Cleaved thin-film probes for scanning tunneling microscopy.

We introduce an alternative type of probe for scanning tunneling microscopy (STM). Instead of using a needle-like tip made from a piece of metallic wire, a sharp-edged cleaved insulating substrate, which is initially covered by a thin conductive film, is used. The sharp tip is formed at the intersection of the two cleaved sides. Using this approach a variety of materials for STM probes can be used, and functionalization of STM probes is possible. The working principle of different probes made of metallic (Pt, Co, and CoB), indium-tin oxide, as well as Cu/Pt and Co/Pt multilayer films are demonstrated by STM imaging of clean Cu(001) and Cu(111) surfaces as well as the epitaxial Co clusters on Cu(111).

Controlling immune response and demyelination using highly potent bifunctional peptide inhibitors in the suppression of experimental autoimmune encephalomyelitis.

In this study, we investigated the efficacy of new bifunctional peptide inhibitors (BPIs) in suppressing experimental autoimmune encephalomyelitis (EAE) in an animal model. BPI [e.g. proteolipid protein-cyclo(1,8)-CPRGGSVC-NH2 (PLP-cIBR)] is a conjugate between the PLP139-151 peptide derived from proteolipid protein (PLP) and the cIBR7 peptide derived from domain-1 (D1) of intercellular adhesion molecule-1 (ICAM-1). PLP-cIBR is designed to bind to major histocompatibility complex (MHC)-II and leucocyte function-associated antigen-1 (LFA-1) simultaneously to inhibit the formation of the immunological synapse and alter the differentiation and activation of a subpopulation of T cells, thus inducing immunotolerance. The results show that PLP-cIBR is highly potent in ameliorating EAE, even at low concentrations and less frequent injections. Mice treated with PLP-cIBR had a higher secretion of cytokines related to regulatory and/or suppressor cells compared to phosphate-buffered saline (PBS)-treated mice. In contrast, T helper type 1 (Th1) cytokines were higher in mice treated with PBS compared to PLP-cIBR, suggesting that it suppressed Th1 proliferation. Also, we observed significantly less demyelination in PLP-cIBR-treated mice compared to the control, further indicating that PLP-cIBR promoted protection against demyelination.

Modulation of melphalan resistance in glioma cells with a peripheral benzodiazepine receptor ligand-melphalan conjugate.

Peripheral benzodiazepine receptors (PBRs) are located on the outer membrane of mitochondria, and their density is increased in brain tumors. Thus, they may serve as a unique intracellular and selective target for antineoplastic agents. A PBR ligand-melphalan conjugate (PBR-MEL) was synthesized and evaluated for cytotoxicity and affinity for PBRs. PBR-MEL (9) (i.e., 670 amu) was synthesized by coupling of two key intermediates: 4-[bis(2-chloroethyl)-amino]-L-phenylalanine ethyl ester trifluoroacetate (6) and 1-(3'-carboxylpropyl)-7-chloro-1,3- dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one (8). On the basis of receptor-binding displacement assays in rat brain and glioma cells, 9 had appreciable binding affinity and displaced a prototypical PBR ligand, Ro 5-4864, with IC50 values between 289 and 390 nM. 9 displayed differential cytotoxicity to a variety of rat and human brain tumor cell lines. In some of the cell lines tested including rat and human melphalan-resistant cell lines, 9 demonstrated appreciable cytotoxicity with IC50 values in the micromolar range, lower than that of melphalan alone. The enhanced activity of 9 may reflect increased membrane permeability, increased intracellular retention, or modulation of melphalan's mechanisms of resistance. The combined data support additional studies to determine how 9 may modulate melphalan resistance, its mechanisms of action, and if target selectivity can be achieved in in vivo glioma models.

Peptides derived from ICAM-1 and LFA-1 modulate T cell adhesion and immune function in a mixed lymphocyte culture.

BACKGROUND: The counter receptors intercellular adhesion molecule (ICAM)-1 and lymphocyte function-associated antigen (LFA)-1 are lymphocyte cell surface adhesion proteins the interaction of which can provide signals for T cell activation. This binding event is important in T cell function, migration, and general immune system regulation. The ability to inhibit this interaction with monoclonal antibodies has proved to be therapeutically useful for several allograft rejection and autoimmune disease models. METHODS: Short peptides representing counter-receptor contact domains of LFA-1 and ICAM-1 were examined for their ability to inhibit T cell adhesion and T cell function. RESULTS: Peptides encompassing amino acids Q1-C21 and D26-K50 of ICAM-1, I237-I261 and G441-G466 of the LFA-1 alpha-subunit, and D134-Q159 of the LFA-1 beta-subunit inhibited LFA-1/ICAM-1-dependent adhesion in a phorbol-12,13-dibutyrate-induced model of tonsil T cell homotypic adhesion. This inhibition was specific to the peptide sequence and occurred without stimulation of T cell proliferation. The peptides also were effective in preventing T cell function using a one-way mixed lymphocyte reaction model for bone marrow transplantation. CONCLUSIONS: Our data suggest that these peptides or their derivatives may be useful as therapeutic modulators of LFA-1/ICAM-1 interaction during organ transplants.

Synergistic inhibitory activity of alpha- and beta-LFA-1 peptides on LFA-1/ICAM-1 interaction.

Interactions of cell-adhesion molecule LFA-1 and its ligand ICAM-1 play important roles during immune and inflammatory responses. Critical residues of LFA-1 for ICAM-1 binding are known to be in the I-domain of the alpha-subunit and the I-like domain of the beta-subunit. On the basis of our previous work demonstrating the inhibitory activity of I-domain cyclic peptide cLAB.L on LFA-1/ICAM-1 interaction, here we have explored the activity of I-like-domain peptide LBE on the binding mechanism of cLAB.L. LBE enhances cLAB.L binding to T-cells and epithelial cells. The adherence of T-cells to epithelial monolayers was suppressed by the two peptides. The addition of LBE to the monolayers prior to the addition cLAB.L produced a better inhibitory effect than the reverse procedure. LBE, but not cLAB.L, changes the ICAM-1 conformation, suggesting that LBE binds to ICAM-1 at sites that are distinct from these of cLAB.L and induces improved conformation in ICAM-1 for binding to cLAB.L.

Linear and cyclic LFA-1 and ICAM-1 peptides inhibit T cell adhesion and function.

Short peptides derived from functional proteins have been used in several instances to inhibit activity of the parent proteins. In some cases, stability and efficacy were found to be increased by cyclization of these peptides. Inhibition of interaction of the two cell adhesion counter receptors leukocyte function-associated antigen (LFA)-1 and intercellular adhesion molecule (ICAM)-1 is being studied as a method for modulating autoimmune diseases such as rheumatoid arthritis and for facilitating organ transplantation. Here, several 10-amino acid peptides derived from the contact domains of LFA-1 and ICAM-1 were evaluated for their ability to interfere with intercellular adhesion by T cells and to inhibit a more biologic, mixed lymphocyte reaction. Both linear and cyclic forms of the peptides were effective at inhibiting intercellular adhesion. Cyclic forms were effective at inhibiting T cell activation and proliferation in the mixed lymphocyte reaction.

Modulation of cellular adhesion in bovine brain microvessel endothelial cells by a decapeptide.

The importance of cell adhesion molecules in maintaining the cellular integrity of the endothelial layer is well recognized, yet their exact participation in regulating the blood-brain barrier (BBB) is poorly understood. Both Ca(2+)-dependent and Ca(2+)-independent cell adhesion molecules are found in endothelial cells. In this study, we used immunofluorescence, ELISA, Western blot and cell adhesion assay to identify a Ca(2+)-dependent cell adhesion molecule, E-cadherin, in bovine brain microvessel endothelial cells (BBMECs). Monoclonal anti-E-cadherin antibody specifically interacted with cultured BBMECs and decorated the cellular junctions with a series of punctate fluorescence spots as seen by indirect immunofluorescence using a confocal microscope. The intensity of these fluorescence spots increased after brief treatment with hIFN-gamma or CPT-cAMP. In the cellular extract of BBMECs, a 120 kDa protein was immunoprecipitated with anti-E-cadherin antibody. BBMECs did not react with anti-N-cadherin antibody, but recognized the FITC-labeled LRAHAVDVNG-NH2, a decapeptide generated from the EC-1 domain of N-cadherin, which decorated the lateral margins of the cells with fluorescence spots. A concentration-dependent binding of this decapeptide was also observed in the flow cytometry assay. BBMECs dissociated with trypsin plus Ca2+ were able to reaggregate only in the presence of Ca2+. However, such cell-cell aggregations of BBMECs were prevented by the presence of either anti-E-cadherin antibody or the decapeptide in the assay medium. These results confirm that BBMECs possess a distinct Ca(2+)-dependent cell adhesion mechanism that can be modulated by the decapeptide. This modulation of cell-cell adhesion in BBMECs by the decapeptide is thought-provoking for creating channels for paracellular drug delivery across the BBB.

Comparison of the solution conformations of a cell-adhesive peptide LBE and its reverse sequence EBL.

T-cell adhesion is mediated by an ICAM-1/LFA-1 interaction; this interaction plays a crucial role in T-cell activation during immune response. LBE peptide, which is derived from the beta-subunit of LFA-1, has been shown to inhibit ICAM-1/LFA-1-mediated T-cell adhesion. In this work, we studied the solution conformations of LBE peptide and its reverse sequence (EBL) by NMR, CD and molecular dynamics simulations. Reverse peptides have been used as controls in biological studies. The effect of reversing the sequence of LBE to EBL peptides on their respective conformations is important in understanding their biological properties in vitro or in vivo. The NMR studies for these peptides were carried out in water and in TFE/water solvent systems. In 40% TFE/water, both peptides exhibited helical conformation. CD studies suggested that the LBE exhibits 30% helical conformation, while the EBL exhibits 20% helical conformation. From the NMR and MD simulation studies, it was evident that the peptides exhibited a stable helical conformation; a stable helical structure was found at Leu6 to Leu15 for LBE and at Gly9 to Leu17 for EBL. The helical conformations of LBE and EBL may be in equilibrium with other possible conformers; the other conformers contain loop and turn structures. Both peptides bind to divalent cations because the LBE is derived from the cation-binding region of the LFA-1. This study shows that reversing the peptide sequence did not alter the secondary structure of the corresponding sequence. Hence, caution must be exercised when using reverse peptides as controls in biological studies. This report will improve our ability to design a better inhibitor of ICAM-1/LFA-1 interaction.

Secondary structure of the HAV peptide which regulates cadherin-cadherin interaction.

Cadherins are calcium-binding proteins which are responsible for cell-cell adhesion in biological systems. Cadherins are involved in embryo compaction, neurite growth, cellular differentiation and formation of biological barriers (i.e., intestinal and blood brain barriers). A short linear peptide, LRAHAVDVNG-NH2 (Peptide 1), which contains His-Ala-Val sequence and is derived from the N-cadherin sequence has been shown to inhibit embryo compaction and neurite growth; this is caused by inhibition of the cadherin-cadherin interactions. Peptide 1 was synthesized and its solution conformation was determined by proton nuclear magnetic resonance, circular dichroism and molecular dynamics simulations. These studies indicated that the peptide has an extended structure from residue Leu1 to Asp7, possibly a beta-sheet structure, followed by a beta-turn from Asp7 to Gly10. The X-ray crystal structure of a sequence similar to that of peptide 1 in hemagglutinin indicated that it also has a beta-sheet structure around the HAV sequence, followed by a beta-turn.

Solution structure of a cyclic RGD peptide that inhibits platelet aggregation.

Peptides containing the Arg-Gly-Asp (RGD) sequence can inhibit platelet aggregation. Incorporation of this sequence into a cyclic peptide results in specific binding to a particular integrin. Studies of cyclic RGD peptides show that residues surrounding the RGD sequence have important effects on the selectivity of the peptide to bind with glycoprotein IIb/IIIa (GPIIb/IIIa). In this paper, we elucidate the conformation of cyclo(2,10)Ac-Gly1-Pen2-Gly3-His4-Arg5-Gly6-Asp7 -Leu8-Arg9-Cys10-Ala11-NH2 (1) by NMR and molecular dynamics simulations. This peptide inhibits platelet aggregation in a manner similar to that reported for cyclo(2,10)Gly1-Pen2-Gly3-His4-Arg5-Gly6-Asp7-Le u8-Arg9-Cys10-Ala11-OH (6) (Cheng, S. et al. J. Med. Chem. 1994, 37, 1-8), which is shown to be selective for the GPIIb/IIIa receptor. The cyclic peptide 1 exhibited a major and a minor conformer in solution. In the major conformer, the His4-Arg5-Gly6-Asp7 segment encompasses a 4-->1 hydrogen bond with a distorted type II beta-turn, and the minor conformer has turn-extended-turn. A comparison between the major conformation of this peptide and those of other cyclic RGD peptides suggests the importance of a hydrophobic residue adjacent to the RGD sequence.

Enhancement of transport of D-melphalan analogue by conjugation with L-glutamate across bovine brain microvessel endothelial cell monolayers.

In this paper, the L-glutamate (L-Glu) transport system was targeted to improve the delivery of a model compound, p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD), through the blood-brain barrier (BBB) in vitro cell culture model. D-MOD is an analogue of an antitumor agent D-melphalan. To target the L-Glu transport system, D-MOD was conjugated to L-Glu to give D-MOD-L-Glu conjugate. D-MOD and D-MOD-L-Glu transport properties were evaluated using the bovine brain microvessel endothelial cell (BBMEC) monolayers. The results suggest that D-MOD-L-Glu conjugate permeates through the BBMEC monolayers more readily than the parent D-MOD. The improvement of transport may be due to the recognition of D-MOD-L-Glu by the L-Glu transport system. The transport mechanism was evaluated using several different experiments including: (a) concentration-dependent studies; (b) temperature-dependent studies; (c) substrate inhibition studies; and (d) metabolic inhibitor studies. The D-MOD-L-Glu transport was inhibited by the change of temperature from 37 degrees C to 4 degrees C. At higher concentrations, the transport of D-MOD-L-Glu reached plateau due to saturation. Furthermore, some amino acids (i.e., L-Glu, L-Asp, D-Asp, and L-Gln) inhibited the transport of D-MOD-L-Glu; presumably the conjugate was competing with these amino acids for the same transport system. Metabolic inhibitors (i.e., 2,4-dinitrophenol and sodium azide) suppressed the transport of the conjugate. However, the conjugate was not transported by monocarboxylic acid, dipeptide and neutral amino acid transporters. In conclusion, the L-Glu transport system can be utilized to facilitate a non-permeable drug across the BBB by conjugating the drug with L-Glu amino acid.

Derivatives of melphalan designed to enhance drug accumulation in cancer cells.

The objective of this study was to develop chemical strategies to improve the uptake and accumulation of melphalan (L-Mel and D-Mel), a cytotoxic agent, into cancer cells. Dipeptides synthesized from L- (or D-) Mel and L-glutamic acid (L-Glu) or L-valine (L-Val) and their methyl or ethyl esters (all compounds were trifluoroacetic acid salts) were evaluated for cytotoxicity and cellular uptake using Caco-2 cells, a human colon carcinoma cell line, and RT-2 cells, a rat brain glioma cell line. Treatment of Caco-2 cells with L-Mel or D-Mel (0.5 mg/ml equivalent of melphalan) for 48 h resulted in approximately 50% cell survival. Treatment of the Caco-2 cells with dipeptide derivatives of L-Mel (or D-Mel) (11c-d, 12c-d and 13) caused similar cytotoxicity effects (approximately 50-70% of cell survival). When the cytotoxicities of the esters of L-Mel, D-Mel and their dipeptide derivatives (11a-b, 12a-b and 14) in Caco-2 cells were determined, less than 10% cell survival was observed. Similar results were observed in RT-2 cells. When the cellular uptake properties of these compounds were determined in Caco-2 cell monolayers, L-Glu-L-Mel (12c), L-Glu-D-Mel (12d), and L-Mel-L-Glu (11c) generated slightly lower intracellular levels of L-Mel or D-Mel than when the cell monolayer was treated with the amino acids (L-Mel or D-Mel). In Caco-2 cells treated with 11c, 12c or 12d, low levels of the dipeptides were also detected. Caco-2 cell monolayers treated with D-Mel-L-Glu (11d) or D-Mel-L-Val (13) showed very low levels of the amino acids (L-Mel or D-Mel), but generally higher levels of the dipeptides. In contrast to the amino acids (L-Mel, D-Mel) or the dipeptide derivatives (11c-d, 12c-d and 13), the ester derivatives of the amino acids [L-Mel(OEt), D-Mel(OEt)] or the dipeptides (11a-b, 12a-b and 14) produced 5-20 times higher intracellular concentrations of potentially cytotoxic metabolites (e.g., L-Mel, D-Mel, Mel-containing dipeptides or Mel-containing dipeptide monoesters). L-Mel(OEt), D-Mel(OEt), L-Glu(OEt)-L-Mel(OEt) (12a), L-Glu(OEt)-D-Mel(OEt) (12b), and L-Mel-L-Glu(OEt)2 (11a) accumulated mainly as either L-Mel or D-Mel, and the percentages of L-Mel or D-Mel were 99%, 99%, 90%, 75% and 98% of the total intracellular concentration of potentially cytotoxic agents, respectively. D-Mel-L-Glu(OEt)2 (11b) accumulated as its monoester (> 95%) and D-Mel-L-Val(OMe) (14) accumulated as its dipeptide metabolite (> 98%). Inclusion of Gly-Pro, carnosine, L-Phe or L-Glu did not inhibit uptake of the dipeptide derivatives of L-Mel (or D-Mel) or their esters. These results suggest that the cellular uptake of the dipeptide derivatives of melphalan and their esters is probably via passive diffusion rather than being facilitated by an amino acid transporter or a di/tripeptide transporter. The higher intracellular levels of cytotoxic agents generated from the ester derivatives of the amino acids and the dipeptides are probably due to their higher lipophilicity and the overall neutral charge of the esters and subsequent intracellular formation of the more polar amino acids (L- or D-Mel) and/or Mel-containing dipeptides. Finally, these studies suggest that dipeptides of D-Mel [11b, 11d, 13] have inherent cytotoxicity properties.

Conjugation with L-Glutamate for in vivo brain drug delivery.

In vitro studies have shown that conjugation of a model compound [p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD)] with L-Glu can improve D-MOD permeation through the bovine brain microvessel endothelial cell monolayers (Sakaeda et al., 2000). The transport of this D-MOD-L-Glu conjugate is facilitated by the L-Glu transport system. In this paper, we evaluate the in vivo brain delivery of model compounds (i.e. D-MOD, p-nitro-D-phenylalanine (p-nitro-D-Phe), 5,7-dichlorokynurenic acid (DCKA) and D-kyotorphin) and their L-Glu conjugates. DCKA was also conjugated with L-Asp and L-Gln amino acids. The analgesic activities of D-kyotorphin and its L-Glu conjugate were also evaluated. The results showed that the brain-to-plasma concentration ratio of D-MOD-L-Glu was higher than the D-MOD alone; however, the plasma concentration of both compounds were the same. The plasma concentration of p-nitro-D-Phe-L-Glu conjugate was higher than the parent p-nitro-D-Phe; however, the brain-to-plasma concentration ratio of p-nitro-D-Phe was higher than its conjugate. On the other hand, both DCKA and DCKA conjugates have a low brain-to-plasma concentration ratio due to their inability to cross the blood-brain barrier (BBB). The L-Asp and L-Glu conjugates of DCKA have elevated plasma concentrations relative to DCKA; however, the DCKA-L-Gln conjugate has the same plasma concentration as DCKA. For D-kyotorphin, both the parent and the L-Glu conjugate showed similar analgesic activity. In conclusion, conjugation of a non-permeable drug with L-Glu may improve the drug's brain delivery; however, this improvement may depend on the physicochemical and receptor binding properties of the conjugate.

Effect of conformation on the rate of deamidation of vancomycin in aqueous solutions.

The instability of vancomycin, a glycopeptide antibiotic, limits its shelf-life because the deamidation of its asparagine residue results in the formation of a zwitterion with limited aqueous solubility. Analysis of the pH-rate profile for vancomycin indicates that the deamidation reaction is notably sensitive to the ionic state of the molecule. This observation results in a hypothesis in which the ionic state of vancomycin may influence the conformation of the molecule and therefore affect its reactivity. Two-dimensional nuclear magnetic resonance (NMR), homonuclear Hartmann-Hahn (HOHAHA) and rotating frame Overhauser enhancement spectroscopy (ROESY) information combined with molecular dynamic simulations were used to estimate the apparent conformation of vancomycin in aqueous solution at pH 4 and pH 9 where the molecule exists primarily as a monocation and monoanion, respectively. The apparent conformation for vancomycin at pH 4 is compact, and the proximity of the backbone amide nitrogen to the side chain carbonyl carbon of asparagine is favorable for the rapid formation of the cyclic imide intermediate, thus increasing its reactivity. The apparent conformation for vancomycin at pH 9, however, is expanded in comparison with the conformation at pH 4, and the increase in distance between the reacting atoms leads to slower cyclic imide formation and thus decreased intrinsic reactivity. That cyclic imide formation was rate limiting at both pH values was confirmed by cyclic imide isolation and stability estimation. It becomes apparent from the analysis of the pH-rate and conformational profiles of vancomycin that the deamidation rate of vancomycin is largely influenced by the ionization state of the N-methyl leucine nitrogen.

Synthesis of an esterase-sensitive cyclic prodrug of a model hexapeptide having enhanced membrane permeability and enzymatic stability using a 3-(2'-hydroxy-4',6'-dimethylphenyl)-3,3-dimethyl propionic Acid promoiety.

One of the major obstacles to the development of biologically active peptides as clinically useful therapeutic agents has been their low permeation through biological barriers (e.g., intestinal mucosa, blood-brain barrier) and their metabolic lability (1,2). Overcoming these problems is a very contemporary issue for the development of peptide pharmaceuticals. In the preceding chapter, we have indicated that masking the C- and N-terminal polar functional groups of a peptide through cyclization with an acyloxyalkoxy linker can greatly enhance the membrane permeation and metabolic stability of the linear peptide (3). In this chapter, we wish to report a method for the preparation of esterase-sensitive cyclic prodrugs of peptides by taking advantage of a unique "trimethyl lock"-facilitated lactonization system (Fig. 1). Substituted phenol propionic acid derivatives such as 2, upon unmasking of the hydroxyl group, undergo a facile spontaneous intramolecular cyclization to release the moieties attached to the carboxyl functional group (Fig. 1) (4-6). The facile cyclization reaction is the result of the "trimethyl lock", which was shown earlier to increase the rate of the cyclization reaction in the order of 10(5-7) (4-7). The result of such facilitation is that compound 2 has a half-life of only approximately 100 s at room temperature in aqueous solution (8,9). Such systems have been used to develop prodrugs of amines and alcohols (8-10) and redox-sensitive protecting groups of amines (11). Fig. 1. The design of an esterase sensitive prodrug system for the cyclic deriva-tization of peptides.

Synthesis of an esterase-sensitive cyclic prodrug of a model hexapeptide having enhanced membrane permeability and enzymatic stability using an acyloxyalkoxy promoiety.

The clinical development of orally active peptide drugs has been limited by their unfavorable physicochemical characteristics (e.g., charge, hydrogen bonding potential, size), which prevent them from permeating biological barriers such as the intestinal mucosa, and also their lack of stability against enzymatic degradation (1-12). Unfortunately, many of the structural features of peptides (i.e., the N-terminal amino group and C-terminal carboxyl group, and side chain carboxyl, amino, and hydroxyl groups) that bestow upon the molecule affinity and specificity for its pharmacological receptor severely restrict its ability to permeate biological barriers and make the molecules substrates for peptidases. Therefore, successful oral delivery of peptides depends on strategies designed to alter the physicochemical characteristics of these potential drugs without changing their biological activity in order to circumvent the intestinal epithelial cells.

Inhibition of the adherence of T-lymphocytes to epithelial cells by a cyclic peptide derived from inserted domain of lymphocyte function-associated antigen-1.

Tissue inflammation is characterized by aggravated leukocyte infiltration into the sites of inflammation. The mechanism requires the interactions of leukocyte adhesion-molecules and their ligands in the inflamed tissues. In this study, we demonstrate that a cyclic peptide cLAB.L [cyclol, 12-PenlTDGEATDSGC], derived from the "inserted" or I-domain of LFA-1 is able to inhibit the adherence of T-lymphocytes to the epithelial cell monolayers. This inhibition has been thought to involve the disruption of LFA-1/ICAM-1 interaction. The heterotypic adhesion of phorbol ester-activated Molt-3 cells and IFN-gamma-induced Caco-2 monolayers was inhibited upon treatment of the monolayers with monoclonal antibodies (MAbs) to adhesion molecules or with cLAB.L peptide. The adhesion can be inhibited by MAbs to ICAM-1, ICAM-2, and VCAM-1, and cLAB.L peptide in a concentration-dependent manner. However, none of the individual uses of these molecules led to a total inhibition. The inhibitory activity of cLAB.L is greatly reduced by low temperature and the absence of cell activation. Treatment of cLAB.L peptide may trigger an early event of apoptosis on activated but not on non-activated Molt-3 cells; no indication of peptide-induced apoptosis was found on Caco-2 cells. Taken together, data from this work suggest that cLAB.L may have applications to direct cell-targeted delivery during tissue inflammation.

Conformational study of cyclo[Gln-Trp-Phe-Gly-Leu-Met] as NK-2 antagonist by NMR and molecular dynamics.

Cyclo[Gln-Trp-Phe-Gly-Leu-Met] (1) is a selective peptide antagonist of NK-2 receptors. The conformational analysis of this peptide was conducted using nuclear magnetic resonance (NMR) and molecular dynamics. This study improves understanding of the neurokinin ligand-receptor interactions. Two-dimensional Homonuclear Hartmann-Hahn (2D-HOHAHA) and rotating frame Overhauser enhancement spectroscopy (2D-ROESY) were used to assign all the protons and to obtain through-space proton-proton interactions. ROE (rotating frame Overhauser enhancement) constraints molecular dynamics were done to find the conformation which is consistent with the NMR data. Two beta I (or beta V') turns around Trp-2-Phe-3 and around Leu-5-Met-6 are found in this peptide which are represented by models. The conformation of this peptide is also compared with the non-peptide NK-2 antagonist SR-48968 (2).

[Alloarthroplastic experience with the Wagner double-cup (author's transl)]. / Alloarthroplastische Erfahrungen mit der Zweischalenprothese nach Wagner.

Today, alloarthroplasty is one of the many standard therapeutical procedures in osteoarthrosis of the hip, or coxarthrosis. With this operation, many patients can be completely relieved from pain and regain their ability to walk to an excellent degree. However, long-term observations have shown that the stability of the prostheses does not always correlate with the life expectation of the operated patient. Dislocations and states of irritation, over and above infections, may necessitate repeat surgery. It must be borne in mind that such repeated operations are by no means certain to produce safe results. Hence, it is recommended to employ for the first implantation a prosthesis model which offers a better chance of interchangeability. This possibility is supplied by the double-cup prosthesis of the type described by Wagner and other authors. Certain limitations are imposed by the prosthetic form and by the surgical approach; these limitations should be taken into account when choosing the double-cup prosthesis.