schwann cell-neuron interactions that occur during neurite outgrowth and myelination, we investigated the expression of a protein-tyrosine kinase, pp60c-src, during peripheral nerve regeneration through a silicone tube. Segments of regenerated nerve, extracted at various times following nerve-transection, showed an induction of in vitro c-src kinase activity as measured by autophosphorylation of immunoprecipitated pp60c-src. This activity occurred at 7 days following nerve transection coincident with the onset of neurite outgrowth in vivo. This kinase activity, which peaked out between 21 and 35 days and decreased thereafter, appeared to be associated with axonal growth and myelination, but not mitogenesis in the tube. Analysis of c-src proteins levels by Western blot showed a similar expression profile as that of the kinase activity. Qualitatively, the expression of an immunoreactive c-src band, migrating slightly slower than pp60, was detected in extracts of regenerating nerve segments as well as in the corresponding L4 and L5 dorsal root ganglia. This protein may be the CNS neuronal-specific form (pp60+) of the c-src protein. In situ hybridization revealed that Schwann cells and sensory and motor neurons associated with the regenerated sciatic nerve were positive for c-src mRNA during regeneration possibly accounting for the increased src protein expression during regeneration. Since the increased expression of pp60c-src in regenerated nerve segments coincides with both axonal sprouting and myelination, our findings suggest that the c-src protein may play a role in Schwann cell-neuron interactions which facilitate the occurrence of these events during regeneration. In addition, although pp60+ is generally not detectable in the mature PNS, our findings show that this protein may be induced during conditions of PNS differentiation which promote neurite outgrowth.

Extracellular fluid conditioned during peripheral nerve regeneration stimulates Schwann cell adhesion, migration and proliferation.

Schwann cell movement and proliferation occur during peripheral nerve regeneration and remyelination. We asked whether soluble factors promoting these activities were present in fluid surrounding rat sciatic nerves regenerating across a 10-mm gap bridged by a silicone tube. In this model, regenerated and remyelinated axons extend across the gap by 28 days following nerve transection and tube implantation. Fluid conditioned by cells participating in nerve regeneration (RCF) was assayed for its ability to promote Schwann cell adhesion, migration and proliferation in vitro. RCFs collected at post-transectional days 1-28 were equally effective in promoting Schwann cell-substratum adhesion. In contrast, the motility-promoting activity of RCF was minimal at 1-2 days following nerve-transection, peaked at 7 days and remained elevated through 21 days. The RCF peak response was 87-fold greater than control. Schwann cell proliferative activity of RCF exhibited peaks of activity at 1 and 14 days post-transection. The biological potency of this fluid for each activity assayed in vitro correlated well with the behavior of Schwann cells chronicled during nerve repair in vivo. These findings suggest that soluble factors promoting Schwann cell adhesion, migration, and proliferation accumulate extracellularly during peripheral nerve regeneration and remyelination.

Ultrastructural and morphometric analysis of long-term peripheral nerve regeneration through silicone tubes.

Light and electron microscopy were used to investigate long-term regeneration in peripheral nerves regenerating across a 10 mm gap through silicone tubes. Schwann cells and axons co-migrated behind an advancing front of fibroblasts, bridging the 10 mm gap between 28 and 35 days following nerve transection. Myelination of regenerated fibres started between 14 and 21 days after transection and occurred in a manner similar to that reported during development. Although these early events were successful in producing morphologically normal-appearing regenerated fibres, complete maturation of many of these fibres was never achieved. Axonal distortion by neurofilaments, axonal degeneration and secondary demyelination were seen at 56 days following nerve transection. These changes progressed in severity with time as more axons advanced through the distal stump towards their peripheral target. Since regeneration occurs in the absence of endoneurial tubes, and because constrictive forces act on the nerve during regeneration, we suggest that these extrinsic factors limit the successful advancement of axons through the distal stump to their target organ.

An altered form of pp60c-src is expressed primarily in the central nervous system.

The expression of two forms of pp60c-src, pp60 and pp60+, was measured in the central nervous system (CNS) and the peripheral nervous system. Both forms were expressed in the CNS, whereas only pp60 was primarily detected in the peripheral nervous system. Our findings suggest that pp60+ may play a role in events important to the CNS.