bluuebunny/crossref_metadata_2025 huggingface dataset:
HLA class I polymorphism, as characterised by PCR‐SSOP, in a Brazilian exogamic population
Authors: Braun‐Prado K. , Vieira Mion A.L. , Farah Pereira N. , Culpi L. , Petzl‐Erler M.L.
Published: March 2003

Acknowledgments:

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and FUNPAR‐PRPPG/UFPR. We thank the Human Molecular Genetics Working Group, University of Paraná, for comments.

HLA‐A, ‐B and ‐C genes were analysed in the population living in the metropolitan region of Curitiba, the main city of Paraná State, southern Brazil, to provide data for studies and applications in HLA‐related fields, and to contribute to the understanding of human microevolution. Heterozygosity is high (95–99%) for all three loci. Frequencies for most alleles and haplotypes of sub‐Saharan African and of European ancestry presented a clear gradient between the White, Mulatto and Black sub‐populations. Among Whites, the four most common haplotypes were A*01‐Cw*07‐B*0801, A*02‐Cw*07‐B*07, A*11‐Cw*0401‐B*35 and A*03‐Cw*0401‐B*35. Their frequencies ranged from 5.6% to 3.0%. In the Mulatto sub‐population, six haplotypes presented very similar frequencies, close to 2.0–2.4%: A*02‐Cw*03‐B*15, A*02‐Cw*0401‐B*35, A*02‐Cw*07‐B*07, A*03‐Cw*0401‐B*35, A*30‐Cw*17‐B*4201, A*68‐Cw*03‐B*15. Haplotype A*30‐Cw*17‐B*4201 was found to be very common (6.6%) in the Black sub‐population. Admixture estimate revealed the relative contributions of Europeans, sub‐Saharan Africans and Amerindians to this populations which were, respectively, 94%, 3% and 3% for the White sub‐population, 57%, 39% and 4% for the Mulatto sub‐population, and 25%, 74% and 1% for the Black sub‐population.
Identification of MICA alleles with a long Leu‐repeat in the transmembrane region and no cytoplasmic tail due to a frameshift‐deletion in exon 4
Authors: Obuchi N. , Takahashi M. , Nouchi T. , Satoh M. , Arimura T. , Ueda K. , Akai J. , Ota M. , Naruse T. , Inoko H. , Numano F. , Kimura A.
Published: March 2003

Abstract: MHC class I chain‐related gene A (MICA) is located close to HLA‐B gene and expressed in epithelial cells. The MICA gene is reported to be highly polymorphic as are the classical class I genes. To further assess the polymorphism in the MICA gene, we analyzed a total of 60 HLA‐homozygous cells for the sequences spanning exons 2–6. In the analysis, four new MICA alleles were identified and six variations were recognized in exon 6. MICA*017, which was identified in three HLA‐B57 homozygous cells (DBB, DEM and WIN), differed from MICA*002 in exon 3 and had a guanine deletion at the 3′ end of exon 4. MICA*015 identified in an HLA‐B45 homozygous cell (OMW) also had the same deletion that causes a frameshift mutation resulting in complete change of the transmembrane region and premature termination in the cytoplasmic tail; these alleles have a long hydrophobic leucine‐rich region instead of the alanine repeat in the transmembrane region and terminate at the second position in the cytoplasmic domain. The frameshift deletion was found only in HLA‐B45‐ or ‐B57‐positive panels tested, suggesting a strong linkage disequilibrium between the deletion and B45 or B57. MICA*048, which was different in exon 5 from MICA*008, was identified in an HLA‐B61 homozygous cell (TA21), while MICA*00901 identified in HLA‐B51 homozygous cells (LUY and KT2) was distinguished from MICA*009 by exon 6.
Allelic distribution of HLA‐B*5 in HLA‐B5‐positive Israeli patients with Behçet’s disease
Authors: Paul M. , Klein T. , Krause I. , Molad Y. , Narinsky R. , Weinberger A.
Published: March 2003

Abstract: In order to investigate the sub‐typing of the B5 antigen in Israeli (Jewish and Arabic) patients with Behçet’s disease (BD) allele‐specific genotyping of B51 and B52 alleles was performed in Israeli BD patients and healthy controls. Among the HLA‐B51‐positive BD patients, B*5101 was found to be the predominant allele, identified in 62% of all BD patients and 78% of Jewish BD patients. HLA‐B*5101 was also the predominant allele in HLA‐B51‐positive healthy controls. HLA‐B*5108 and B*5104 alleles were identified in 23% and 15% of B51‐positive BD patients, respectively. The HLA‐B*5201 allele was identified in all HLA‐B52‐positive patients and controls. Our study suggests that both HLA‐B*5101 and HLA‐B*5201 are the dominant alleles of HLA‐B5 in Israeli BD patients.
Modulation of T cell response to hGAD65 peptide epitopes
Authors: Masewicz S. A. , Papadopoulos G. K. , Swanson E. , Moriarity L. , Moustakas A. K. , Nepom G. T.
Published: March 2003

Abstract: Human CD4 T cell responses to an epitope of hGAD65 (GAD = glutamic acid decarboxylase), residues 555–567, are modulated by interaction with an altered peptide ligand containing modifications at TCR contact residues. Using different HLA‐DR4 molecules with polymorphisms at sites corresponding to peptide binding pockets p1 and p9, we tested the effect of additional modifications in the altered peptide ligand (APL) designed to increase the avidity of the MHC–peptide interaction and therefore the efficiency of TCR signaling. Modification of the peptide or the MHC molecule which enhanced the p1 interaction also enhanced the antagonist activity of the modified APL. In contrast, modifications at p9 led to a reversal in APL function, resulting in agonist activity. Molecular homology modeling of these MHC–peptide interactions suggests a structural basis for this functional dichotomy in which topographically remote variations lead to unique interaction effects.
Sequencing‐based typing of HLA‐B*51 alleles and the significant association of HLA‐B*5101 and ‐B*5108 with Behçet's disease in Greek patients
Authors: Mizuki N. , Ota M. , Katsuyama Y. , Yabuki K. , Ando H. , Shiina T. , Palimeris G.D. , Kaklamani E. , Ito D. , Ohno S. , Inoko H.
Published: March 2003

Abstract: Behçet's disease (BD) is widely known to be strongly associated with human leukocyte antigen (HLA) B51 in many different ethnic groups.Recently, HLA‐B51 allele typing of Greek BD patients was performed to study the distribution of B*5101‐B*5107 alleles in this Greek population, the B51 antigen strongly associated with BD was found to be predominantly encoded by allele B*5101.As it is now known that the B51 antigen can be encoded by 21 alleles, B*5101‐B*5121, we performed HLA‐B*51 allele genotyping among 58 Greek patients with BD.After serological HLA typing, typing of HLA‐B*51 alleles was performed using the polymerase chain reaction‐sequencing‐based typing (PCR‐SBT) method.The frequency of the B51 antigen was found to be significantly higher in the patient group as compared with the control group (75.9% of patients vs 22.0% of controls. In the genotyping of B51 alleles, 34 out of 44 B51‐positive patients possessed B*5101, 13 out of the 44 carried B*5108.In contrast, all of the 9 B51‐positive normal controls carried B*5101.This study revealed a strong association between Greeks with BD, both B*5101, B*5108, provided important insights into the molecular mechanism underlying the association between HLA status, this disease.
Feasibility of using genetic linkage analysis to identify the genes encoding T cell‐defined minor histocompatibility antigens
Authors: Warren E.H. , Otterud B.E. , Linterman R.W. , Brickner A.G. , Engelhard V.H. , Leppert M.F. , Martin P.J. , Riddell S.R.
Published: March 2003

Abstract: We have evaluated the utility of genetic linkage analysis to identify genes that encode minor histocompatibility antigens using vaccinia virus vectors as a simple and convenient method for transient expression of class I MHC molecules in lymphoblastoid cell lines. As a test case, we used a CTL clone that recognizes HA‐8, a minor histocompatibility antigen encoded by the KIAA0020 gene and presented by HLA‐A*0201. EBV‐transformed B cell lines from individuals in three large pedigrees from the CEPH reference family collection were infected with a recombinant vaccinia virus vector encoding an HLA‐A*0201 transgene, which led to high level expression of the MHC restricting allele HLA‐A*0201 on the cell surface. HA‐8 expression in the vaccinia‐infected target cells was then determined using standard in vitro cytotoxicity assays. Pairwise linkage analysis of the segregation of HA‐8 expression in these pedigrees demonstrated that the HA‐8 gene was tightly linked with a cluster of marker loci located on the distal portion of chromosome 9p. Analysis of 9p marker haplotypes for individuals in the three families identified several individuals with recombinant haplotypes, and these recombination events were used to refine the precision of the HA‐8 gene localization further. The data collectively indicate that the HA‐8 gene is localized to a 10.3 cM (corresponding to 3.9 Mb) interval of distal 9p that is thought to encode at least 11 genes, including KIAA0020. These results demonstrate that linkage analysis can be used to map minor histocompatibility genes with high precision and accuracy. Over the next years, refinement and annotation of the human genome sequence will undoubtedly increase the utility of linkage analysis as a tool for identifying minor histocompatibility antigen genes.
Identification and sequencing of HLA‐B*0714 and B*2718 alleles and novel exon 1 sequences of B*0709 and B*2714 alleles in potential bone marrow donors
Authors: Feldman D. , Kearns J. , Wu J. , Bassinger S. , Montoya G.D. , McKeen M. , Birkos S. , Williams T.M. , Kamoun M.
Published: March 2003

Abstract: Sequence specific oligonucleotide probe hybridization and sequence specific primer PCR typing of volunteer bone marrow donors suggested the presence of variants of known HLA‐B alleles in two individuals. PCR products encompassing HLA‐B locus exons 1, 2, and 3 were prepared, subcloned and sequenced. A Hispanic individual had a novel B*07 allele (B*0714) and a Chinese individual had a novel B*27 allele (B*2718). In two other individuals, a previously unknown sequence of exon 1 was determined for HLA‐B*0709 (African American) and B*2714 (Native American). These findings further illustrate the substantial genetic variation present at the HLA‐B locus within human populations. We discuss the structural variation in the protein sequence for these HLA‐B alleles and its potential functional effects.
The HLA‐DOB gene displays limited polymorphism with only one amino acid substitution
Authors: Naruse T.K. , Kawata H. , Inoko H. , Isshiki G. , Yamano K. , Hino M. , Tatsumi N.
Published: March 2003

Abstract: The HLA‐DO molecule is a non‐classical class II heterodimer composed of α and β chains. We have previously recognized that all eight of the allelic variations of the HLA‐DOA gene represent non‐synonymous amino acid substitution. In the present study, to analyze genetic polymorphism and allelic variation of the HLA‐DOB gene which may affect the efficiency of class II restricted antigen presentation thereby being involved in the susceptibility of HLA associated diseases, we conducted direct DNA sequencing of HLA‐DOB in 36 HLA class II homozygous typing cells and identified six new allelic variations (DOB*0101101, *0101102, *01012, *01022, *0104101 and *0104102) including five single nucleotide polymorphisms with only one amino acid substitution. Furthermore, strong linkage disequilibrium was detected between DOB*01022 and DRB1*1502 only, with no linkage disequilibrium between the DOA and the DOB genes. The HLA‐DOB gene has been identified in other mammals, and their nucleotide sequences are well conserved. These facts suggest that limited polymorphism in the DOB gene is profitable to execute their unique function as a co chaperone and so strong selective pressure is operating to prevent generic variation against the DOB molecule interacting with the DM molecule and thus maintaining the specified immunological function of regulating antigen presentation.
Sequence analysis of exons 1, 2, 3, 4 and 5 of the HLA‐B5/35 cross‐reacting group
Authors: Swelsen W.T.N. , Voorter C.E.M. , Van Den Berg‐Loonen E.M.
Published: March 2003

Abstract: The HLA‐B5/35 cross‐reacting group (CREG) is a set of closely related antigens including HLA‐B35, B51, B52, B53 and B78. The nucleotide sequences of exon 1 through 5 of the B5/35 CREG were determined to assess the level of polymorphism. For exons 2 and 3, the previously described sequence‐based typing (SBT) strategy was applied, the nucleotide sequences of exon 1, 4 and 5 were determined by allele‐specific sequencing. A total of 225 unrelated individuals were HLA‐B typed by heterozygous sequencing of exons 2 and 3. In the B5/35 CREG, 26 different alleles were identified, whereas 63 non‐B5/35 CREG alleles were sequenced. The SBT strategy was proven to be reliable and efficient for high resolution typing of the B5/35 CREG. The nucleotide sequences of exon 1, 4 and 5 were determined for the 26 different B5/35 CREG alleles to establish the level of polymorphism. For seven different alleles, of which the exon 1, 4 and 5 sequences were hitherto unknown, the sequences were elucidated and in agreement with the known B5/35 sequences. Nineteen HLA‐B5/35 CREG alleles with previously published exon 1, 4 and 5 sequences were sequenced in at least two individuals. Three new alleles were identified. The first, B*5204, showed a difference at position 200 compared to B*52011, which was previously considered a conserved position. The other two alleles, B*3542 and B*51015, showed exon 2 and 3 sequences identical to B*35011 and B*51011, but differences in exons 1 and 4, respectively. B*3542 had differences at position 25 and 72 and B*51015 showed a difference at position 636. More polymorphism might be present outside exons 2 and 3 than previously thought.
Cloning and sequencing full‐length HLA‐B and ‐C genes
Authors: Cox S.T. , McWhinnie A.J. , Robinson J. , Marsh S.G.E. , Parham P. , Madrigal J.A. , Little A‐M.
Published: March 2003

Abstract: Currently most available HLA‐A, ‐B and ‐C DNA sequences cover exons 2 and 3 with a limited number extending to include other exons and introns. We have developed a method for the accurate determination of full‐length genomic DNA sequences for HLA‐A, ‐B and ‐C alleles. The method involves cloning of PCR amplified full‐length HLA genes to separate alleles at heterozygous loci. The approach avoids any ambiguities from sequencing heterozygous PCR products directly and also avoids ambiguities from sequencing overlapping PCR products to achieve full‐length sequence. To date we have sequenced full‐length genomic sequences from representatives of all the major HLA‐B and ‐C allele groups.
Conformational analyses by 1H NMR and computer simulations of cyclic hexapeptides related to somatostatin containing acidic and basic peptoid residues
Authors: Mattern R.‐H. , Tran T.‐A. , Goodman M.
Published: March 2003

We report the conformational analysis by 1H NMR in DMSO and computer simulations involving distance geometry and molecular dynamics simulations at 300K of peptoid analogs of the cyclic hexapeptide c‐[Phe11‐Pro6‐Phe7‐D‐Trp8‐Lys9‐Thr10]. The analogs c‐[Phe11‐Nasp6‐Phe7‐D‐Trp8‐Lys9‐Thr10] (1), c‐[Phe11‐Ndab6‐Phe7‐D‐Trp8‐Lys9‐Thr10] (2) and c‐[Phe11‐Nlys6‐Phe7‐D‐Trp8‐Lys9‐Thr10] (3) where Nasp denotes N‐(2‐carboxyethyl) glycine, Ndab N‐(2‐aminoethyl) glycine and Nlys N‐(4‐aminobutyl) glycine are subject to conformational studies. The results of free and restrained molecular dynamics simulations at 300K are reported and give insight into the conformational behaviour of these analogs. The compounds show two sets of nuclear magnetic resonance signals corresponding to the cis and trans orientations of the peptide bond between residues 11 and 6. The backbone conformation of the cis isomers that we believe are the bioactive isomers of the three compounds are very similar to each other while there are larger variations amongst the trans isomers. The binding data to the isolated receptors show that the introduction of the Nlys residue in analog 3 leads to an enhancement of binding potency to the hsst5 receptor compared with analog 2 while maintaining identical binding potency to the hsst2 receptor. The Nasp6 analog 1 binds weakly to the hsst2 and is essentially inactive towards the other receptors. Comparison of the conformations and binding activities of these three analogs indicates that the Nlys residue extends sufficiently far to allow binding to a negatively charged binding domain on the hsst5 receptor. According to this model, the Ndab analog 2 cannot extend far enough to allow for binding to the receptor pocket. The loss of activity observed for the Nasp6 compound 1 indicates that the presence of a negatively charged residue in position 6 is unfavorable for binding to the hsst receptors.
Conformational analysis of biologically active truncated linear analogs of endothelin‐1 using NMR and molecular modeling
Authors: Boulanger Y. , Biron E. , Khiat A. , Fournier A.
Published: March 2003

Some linear truncated analogs of endothelin‐1 display potent agonistic activity at the ETB receptor, especially when the side chain of Trp21 is N‐formylated. Then, the three‐dimensional arrangements of six structurally reduced linear analogs, three formylated and three nonformylated, have been investigated by high resolution NMR spectroscopy and molecular modeling, in order to pinpoint the conformational features related to the biological activity. Two‐dimensional double‐quantum‐filtered correlation spectroscopy (DQFCOSY), total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY) were recorded and analyzed for each molecule. Interspatial distance constraints were derived from the intensity of the NOESY connectivities. The formation of hydrogen bonding was monitored from the temperature dependence of the NH chemical shifts. Molecular models calculated by means of distance geometry, simulated annealing and energy minimization, using the NMR constraints, strongly suggested a global elongated structure for the formylated analogs exhibiting biological activity, and a folded arrangement for the unformylated derivatives. Homology comparisons allowed the identification of a β‐turn‐like folding of the C‐terminal segment Asp18‐Trp21 as a probable key‐factor for activity.
Solid‐phase synthesis and application of double‐fluorescent‐labeled lipopeptides, containing a CTL‐epitope from the measles fusion protein
Authors: Hoogerhout P. , Stittelaar K.J. , Brugghe H.F. , Timmermans J. A. M. , Ten Hove G.J. , Jiskoot W. , Hoekman J. H. G. , Roholl P. J. M.
Published: March 2003

Abstract: The mechanism which enables lipopeptides to induce cytotoxicity is not known. By preparing fluorescent‐labeled lipopeptides one might unravel the mechanism of their entry into the cell and their intracellular pathway. A method of preparing double‐fluorescent‐labeled peptides by solid‐phase chemistry is described. As model peptides we have chosen analogs of the sequence RRYPDAVYL, which occurs in the measles fusion protein (F438–446) and is an epitope for cytotoxic T lymphocytes. The peptides Pal‐KTMRKKKRRYPDAVKFLL (7) and Pal‐KFLKKKRRYPDAVKTMRL (8), in which Pal is palmitoyl and KTMR and KFL are Nε‐carboxytetramethylrhodamine‐ and Nε‐carboxyfluorescein‐labeled lysyl residues, respectively, were prepared and obtained in ≈ 30% yield after purification by high‐performance liquid chromatography. The fluorescence of fluorescein and tetramethylrhodamine in lipopeptide Pal‐KTMRKKKRRYPDAVKFLL (7) was quenched to 98–99% due to intramolecular interaction of the labels. On incubation with trypsin (i.e. cleavage at the KKKRR‐site) the fluorescence of both labels was restored. The intracellular routing of lipopeptide Pal‐KTMRKKKRRYPDAVKFLL was studied with human melanoma cell line, Mel/J, which was transfected with human leukocyte antigen B*2705. It appeared that the double‐fluorescent‐labeled lipopeptide was able to induce antigen‐specific cytotoxicity. Furthermore, preliminary confocal microscopical studies indicated that this lipopeptide is observed intracellularly.
Efficient solid‐phase synthesis of Vpr from HIV‐1 using low quantities of uniformly 13C‐, 15N‐labeled amino acids for NMR structural studies
Authors: Cornille F. , Wecker K. , Loffet A. , Genet R. , Roques B.
Published: March 2003

Abstract: The 96‐amino acid protein Vpr functions as a regulator of cellular processes involved in the human immunodeficiency virus, type 1 (HIV‐1) life cycle, including cell‐cycle arrest at the G2/M check point, promotion of the HIV‐1 preintegration complex for nuclear transport, induction of apoptosis and transcriptional activation of a variety of viral and cellular promoters. Preliminary 1H NMR experiments performed on Vpr fragments showed the presence of several helical regions. However, the assignment of many protons in the amide region of the complete sequence of Vpr proved to be impossible due to the overlap of multiple NOE cross peaks. Moreover, because of its cytotoxicity, it is difficult to produce large quantities of 15N‐ and 13C‐labeled Vpr using molecular biology approaches. Therefore, the solid‐phase peptide synthesis of (1–96)Vpr, labeled at 22 selected positions, using recently commercially available uniformly 13C‐, 15N‐labeled fmoc amino acids, has been optimized to produce large quantities (104 mg, 15% yield) of pure compound, while minimizing the quantity of labeled amino acids used for each coupling. As expected two‐dimensional heteronuclear NMR experiments performed with this protein allowed the unequivocal assignments of all the proton signals. This study shows that introduction of few labeled 13C/15N labeled amino acids in selected positions facilitates the determination of structure solution of small protein accessible by solid‐phase peptide synthesis, and could allow dynamic studies of their conformational behavior to be carried out.
Structure of a paralytic peptide from an insect, Manduca sexta
Authors: Yu X.‐Q. , Prakash O. , Kanost M.R.
Published: March 2003

Abstract: Paralytic peptide 1 (PP1) from a moth, Manduca sexta, is a 23‐residue peptide (Glu‐Asn‐Phe‐Ala‐Gly‐Gly‐Cys‐Ala‐Thr‐Gly‐Tyr‐Leu‐Arg‐Thr‐Ala‐Asp‐Gly‐Arg‐Cys‐Lys‐Pro‐Thr‐Phe) that was first found to have paralytic activity when injected into M. sexta larvae. Recent studies demonstrated that PP1 also stimulated the spreading and aggregation of a blood cell type called plasmatocytes and inhibited bleeding from wounds. We determined the solution structure of PP1 by two‐dimensional1H NMR spectroscopy to begin to understand structural–functional relationships of this peptide. PP1 has an ordered structure, which is composed of a short antiparallel β‐sheet at residues Tyr11‐Thr14 and Arg18‐Pro21, three β turns at residues Phe3‐Gly6, Ala8‐Tyr11 and Thr14‐Gly17, and a half turn at the carboxyl‐terminus (residues Lys20‐Phe23). The well‐defined secondary and tertiary structure was stabilized by hydrogen bonding and side‐chain hydrophobic interactions. In comparison with two related insect peptides, whose structures have been solved recently, the amino‐terminal region of PP1 is substantially more ordered. The short antiparallel β‐sheet of PP1 has a folding pattern similar to the carboxyl‐terminal subdomain of epidermal growth factor (EGF). Therefore, PP1 may interact with EGF receptor‐like molecules to trigger its different biological activities.
Study on the cyclization tendency of backbone cyclic tetrapeptides
Authors: Besser D. , Reissmann S. , Olender R. , Rosenfeld R. , Arad O.
Published: March 2003

Abstract:The cyclization kinetics of five backbone‐cyclic tetrapeptides was investigated both experimentally and computationally. The aim was to both accurately measure the cyclization rates in solution and develop a method that efficiently estimates the relative cyclization tendencies computationally. Progression of the cyclization reaction was monitored directly, yielding the kinetics of changes in the amounts of the linear precursor and the products. These measurements were used to calculate the reaction rates; the results were consistent with a first‐order reaction kinetics. In order to predict the cyclization rates computationally, the conformation space of the linear precursors was mapped and used to construct an approximate partition function. We assumed that the cyclization tendency was correlated with the relative probability of being found in a cyclization‐prone conformation of the backbone, this probability was estimated from the partition function. The results supported this assumption and demonstrated that, within reasonable accuracy, we are able to predict the relative cyclization tendencies of the peptides measured.
Theoretical conformational analysis of six arginine vasopressin analogs with the l‐naphthylalanine in position 3
Authors: Ślusarz R. , Kaźmierkiewicz R. , Lammek B.
Published: March 2003

Abstract: Introduction of the naphthylalanine residue into either position 3 of arginine vasopressin (AVP), or its analogs results in peptides with interesting pharmacological properties. The single substituted analog of AVP with l‐2‐Nal in position 3 causes moderate antiduretic activity, whereas [Mpa1, (l‐1‐Nal)3, (d‐Arg)8] VP and [Mpa1, (l‐2‐Nal)3, (d‐Arg)8] VP are potent and selective V2 agonists. Moreover [(l‐2‐Nal)3, (d‐Arg)8] VP is among the most potent and selective antagonists of V1a receptors. In this study we carried out conformational calculations on [(l‐1‐Nal)3] AVP, [(l‐2‐Nal)3] AVP, [(l‐1‐Nal)3, (d‐Arg)8] VP, [(l‐2‐Nal)3, (d‐Arg)8] VP, [Mpa1, (l‐1‐Nal)3, (d‐Arg)8] VP, [Mpa1, (l‐2‐Nal)3, (d‐Arg)8] VP, using the ECEPP/3 force field with and without including hydration to simulate aqueous and nonpolar environments. It was found that in all six compound studied, the low‐energy conformations have common geometry and relative energies. Therefore, the modifications of the Phe in position 3 influence the binding to the receptor by changing the size of the third residue, rather than by changing the conformational space. The lowest‐energy conformations in the presence and absence of water had β‐turns at residues Phe3‐Gln4 and Gln4‐Asn5 and Gln4‐Asn5, respectively. The conformation at the Gln4‐Asn5 turn was most similar to the crystal structure of the pressinoic acid (the cyclic moiety of vasopressin).
The Chemical Shift Index method applied to resin‐bound peptides
Authors: Fruchart J.‐S. , Lippens G. , Warrass R. , Seetharaman C. , Dhalluin C. , Boutillon C.
Published: March 2003

Abstract: The Chemical Shift Index (CSI) method proposed by Wishart et al. [Biochemistry (1992) 31, 1647–1651] to evaluate the secondary structure of peptides in aqueous solution uses as its reference the chemical shift values of each of the 20 natural amino acids (X) in a typical nonstructured sequence GGXAGG (17–20). In order to apply the CSI method to protected resin‐bound peptides, we established a new database of chemical shift values for the same GGXAGG sequences in their protected form and anchored to a polystyrene resin swollen in DMF‐d7. The predictive value of this new reference set in the CSI protocol was tested on different resin‐bound peptides that were previously characterized by a full NOE analysis.
Design of the Y1‐receptor‐selective cyclic peptide based on the C‐terminal sequence of neuropeptide Y
Authors: Takebayashi Y. , Koga H. , Kurihara H. , Furuya T. , Tanaka A. , Togami J. , Inui A. , Koshiya K. , Murase K.
Published: March 2003

Abstract: We designed four cyclic peptides which are mimics of the C‐terminal region of human neuropeptide Y (NPY) on the basis of the structural model of NPY. One of these cyclic peptides, c[D‐Cys29‐L‐Cys34]NPY Ac‐29‐36 (YM‐42454), exhibited significantly higher affinity for the Y1‐receptor than the corresponding C‐terminal linear fragment, NPY Ac‐28‐36. Interestingly, YM‐42454 showed binding affinity for the Y1‐receptor in spite of the lack of the N‐terminal sequence of NPY, whereas it did not show any binding affinity for the Y2‐receptor. This conformationally restricted Y1‐selective peptide would provide some insights into the bioactive conformation of the C‐terminal region of NPY.
An internal histidine residue from the bacterial surface protein, PAM, mediates its binding to the kringle‐2 domain of human plasminogen
Authors: Schenone M.M. , Warder S.E. , Martin J.A. , Prorok M. , Castellino F.J.
Published: March 2003

Abstract: The determinants of binding of a peptide lacking C‐termini‐exposed lysine residues to a kringle domain were investigated using an up‐regulated lysine binding kringle (K2Pg[C4G/E56D/K72Y]) of plasminogen and a peptide (a1‐PAM) with a sequence derived from a surface‐exposed M‐like streptococcal protein. Significant kringle‐induced chemical shifts in a His side‐chain of a1‐PAM were revealed by two‐dimensional NMR. Further studies using isothermal titration calorimetry (ITC) provided support for the involvement of His12 in the peptide/protein complex. In an effort to screen a1‐PAM‐derived truncation peptides, a combinatorial mixture, a1Δa2‐PAM[H12X] (where X = Pro, Arg, His, Trp, Lys, Ala, Phe, Asp and Gly), was analyzed using the surface‐enhanced laser desorption ionization time‐of‐flight mass spectrometry (SELDI) platform. The major peptide that remained bound to the surface of the K2Pg[C4G/E56D/K72Y]‐containing chip was that containing His12, corresponding to the wild‐type sequence. Minor peaks, representing binding, were obtained for Lys12‐, Arg12‐ and Trp12‐containing peptides. Individual peptides containing these amino acids were then examined using ITC and the binding constants obtained correlated with the relative strengths of binding estimated from the SELDI‐based screen.
Transport characteristics of peptides and peptidomimetics: II. Hydroxyethylamine bioisostere‐containing peptidomimetics as substrates for the oligopeptide transporter and P‐glycoprotein in the intestinal mucosa
Authors: Gao J. , Winslow S.L. , Vander Velde D. , Borchardt R.T. , Aubé J.
Published: March 2003

Abstract: Peptide bond bioisosteres, such as hydroxyethylamine (Hea), have frequently been used to stabilize metabolically labile peptide bonds in peptidomimetic drug design in an effort to increase the oral bioavailability of drug candidates. However, the impact of the peptide bond bioisosteres on the cell permeation characteristics of peptidomimetics is not well understood, particularly with respect to the effects on the substrate activity for proteins that can restrict (e.g. P‐glycoprotein, P‐gp) or facilitate (e.g. the oligopeptide transporter, OPT) intestinal mucosal permeation of peptidomimetics. In this study, terminally free and terminally modified (N‐acetylated and C‐amidated) peptidomimetics of H‐Ala‐Phe‐OH and H‐Ala‐Phe‐Ala‐OH with the Ala‐Phe peptide bonds replaced by Hea bioisosteres were synthesized. Transport characteristics of these peptidomimetics were investigated using Caco‐2 cell monolayers as an in vitro model of the intestinal mucosa. The study showed that the Hea bioisostere stabilized the peptidomimetics to protease metabolism in Caco‐2 cells. All terminally free peptidomimetics showed significant affinity and substrate activity for OPT. The affinity and substrate activity for OPT were stereoselective for peptidomimetics containing an S,S‐configuration for the two adjacent chiral centers related to the Hea bioisostere. Three of the four terminally modified peptidomimetics showed significant substrate activity for P‐gp and, interestingly, the substrate activity for P‐gp was also stereoselective; however, it was in favor of an R,R‐configuration for the two adjacent chiral centers related to the Hea bioisostere.
Plasmodium falciparum: binding studies of peptide derived from the sporozoite surface protein 2 to Hep G2 cells
Authors: López R. , Curtidor H. , Urquiza M. , Garcia J. , Puentes A. , Suarez J. , Ocampo M. , Vera R. , Rodriguez L.E. , Castillo F. , Cifuentes G. , Patarroyo M.E.
Published: March 2003

Abstract: Plasmodium falciparum sporozoite surface protein 2 (Pf SSP2), also called thrombospondin related anonymous protein (TRAP), is involved in the process of sporozoite invasion of hepatocytes. Pf SSP2/TRAP possesses two different adhesion domains sharing sequences and structural homology with von Willebrand factor A‐domains and human repeat I thrombospondin (TSP). Pf SSP2/TRAP has also been implicated in sporozoite mobility and in mosquito salivary gland invasion processes. We tested 15‐mer long synthetic peptides having five overlapping residues covering the complete protein Pf SSP2 sequence in binding assays to Hep G2 cells. In these 57 peptides, 21 high‐activity binding peptides (HABPs) were identified; five were in the adhesion domains already described and 16 were in two regions toward the protein's carboxy and middle terminal part. Six HABPs showed conserved amino acid sequences: 3243 (21FLVNGRDVQNNIVDE35), 3279 (201FLVGCHPSDGKCNLY215), 3287 (241TASCGVWDEWSPCSV255), 3289 (251SPCSVTCGKGTRSRK265), 3327 (441ERKQSDPQSQDNNGNY455) and 3329 (451DNNGNRHVPNSEDREY465). The HABPs show saturable binding and dissociation constants between 140 and 900 nm with 40 000–855 000 binding sites per cell. The 3279 (201FLVGCHPSDGKCNLY215), 3323 (421NDKSDRYIPYSPLSP435) and 3331 (461SEDRETRPHGRNNENY475) HABPs have B epitopes in their sequences; these have previously been recognized by antibodies partially inhibiting hepatocyte invasion and development of the hepatic state. The 3287 (241TASCGVWDEWSPCSV255) and 3289 (251SPCSVTCGKGTRSRK265) HABPs share common sequences with the Pf SSP2/TRAP region II plus, which is present in a great number of adhesion proteins. Based on this information, six new peptides covering the high binding regions identified previously were synthesized and, using a competition assay, the amino acid involved in the binding were determined.
A novel approach to the synthesis of EGF‐like domains: a method for the one‐pot regioselective formation of the three disulfide bonds of a human blood coagulation Factor VII EGF‐1 analogue
Authors: Husbyn Mette , Cuthbertson Alan
Published: March 2003

Abstract: The study of EGF‐like domains is of great general interest in protein science because of their participation in a multitude of protein–protein interactions. A common structural feature of EGF‐like modules is the presence of three disulfide bonds, the regioselective formation of which still remains a challenge to peptide chemists. We report here on a method for the one‐pot regioselective synthesis of an analogue of the EGF‐1 domain of human coagulation Factor VII (residues 45–83) comprising an Asn57β–Asp substitution. The cysteine protecting groups trityl, t‐butyl and acetamidomethyl were chosen for the three disulfide bond pairings. All three disulfide bridges were prepared directly from the crude starting product, obviating the need for the timely and costly purification of the intermediate folded products. The fully folded product was purified by preparative high‐pressure liquid chromatography prior to evaluation of its biological activity in an assay to detect inhibition of FVII/TF complex formation. In addition circular dichroism spectroscopy was employed to elucidate the main structural similarities between this peptide analogue and the native human Factor VII EGF‐1 domain.
pH dependence of the relative hydrophobicity and lipophilicity of amino acids and peptides measured by aqueous two‐phase and octanol–buffer partitioning
Authors: Gulyaeva N. , Zaslavsky A. , Lechner P. , Chait A. , Zaslavsky B.
Published: March 2003

Abstract: Partitioning of a series of free amino acids, their derivatives, and homo‐oligopeptides in aqueous dextran–PEG two‐phase systems and octanol buffer systems was examined at pH values from 2.0 up to 12.5. The pH‐dependent partition behavior of free amino acids and peptides in the two‐phase systems was compared with that of monofunctional drug‐like compounds and found to be clearly different. The differences observed indicate that the information provided by the techniques of partitioning in octanol buffer (logDpH) and in aqueous two‐phase systems, N(CH2), is different. It is suggested that the combination of the two descriptors, logD (lipophilicity) and N(CH2) (relative hydrophobicity), may be useful for QSAR analysis of the biological activities involving distribution and/or transport of chemical compounds in biological systems.
Restriction fragment‐length polymorphism analysis of 16S ribosomal RNA genes amplified by polymerase chain reaction for rapid identification of cultivable oral treponemes
Authors: Sato T. , Kuramitsu H. K.
Published: March 2003

Although oral treponemes are among the most frequently found bacteria in periodontal pockets, identification of these organisms can be difficult. In this study, restriction fragment‐length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)–amplified 16S ribosomal RNA genes (16S rRNA gene PCR‐RFLP) was used to generate restriction profiles of reference strains of oral treponemes including Treponema denticola, Treponema socranskii, Treponema vincentii, Treponema pectinovorum and Treponema medium as well as for Treponema phagedenis and Treponema pallidum and five treponeme strains isolated from human periodontal pockets. Before RFLP analysis, the 16S rRNA gene sequences were obtained from the GenBank database, and the analysis of the theoretical banding patterns for HpaII suggested good species discrimination. 16S rRNA gene sequences were amplified from isolated genomic DNA samples by PCR with spirochete‐specific primers. The PCR products were then purified and characterized by single digestion with restriction endonuclease HpaII, and this allowed discrimination between the respective reference strains. Five clinical isolates, four T. denticola and one T. socranskii, were assigned on the basis of their restriction profiles by digestion with HpaII. 16S rRNA gene PCR‐RFLP using HpaII is a rapid and reliable method for differentiation of cultivable oral treponemes.
Growth and hydrolytic enzyme production of Capnocytophaga gingivalis on different protein substrates
Authors: Spratt D. A. , Greenman J. , Schaffer A. G.
Published: March 2003

Capnocytophaga gingivalis was grown with proteins (albumin, collagen, mucin and hemoglobin) as carbon and energy sources in chemostat culture. The μmax (0.34 h−1) and biomass yield (0.96 g · l−1) were as high with hemoglobin (3 g · l−1) as with glucose (3 g · l−1) (
20). Albumin, collagen and mucin also supported an increased μmax, or yield or both, in comparison with basal (tryptone/thiamine) medium. In steady‐state, trypsin‐like protease specific activity increased 3‐ to 5‐fold in the presence of albumin, collagen and hemoglobin; whereas the greatest increase (21‐fold) in α‐glucoside activity was in the presence of mucin. There were significant, but less substantial changes in other hydrolytic enzymes (aminopeptidase, acid and alkaline phosphatases). The bulk of the detected hydrolytic activity (>66%) was associated with the cells. The data indicate that C. gingivalis regulates its production of hydrolytic enzymes in response to environmental conditions.
Serotype distribution and secretory acid proteinase activity of Candida albicans isolated from the oral mucosa of patients with denture stomatitis
Authors: McMullan‐Vogel C. G. , Jüde H. D. , Ollert M. W. , Vogel C.‐W.
Published: March 2003

Denture stomatitis is the most common form of oral Candida infection in humans. In the current study, the distribution of Candida albicans serotype A and B as well as the activity of the secreted acid proteinase were determined in clinical isolates from patients with denture stomatitis. It was found that 70% of individuals with clinical signs of denture stomatitis exhibited fungal growth, with C. albicans representing the most frequently isolated species (75%). Of the C. albicans isolates, 75% were serotype A and 25% were serotype B, representing a significant increase of serotype B compared to a control group of non‐denture‐wearing HIV‐seronegative individuals with oral candidiasis, but no significant difference compared with isolates from HIV‐seropositive patients, who also exhibited a high percentage of serotype B. The mean secretory acid proteinase activity of C. albicans isolates from denture stomatitis patients (2796±819 U/l) was statistically not different from the mean secretory acid proteinase activity in non‐denture‐wearing HIV‐seronegative individuals (2324±1487 U/l). Both values were significantly lower than the mean secretory acid proteinase activity of C. albicans from HIV‐seropositive individuals (4256±2372 U/l). No correlation exists between the C. albicans serotype and the amount of secreted acid proteinase, indicating that serotype and secretory acid proteinase expression are two independent pathogenetic factors in oral candidiasis. These results indicate that there seems to be strain selection for C. albicans serotype B in denture stomatitis. These results further indicate that increased secretion of the acid proteinase seems to be of pathogenetic significance in the candidiasis of HIV‐seropositive patients but not in denture stomatitis. Nevertheless, the secretory acid proteinase is likely to be an important pathogenetic factor also in denture stomatitis, where an increased secretion of the acid proteinase may not be required because of decreased salivary flow and a low pH under the denture, which will result in a high enzymatic activity.
Mutans streptococci and other caries‐associated acidogenic bacteria in five‐year‐old children in South Africa
Authors: Toi C. S. , Cleaton‐Jones P. E. , Daya N. P.
Published: March 2003

Correlations between mutans streptococci and caries are inconsistent; some studies show that early caries may occur with other bacterial species possessing the ability to survive, interact and compete with mutans streptococci. This study looked at the relationship between mutans streptococci, Lactobacillus, Actinomyces and Veillonella in saliva and plaque collected from four tooth sites in two ethnic groups, with and without caries. All samples were collected and cultured onto selective media. Colonies were enumerated and identified using biochemical tests. Pearson correlations showed low yet statistically significant correlations between plaque mutans streptococci counts and the number of decayed surfaces (ds) and decayed, missing and filled surfaces (dmfs) of primary teeth in blacks and in coloreds (historical race classification). Salivary mutans streptococci counts correlated with ds and dmfs in coloreds. No relationship was found between ds, dmfs and Lactobacillus, Actinomyces or Veillonella numbers. Significant associations were evident between plaque mutans streptococci and Veillonella and Lactobacillus in black children with caries and between plaque mutans streptococci and Actinomyces and Lactobacillus and Veillonella in colored children. Hence, oral microbes are in constant flux but have interrelationships in children with caries.
Antibodies to Treponema pallidum in serum from subjects with periodontitis: relationship to pathogen‐related oral spirochetes
Authors: Tzagaroulaki E. , Riviere G.
Published: March 2003

Pathogen‐related oral spirochetes were identified in dental plaque using monoclonal antibodies to putative Treponema pallidum–specific proteins, and serum from subjects with necrotizing ulcerative gingivitis contained immunoglobulin G to molecules thought to be restricted to T. pallidum. The purpose of this study was to determine whether subjects with periodontitis were more likely to have serum antibodies to T. pallidum if pathogen‐related oral spirochetes were present. Pathogen‐related oral spirochetes were detected in subgingival plaque from 27 of 40 subjects, and 33 subjects had serum antibodies that bound T. pallidum proteins in immunoblots. Subjects with pathogen‐related oral spirochetes were no more likely to have IgA, IgG or IgM to 15‐, 37‐ or 47‐kDa proteins than were subjects without pathogen‐related oral spirochetes. In contrast to subjects with necrotizing ulcerative gingivitis, subjects with periodontitis had no detectable antibodies to 37‐ or 12‐kDa proteins. Further research is needed to identify the stimulus for antibodies that cross‐react with T. pallidum proteins.
Genetic heterogeneity in Prevotella intermedia, Prevotella nigrescens, Prevotella corporis and related species isolated from oral and nonoral sites
Authors: Pearce M. A. , Devine D. A. , Dixon R. A. , Van Steenbergen T. J. M.
Published: March 2003

Prevotella intermedia (43 isolates), Prevotella nigrescens (55) and Prevotella corporis (8) from oral and nonoral sites were distinguished by species‐specific DNA fragments, after hybridization of DNA fragments with ribosomal RNA (ribotyping). Eight strains previously identified as P. intermedia did not have these specific fragments. P. nigrescens, P. intermedia and P. corporis formed separate clusters in dendrograms constructed using clustering with an unweighted pair group method with arithmetic averages of similarity values derived from ribotype patterns, with 10 subclusters in P. intermedia isolates and 26 in P. nigrescens. Nine groups of P. intermedia isolates and 6 of P. nigrescens shared identical patterns. Specific ribotypes or species were not associated with particular diseases when all isolates were analyzed. However, results from organisms isolated by one laboratory using consistent clinical reporting indicated that P. intermedia was associated with more severe forms of periodontitis and P. nigrescens with mild to moderate disease.
Xylitol disturbs protein synthesis, including the expression of HSP‐70 and HSP‐60, in Streptococcus mutans
Authors: Hrimech M. , Mayrand D. , Grenier D. , Trahan L.
Published: March 2003

Xylitol, a natural sugar alcohol and a caries‐preventive carbohydrate sweetener, inhibits xylitol‐sensitive wild‐type Streptococcus mutans but also selects for its natural xylitol‐resistant mutants. The aim of the work was to verify the influence of xylitol on heat shock proteins HSP‐60 (GroEL‐like) and HSP‐70 (DnaK‐like) in xylitol‐sensitive and xylitol‐resistant strains. Cells from fresh isolate S. mutans 123.1 were grown at 37°C and constant pH 7.0. The cell culture was stressed by raising the temperature to 43°C or adding xylitol (4% final). Cell proteins labeled with a cocktail of 14C‐amino acids were analyzed by SDS‐PAGE and autoradiography whereas HSP‐60 and HSP‐70 were visualized using Western immunoblotting. In both xylitol‐sensitive and xylitol‐resistant strains, heat stress was associated with an increase of both HSP‐60 (63 kDa) and HSP‐70 (71 kDa) and a decrease in the intensity of a number of other protein bands compared with cells maintained at 37°C. Exposure to xylitol but not to other polyols induced a decrease of both these heat shock proteins in the xylitol‐sensitive strain but did not modify them in the xylitol‐resistant mutant. It also decreased all protein bands above 60 kDa together with a 53 kDa protein and increased the amount of 57‐, 50‐ and 40‐kDa proteins in the xylitol‐sensitive strain whereas the proteins of the xylitol‐resistant strain remained unchanged. The results suggest that xylitol is a strong metabolic inhibitor that disturbs protein synthesis and reduces the expression of HSP‐70 and HSP‐60 proteins in the wild‐type xylitol‐sensitive S. mutans but not in the xylitol‐resistant natural mutant strain.