bluuebunny/crossref_metadata_2025 huggingface dataset:
Exon 2 sequence analysis of a novel HLA‐DRB1 allele, DRB1*0314
Authors: Koninkx H.J.L. , Tijssen H.J. , Joosten I.
Published: March 2003

Abstract: We describe the identification of a new DRB1*03 variant in a Caucasian volunteer bone‐marrow donor (DR’KW’). The indication for the existence of this new variant arose from contradictory results obtained by serological and several DNA typing methods (SSP, SSOP). Analysis of the exon 2 sequence revealed that the DRB1*0314 contained the characteristic DR3 specific sequence motifs, except for codon 77 (AAC→ACC) where threonine is substituted for asparagine.
THE RELATIONSHIP BETWEEN HLA TYPING AND HCV INFECTION AND OUTCOME OF RENAL TRANSPLANTATION IN HCV‐POSITIVE PATIENTS
Authors: Hadhoud A , Abdulaziz AM , Al Menawi L , Shaheen FA , Abdulghaffar A , Al Abas F , Al Mobrak MF
Published: March 2003

The role of HLA‐antigens in susceptibility to hepatitis C virus (HCV) infection is still being debated. We analyzed HLA phenotype frequencies in two major ethnic groups, namely Egyptian and Saudi nationals. The Egyptian group included 110 patients of whom 55 were HCV positive and the other 55 were HCV negative (control group). The Saudi group included 146 HCV‐positive patients and 122 HCV‐negative individuals (control group). The results for the Egyptian population revealed increased frequencies of some HLA phenotypes and decreased frequencies of others but without any statistically significant difference. In contrast, in the Saudi population, the HLA‐A19 phenotype was significantly increased in the HCV‐positive patients when compared with the control group, while significantly decreased frequencies were found for HLA‐B8, HLA‐DRI and HLA‐DR3. Our data suggest that there was no significant association between the HLA phenotypes and the susceptibility to HCV infection among the Egyptian population, while the overall data of the Saudi population seem to indicate that the expression of particular HLA‐alleles could be associated with the susceptibility or resistance to the HCV infection. Further studies on larger numbers of patients are needed to support the role of the HLA system in HCV infection. A total of 108 HCV‐positive patients underwent renal transplantation at the Jeddah Kidney Center, and the results were compared with 100 age‐ and sex‐matched controls. Graft survival at 36 months was 82% and 86% for the HCV positive and control subjects, respectively, while the patient survival rate was, respectively, 90% and 91%. Our data suggest that the outcome, at least in the short‐term, of renal transplantation in HCV‐positive patients is very good.
POST‐TRANSPLANT C‐REACTIVE PROTEIN MONITORING PREDICTS CHRONIC REJECTION AND RENAL ALLOGRAFT SURVIVAL
Authors: Sezer S , Özdemir FN , Akçay A , Külah E , Arat Z , Erdal R , Haberal M
Published: March 2003

Chronic allograft nephropathy (CAN) is a major problem after renal transplantation, and chronic inflammation is an important element of this condition. C‐reactive protein (CRP) is an important marker of inflammation and atherosclerosis. This retrospective study analyzed the value of post‐transplantation CRP monitoring in the prediction of CAN and graft failure in kidney transplantation.

The study included 125 renal transplant patients whose CRP levels were in the normal range (<10 mg/l) at the time of transplantation. Serum CRP levels before transplantation, at 1 month and at 1 year post‐transplantation, at the time of CAN diagnosis, and the mean level throughout follow up in patients without CAN were recorded. The CRP findings were compared in patients grouped according to whether or not they developed CAN. Chronic rejection was diagnosed in 40.8% of the patients, and 82.4% of this group progressed to graft failure at a mean of 45.8±30.4 months after the diagnosis of CAN. When the group CRP results were compared, the levels before transplantation were similar, but at 1 month post‐transplantation the levels in the group with CAN were significantly higher (P<0.04). The mean CRP level at 1 year was also higher in the CAN group, but this difference was not significant. The mean level at the time of the CAN diagnosis was significantly higher than the mean level during follow up in the group without CAN (P<0.01). Thus, the CRP levels were significantly higher in CAN group than in the group without CAN at two of the timepoints tested: immediately after transplantation and at the time of CAN diagnosis. Regression analysis showed that the high CRP level at 1 month post‐transplantation and at the time of the CAN diagnosis significantly influenced the allograft failure (P<0.005 and P<0.01, respectively).

In conclusion, post‐transplantation CRP monitoring provides important information concerning the risks of CAN and graft failure. Testing of CRP levels should be part of the follow‐up assessment after renal transplantation.
Specific amplification of the HLA‐DRB4 gene from c‐DNA. Complete coding sequence of the HLA alleles DRB4*0103101 and DRB4*01033
Authors: De Pablo R. , Solís R. , Balas A. , Vilches C.
Published: March 2003

Abstract: We present the complete coding sequence of the HLA alleles DRB4*0103101 and DRB4*01033 derived from the lymphoblastoid cell line G081, established from an individual of Spanish Gypsy ethnic origin. This cell was typed by PCR‐SSP and reverse SSO as DRB4*0103101 but further characterization of the DRB4 gene by sequence‐based typing (SBT) demonstrated heterozygosity at codon 78 (TAC, TAT). With the aim of confirming this polymorphism, RNA isolated from G081 was subjected to RT‐PCR using primers designed to recognize specifically the 5′ and 3′ UT regions of HLA‐DRB4 and the product was cloned and sequenced. Nucleotide sequences derived from seven clones confirmed the heterozygosity of G081, as they corresponded to two open reading frames of 801 nucleotides that matched either DRB4*0103101 or the recently described DRB4*01033, for which a partial sequence, spanning exons 2 and 3, has been reported. The phenotype of G081 (A*01; B*0702, *1302/1303; Cw*0602, *07; DRB1*0403, *0701; DRB4*0103101, *01033; DQB1*0202, *0302; DQA1*0201, *0301) is consistent with a proposed association of DRB4*01033 with DRB1*0403 and DQB1*0302.
Relationship of polymorphisms of the Interleukin‐1 gene cluster to occurrence and severity of rheumatoid arthritis
Authors: Kaijzel E. L. , Van Dongen H. , Bakker A. M. , Breedveld F. C. , Huizinga T. W. J. , Verweij C. L.
Published: March 2003

Abstract: Interleukin‐1 (IL‐1) has been implicated in the pathogenesis of rheumatoid arthritis (RA). We investigated whether ΙL‐1 gene locus polymorphisms are associated with susceptibility to or severity of RA. Genotyping for IL‐1α, IL‐1β and IL‐1Ra single nucleotide polymorphisms (SNPs) performed in a cross‐sectional group of 312 consecutive RA patients (RA‐group 1) and a cohort of 94 incident female RA patients (RA‐group 2) revealed that the rare IL‐1RN + 2017 C allele was significantly increased in RA compared to controls (n = 245). A retrospective analysis in RA‐group 1 showed no significant associations between IL‐1 genotypes and disease severity. A prospective study in RA‐group 2 demonstrated that the extent of joint destruction over 12 years was higher in patients genotyped heterozygous for the IL‐1 A + 4845, IL‐1B + 3953 and IL‐1RN + 5111 SNPs compared to homozygous wildtype patients, although differences did not reach statistical significance. These data indicate that the IL‐1RN + 2017 polymorphism is associated with susceptibility to RA.
HLA‐A and HLA‐B in Kenya, Africa:  Allele frequencies and identification of  HLA‐B*1567 and HLA‐B*4426
Authors: Luo M. , Embree J. , Ramdahin S. , Ndinya‐Achola J. , Njenga S. , Bwayo J.B. , Pan S. , Mao X. , Cheang M. , Stuart T. , Brunham R.C. , Plummer F.A.
Published: March 2003

Abstract: HLA‐A and HLA‐B alleles of a population from Kenya, Africa were examined by sequencing exon 2 and exon 3 DNA and typing using a Taxonomy‐based Sequence‐analysis (TBSA) method. Extensive diversities were observed at both HLA‐A and HLA‐B loci in this population. Forty‐one HLA‐A alleles were identified from 159 unrelated individuals. The most frequently observed alleles were A*6802 (11.64%), A*02011/09 (9.75%), A*7401/02 (9.43%), A*3001 (7.86%), A*3002 (7.23%) and A*3601 (6.6%). Forty‐nine HLA‐B alleles were identified in 161 unrelated individuals, including two novel alleles, B*1567 and B*4426. The most frequently observed HLA‐B alleles were B*5301 (9.01%), B*5801 (8.38%), B*4201 (7.76%), B*1503 (7.14%), B*1801 (6.21%), and B*5802 (5.90%). The most frequently observed HLA‐A‐B haplotypes were A*3601‐B*5301 (3.55%) and A*3001‐B*4201 (3.19%), followed by A*7401/02‐B*5801 (2.84%), A*7401/02‐B*5802 (2.84%) and A*02011/09‐B*1503 (2.13%). Linkage disequilibrium and χ2 analysis showed the association of these HLA‐A‐B haplotypes at the antigen level to be significant. The frequencies of HLA‐A and HLA‐B alleles from the Kenyan population were compared with that of a population from Cameroon. The difference in allele and haplotype frequency distributions partly reflected the different ethnic composition of these two African populations.
HLA‐DRB1 and HLA‐DQB1 polymorphisms in Pacific Islands populations
Authors: Velickovic Z.M. , Delahunt B. , Carter J.M.
Published: March 2003

Abstract: Allele frequency distributions of the HLA‐DRB1 and HLA‐DQB1 genes were investigated in four Pacific Islands populations from the Cook Islands, Samoa, Tokelau and Tonga. Limited diversity was observed for both the HLA‐DRB1 and HLA‐DQB1 loci. Five HLA‐DRB1 alleles were observed to be the most frequent amongst all the studied Pacific Islands populations. They were: HLA‐DRB1*0403, HLA‐DRB1*08032, HLA‐DRB1*09012, HLA‐DRB1*11011 and HLA‐DRB1*1201. Cook Islanders had the largest number of low frequency DRB1 alleles followed by Samoans, Tokelauans and Tongans, most of which may be attributed to reported non‐Polynesian admixture. The most frequently observed DQB1 alleles in the four studied Pacific Islands populations were those of the DQ3 subgroup of alleles HLA‐DQB1*03011, HLA‐DQB1*0302 and HLA‐DQB1*03032 as well as HLA‐DQB1*05031 and HLA‐DQB1*06011. Cook Islanders had the highest number of rare HLA‐DQB1 alleles, the distibution being similar to that of the HLA‐DRB1 allele. While, in general, the values of homozygosity for DRB1 and DQB1 were observed to be lower then expected under neutrality, a statistical significance was observed in Tongans, Samoans and Tokelauans for the DQB1 locus and in Tongans for the DRB1 locus. Differences were observed between allele frequency distributions for Tokelauans compared to the other three populations. This was also demonstrated by principal component analysis of DRB1 and DQB1 allele frequencies, which separated the Tokelauan population from Cook Islanders, Tongans and Samoans. Tongans and Samoans were separate from the other Polynesian populations in the phylogenetic trees. Observed allele and haplotype frequencies were found to be in agreement with previously published HLA‐DRB and HLA‐DQB Polynesian data.
Similar incidence of type 1 diabetes in two ethnically different populations (Italy and Slovenia) is sustained by similar HLA susceptible/protective haplotype frequencies
Authors: Petrone A. , Battelino T. , Krzisnik C. , Bugawan T. , Erlich H. , Di Mario U. , Pozzilli P. , Buzzetti R.
Published: March 2003

Abstract: The incidence of type 1 diabetes (T1DM) seems to depend in part on the population frequencies of susceptible and protective HLA haplotypes. The present study aimed to (i): characterize the genetic susceptibility to T1DM in the Slovenian population, (ii) test the general hypothesis that T1DM incidence is related to the frequencies of susceptible/protective haplotypes, (iii) compare allele, haplotype and genotype frequencies in Slovenians and Italians that represent two white populations with a similar incidence of T1DM (7.9/100,000/year and 8.1/100,000/year, respectively). The haplotype found most frequently among Slovenian T1DM patients was DRB1*0301‐DQA1*0501‐DQB1*0201 (53%). The DR4‐DQA1*0301‐DQB1*0302 haplotypes conferring susceptibility to T1DM were those bearing DRB1*0401 (OR = 12), DRB1*0404 (OR = 4.7) and DRB1*0402 (OR = 4.5). Negative associations with the disease were found for the following haplotypes: DRB1*1501‐DQA1*0102‐DQB1*0602, DRB1*1301‐DQA1*0102‐DQB1*0603, DRB1*1101/1104‐DQA1*0501‐DQB1*0301, and DRB1*1401‐DQA1*0101‐DQB1*0503. Our findings indicate that the low frequencies of susceptible genotypes, in particular, DR3‐DQA1*0501‐DQB1*0201/DR4‐DQA1*0301‐DQB1*0302, together with a high frequency of protective haplotypes, could in part explain the low incidence of T1DM in the Slovenian population. The combined frequencies of susceptible genotypes were similar in the two populations (Slovenia = 19.2%, Italy = 17.6%), and the 95% confidence limits of the OR values for each genotype in the two populations overlapped, indicating no significant differences between the values. We conclude that the similar incidences of T1DM in Italian and Slovenian populations are in part a reflection of similar frequencies of HLA susceptible/protective haplotypes.
A nonsense mutation in Exon 3 results in the HLA‐B null allele B*5127N
Authors: Hou J.Y. , Luning Prak E. , Kearns J. , Wu J. , Bassinger S. , Birkos S. , Williams T.M. , Kamoun M.
Published: March 2003

Abstract: A new HLA‐B null allele has been identified within the B*51 group by combined serological and molecular typing of an Italian Caucasoid family. Serological data indicated that the proband typed homozygous for A2 and B60. Confirmatory typing using sequence specific oligonucleotide hybridization (SSPOH) detected a second B allele within the B*51 group. Allele specific typing (SSP) for B*51 subtypes, including the known B*5111N allele, was performed, and typing results were consistent with B*5101, suggesting the presence of a new null variant. Cloning and sequencing of this allele identified a B*5101 variant with a nonsense mutation in exon 3. This new null allele has been designated B*5127N. The combined use of serologic and DNA‐based typing methods facilitates the identification of null and low‐expression alleles. An overview of null alleles of class I HLA is presented.
Chromogenic and fluorogenic glycosylated and acetylglycosylated peptides as substrates for serine, thiol and aspartyl proteases
Authors: Juliano M.A. , Del Nery E. , Juliano L. , Filira F. , Gobbo M. , Rocchi R.
Published: March 2003

We synthesized short chromogenic peptidyl–Arg–p‐nitroanilides containing either (Galβ)Ser or (Glcα,β)Tyr at P2 or P3 sites as well as O‐acetylated sugar moieties and studied their hydrolysis by bovine trypsin, papain, human tissue kallikrein and rat tonin. For comparison, the susceptibility to these enzymes of Acetyl–X–Arg–pNa and Acetyl–X–Phe–Arg–pNa series, in which X was Ala, Phe, Gln and Asn were examined. We also synthesized internally quenched fluorescent peptides with the amino acid sequence Phe8–His–Leu–Val–Ile–His–Asn14 of human angiotensinogen, in which [GlcNAcβ]Asn was introduced before Phe8 and/or after His13 and ortho‐aminobenzoic acid (Abz) and N‐[2‐, 4‐dinitrophenyl]–ethylenediamine (EDDnp) were attached at N‐ and C‐terminal ends as a donor/receptor fluorescent pair. These peptides were examined as substrates for human renin, human cathepsin D and porcine pepsin. The chromogenic substrates with hydrophilic sugar moiety increased their susceptibility to trypsin, tissue kallikrein and rat tonin. For papain, the effect of sugar depends on its position in the substrate, namely, at P3 it is unfavorable, in contrast to the P2 position that resulted in increasing affinity, as demonstrated by the higher inhibitory activity of Ac–(Galβ)Ser–Arg–pNa in comparison to Ac–Ser–Arg–pNa, and by the hydrolysis of Ac–(Glcα,β)Tyr–Arg–pNa. On the other hand, the acetylation of sugar hydroxyl groups improved hydrolysis of the susceptible peptides to all enzymes, except tonin. The P′4 glycosylated peptide [Abz–F–H–L–V–I–H–(GlcNAcβ)N–E–EDDnp], that corresponds to one of the natural glycosylation sites of angiotensinogen, was shown to be the only glycosylated substrate susceptible to human renin, and was hydrolysed with lower Km and higher kcat values than the same peptide without the sugar moiety. Human cathepsin D and porcine pepsin are more tolerant to substrate glycosylation, hydrolysing both the P′4 and P4 glycosylated substrates.
Synthesis and fluorescence properties of intramolecularly quenched fluorogenic p‐nitroanilides containing coumarin or quinolinone derivatives as fluorophores
Authors: Charitos C. , Tzougraki C. , Kokotos G.
Published: March 2003

Abstract: Nine model intramolecularly quenched fluorogenic substrates (IQFS) of the general structure F‐Phe‐NH‐Np, containing coumarin or quinolinone derivatives as fluorophores (F) and the p‐nitroanilide group (Np) as quencher, were synthesized. The study of the fluorescence properties of the substrates synthesized and the corresponding fluorophores showed that efficient quenching of fluorescence (> 89%) was observed in all cases. The combination of 7‐glutarylamido‐4‐methyl‐coumarin (Mec‐NH‐Glt‐OH) or 7‐methoxy‐4‐coumaryl‐acetic acid (Mca) with the p‐nitroanilide group gave the best results (97.2 and 98.8% quenching, respectively). These fluorophores can be used to convert peptide p‐nitroanilides into IQFS, which, retaining their chromogenic properties, may be applied in both fluorometric and colorimetric assays.
Systematic solid‐phase synthesis of linear pseudooligolysines containing multiple adjacent CH2NH amide bond surrogates: potential agents for gene delivery
Authors: Fridkin G. , Gilon C. , Gilon T. , Loyter A.
Published: March 2003

Abstract: Solid‐phase methodology was used to synthesize a series of fully reduced linear oligolysines (pseudooligolysines, abbreviated herein as PLs) containing up to five adjacent CH2NH peptide bond isosteres. The reduced peptide bonds were introduced by the reductive alkylation reaction between Fmoc‐Lys‐(Boc)‐al and a free α‐amine moiety on the pseudopeptidyl resin, using sodium cyanoborohydride in an acidified mixture of NMP/CH3OH (1 : 1 v/v). The oligomeric molecules, which can be regarded as polyethylene imine and spermine analogs, possess multiple positive charges under physiological conditions and form tight complexes with plasmid DNA. These characteristics and the increased resistance to hydrolysis by trypsin make these molecules potential candidates for future use as DNA carriers in gene delivery.
Synthesis, isolation and characterization ofPlasmodium falciparumantigenic tetrabranched peptide dendrimers obtained by thiazolidine linkages
Authors: Chaves F. , Calvo J.C. , Carvajal C. , Rivera Z. , Ramírez L. , Pinto M. , Trujillo M. , Guzmán F. , Patarroyo M.E.
Published: March 2003

Abstract:Different chemical alternatives were evaluated for obtaining immunogenic polypeptidic macromolecules which could then be used as vaccines. These were based on the ligation reaction between an unprotected immunogenic peptide and an unprotected multifunctional core peptide; polyantigens, designated dendrimers because their form resembles that of dendritic cells, were thus obtained. The antigen‐core ligation alternatives, studied by indirect synthesis, were the formation of oxime, hydrazone and thiazolidine linkages, making use of the reaction between a weak base (acting as nucleophile) and an alkyl aldehyde. The other alternative was the formation of a thioether linkage between a sulfydryl and an alkyl halide. Finally, a multiple antigen peptide (MAP) was synthesized by direct synthesis. All reactions were monitored by SEC‐HPLC and SDS−PAGE. Dendrimer molecular mass obtained was confirmed by MS MALDI‐TOF. Dendrimer purification was first carried out by concentrating crude reaction products with CP‐5000 centricons and (using SEC‐HPLC) pure tetramers were then obtained. A 20‐residue 9376 immunogenic sequence, fromPlasmodium falciparumapical merozoite antigen protein (AMA‐1), was used to study the best alternative for chemical ligation. It was observed that thiazolidine formation proceeded with greater yield and in less time than the others. A tetramer has been simultaneously synthesized via thiazolidine with the SPf‐66 antimalarial vaccine 45‐residue monomer, proving the technique's versatility. The 9376 peptide disulfide bound polymer and SPf‐66 (as well as their tetrameric thiazolidine dendrimers) were inoculated in rabbits to evaluate their antibody response. It was observed that titers for tetrameric thiazolidine dendrimers were not just greater but were also sustained over time. Western blot for pre‐immune and immune sera showed that dendrimer sera recognized specificPlasmodium falciparumproteins as well as disulfide‐bound polymers.
Chemical synthesis of β‐defensins and LEAP‐1/hepcidin
Authors: Klüver E. , Schulz A. , Forssmann W.‐G. , Adermann K.
Published: March 2003

Abstract: A large and steadily growing subfamily of antimicrobially active peptides of animals and plants is formed by the defensins, which are highly disulfide‐bonded, cationic peptides with a molecular mass of about 4 kDa. The synthesis of the human β‐defensins 1 and 2 (hBD‐1, hBD‐2) as well as of the novel murine β‐defensins 7 and 8 (mBD‐7 and mBD‐8) is reported. The peptides were synthesized by solid‐phase peptide synthesis using fluorenylmethoxycarbonyl chemistry. The linear products were oxidized in the presence of the cysteine/cystine redox system to the biologically active molecules. The correct disulfide connectivity of the resulting cyclic products was partly verified by mass spectrometry and sequence analysis of the fragments obtained after tryptic cleavage. In addition, the recently discovered antimicrobially active human peptide LEAP‐1/hepcidin, which contains four disulfide bonds, was successfully synthesized and subsequently oxidized. For Liver‐expressed anti microbial peptide (LEAP)‐1/hepcidin and hBD‐1, the identity of native and synthetic peptides was demonstrated by high‐pressure liquid chromatography and capillary electrophoretic analysis. The general synthetic procedure is suitable to rapidly perform the total chemical synthesis of novel fully bioactive defensins, which are expected to be identified soon, as well as of structurally modified analogs.
Analogs of Substance P modified at the C‐terminus which are both agonist and antagonist of the NK‐1 receptor depending on the second messenger pathway
Authors: Sachon E. , Girault‐Lagrange S. , Chassaing G. , Lavielle S. , Sagan S.
Published: March 2003

Abstract: The initial goal of this study was to analyze, using photolabeling, the interactions between Substance P and its tachykinin NK‐1 receptor. Therefore, the photoreactive amino acid para‐benzoyl‐phenylalanine (pBzl)Phe was incorporated into the Substance P sequence from position 4 to 11 leading to Bapa0[(pBzl)Phex]SP analogs. Biotinyl sulfone‐5‐aminopentanoic acid (Bapa) was introduced in order to purify the covalent complex. These photoreactive SP analogs were first assayed for their affinity for the two binding sites associated with the NK‐1 receptor, as well as for their potency in activating the phospholipase C and adenylate cyclase pathways. All analogs photoreactive from position 4 to 11 have moderate to high affinity for the two NK‐1 receptor‐binding sites, except for the analog modified at position 7. This affinity could be correlated to their potency to activate the phospholipase C and adenylate cyclase pathways, except for the analog photoreactive at position 11. Bapa0[(pBzl)Phe11]SP was found to be an agonist in the phospholipase C pathway and an antagonist in the adenylate cyclase pathway, other analogs modified at position 11 were therefore analyzed. Among these, Bapa0[Pro9, (pBzl)Hcy(O2)11]SP is a partial agonist, whereas Bapa0[Hcy(ethylaminodansyl)11]SP is a full agonist in the phospholipase C pathway, the two analogs being antagonist in the adenylate cyclase pathway. These results show that analogs of SP can be simultaneously agonist at one binding site and antagonist at the other binding site associated with the NK‐1 receptor.
Biological variation responses in fMLP‐OMe analogs, introducing bulky protecting groups on the side‐chain of hydrophilic residues at position 2
Authors: Cavicchioni G. , Turchetti M. , Spisani S.
Published: March 2003

Abstract:  for‐Met‐Ser(Bzl)‐Phe‐OMe, for‐Met‐Cys(Bzl)‐Phe‐OMe, for‐Met‐Tyr(Bzl)‐Phe‐OMe and for‐Met‐Lys(Z)‐Phe‐OMe were synthesized to investigate the importance of a bulky protecting group on the side‐chain of a hydrophilic residue at position 2 on the biological activities of human neutrophils. Our results indicate that these compounds do not trigger a good chemotactic response, which, in any case, is not improved with respect to that induced by the analogs with the unprotected residues. Instead, both superoxide anion production and, particularly, lysozyme release are more efficient.
Development and validation of opioid ligand–receptor interaction models: The structural basis of mu vs. delta selectivity
Authors: Mosberg H.I. , Fowler C.B.
Published: March 2003

Abstract: Opioid receptor binding conformations for two structurally related, conformationally constrained tetrapeptides, JOM‐6 (µ receptor selective) and JOM‐13 (δ receptor selective), were deduced using conformational analysis of these ligands and analogs with additional conformational restrictions. Docking of these ligands in their binding conformations to opioid receptor structural models, based upon the published rhodopsin X‐ray structure, implicates specific structural features of the µ and δ receptor ligand binding sites as forming the basis for the µ selectivity of JOM‐6 and the δ selectivity of JOM‐13. In particular, the presence of E229 in the µ receptor (in place of the corresponding D210 of the δ receptor) causes an adverse electrostatic interaction with C‐terminal carboxylate‐containing ligands, resulting in the observed preference of ligands with an uncharged C‐terminus for the µ receptor. In addition, the requirement that the Phe3 side chain of JOM‐13 assume a gauche orientation for optimal δ binding, whereas the Phe3 side chain of JOM‐6 must be in a trans orientation for high‐affinity µ binding can be largely attributed to the steric effect of replacement of L300 of the δ receptor by W318 of the µ receptor. Testing this hypothesis by examining the binding of JOM‐6 and several of its key analogs with specific µ receptor mutants is described. Our initial results are consistent with the proposed ligand–receptor interaction models.
Activation of the cannabinoid CB1 receptor may involve a W6.48/F3.36 rotamer toggle switch
Authors: Singh R. , Hurst D. P. , Barnett‐Norris J. , Lynch D. L. , Reggio P. H. , Guarnieri F.
Published: March 2003

Abstract:  The cannabinoid CB1 receptor, a member of the Rhodopsin (Rho) family of G protein coupled receptors (GPCRs), exhibits high levels of constitutive activity. In contrast, Rho exhibits an exquisite lack of constitutive activity. In Rho, W6.48(265) on transmembrane helix 6 (TMH6) is flanked by aromatic residues at positions i‐4 (F6.44) and i + 3 (Y6.51), while in CB1 the residues i‐4 and i + 3 to W6.48 are leucines (L6.44 and L6.51). Based upon spectroscopic evidence, W6.48 has been proposed to undergo a rotamer switch (χ1 g+ →trans) upon activation of Rho. In the work reported here, the biased Monte Carlo method, Conformational Memories (CM) was used to test the hypothesis that the high constitutive activity exhibited by CB1 may be due, in part, to the lack of aromatic residues i‐4 and i + 3 from W6.48. In this work, the W6.48 rotamer shift (χ1 g+ →trans) was used as the criterion for activation. Conformational Memories (CM) calculations on WT CB1 TMH6 and L6.44F and L6.51Y mutant TMH6s revealed that an aromatic residue at 6.44 tends to disfavor the W6.48 χ1 g+ →trans transition and an aromatic residue at 6.51 would require a concomitant movement of the Y6.51 χ1 from trans→g+ when the W6.48 χ1 undergoes a g+ →trans shift. In contrast, CM calculations on WT CB1 TMH6 revealed that the presence of leucines at 6.44 and 6.51 provide W6.48 with greater conformational mobility, with a W6.48 transχ1 preferred. Conformational Memories calculations also revealed that the W6.48 χ1 g+ →trans transition in WT CB1 TMH6 is correlated with the degree of kinking in TMH6. The average proline kink angles for TMH6 were higher for helices with a W6.48 g+ χ1 than for those with a W6.48 transχ1. These results are consistent with experimental evidence that TMH6 straightens during activation. Transmembrane helix (TMH) bundle models of the inactive (R) and active (R*) states of CB1 were then probed for interactions that may constrain W6.48 in the inactive state of CB1. These studies revealed that F3.36 (transχ1) helps to constrain W6.48 in a g+ χ1 in the inactive (R) state of CB1. In the R* state, these studies suggest that F3.36 must assume a g+ χ1 in order to allow W6.48 to shift to a transχ1. These results suggest that the W6.48/F3.36 interaction may act as the ‘toggle switch’ for CB1 activation, with W6.48 χ1 g+/F3.36 χ1 trans representing the inactive (R) and W6.48 χ1 trans/F3.36 χ1 g+ representing the active (R*) state of CB1.
Probing the conformational and dynamical effects of O‐glycosylation within the immunodominant region of a MUC1 peptide tumor antigen
Authors: Schuman J. , Campbell A.P. , Koganty R.R. , Longenecker B.M.
Published: March 2003

Abstract: MUC1 mucin is a large transmembrane glycoprotein, the extracellular domain of which is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is under‐glycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above), as well as in the exposure of normally cryptic core Tn (GalNAc), STn (sialyl α2–6 GalNAc) and TF (Gal β1–3 GalNAc) carbohydrates. Here, we report the results of 1H NMR structural studies, natural abundance 13C NMR relaxation measurements and distance‐restrained MD simulations designed to probe the structural and dynamical effects of Tn‐glycosylation within the PDTRP core peptide epitope. Two synthetic peptides were studied: a nine‐residue MUC1 peptide of the sequence, Thr1‐Ser2‐Ala3‐Pro4‐Asp5‐Thr6‐Arg7‐Pro8‐Ala9, and a Tn‐glycosylated version of this peptide, Thr1‐Ser2‐Ala3‐Pro4‐Asp5‐Thr6(αGalNAc)‐Arg7‐Pro8‐Ala9. The results of these studies show that a type I β‐turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence. The existence of a similar β‐turn within the PDTRP core peptide epitope of the under‐glycosylated cancer‐associated MUC1 mucin protein might explain the immunodominance of this region in vivo, as the presence of defined secondary structure within peptide epitope regions has been correlated with increased immunogenicity in other systems. Our results have also shown that Tn glycosylation at the central threonine within the PDTRP core epitope region shifts the conformational equilibrium away from the type I β‐turn conformation and toward a more rigid and extended state. The significance of these results are discussed in relation to the possible roles that peptide epitope secondary structure and glycosylation state may play in MUC1 tumor immunogenicity.
O‐Linked glycosylation occurs on basic parotid salivary proline‐rich proteins
Authors: Carpenter G. H. , Proctor G. B.
Published: March 2003

Interactions between salivary glycoproteins and many oral bacteria have been shown to depend on O‐linked glycans on salivary glycoproteins. Basic proline‐rich proteins form the largest group of proteins within human parotid saliva. In the present study human parotid salivary glycoproteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis or two‐dimensional electrophoresis, electroblotted onto nitrocellulose and probed with two biotin‐labelled lectins from Maclura pomifera (MPA) and Arachis hypogaea (PNA) which are specific for O‐linked (galactose β1,3 N‐Acetylgalactosamine) glycans. Lectin binding was detected with avidin‐biotin complex and enhanced chemiluminescence. Two‐dimensional electrophoresis in combination with lectin binding indicated that only basic parotid salivary glycoproteins bind the lectin MPA. Following removal of terminal sialic acid residues by sialidase digestion the same glycoproteins were detected by the lectin PNA. Glycosidase digestion with endo‐α‐N‐acetylgalactosaminidase (O‐glycanase) in conjunction with sialidase eliminated MPA binding. Taken together these results indicate that many basic parotid salivary glycoproteins contain O‐glycans, all of which are sialylated.
In vitro evaluation of the retention of three species of pathogenic microorganisms by three different types of toothbrush
Authors: Bunetel L. , Tricot‐Doleux S. , Agnani G. , Bonnaure‐Mallet M.
Published: March 2003

The retention and survival of microorganisms on toothbrushes pose a threat of recontamination for certain patients at risk. In order to measure the influence of brush design and optimize the choice of toothbrush model for complementary studies, the in vitro retention of three microbial species (Porphyromonas gingivalis ATCC 33277, Streptococcus mutans ATCC 25175 and Candida albicans ATCC 26555) was evaluated for three types of toothbrush. Two series of standardized experiments were carried out for each brush and microorganism. The first series tested the retention of the microorganisms on the head portion of the brush, while the second measured retention on the head of the brush and the part of the handle inserted in the mouth during brushing. For each series, the microorganisms were counted at T0 and T24 (after storage of the brushes at room temperature for 24 h). Depending on the microorganism studied, from 0.2% to 2% of the initial inoculum was retained on the brush. The number detected increased with the size of the exposed area. After 24 h, P. gingivalis and S. mutans were found on only one type of brush. C. albicans survived on all three. These results confirm that microorganisms can quickly colonize toothbrushes.
Porphyromonas gingivalisplatelet aggregation activity: outer membrane vesicles are potent activators of murine platelets
Authors: Sharma A. , Novak E. K. , Sojar H. T. , Swank R. T. , Kuramitsu H. K. , Genco R. J.
Published: March 2003

Recent evidence has established an association between chronic periodontitis and cardiovascular disease. Periodontitis is a chronic inflammatory disease caused by a small group of gram‐negative bacteria, of whichPorphyromonas gingivalisis considered an important causative agent. It has been proposed that dental plaque bacteria and their products can disseminate into the bloodstream from the site of infection and promote thromboembolic events associated with atherosclerosis and myocardial infarction. In this regard,Streptococcus sanguisandP. gingivalishave been shown to induce platelet aggregationin vitro. Here we report thatP. gingivaliswas able to induce platelet aggregation, and that oral strains ofActinobaillus actinomycetemcomitans,Bacteroides forsythus,Campylobacter rectus,Fusobacterium nucleatum,Prevotella intermediaandTrepenoma denticolafailed to aggregate platelets when tested for platelet aggregation activity under similar conditions. Additionally, we show that vesicles (outer membrane evaginations that are shed into the environment by the bacteria) ofP. gingivalisare potent inducers of mouse platelet aggregationin vitro. In summary, our data show that i) initial adherence of the bacterium to platelet may be facilitated byP. gingivalisfimbriae and ii)P. gingivalisvesicles possess platelet aggregation–inducing activity.
Effects of the oral spirochete Treponema denticola on interleukin‐8 expression from epithelial cells
Authors: Deng Q.‐D. , Han Y. , Xia X. , Kuramitsu H. K.
Published: March 2003

This communication demonstrates that the interaction of the oral spirochete Treponema denticola 35405 with KB epithelial cells does not lead to the induction of interleukin‐8 production as occurs with a variety of other bacteria. Utilizing the dentilisin protease mutant K1 of T. denticola, this property was demonstrated to be primarily a function of the expression of the protease by strain 35405.
Alteration of the coadherence of Candida albicans with oral bacteria by dietary sugars
Authors: Nikawa H. , Egusa H. , Makihira S. , Yamashiro H. , Fukushima H. , Jin C. , Nishimura M. , Pudji R. R. , Hamada T.
Published: March 2003

Interactions between bacterial oral flora and Candida albicans are important in denture plaque formation. This study therefore first aimed to quantify the coadherence of C. albicans and bacteria by the use of a bioluminescent adenosine triphosphate (ATP) assay based on the firefly luciferase‐luciferin system. The second aim was to examine the effect of i) dietary sugars (used for preculture) and ii) enzymatic digestion of fungi on the coadherence. When yeast was preincubated in yeast nitrogen base medium (YNB) supplemented with 250 mM glucose, the yeast coadhered with all isolates of Streptoccus mutans and Streptococcus sanguis, and no significant coadhesion was observed with the isolates of Streptococcus sobrinus, Streptococcus salivarius, Lactobacillus and Actinomyces. However, when the yeast was precultured in YNB supplemented with 500 mM galactose, the yeast coadhered with S. salivarius and Actinomyces, which was not observed when the yeast was grown in YNB with glucose. In addition, the coadherence of the yeast with the isolates of S. sanguis was significantly reduced. Enzymatic digestion of yeast and a reverse transcription polymerase chain reaction assay revealed that expression of at least two types of proteinaceous adhesins are involved in these phenomena.
Phenotypic and genotypic diversity of Streptococcus sanguis in infants
Authors: Pan Y. P. , Li Y. , Caufield P. W.
Published: March 2003

Streptococcus sanguis comprises a heterogeneous group of oral streptococci indigenous to the oral cavity of humans. A total of 289 isolates from an infant population (n=37) were tentatively identified as S. sanguis on the basis of the distinctive colony morphology as shown on MM10‐sucrose non‐selective medium. These isolates were divided into four biovars of S. sanguis as determined by an extended panel of biochemical attributes. Chromosomal DNA was extracted from each isolate, and an AP‐PCR fingerprint profile was obtained to allow study of the diversity within and among the infants. In this study, all four biovars of S. sanguis were detected in the infants. A wide genotypic diversity of S. sanguis was observed among these isolates; on average, each infant harbored 2.7 unique amplitypes as shown by the AP‐PCR fingerprints. To explore the phylogenic relationship among these S. sanguis isolates, 20 strains representing the four biovars were selected at random for sequencing of their 16S rDNA and 16S‐23S rDNA intergenic spacer chromosomal loci. Two major sequence patterns were identified within the 16S rDNA sequences. A phylogenic analysis showed that members from each of the four biovars of S. sanguis bore close relationship with the type‐strain ATCC 10556 sequence, and that all of the isolates representing the four biovars could be clustered into two main phylotypes. The biovars were distributed throughout the phylotypes, indicating no correlation between the genetic and phenotypic groupings.
Antimicrobial susceptibility of major pathogens of orofacial odontogenic infections to 11  β‐lactam antibiotics
Authors: Kuriyama T. , Karasawa T. , Nakagawa K. , Nakamura S. , Yamamoto E.
Published: March 2003

In this study, we evaluated the current effectiveness of 11 β‐lactam antibiotics for treatment of orofacial odontogenic infections by determining the antimicrobial susceptibility of the major pathogens. The antimicrobial susceptibilities of viridans streptococci (n = 47), Peptostreptococcus (n = 67), Porphyromonas (n = 18), Fusobacterium (n = 57), black‐pigmented Prevotella (n = 59) and non‐pigmented Prevotella (n = 47) isolated from pus specimens of 93 orofacial odontogenic infections to penicillin G, cefmetazole, flomoxef, cefoperazone, cefoperazone/sulbactam, ceftazidime, cefpirome, cefepime, cefoselis, imipenem and faropenem were determined using the agar dilution method. Penicillin G, most cephalosporins, imipenem and faropenem worked well against viridans streptococci, Peptostreptococcus, Porphyromonas and Fusobacterium. Penicillin G and most cephalosporins, including fourth‐generation agents, were not effective against β‐lactamase‐positive Prevotella, though they were effective against β‐lactamase‐negative strains. Cefmetazole, cefoperazone/sulbactam, imipenem and faropenem expressed powerful antimicrobial activity against β‐lactamase‐positive Prevotella. In conclusion, penicillins have the potential to be first‐line agents in the treatment of orofacial odontogenic infections. Most of the other β‐lactam antibiotics, including fourth‐generation cephalosporins, were not found to have greater effectiveness than penicillins. In contrast, cefmetazole, cefoperazone/sulbactam, imipenem and faropenem were found to have greater effectiveness than penicillins.
Can immunization affect the development of allergy?
Authors: Pershagen Göran
Published: March 2003

A changing pattern of infections may be of importance for the increase in prevalence of asthma and other allergic diseases in developed countries during recent decades. The clear inverse relation between number of siblings and atopy observed in several studies may be related to a protective role of infections, although specific information is inconclusive. A recent study showed that positive tuberculin responses in schoolchildren correlated with a lower prevalence of atopic disorders, but other studies did not find a relation between BCG vaccination and allergic disease or sensitization. Transient production of IgE antibodies to pertussis toxin has been demonstrated after pertussis immunization; however, randomized clinical trials involving both whole cell and acellular pertussis vaccines have failed to show any enhancement of atopic manifestations in children. Epidemiologic investigations indicate that viral infections may either promote (RSV) or inhibit (hepatitis A, measles) atopy, although data are scarce. In conclusion, the evidence is limited regarding a direct role of vaccinations for development of atopic manifestations, but speaks against a major effect of some types of vaccinations. On the other hand, since some infections may offer protection in relation to allergy, vaccination could result in an increased risk.
Puberty and prognosis of asthma and bronchial hyper‐reactivity
Authors: Nicolai T. , Illi S. , Tenbörg J. , Kiess W. , Mutius E. V.
Published: March 2003

It is a commonly held view that pediatric asthma frequently abates during puberty. However, little data are available that associate the stage of puberty with the prognosis of asthma and bronchial hyper‐reactivity (BHR). In this study, 155 children with active asthma at 10 years of age (60 girls [38.7%], 95 boys [61.3%]) were followed‐up until they reached 14 years of age. The stage of puberty was assessed by parental questionnaire; in addition, serum 3‐α‐androstanediolglucuronide, as an endocrinological marker for peripheral androgen status, was measured in 107 subjects. Persistence of asthma was determined via questionnaire, lung function testing, and bronchial provocation (hyperventilation of cold, dry air). At 14 years of age, 73.3% of girls were reported to have had menarche and 40.8% of boys a voice change, and only 35.5% of the subjects had experienced acute asthma symptoms during the last 12 months, with an almost unchanged gender ratio (19 girls [34.5%], 36 boys [65.5%]) vs. that recorded at 10 years of age. The level of androstanediolglucuronide was higher in the children who reported puberty (mean ± SD): 3.03 ± 2.13 nmol/l vs. 1.89 ± 1.26 nmol/l, p = 0.003. No statistically significant relationship was found between the reported signs of late puberty and loss of asthma or BHR. Likewise, no significant association was found between asthma persistence and the level of androstanediolglucuronide (2.39 ± 1.75 nmol/l vs. 2.44 ± 1.82 nmol/l, p = 0.84), or BHR and the level of androstanediolglucuronide (3.02 ± 1.97 nmol/l vs. 2.28 ± 1.67 nmol/l, p = 0.13), at 14 years of age, in girls or boys. At 14 years of age, no change in the gender ratio of children with active asthma had occured. These results may indicate that the change in gender predominance of asthma through the second decade of life is not caused by increased loss of established asthma in boys between 10 and 14 years of age.
Development of specific immunoglobulin E in coughing toddlers: A medical records review of symptoms in general practice
Authors: Eysink Petra , Bindels Patrick , Huisman Jente , Bottema Ben , Aalberse Rob , Schadé Bert
Published: March 2003

The aim of this study was to study whether young children, originally immunoglobulin E (IgE) negative and who became sensitized to specific inhalation allergens, presented more frequently to their general practi‐tioner (GP) with other allergy‐ and asthma‐related symptoms than children who remained IgE negative. It was also investigated whether asthma was diagnosed more often in children who developed IgE to inhalant allergens. Coughing children, 1–5 years of age, visiting the participating GPs, were tested for IgE antibodies to mites, dogs, and cats by using radioallergosorbent testing (RAST). All IgE‐negative (RAST < 0.2 IU/ml) children were re‐tested after 2 years. The medical records of 162 children were reviewed on asthma‐ and allergy‐related symptoms and on prescribed medication. After 30 months, 27 of the 162 children (17%) had become IgE positive for one or more allergens. Most children (93%) had visited their GP for treatment of respiratory symptoms during this period. However, the children who had become IgE positive had visited their GP more often than the children who remained IgE negative. Differences in visits were seen for: shortness of breath (52% IgE‐positive vs. 19% IgE‐negative children, respectively), wheeze (37% vs. 17%), allergic rhinitis (33% vs. 16%), and pneumonia (22% vs. 8%), but not for coughing (89% vs. 88%). The IgE‐positive children were more frequently diagnosed by their GP as having asthma (48%) than were the IgE‐negative children (23%). In a multivariate analysis, indicators of becoming IgE positive were: a visit for shortness of breath (odds ratio [OR] = 6.9; 95% confidence interval [CI] = 2.1–23.1) and two or more visits for wheeze (OR = 6.0; 95% CI = 1.9–19.2), adjusted for breast‐feeding, age, and asthma or allergy in the family. The positive predictive value (PPV) of being IgE positive with a diagnosis of asthma was 90% (whereas the negative predictive value was 48.0%) for a child attending their GP for treatment of wheeze. For recurrent coughing (six or more visits) and shortness of breath, the PPVs were 73% and 71%, respectively. The development of sensitization to common inhalant allergens is associated with specific allergy and asthma‐related symptoms in young children. IgE‐positive children were more frequently diagnosed as having asthma by their GP. This implies that in general practice it is possible to detect children at high risk for developing allergic asthma early in life by their respiratory symptoms and by subsequent testing for specific IgE to inhalant allergens.