Biomedical subjects

H Kessler

Publications and source records attributed to H Kessler.

At least 19 recordsLinked to original sources

PubMed1996

Systematic mutational analysis of the receptor-binding region of the human urokinase-type plasminogen activator.

The amino-terminal fragment of human uPA (ATF; amino acids 1-135), which contains the binding site for the uPA receptor (uPAR, CD87) was expressed in the yeast Saccharomyces cerevisiae. Recombinant yeast ATF, modified and extended by an amino-terminal in-frame insertion of a His6 tract, was purified from total protein extracts by nickel chelate affinity chromatography and shown to be functionally active since it efficiently competes with uPA for binding to cell-surface-associated uPAR. The ATF expression plasmid served as a template for the construction of a series of site-directed mutants in order to define those amino acids that are important for binding to uPAR. All mutant ATF proteins but one (deletion of Ser26) were expressed in a stable form (about 20-30 ng/mg total protein) and the binding capacity of each mutant was tested by a uPA-ligand binding assay employing recombinant uPAR immobilized to a microtiter plate. Each of the 11 amino acids of loop B of the binding region of uPA (amino acids 20-30) were individually substituted with alanine. Lys23, Tyr24, Phe25, IIe28, and Trp30 were important determinants for uPAR binding. A systematic alanine scan was also performed with chemically synthesized linear peptides spanning amino acids 14-32 of ATF. Comparable results to those with the yeast ATF mutants were obtained. In a different set of experiments, those amino acids of the uPAR-binding region of uPA that are only conserved between man and baboon but not in other species were altered: whereas substitution of Thr18 by alanine or Asn32 by serine had hardly any effect, replacement of Asn22 by tyrosine and Trp30 by arginine (both positions are strictly conserved in other mammals) led to ATF variants incapable of interacting with human uPAR. Deletion of either Val20, Ser21, Lys23, His29 or Val20 plus Ser21, respectively, also generated non-reactive ATF mutants. Finally, Lys23 in ATF was substituted with certain amino acids: whereas the replacement of Lys23 by alanine, histidine or glutamine generated ATF variants with moderate uPAR-binding activity, the introduction of a negatively charged amino acid (exchange of Lys23 by glutamic acid) completely abolished uPAR-binding activity. The results presented for the ATF mutants and uPA-derived peptides may provide clues necessary to establish the nature of the physical interaction of uPA with its receptor and may help to develop uPA-derived peptide analogues as potential therapeutic agents to block tumor cell-associated uPA/uPAR interaction.

Amino Acid Sequence↗

PubMed1996

In vitro restoration of T cell immune function in human immunodeficiency virus-positive persons: effects of interleukin (IL)-12 and anti-IL-10.

Cells from human immunodeficiency virus (HIV)-positive patients were evaluated for their in vitro responsiveness to recall antigen, alloantigen, and phytohemagglutinin (PHA) following the in vitro addition of interleukin (IL)-12 or anti-IL-10. Three-color flow cytometric analysis of CD4 and CD8 subsets was done to determine whether specific in vivo alterations in cell surface markers are associated with in vitro function changes. The results demonstrated a hierarchical response pattern to recall antigens versus alloantigen versus PHA, and these in vitro responses were associated with the number and activation status of CD4 cells. The in vitro addition of IL-12 or anti-IL-10 could restore antigen responses (HIV envelope peptides or influenza) in patients with 200-500 CD4 cells/microL; however, in patients with < 200 CD4 cells/microL, this improved response was limited to the influenza response. Studies of this nature may provide important insights into the role of cytokines in the natural history of HIV disease, and they suggest that immune therapy of this type may be most effective in patients who have more preserved immune systems.

Antibodies↗

PubMed1996

Expression of c-erbB2 oncogene product in different tumours and its standardised evaluation.

In order to evaluate the reliability and reproducibility of the immunohistochemical demonstration of the c-erbB2 oncoprotein and define criteria for positivity assessment in paraffin-embedded material from different tumours, we examined immunohistochemically 79 gastric, 100 colorectal and 20 bladder carcinomas using 5 different c-erbB2 antibodies. To determine criteria for the evaluation of c-erbB2 positivity, the observed immunohistochemical staining was compared with the actual protein prevalence, determined by Western blot analysis of each tumour. The comparison of protein levels obtained from Western blot with corresponding immunohistochemistry revealed that positive staining can only be considered to be specific for c-erbB2 oncoprotein if: (i) there is a definite staining along the tumour cell membrane with no predominant immunoreactivity inside the tumour cell cytoplasm, (ii) the intensity of immunoreactivity is comparable with a positive standard (e.g. in this study breast carcinoma) and (iii) the surrounding normal, non-neoplastic tissue shows no immunoreactivity. The graduation of staining intensities did not correlate to the actual amount of protein detected in Western blots. This study stresses the importance of defined and controlled criteria for evaluation of c-erbB2 oncoprotein positivity in immunohistochemistry.

Blotting, Western↗

PubMed1995

The tolerability and pharmacokinetics of N-butyl-deoxynojirimycin in patients with advanced HIV disease (ACTG 100). The AIDS Clinical Trials Group (ACTG) of the National Institute of Allergy and Infectious Diseases.

Twenty-nine patients were enrolled in a phase I dose-escalating tolerance trial of N-butyl-deoxynojirimycin, an alpha-glucosidase I inhibitor that inhibits human immunodeficiency virus (HIV)-1 replication by altering glycosylation of gp120. Dosing was begun at 8 mg/kg/day and subsequent doses were 16, 32, 48, and 64 mg/kg/day. The maximum tolerated dose was not achieved because of slow accrual and because the study was stopped after the finding of cataracts in initial long-range rat toxicology studies. These cataracts were later shown to be transient and not found in other animals. The most common side effects were gastrointestinal, with diarrhea and flatulence occurring in most subjects, which seemed to partially improve on a modified diet that excluded complex carbohydrates. Grade III elevations in liver function tests were seen in two patients. Grade III leukopenia and neutropenia were seen in seven patients, but were only severe enough in two to require discontinuation. No significant trends in CD4 cell counts or HIV-1 p24 levels were noted.

1-Deoxynojirimycin↗

PubMed1995

Influence of serine in position i on conformation and dynamics of reverse turns.

NMR spectroscopy has been employed for the conformational analysis of the cyclic hexapeptide cyclo(-D-Pro1-Ala2-Ser3(Bzl)-Trp4-Orn5(Z)-Tyr 6-) with and without protecting groups on Ser3 and Orn5. This peptide sequence was derived from the active loop sequence of the alpha-amylase inhibitor Tendamistat (HOE 467). The aim was to investigate the role of serine in position i of a standard beta-turn on the conformation and stabilization of this turn. Based on distance and torsion constraints from 2D NMR spectroscopic measurements in DMSO-d6 solution, structure refinement was accomplished by restrained molecular dynamics (MD) simulations in vacuo and in DMSO. The analysis of both structures in solution reveals a considerable effect of the unprotected serine sidechain on the adjacent beta-turn conformation. While in the protected peptide with Ser3(Bzl) a beta II-turn is observed between Trp4 and Orn5, the deprotected compound reveals a beta I-turn in this region. The beta I-turn is stabilized by a backbone-sidechain hydrogen bond from Orn5N alpha H to Ser3O gamma. Comparisons with other NMR-derived solution structures of cyclic model peptides and in some protein structures from literature reveal a general structural motif in the stabilization of beta I-turns by serine in the i position through backbone-sidechain interactions.

Amino Acid Sequence↗

PubMed1994

Selective recognition of cyclic RGD peptides of NMR defined conformation by alpha IIb beta 3, alpha V beta 3, and alpha 5 beta 1 integrins.

The binding of purified fibrinogen receptor alpha IIb beta 3, vitronectin receptor alpha V beta 3, and fibronectin receptor alpha 5 beta 1 to their corresponding ligands in solid-phase binding assays was used to examine the inhibitory activity of various linear and cyclic penta- and hexapeptides of different conformation containing RGD or RAD sequences. Cyclic peptides with different defined backbone conformations were designed by introducing a single D-amino acid or a proline at different positions in the ring. The data were calibrated for alpha IIb beta 3 integrin incorporated into a planar lipid bilayer by a physical method (total internal reflection fluorescence microscopy) which yielded KD = 1.7 microM for a linear RGD peptide and KD = 0.03 microM for fibrinogen. With this integrin, three cyclic hexapeptides ([GRGDFL], [ARGDFV], [GRGDFV]) were 2-4-fold more inhibitory than the linear GRGDS peptide in solid-phase assays and showed similar inhibition as the fibrinogen ligand. Six peptides had the same or a 2-fold lower activity as the linear reference peptide, and three peptides were up to 7-fold less active. Replacement of Arg or Asp by their stereoisomers or Gly by Ala resulted in a 100-1000-fold reduction in activity. With the two other integrins, a single cyclic pentapeptide [RGDFV] was 10-fold more active (alpha V beta 3) or equal in activity (alpha 5 beta 1) to linear GRGDS, while all of the other cyclic peptides were moderately or distinctly less active. Changes in the RGD sequence caused a less dramatic reduction in binding strength for alpha V beta 3 and alpha 5 beta 1 than for alpha II beta 3. Inhibitory activity was compared with the distance between the C beta atoms of Arg and Asp residues as determined by NMR and indicated that the optimum distance is in the range of 0.75-0.85 nm for alpha IIb beta 3 and at or below 0.67 nm for alpha V beta 3 and alpha 5 beta 1. This indicates that alpha IIb beta 3 less sensitive to variations in the RGD backbone structure and can accommodate a larger distance than the integrins alpha V beta 3 and alpha 5 beta 1.

Amino Acid Sequence↗

PubMed1994

Structure of cobra cardiotoxin CTX I as derived from nuclear magnetic resonance spectroscopy and distance geometry calculations.

The structure of cardiotoxin CTX I from Naja naja atra has been investigated by NMR spectroscopy. Sequence specific resonance assignments have been obtained for all backbone protons as well as for most side-chain protons. Distance geometry calculations were carried out using a metric matrix DG program. A total of 715 NOE constraints, 27 phi angle constraints and a list of the hydrogen bond donors were used for the metric matrix DG calculations and refinement. The average pairwise r.m.s.d. of the resulting structures was 1.01 A for the backbone heavy atoms, and 1.69 A for all heavy atoms. The protein is rich in beta structure and consists of a large triple-stranded, antiparallel beta sheet as well as a short double-stranded, antiparallel beta sheet. Non-regular hydrogen bonding is found between side-chains of the carboxy-terminal end and the rest of the core region. The structure is discussed in terms of evolutionary aspects as well as recent investigations about the biological function and active site.

Amino Acid Sequence↗

PubMed1994

Comparison of the conformation of active and nonactive backbone cyclic analogs of substance P as a tool to elucidate features of the bioactive conformation: NMR and molecular dynamics in DMSO and water.

The conformations of two backbone-cyclized substance P analogs as derived from 1H NMR and molecular dynamics simulations carried out in DMSO and water are described. The method of floating chiralities is used in the simulations to facilitate the diastereotopic assignment of methylene protons. One of the analogs, cyclo-[-(CH2)3-NH-CO-(CH2)4-Arg-Phe-Phe-N-]-CH2-CO-Leu-Met-NH2, is a highly active, selective agonist for the NK-receptor, while the other, cyclo[-(CH2)2-NH-CO-(CH2)2-Gly-Arg-Phe-Phe-N-]-CH2-CO-Leu-Met-NH2, is inactive. Both analogs contain cyclic ring systems of the same size, varying in only the number of amide linkages. From the conformational analysis, the lack of activity can be attributed to the introduction of too much constraint into the ring system. This has an effect on the topological array of the important residues Arg-Phe-Phe. The results presented here are compared with biologically active analogs previously examined. The differences between conformations of active and inactive compounds are used to develop insight into the conformational requirements for biological activity.

Amino Acid Sequence↗

PubMed1994

Mannose transporter of Escherichia coli. Backbone assignments and secondary structure of the IIA domain of the IIABMan subunit.

The mannose transporter of Escherichia coli consists of two transmembrane and one peripheral protein subunit. The complex acts by a mechanism which couples translocation of the substrate with substrate phosphorylation. The peripheral IIABMan is a homodimer. The IIABMan monomer itself contains two domains which are linked by an Ala-Pro-rich hinge and which are both transiently phosphorylated at histidyl residues. The IIA and IIB domains can be separated by limited proteolysis. The IIA domain has a dimer molecular mass of 2 x 14 kDa. Almost complete 1H, 13C, and 15N NMR assignments of the backbone resonances of IIAMan have been achieved using 3D and 4D double-and triple-resonance techniques. Secondary structure elements were derived from NOE data. The IIA domain consists of a central beta-sheet of four parallel and one antiparallel strand (strand order 5 4 3 1 2) with helices on both sides of the sheet. The active-site His-10 is located in a loop at the C-terminus of beta-strand 1. This loop and the loop after strand 3 are at the topological switch point of the sheet.

Amino Acid Sequence↗

PubMed1994

The glucose transporter of Escherichia coli. Assignment of the 1H, 13C and 15N resonances and identification of the secondary structure of the soluble IIB domain.

The IICBGlc subunit of the Escherichia coli glucose transporter consists of two domains, the membrane-spanning IIC domain, and the hydrophilic IIB domain which contains the phosphorylation site (Cys421). A functional form of the IIB domain was over-expressed separately and isotopically labelled with 13C and 15N. A variety of 15N-edited and 13C, 15N triple-resonance NMR experiments yielded a nearly complete assignment of the 1H, 13C and 15N resonances. Based on the evaluation of conformationally sensitive parameters including NOE effects, scalar couplings and chemical shifts, the secondary structure of the IIB domain is presented. The protein is comprised of four beta-strands forming an antiparallel beta-sheet, two larger alpha-helices at the N- and C-termini and a smaller helical structure of residues 52-58.

Amino Acid Sequence↗

PubMed1994

Pharmacophore refinement of gpIIb/IIIa antagonists based on comparative studies of antiadhesive cyclic and acyclic RGD peptides.

Structurally guided design approaches to low-molecular-weight platelet aggregation antagonists addressing the platelet-associated heterodimeric cell surface receptor gpIIb/IIIa rely on comparative studies of an ensemble of conformationally and biologically characterized compounds, since no high-resolution structure of the receptor system is available. We report a classical indirect and comparative pharmacophore refinement approach based on a series of small cyclic Arg-Gly-Asp (RGD) peptides as gpIIb/IIIa-fibrinogen interaction antagonists. These peptides have previously been investigated as potent and selective tumor cell adhesion inhibitors. The definition of geometrical descriptors classifying the RGD peptide conformations and their subsequent analysis over selected RGD- and RXD-containing protein structures allows for a correlation of distinct structural features for platelet aggregation inhibition. An almost parallel alignment of the Arg and Asp side chains was identified by a vector analysis as being present in all active cyclic hexa- and pentapeptides. This orientation is induced mainly by the constraint of backbone cyclization and is not of any covalent tripeptide-inherent origin, which was rationalized by a 500 ps high-energy MD simulation of a sequentially related linear model peptide. The incorporation of the recognition tripeptide Arg-Gly-Asp into the cyclic peptide templates acted as a filter mechanism, restricting the otherwise free torsional relation of both side chains to a parallel orientation. Based on the derived results, several detailed features of the receptor binding site could be deduced in terms of receptor complementarity. These findings should govern the design of next-generation compounds with enhanced activities. Furthermore, the complementary stereochemical characteristics of the substrate can be used as boundary conditions for pseudoreceptor modelling studies that are capable of reconstructing a hypothetical binding pocket, qualitatively resembling the steric and electronic demands of gpIIb/IIIa. It is interesting to note that these features provide clear differentiation to requirements for inhibition of alpha v beta 3 substrate binding. This can account for the extremely high selectivity and activity of some of our constrained peptides for either the alpha IIb beta 3 or the alpha v beta 3 receptor.

Amino Acid Sequence↗

PubMed1994

Coupling constants again: experimental restraints in structure refinement.

Utilization of coupling constants as restraints in computational structure refinement is reviewed. In addition, we address the effect of conformational averaging and examine different approaches to apply the restraints when the experimental observable is obviously a result of averaging. Here, two different computational methods are compared. The simulation of a single structure with time-dependent restraints produces results very similar to those obtained with the calculation of numerous copies of the molecule (an ensemble of structures) and ensemble averaging. The advantages and disadvantages of the two methods are illustrated with simulations of cyclosporin A, for which 117 NOEs and 62 homo- and heteronuclear coupling constants have been measured.

Chemical Phenomena↗

PubMed1994

Enhanced sensitivity of rapidly exchanging amide protons by improved phase cycling and the constructive use of radiation damping.

Two alternative, general methods are presented that lead to enhanced signal intensity of rapidly exchanging protons. Both methods work by avoiding saturation of the water resonance, and are convenient to implement since they do not use any selective pulses. One method carefully chooses proton pulse phases and gradient strength and position in such a way that the water is realigned along the +z axis at the beginning of the acquisition time. An alternative method is proposed for cases where the pulse sequence does not allow such phase cycling. The latter uses radiation damping to bring water back to the +z axis 20-30 ms after acquisition. The methods are applied to the triple-resonance experiments HNCA, HNCO and HN(CO)CA. Both methods require pulsed B0 field gradients and can result in higher signal intensity by a factor of two or more.

Magnetic Resonance Spectroscopy↗

PubMed1994

Use of 19F nuclear magnetic resonance spectroscopy for probing substitution of Al for Ga in cloverite.

The synthesis of gallophosphate cloverite in the presence of aluminium leads to a partial substitution of Ga by Al. 27Al and 19F magic-angle spinning nuclear magnetic resonance (MAS NMR) studies on this material confirm this substitution. 27Al MAS NMR shows a broad line at 30 ppm which can be attributed to tetrahedrally coordinated Al in the solid. The 19F MAS NMR results are more complex. The spectrum presents five lines between -68 ppm and -95 ppm which can be assigned to fluorine in D4Rs with variable composition in Ga and Al. Two other lines, at -145.6 ppm and -178.2 ppm, may correspond to impurities and to a fluorine atom in a pseudo-sodalite cage environment, respectively.

Aluminum Compounds↗

PubMed1994

Cyclic RGD peptides ameliorate ischemic acute renal failure in rats.

Renal tubular obstruction is an important contributor to the pathophysiology of acute renal failure. Based on the previous findings of the role played by arginine-glycine-aspartic acid (RGD) recognizing integrins in tubular obstruction, this study examined the effect of RGD peptides on the course of ischemic acute renal failure in rats. For in vivo studies, animals were subjected to 45 minutes of unilateral renal ischemia with contralateral nephrectomy, and cyclic RGD peptides or a linear biotinylated RGD peptide were injected systemically after the release of renal artery clamp. In vitro studies compared the potency of the peptides in inhibiting BS-C-1 cell-matrix and cell-cell adhesion. Two novel cyclic RGD peptides utilized in these studies showed different inhibitory potency in preventing cell-matrix adhesion: cyclic RGDDFV was a highly potent in vitro inhibitor of BS-C-1 cell-matrix adhesion, whereas cyclic RGDDFLG was less potent. In cell-cell adhesion assays, however, both peptides were equipotent. Despite the differences in inhibiting cell-matrix adhesion, a single systemic administration of either peptide improved creatinine clearance postoperatively and accelerated recovery of renal function with a rank order: cyclic RGDDFV > or = RGDDFLG >> RDADFV (inactive control). These findings represent the first in vivo demonstration of the effectiveness of cyclic RGD peptides in ameliorating ischemic acute renal failure, and suggest that in this setting RGD peptides predominantly inhibit cell-cell adhesion, whereas inhibition of cell-matrix adhesion is of lesser significance.

Acute Kidney Injury↗

PubMed1994

Synthesis and solution structure of an S-glycosylated cyclic hexapeptide. Evidence for conformational change induced by glycosylation.

Synthesis and conformational analysis of the S-glycosylated cyclic hexapeptide cyclo(-D-Pro1-Phe2-Cys3(tetra-O-acetyl-beta-D-galactopyranosyl)-++ +Trp4-Lys(Z)5- Phe6-) I was carried out to examine the influence of a saccharide residue in position i of a standard beta-turn on the formation of reverse turns and on the biological activity. Synthesis was carried out in the liquid phase employing a galactosylated cysteine building block. The cyclization reagents DPPA/NaHCO3 avoided high dilution conditions. Spectroscopic data were extracted from homo- and heteronuclear 2D-NMR techniques (TOCSY, NOESY, HMQC, HMQC-TOCSY, HMBCS-270). For structural refinement restrained molecular dynamics (MD) simulations in vacuo and with explicit DMSO as solvent were performed. Finally, simulations in DMSO without experimental restraints provided insight in stability and dynamics of the structural model. A comparison of the S-glycosylated Cys3 peptide with the analogous Thr3 peptide exhibits a similar overall conformation of the hexapeptide [beta II' D-Pro-Phe and another beta-turn about Trp4-Lys5(Z)]. However, the latter shows a distinct dynamic flip beta I, beta II in the glycopeptide, whereas the Thr-analogue only populates beta I. This influence is attributed to a beta I stabilizing effect of a hydrogen bridge of Thr-O gamma in position i to the NH of the amino acid in position i + 2, which is lacking in the glycosylated compound.

Amino Acid Sequence↗

PubMed1994

Coupling constants and hydrogen bonds as experimental restraints in a distance geometry refinement protocol.

A refinement procedure commonly used after distance geometry calculations has been modified to include the use of experimental restraints from coupling constants and hydrogen bonds. Fewer experimental distance constrains (NOEs) are available for peptides as compared to proteins; therefore it is important to incorporate other conformational restraints into refinement methods. The procedure was applied to a cyclic hexapeptide containing two thioamide substitutions, cyclo(-Gly1-Pro2-Phe3 psi [CS-NH]Val4-D-Phe5-Phe6 psi [CS-NH]-). Distance geometry was used to study this peptide, since no potential energy parameters, required in molecular mechanics or dynamics calculations, are available for the thioamide. This is a general problem in the study of peptidomimetics; physiochemical properties of heteroatoms are required within a self-consistent force field. Here, we illustrate the use of metric matrix distance geometry followed by refinement with distance and angle driven dynamics (DADD). We also introduce a new way to handle intramolecular hydrogen bonds by an additional very small and flexible restraint. This method is a viable alternative for the conformational examination of peptides and peptidomimetics. The modifications described here should also find use in the conformational determination of flexible regions of proteins, where the number of NOEs are limited.

Amino Acid Sequence↗

PubMed1994

The pseudo-beta I-turn. A new structural motif with a cis peptide bond in cyclic hexapeptides.

Synthesis and conformational analysis of three cyclic hexapeptides cyclo(-Gly1-Pro2-Phe3-Val4-Xaa5-Phe6), Xaa = Phe (I), D-Phe (II) and D-Pro (III), were carried out to examine the influence of proline on the formation of reverse turns and the dynamics of hydrophobic peptide regions. Assignment of all 1H and 13C resonances was achieved by homo- and heteronuclear 2D-NMR techniques (TOCSY, ROESY, HMQC, HMQC-TOCSY and HMBCS-270). The conformational analysis is based on interproton distances derived from ROESY spectra and homo- and heteronuclear coupling constants (E.COSY, HETLOC and HMBCS-270). For structural refinements restrained molecular dynamics (MD) simulations in vacuo and in DMSO were performed. Each peptide exhibits two conformations in DMSO solution due to cis-trans isomerism about the Gly-Pro peptide bond. Surprisingly the cis-Gly-Pro segment in the minor isomers is not involved in a beta VI-turn, but forms a turn structure with cis-Gly-Pro in the i and i + 1 positions. Although no stabilizing hydrogen bond is found in this turn, the phi- and psi-angles closely correspond to a beta I-turn [Pro2: phi(i + 1) -60 degrees, psi(i + 1) -30 degrees; Phe3: phi(i + 2) -100 degrees, psi(i + 2) -50 degrees]. Hence we call this structural element a pseudo-beta I-turn. As expected, in the dominating all-trans isomers proline occupies the i + 1 position of a standard beta I-turn. Therefore, cis-trans isomerization of the Gly1-Pro2 amide bond only induces a local conformational rearrangement, with minor structural changes in other parts of the molecule. However, the geometry of the other regions is affected by the chirality of the i + 1 amino acid for both isomers (beta I for Phe5, beta II' for D-Phe5 or D-Pro5).

Computer Simulation↗