bluuebunny/crossref_metadata_2025 huggingface dataset:
Non‐expression of HLA‐B*5111N is caused by an insertion into the cytosine island at exon 4 creating a frameshift stop codon
Authors: Elsner H.‐A. , Drábek J. , Rebmann V. , Ambruzova Z. , Grosse‐Wilde H. , Blasczyk R.
Published: March 2003

Abstract: The identification of the “blank” allele HLA‐B*5111N, which was detected in German and Czech individuals, is described. In the pedigree analysis this new allele segregates with the serological haplotype HLA‐A2; B‐; DR4 which is frequent in Czech population. The non‐expression of B*5111N is caused by the insertion of an additional cytosine molecule at the cytosine island between the nucleotides 621–626 (codons 183–185, first three codons of exon 4) leading to a frame shift that creates a stop codon at codon 196. This insertion may be explained either by conversion with the pseudogene HLA‐J or by slipped‐strand mispairing. In order not to overlook the presence of alleles with altered expression in case of hematopoietic stem cell transplantation, both serological and DNA‐based typing should be performed (Note).
Genetic variability and linkage disequilibrium within the HLA‐DP region: analysis of 15 different populations
Authors: Begovich A.B. , Moonsamy P.V. , Mack S.J. , Barcellos L.F. , Steiner L.L. , Grams S. , Suraj‐Baker V. , Hollenbach J. , Trachtenberg E. , Louie L. , Zimmerman P. , Hill A.V.S. , Stoneking M. , Sasazuki T. , Konenkov V.I. , Sartakova M.L. , Titanji V.P.K. , Rickards O. , Klitz W.
Published: March 2003

Abstract: In order to understand the forces governing the evolution of the genetic diversity in the HLA‐DP molecule, polymerase chain reaction (PCR)‐based methods were used to characterize genetic variation at the DPA1 and DPB1 loci encoding this heterodimer on 2,807 chromosomes from 15 different populations including individuals of African, Asian, Amerindian, Indian and European origin. These ethnically diverse samples represent a variety of population substructures and include small, isolated populations as well as larger, presumably admixed populations. Ten DPA1 and 39 DPB1 alleles were identified and observed on 87 distinct DP haplotypes, 34 of which were found to be in significant positive linkage disequilibrium in at least one population. Some haplotypes were found in all ethnic groups while others were confined to a single ethnic group or population. Strong positive global linkage disequilibrium (Wn) between DPA1 and DPB1 was present in all 15 populations. The African populations displayed the lowest values of Wn whereas the Amerindian populations displayed near absolute disequilibrium. Analysis of the distribution of haplotypes using the normalized deviate of the Ewens‐Watterson homozygosity statistic, F, suggests that DP haplotypes encoding the functional heterodimer are subject to much lower degrees of balancing selection than other loci within the HLA region. Finally, neighbor joining tree analyses demonstrate the power of haplotype diversity for inferring the relationships between the different populations.
Intron sequences of HLA‐B*73
Authors: Voorter C.E.M. , Swelsen W.T.N. , Van Den Berg‐Loonen E.M.
Published: March 2003

Abstract: Molecular typing methods of HLA‐B, like sequence‐specific oligonucleotide hybridization and sequence‐based typing, are based on gene‐specific amplifications of exons 2 and 3 followed by probe hybridization or sequence determination. The necessary gene‐specific amplification primers are often located in rather conserved regions of the introns. In several of these procedures HLA‐B*73 was not amplified, resulting in drop‐out of the allele. To investigate the reason for the allelic drop‐out, the sequences of introns 1, 2 and 3 of HLA‐B*7301 were determined. Comparison of the intron sequence of B*7301 with other HLA‐B and HLA‐C alleles revealed several remarkable features. The overall sequence resembles the sequence of other HLA‐B alleles, although 35 differences were found with a consensus intron sequence. The insertions and deletions shown in intron 2 of B*73 were strikingly similar with the sequences of the HLA‐C alleles, as was the 5′ end of intron 3. Furthermore, a unique deletion was observed in the middle of intron 3, not noticed in other HLA‐B or C alleles. The HLA‐B‐specific primers, widely used for sequence‐specific oligonucleotide hybridization and sequence‐based typing purposes, showed mismatches with the B*73 intron sequences, causing the allelic drop‐out. Correct amplification of complete exons 2 and 3 of B*7301 was enabled by the design of new primers in intron 2 and 3.
Identification of a new HLA‐DRB1 allele (DRB1*0318) in three members of a Caucasian Spanish family
Authors: Ercilla M.G. , Guardia A. , Suárez B. , Arias M.T. , Fabregat V. , Costa M. , Salinas I. , Martorell J. , Vives J. , Lozano F.
Published: March 2003

Abstract: This communication reports the identification of a new allele HLA‐DRB1*03 in three members of a Caucasian Spanish family. The new allele has been officially named HLA‐DRB1*0318 by the World Health Organization Nomenclature Committee. The exon 2 sequence of this new allele is identical to that of DRB1*03011 except for the first nucleotide of codon 45. The nucleotide change (C replacing G) leads to the amino acid substitution of glycine to arginine (GGG→CGG) at position 45. This position of the β1 domain shows very little polymorphism among DRB1* alleles (nucleotide changes at this position have only been reported for DRB1*1436 and DRB1*0105) and locates in the vicinity of the highly polymorphic position 47, which is a constituent of the groove’s pocket interacting with the amino acid position 7 of the antigen peptide. The familial study showed that the new allele was maternally transmitted into the HLA‐A*3002, ‐B*1801, ‐Cw*0501, ‐DRB1*0318, ‐DRB3*0202, ‐DQB1*0201 haplotype. Interestingly, the two siblings of the family, which were HLA identical and suffered of insulin‐dependent diabetes mellitus (IDDM), were carriers of the two HLA haplotypes (DRB1*03/DQB1*0201 and DRB1*04/DQB1*0302) reported as susceptibility markers to IDDM in Caucasians (Note).
Genomic analysis of MIC genes in rhesus macaques
Authors: Seo J.W. , Walter L. , Günther E.
Published: March 2003

Abstract: MIC genes map to the major histocompatibility complex (MHC) and are distantly related to MHC class I genes. Recently, MICA/MICB‐like genes have been described in nonhuman primates. In Macaca mulatta, three MICA/B‐like genes could be identified: Mamu‐MIC1, Mamu‐MIC2, and Mamu‐MIC3. We show here the isolation and characterization of rhesus macaque cosmid clones which carry the Mamu‐MIC2 and Mamu‐MIC3 genes. Neither the MIC2‐ and MIC3‐coding sequences nor respective flanking sequences can be aligned unambiguously to either the human HLA‐MICA or ‐MICB subregions, although MIC2 was found at a similar distance to the BAT1 gene as known for MICB in human. Thus, the characteristics allowing for a classification of primate MIC genes as being of the MICA or MICB types appear to have evolved after the separation of humans and rhesus monkeys from a common ancestor. Furthermore, also Mamu‐MICD‐containing cosmids could be isolated. In contrast to Mamu‐MIC2 and Mamu‐MIC3, the Mamu‐MICD gene and its flanking sequences are highly conserved and orthologous to the human MICD subregion.
High‐resolution sequence‐based typing strategy for HLA‐DQA1 using SSP‐PCR and subsequent genotyping analysis with novel spreadsheet program
Authors: Cordovado S.K. , Simone A.E. , Mueller P.W.
Published: March 2003

Abstract: We present a new sequence‐based typing (SBT) strategy for the polymorphic HLA‐DQA1 locus that is based on sequence‐specific primer – polymerase chain reaction (SSP‐PCR) amplification from genomic DNA. This method allows high‐resolution genotyping in the second exon of the DQA1 gene. This gene presents a unique situation in which half of the known alleles contain an inframe three base pair deletion of codon 56. This deletion confounds direct SBT methodologies of heterozygous individuals containing both a deletion and nondeletion allele. The primary HLA haplotype associated with type 1 diabetes susceptibility is DR3/DR4. The DQA1 genotype for these two haplotypes are DQA1 *0501, a non‐deletion allele and *0301, a deletion allele, thus creating a situation that cannot be resolved using a direct sequencing approach. Our group‐specific SBT strategy isolates the deletion alleles from the nondeletion alleles, allowing them to be resolved by direct sequencing. Additionally, we present a novel spreadsheet program that accurately assigns the genotype of both homozygous and heterozygous persons.
Identification and sequencing of the HLA‐B*4106 allele
Authors: Vidal S. , Morante M.P. , Mosquera A.M. , Moga E. , Querol S. , García J. , Rodríguez‐Sánchez J.L.
Published: March 2003

Abstract: We report a novel HLA‐B*41 allele (HLA‐B*4106) initially detected by an unusual sequence‐specific oligonucleotide (SSO) hybridization pattern and identified by sequence‐based HLA typing. Molecular cloning and sequencing determined that the new HLA‐B allele was identical to the HLA‐B*4101 in exon 2 and 3 except for a single nucleotide substitution in the exon 3 changing codon 204 from Glu to Gln (GAG→CAG). In addition, intron 2 was identical to the published HLA‐B*4104 intron 2, except for the base 509 (C→G).
Strong association of a tumor necrosis factor‐α promoter allele with cerebral malaria in Myanmar
Authors: Ubalee R. , Suzuki F. , Kikuchi M. , Tasanor O. , Wattanagoon Y. , Ruangweerayut R. , Na‐Bangchang K. , Karbwang J. , Kimura A. , Itoh K. , Kanda T. , Hirayama K.
Published: March 2003

Abstract: To investigate the host genetic factors affecting the clinical course of falciparum malaria, polymorphism of the tumor necrosis factor‐α (TNF‐α) promoter region was analyzed in patients with cerebral malaria. Two hundred and forty‐three Myanmar patients with falciparum malaria at Mae Sot Malaria clinic and Mae Sot General Hospital located at the border between Thailand and Myanmar, were included in this study. Among the patients (128 from Karen, 115 from Burma), 200 were uncomplicated and 43 had cerebral malaria. The TNF‐α 5′‐ flanking region showed biallelic polymorphic sites at −238, −308, −857, −863, −1031, and there were 7 alleles (TNFP‐A, B, C, D, M1, M4, M7) found in the patients from Myanmar. We found that the TNFP‐D allele was significantly associated with cerebral malaria in the populations from Karen (Pc<0.0001, OR=124.86) and Burma (Pc<0.0001, OR=34.50). TNFP‐D showed no significant linkage disequilibrium with any alleles of HLA‐B or HLA‐DRB1, suggesting that TNFP‐D was primarily associated with cerebral malaria in Myanmar.
GASTRIC EMPTYING TIME IN RENAL TRANSPLANT RECIPIENTS TREATED WITH CYCLOSPORINE‐A
Authors: Ozkaya O , Derici U , Buyan N , Dalgic A , Dalgic B , Cingi E , Kitapci M , Sindel S , Hasanoglu E
Published: March 2003

Gastric emptying time (GET) appears to be a rate‐limiting factor in the absorption of cyclosporine‐A (CsA), and may be responsible for intra‐ and interpatient variabilities of CsA bioavailability. Few studies have assessed gastric motility after renal transplantation. The purpose of this study was to evaluate gastric emptying of semisolid material in stable renal transplant patients, and to assess whether it is associated with blood CsA levels. Gastric emptying time of semisolids (GET1/2: half emptying time) was measured in 16 transplant recipients who were taking CsA (Neoral®), prednisolone and azathioprine (or mycophenolate mofetil). None of the subjects had a history of gastrointestinal, metabolic, or systemic disease or surgery, and none was taking any drugs at the time of the study that affect gastrointestinal motility. Gastric emptying time (t=1/2) was measured by radionuclide methods. Data concerning the daily CsA dose, plasma levels of CsA (C0), and serum creatinine levels were also recorded. The mean GET (t=1/2) was 89.1±26.4 min. Twelve of the patients exhibited delayed gastric emptying. The mean plasma CsA level for the group was 171.8±56 ng/ml, and the mean CsA dose was 4.1±1.1 mg/kg/day. Gastric emptying time (t=1/2) was not significantly correlated with the serum creatinine levels, the time since transplantation, or the plasma levels of CsA. There was a weak‐positive, but statistically insignificant, correlation between the mean daily CsA dose and the GET (t=1/2; r=0.33, P=0.2). In conclusion, we cannot speculate whether a higher dose of CsA causes delayed gastric emptying or whether patients with delayed emptying require higher doses of CsA. However, we believe that determining GET after transplantation is important for adjusting the immunosuppressant dosage.
Identification of a novel HLA‐B*07 allele – HLA‐B*0726
Authors: Herbut B. , Woodman R. , Kowalick L. , Malkki M. , Petersdorf E.W. , McKenna R.
Published: March 2003

Abstract: We describe here, the identification of a novel HLA‐B*07 allele named HLA‐B*0726. This allele was found in a Caucasian individual serologically typed as HLA‐B7, B35. Novel DNA probe patterns for the HLA‐B*07 allele were found using HLA‐B specific reverse sequence‐specific oligonucleotide probe (SSOP) and sequence‐specific primer (SSP) typing. DNA sequencing demonstrated the presence of a new HLA‐B*07 sequence variant encoding a single nucleotide substitution from a G to a T at nucleotide 539 in exon 3. This results in an amino acid substitution from arginine to leucine at residue 156 in exon 3.
Description of fourteen new DRB alleles found in a stem cell donor registry
Authors: Tang T.F. , Lin Y.‐S. , Robbins F.‐M. , Li L. , Sintasath D. , Coquillard G. , Huang A. , Heine U. , Ng J. , Hartzman R.J. , Hurley C.K.
Published: March 2003

Abstract: Fourteen DRB alleles, DRB1*0705, DRB1*11014, DRB1*1134, DRB1*1136, DRB1*1141, DRB1*1335, DRB1*1337, DRB1*1338, DRB1*1342, DRB1*1343, DRB1*1349, DRB1*1510, DRB3*0105, and DRB5*0103, are described. Among them, eleven are variants which differ by only one nucleotide from previously described alleles, including one silent variant (DRB1*11014). Alleles, DRB1*0705, DRB1*1335 and DRB3*0105, display unique sequence motifs that have never been observed in DRB alleles.
The IL12B gene does not confer susceptibility to coeliac disease
Authors: Louka A.S. , Torinsson Naluai Å. , D'Alfonso S. , Ascher H. , Coto I. , Ek J. , Giordano M. , Gudjónsdóttir A.H. , Mellai M. , Momigliano‐Richiardi P. , Percopo S. , Samuelsson L. , Wahlström J. , Greco L. , Sollid L.M.
Published: March 2003

Abstract: Coeliac disease (CD) is a chronic inflammatory disorder where dietary gluten is not tolerated. In the lesion there are gluten reactive T cells predominantly secreting γ‐interferon. Both HLA and non‐HLA genes contribute to CD susceptibility. Interleukin‐12 (IL‐12) regulates γ‐interferon production. The IL12B gene is located in a region (5q31.1–33.1) where there is evidence for linkage with CD. Allele 1 of an IL12B 3′UTR single‐nucleotide polymorphism leads to increased expression of IL‐12, and was recently implicated in susceptibility for type 1 diabetes (T1D). We found no evidence for association of allele 1 to CD by the transmission/disequilibrium test or case‐control approach. No increased frequency was observed in patients belonging to families where the disease was linked to markers on chromosome 5q. Unlike T1D, allele 1 does not appear to confer susceptibility to CD.
Sequence of a new DR12 allele with two silent mutations that affect PCR‐SSP typing
Authors: Zanone R. , Bettens F. , Tiercy J.‐M.
Published: March 2003

Abstract: A new HLA‐DR12 allele has been identified in a European Caucasoid bone marrow donor. The DRB1*12012 allele differs from DRB1*12011 by two silent substitutions at codons 72 and 78, two polymorphic positions used for DNA subtyping of the DR12 serotype. The co‐occurence of the two nucleotide changes is unique to the DR12 group and results in a new PCR‐SSP typing pattern. The complete HLA type of the donor is A24, A68; B55, B61; Cw*01, Cw*0304; DRB1*12012, DRB1*1402; DRB3*0101, DRB3*0202; DQB1*0301. HLA‐DRB1*12012 is a rare allele as it occurs in < 0.2% of DR12 donors.
Identification of a new MICA allele, MICA*047
Authors: Pérez‐Rodríguez M. , Raimondi E. , Marsh S. G. E. , Madrigal J. A.
Published: March 2003

Abstract: The MHC Class I related (MIC) gene family has been shown to be very polymorphic with 46 different MICA alleles being officially named by the WHO Nomenclature Committee for factors of the HLA system to date. We have identified a novel MICA allele, MICA*047, in a Coya American Indian individual from the Jujuy province of north‐western Argentina. The novel MICA*047 allele differs from the MICA*030 allele by a single non‐synonymous substitution in exon 2, condon 26 GTA→GGA, Valine to Glycine1.
Identification and nucleotide sequence of a new null allele, HLA B*3540N
Authors: Dunne C. , Little A.M. , Cox S.T. , Masson D. , Crowley J. , Barnes T. , Marsh S.G.E. , Rooney G. , Hagan R. , Lawlor E. , Madrigal J.A.
Published: March 2003

Abstract: We report the definition of an HLA class I null allele that has been identified within the B35 group by a combination of serological and molecular typing. This allele, which has been named B*3540N, was detected in a French, potential unrelated hematopoietic stem cell donor of unknown ethnic origin, selected as a probable match for an Irish patient. The presence of the null allele was initially determined by the absence of B35 reactivity by serological typing, in contrast to positive reactions by PCR‐SSP and PCR‐SSO typing. Subsequent sequencing of clones containing the full genomic sequence of the B*35 allele identified a single nucleotide deletion within exon 4 which resulted in the introduction of a stop codon downstream within exon 4.
New HLA‐A*11 allele, A*1112, identified by sequence‐based typing
Authors: Garino E. , Belvedere M. , Berrino M. , Bertola L. , Dall'Omo A.M. , Mazzola G. , Rossetto C. , Carcassi C. , Lai S. , Gay E. , Ricotti M. , Curtoni E.S.
Published: March 2003

Abstract: In this report, we describe the identification of HLA‐A*1112, a novel HLA‐A*11 allele found in two Italian families. The new allele was detected during routine HLA typing by a polymerase chain reaction sequence‐specific primer and was confirmed by high‐resolution sequencing‐based typing. The nucleotide sequences of HLA‐A*1112 exons 2 and 3 are identical to HLA‐A*11011 except for a single nucleotide substitution in codon 90 (GAC→GCC).
Identification of feline MAGE‐1 gene product by monoclonal antibodies
Authors: Ma ZY. , Khatlani T.S. , Sasaki K. , Okuda M. , Inokuma H. , Onishi T.
Published: March 2003

Abstract: Melanoma antigens (MAGE) are thought to induce a tumor‐specific immune response and to be potential therapeutical targets for cancer immunotherapy. We have earlier identified the cDNA of feline melanoma antigen 1 (fMAGE‐1), but its product was not characterized in detail. We have expressed the recombinant fMAGE‐1 protein and have generated monoclonal antibodies (mAbs) against it, to identify the native fMAGE‐1 protein in feline lymphoma cell lines and tumor tissues. The fMAGE‐1 protein was found to be approximately 39 kDa in molecular mass on sodium dodecyl‐sulphate‐polycrylamide gel electrophoresis (SDS‐PAGE), and it was found to be located in the cytoplasm of the cells by immunofluorescence. Immunoblotting analysis detected the fMAGE‐1 gene product in the fMAGE‐1‐mRNA‐positive cells, but not in the fMAGE‐1‐mRNA‐negative cells. An interesting finding of the present study was the distribution of the fMAGE‐1 protein, which was found to have a spindle‐like distribution, with filaments twining around the nucleus, suggesting that the fMAGE‐1 protein may be associated with or form some cytoplasmic filaments. This type of finding is so far the first report of its kind, and to the best of our knowledge it has not been reported in either human or mouse MAGE proteins until now. It most probably implies the major diversity of the MAGE family genes.
Frequency and genetic basis of MHC, beta‐2‐microglobulin and MEMO‐1 loss of heterozygosity in sporadic breast cancer
Authors: McEvoy C.R.E. , Seshadri R. , Morley A.A. , Firgaira F.A.
Published: March 2003

Abstract: The Human Leukocyte Antigen (HLA) class I molecules are critical factors in T cell recognition of abnormal, including neoplastic, cells. Loss of HLA class I expression phenotypes, as defined by immunohistochemistry‐based tests, have been previously described in many types of cancer. Here we describe a microsatellite marker DNA‐based loss of heterozygosity (LOH) analysis of three distinct chromosomal regions which have been implicated in HLA class I expression on a cohort of 99 unselected sporadic breast cancer samples. These regions comprise the 4Mb major histocompatibility complex (MHC) region on chromosome 6p, which contains the HLA class I heavy chain loci and other genes responsible for antigen processing, the HLA class I light chain (beta‐2‐microglobulin, β2m) gene on chromosome 15q, and the putative HLA class I modifier of methylation gene (MEMO‐1) on chromosome 1p. Additional chromosome 6 markers were also employed to determine the likely genetic mechanism for MHC loss. We show that 25/99 (25%) of samples show allelic loss within the MHC, 28/95 informative samples (29%) show allelic loss of β2m and 21/76 informative samples (28%) show allelic loss of MEMO‐1. Approximately half of the samples are predicted to have compromised HLA class I gene expression due to LOH at one and/or other of these three loci. Sequencing of the remaining β2m allele in samples displaying β2m LOH failed to detect any additional intragenic mutations. Analysis of the frequency of samples showing LOH at either 0, 1, 2 or 3 of the genomic regions analyzed suggested clustering of tumors into either ‘no loci loss’ or ‘3 loci loss’ categories. These results reveal major underlying genetic causes for the high level of HLA class I expression loss seen in breast cancer.
Point mutation in the β sheet of the HLA‐C α2 domain generates a novel HLA‐C allele, HLA‐Cw*08012, in a Puyuma Aboriginal individual in Taiwan
Authors: Wu S. , Lai C.‐Y. , Chou F.‐C. , Lai S.‐M. , Chen S.‐P. , Shiao Y.‐M.
Published: March 2003

Abstract: We report herein the identification of a new HLA‐C allele using sequence‐based typing (SBT). This novel allele, HLA‐Cw*08012, was found in an Aboriginal individual from the Puyuma tribe in the southern part of Taiwan. This individual was typed by the SBT method as having an HLA genotype of HLA‐A*2402/2402, HLA‐B*1502/4801, HLA‐Cw*08011/08012, HLA‐DRB1*15011/08032, HLA‐DRB5*01011, and DPB1*0501/1401. This new allele differs from HLA‐Cw*08011 in one of the nucleotides of the polymorphic exon 3 at codon 99 [TAT→TAC; both code for tyrosine]. This residue is located in the β sheet of the HLA‐C α2 domain. This new allele was detected in a few individuals of the Puyuma tribe in Taiwan, but has not yet been observed in other populations in Taiwan.
Evidence for discoordinate regulation of the HLA‐DPB1 gene
Authors: Coiras M.T. , Álvarez‐Barrientos A.M. , Díaz G. , Arroyo J. , Sánchez‐Pérez M.
Published: March 2003

Abstract: Characterization of cell lines derived from patients with type II bare lymphocyte syndrome, a pathological state in which the constitutive and inducible expression of HLA class II antigens is lacking, has permitted the identification of several trans‐acting factors involved in the coordinated regulation of HLA class II genes. Although an increasing body of evidence has pointed to the existence of a discoordinate regulation of HLA class II loci, the mechanisms underlying such regulation are essentially unknown. In the present study, 45.EM2, a mutant lymphoblastoid cell line with a new pattern of HLA discoordinate expression is characterized. 45.EM2 expresses HLA‐DR and ‐DQ but fails to express HLA‐DP. The absence of HLA‐DP expression in 45.EM2 is the result of a transcriptional defect, leading to a lack of DPB1 mRNA. By contrast, DPA1 transcription in this LCL is not impaired. The characteristics of 45.EM2 described here suggest the existence of a specific trans‐acting factor involved in the control of DPB1 gene expression.
IMMUNOSUPPRESSIVE THERAPY AND KAPOSI'S SARCOMA AFTER KIDNEY TRANSPLANTATION
Authors: Haberal M , Moray G , Çolak T , Yagmurdur MC
Published: March 2003

Immunosuppressive therapy for organ transplant recipients is complicated by high rates of malignant diseases, one of these being Kaposi's sarcoma (KS). We reviewed the records of 1075 patients who underwent kidney transplantation at our center between October 1985 and May 2002. A total of 52 malignant diseases were observed in 50 patients (4.7%), during the post‐transplantation period. Kaposi's sarcoma was identified in 16 of these 50 individuals. Of the 16 recipients, six were male and 10 were female. The mean age in the group was 39±9 years (range: 10–62 years). At the time of the KS diagnosis, 14 of the recipients were taking triple‐drug immunosuppressive therapy consisting of cyclosporine A, azathioprine, and prednisolone. The other two individuals were receiving azathioprine and prednisolone. The mean time from transplantation to diagnosis with KS was 24±15.2 months (range: 8–74 months). One recipient had concomitant lymphoma and KS. The lesions of seven patients were limited to the skin, five cases involved the skin and gastrointestinal tract, and four patients had disseminated disease. After KS was confirmed, the first‐line of treatment was to withdraw cyclosporine A and azathioprine, and taper the prednisolone. The tumors were managed with appropriate surgical and/or medical therapy. At the time of writing, nine individuals were still alive: four had normal renal function and five had lost their grafts as a result of chronic rejection. We have found the combination of immunosuppressive drug withdrawal and chemotherapy to be very effective in patients with limited disease, but the results in cases of generalized disease have been poor.
Mutations in the gene for toll‐like receptor 4 and multiple sclerosis
Authors: Reindl M. , Lutterotti A. , Ingram J. , Schanda K. , Gassner C. , Deisenhammer F. , Berger T. , Lorenz E.
Published: March 2003

Abstract: Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system with heterogeneous pathological features, disease courses and genetical backgrounds. In this study we determined whether genetic variants of toll‐like receptor (TLR) 4, which confer substantial differences in the inflammation elicited by bacterial lipopolysaccharide, are related to the development of MS. We found no differences in the frequencies of the cosegregating TLR4 Asp299Gly and Thr399Ile polymorphisms between Austrian MS patients (11.6%) and age‐matched controls (13.7%). Furthermore, we could not detect any influence of these mutations on clinical parameters and serum levels of soluble adhesion molecules of MS patients. Our data indicate that these TLR4 polymorphisms have no influence on the incidence, progression and inflammatory parameters of MS.
Evidence for helicity in insect diuretic peptide hormones: computational analysis, spectroscopic studies, and biological assays
Authors: Nittoli T. , Sieburth S.McN. , Coast G.M.
Published: March 2003

The conformation of four insect diuretic hormones has been analyzed computationally using secondary structure prediction routines and comparison with structures in the Brookhaven Protein Databank. Based on this analysis, a common seven‐residue peptide fragment (DVLRQRL) had a high probability of forming an α‐helix. Circular dichroism (CD) studies found that addition of trifluoroethanol (TFE) to an aqueous solution of the seven‐residue fragment induces a change from random coil to helix. Subsequent NMR studies in water‐TFE (1 : 1) produced nOe values and 3JαNH coupling constants confirming a helical conformation: 3JαNH coupling constants for the first five residues (D1 to Q5) were all ≤ 6.0 Hz and two medium‐range nOe values (dαN (i,i+3)) were observed between V2 and Q5, and R4 and L7. The longer fragments PLDVLRQRL in water‐TFE and Lom‐DH 1–26 in water alone, both containing the DVLRQRL sequence of the locust (Locusta migratoria) diuretic hormone, maintained the helicity as determined by CD analysis. However, the remaining 20 residues of the locust diuretic hormone did not maintain the same amount of helicity in water and all of the truncated fragments were not biologically active.
Conformational restriction of the Tyr53 side‐chain in the decapeptide HEL[52‐61]: effects on binding to MHC‐II I‐Ak molecule and TCR recognition
Authors: Casimir J.R. , Iterbeke K. , Van Den Nest W. , Tourwé D. , Trescol‐Biémont M.‐C. , Rabourdin‐Combe C. , Dumortier H. , Muller S. , Gerlier D. , Paris J.
Published: March 2003

Abstract  A series of conformationally restricted analogs of the hen egg lysozyme (HEL) decapeptide 52‐61 in which the conformationally flexible Tyr53 residue was replaced by several more constrained tyrosine and phenylalanine analogs was prepared. Among these tyrosine and phenylalanine analogs were 1,2,3,4‐tetrahydro‐7‐hydroxyisoquinoline‐3‐carboxylic acid (Htc), 1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid (Tic), 4‐amino‐1,2,4,5‐tetrahydro‐8‐hydroxy‐2‐benzazepine‐3‐one (Hba), 4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepine‐3‐one (Aba), 2‐amino‐6‐hydroxytetralin‐2‐carboxylic acid (Hat) and 2‐amino‐5‐hydroxyindan‐2‐carboxylic acid (Hai) in which the rotations around Cα‐Cβ and Cβ‐Cγ were restricted because of cyclization of the side‐chain to the backbone. Synthesis of Pht‐Hba‐Gly‐OH using a modification of the Flynn and de Laszlo procedure is described. Analogs of β‐methyltyrosine (β‐MeTyr) in which the side‐chains were biased to particular side‐chain torsional angles because of substitution at the β‐hydrogens were also prepared. These analogs of HEL[52‐61] peptide were tested for their ability to bind to the major histocompatibility complex class II I‐Ak molecule and to be recognized in this context by two T‐cell hybridomas, specific for the parent peptide HEL[52‐61]. The data showed that the conformation and also the configuration of the Tyr53 residue influenced both the binding of the peptide to I‐Ak and the recognition of the peptide/I‐Ak complex by aT‐cell receptor.
DiSSiMiL: Diverse Small Size Mini‐Libraries applied to simple and rapid epitope mapping of a monoclonal antibody
Authors: Burgess K. , Han I. , Zhang A. , Zheng W.‐H. , Saragovi H.U. , Shanmugam K. , Featherstone M.S.
Published: March 2003

Abstract: Methods for screening protein–protein interactions are useful in protein science and for the generation of drug leads. We set out to develop a simplified assay to rapidly test protein–protein interactions, with a library of 400 pentapeptides comprising the 20 natural amino acids at two variable positions followed by three glycines (NH2‐X1X2GGG). The library was used to identify the epitope of monoclonal antibody (mAb) 10D11 directed against the HOXD4 protein. Three pentapeptide ‘hits’ were selected (VYGGG, PWGGG and WKGGG) from direct binding assays screening for pentapeptide•mAb interactions; and from assays using pentapeptides in solution to competitively block HOXD4•mAb interactions. Alignment of the three ‘hit’ pentapeptides to the HOXD4 sequence predicts the mAb 10D11 epitope as NH2‐VYPWMK. Synthesis of NH2‐VYPWMK hexapeptide confirmed this prediction; and an alanine scan of HOXD4 ablated binding by mAb 10D11 when amino acids in the putative epitope were mutated. We propose that these simplified but diverse libraries can be used for rapid epitope mapping of some mAbs, and for generating lead small peptide analogs that interfere with receptor–ligand or other protein–protein interactions, or with enzymatic activity.
NMR and CD conformational studies of the C‐terminal 16‐peptides of Pseudomonas aeruginosa c551 and Hydrogenobacter thermophilus c552 cytochromes
Authors: Bouchayer E. , Stassinopoulou C.I. , Tzougraki C.H. , Marion D. , Gans P.
Published: March 2003

Abstract: The 16‐amino acid sequences of the C‐terminal helices of the homologous bacterial cytochromes c551 from Pseudomonas aeruginosa and c552 from Hydrogenobacter thermophilus were synthesized and their solution structure studied. Circular dichroism and NMR experiments in aqueous solution have shown the presence of α‐helices and 310‐helices. The populations of helical structures in phosphate buffer, pH 3.5, 293 K, were 21% for c551 and 20% for c552, but increased to 56.7 and 48%, respectively, in 50% aqueous 2,2,2‐trifluoroethanol. An isodichroic point was observed at 203 nm in CD spectra for the helix/coil transition in mixtures of water/2,2,2‐trifluoroethanol. NMR spectra in phosphate buffer show the presence of both α‐ and 310‐helical structures. In water/2,2,2‐trifluoroethanol (50 : 50) α‐helices are predominant. CD temperature‐dependency studies indicate that both peptides exhibit the same cooperativity for the transition in water/2,2,2‐trifluoroethanol (50 : 50). The experimental data show that the amino acid substitutions do not favor heat resistance of the secondary structure of the c552 C‐terminal helix at the local level. Instead, they optimize nonlocal contacts of the polypeptide chain, which stabilize the tertiary structure in the native protein.
Synthesis and conformational analysis of the insulin‐like 4 gene product
Authors: Büllesbach E.E. , Schwabe C.
Published: March 2003

Abstract: Insulin‐like 4 (INSL‐4) is a protein expressed in the early placenta. Its primary structure is insulin‐like with reference to the distribution of cysteine residues and the single chain pro‐form. Insulin‐like 4 was generated by solid‐phase peptide synthesis of the two chains followed by the sequential synthesis of the three disulfide bonds. Two disulfide isomers were produced, one with an insulin‐like disulfide bonding pattern and the other with a reversed chain orientation. The CD spectra of the two disulfide isomers were indistinguishable without any features produced by periodic structures. In addition, the hydrodynamic properties of the two isomers were identical which implied a very open structure of the disulfide‐bonded two‐chain molecules. It appears that insulin‐likeness cannot be defined solely on the basis of the primary structure of cDNA.
Synthesis and biological evaluation on hMC3, hMC4 and hMC5 receptors of γ‐MSH analogs substituted with l‐alanine
Authors: Grieco P. , Balse‐Srinivasan P. , Han G. , Hruby V.J. , Weinberg D. , MacNeil T. , Van der Ploeg L. H. T.
Published: March 2003

Abstract:  To elucidate the molecular basis of the interaction of the native dodecapeptide γ‐MSH with the melanocortin receptors, we performed a structure−activity study in which we systematically replaced l‐Ala in each position of this peptide. Here we report the binding affinity and agonist potency on human MC3R, MC4R and MC5R. Intracellular cAMP concentration was measured on CHO cells, and binding assays were carried out using membranes prepared from these cell lines which stably express hMC3R, hMC4R and hMC5R. Our results indicate that the last four amino acids in the C‐terminal region of γ‐MSH are not important determinants of biological activity and selectivity at human melanocortin receptors. Interesting results were obtained when l‐Ala was substituted for His6, Phe7, Arg8 and Trp9. For these peptides, the affinity and activity at all three human receptors (MC3R, MC4R and MC5R) decreased significantly, demonstrating that the His‐Phe‐Arg‐Trp sequence in γ‐MSH is important for activity at these three melanocortin receptors. Similar results were obtained when Met3 was replaced with l‐Ala, suggesting the importance of this position in the interaction with all three receptors. This study highlights the role played by the His‐Phe‐Arg‐Trp sequence in receptor binding and in agonist activity of γ‐MSH.
An artificial peptide with corticotropin‐releasing factor receptor‐2 (CRF‐R2) selective properties: the role of primary structure in the induction of signal transduction pathways
Authors: Tellam D. J. , Lovejoy D. A. , Smart D. , Qian X. , Lovejoy D.A.
Published: March 2003

Abstract:  Considerable plasticity can occur within the amino acid sequence of amphiphilic peptide hormones. This is particularly evident within the corticotropin‐releasing factor (CRF) family of peptides where, despite less than 15% sequence similarity among the four paralogous lineages, all are capable of acting as high affinity ligands to members of the CRF receptor family. This suggests that these peptides could undergo many mutational changes and remain as high affinity ligands to their receptors as long as the functional motifs do not change radically. Because paralogous peptide lineages are a product of genome duplications, additional genes encoding peptide‐like sequences, which through mutation have lost their functional integrity, may exist. Function to these sequences may be restored if the appropriate motifs are reinserted into the primary structure. We screened rat genomic DNA with highly degenerate polymerase chain reaction (PCR) primers targeted to hybridize with the termini of CRF‐related sequences. One set of sauvagine‐based primers hybridized with a 120‐bp sequence. The theoretical peptide sequence (SV4) showed similarity to the CRF family of peptides at the primary structure level. The encoded sequence was prepared by solid‐phase synthesis and its activity assayed against mouse R1 and human R1/R2 receptors. SV4 did not bind to either mouse or human variants of the R1 receptor, but did bind to the R2 receptor with an affinity comparable to human CRF. SV4 exhibited a similar efficacy of cellular activation as CRF in trials quantifying the acidification rate of human R2α‐transfected Chinese hamster ovary (CHO) cells, but not R1‐transfected cells. SV4 utilizes adenylate cyclase as the principal secondary messenger of R2 signal transduction but, unlike urocortin or sauvagine, does not activate guanylate cyclase‐, calcium‐ or mitogen‐activated protein (MAP) kinase‐mediated pathways. These data suggest that this artificial peptide may be useful to understand the cyclic adenosine monophosphate (cAMP)‐dependent component of the CRF‐R2 signal transduction cascade, and that additional sequences in the genome may be used to engineer bioactive peptides.
Conformational studies of antimetastatic laminin‐1 derived peptides in different solvent systems, using solution NMR spectroscopy
Authors: Jaseja M. , Copié V. , Starkey J.
Published: March 2003

Summary Due to its critical role in cancer progression, interactions between laminin‐1 and the 67 kDa Laminin‐Binding Protein (the 67 kDa LBP) have been the focus of a number of structural and biological studies. As laminin‐1 is such a large and complex molecule, research interests have turned to the investigation of bioactive peptides derived from binding domains of laminin‐1. Two peptides of interest, CDPGYIGSR (peptide 11) and YIGSR, both derived from the β1 chain of laminin‐1, have been shown to block invasion of basement membranes by tumor cells. Substituting the C‐terminal arginine to lysine, a conservative substitution, results in a loss of peptide antimetastatic activity. This difference in bioactivity has been attributed, based on numerous modeling studies of free peptide conformations, to structural differences between YIGSR and YIGSK. Yet the nature of the ‘active’ free peptide backbone conformation has been a matter of debate and controversy. In order to test the validity of the structural modeling claims, we have undertaken detailed conformational studies of the two laminin‐1 derived peptides YIGSR and CDPGYIGSR along with the biologically inactive YIGSK analog by two‐dimensional solution 1H NMR spectroscopy in three different solvent systems. Herein we report that although both the active (YIGSR, CDPGYIGSR) and the inactive (YIGSK) peptides can adopt several closely related conformations in solution, the two peptides share similar conformational preferences, and there are no significant structural differences between the active and inactive peptides, contrary to previously reported modeling data. We conclude that the basis of the peptide biological activity, in contrast to published models, cannot be attributed to well‐defined structural preferences of the free peptides. We infer that the difference in bioactivity observed between YIGSR and YIGSK originates primarily from the chemical nature of the arginine versus lysine sidechain substitution, rather than being due to a structural change in the free peptide conformations.
Regression coefficient analysis of neutrophil chemotaxis in periodontitis
Authors: Meyle J. , Axmann‐Krcmar D.
Published: March 2003

Micropore‐filter techniques have been widely used to assess motility and chemotaxis of neutrophils. In some cases the results were impaired by inadequate methods of evaluation and failures of the test system. For clinical purposes the mode of test evaluation should be able to differentiate between failures of the test system (false‐negative) and real functional defects (true‐negative). A new method has been developed to check parallel assays for homogeneity. Based on earlier procedures this type of evaluation uses the comparison of regression coefficients to identify false‐negative test results within the cell population of an individual. The newly developed method was used to study the chemotactic response in a group of 49 patients and 22 healthy volunteers. Intraclass correlation coefficients of the regression coefficient analysis and the leukotactic index were calculated and compared. It could be demonstrated that the regression coefficient analysis had the same sensitivity as the leukotactic index. Slight variations in the cell counts at a single plane of the chemotactic filter affecting the leukotactic index by 18% had no measurable influence on the calculated regression coefficient.
Soluble products from Eikenella corrodens stimulate oral epithelial cells to induce inflammatory mediators
Authors: Yumoto H. , Nakae H. , Yamada M. , Fujinaka K. , Shinohara C. , Ebisu S. , Matsuo T.
Published: March 2003

In the inflammatory response elicited by bacterial colonization in periodontal pockets, pocket epithelial cells not only serve as a barrier to isolate the pocket microenvironment from external stimuli but also regulate the functions of neighboring cells including fibroblasts and inflammatory cells. To elucidate this mechanism, we characterized the effects of periodontopathic bacterium Eikenella corrodens 1073 components on the production of some inflammatory mediators in a human oral epithelial cell line (KB). In enzyme‐linked immunosorbent assay (ELISA), the E. corrodens supernatant induced interleukin‐6 (IL‐6), IL‐8 and prostaglandin E2 but not interferon‐γ (IFN‐γ) production by KB cells. After incubation with E. corrodens supernatant, KB cells showed a marked increase in the levels of IL‐6, IL‐8 and PG G/H synthase (cyclooxygenase)‐2, but not IFN‐γ, gene expression by reverse‐transcriptase polymerase chain reaction. All these E. corrodens products responsible for production of these inflammatory mediators resisted freezing and boiling and were present in a 10‐kDa filtrate. These results imply that these soluble small‐molecular‐mass products from E. corrodens stimulate various inflammatory mediator productions by human oral epithelial cells and may play a role in the initiation of periodontal inflammation and subsequently perpetuate the inflammatory response during chronic infection.
Detection of cytolethal distending toxin activity and cdt genes in Actinobacillus actinomycetemcomitans isolates from geographically diverse populations
Authors: Fabris A. S. , DiRienzo J. M. , Wïkstrom M. , Mayer M. P. A.
Published: March 2003

A cytolethal distending toxin (CDT) found in Actinobacillus actinomycetemcomitans inhibits the eukaryotic cell cycle, which may contribute to the pathogenic potential of the bacterium. The presence of the cdtABC genes and CDT activity were examined in 40 clinical isolates of A. actinomycetemcomitans from Brazil, Kenya, Japan and Sweden. Thirty‐nine of 40 cell lysates caused distension of Chinese hamster ovary cells. At least one of the cdt genes was detected in all strains examined. The three cdt genes were detected, by PCR, in 34 DNA samples. DNA from one strain from Kenya did not yield amplicons of the cdtA and cdtB genes and did not express toxic activity. Restriction analysis was performed on every amplicon obtained. PCR‐RFLP patterns revealed that the three cdt genes were conserved. These data provided evidence that the cdt genes are found and expressed in the majority of the A. actinomycetemcomitans isolates. Although a quantitative difference in cytotoxicity was observed, indicating variation in expression of CDT among strains, no clear relationship between CDT activity and periodontal status was found.
Subgingival microbiota of renal transplant recipients
Authors: Leung W. K. , Yau J. Y. Y. , Jin L. J. , Chan A. W. K. , Chu F. C. S. , Tsang C. S. P. , Chan T. M.
Published: March 2003

Renal transplant patients undergoing immunosuppressive therapy may experience periodontal side‐effects such as gingival overgrowth. This study evaluated the subgingival microbiota of renal transplant recipients with or without periodontal tissue destruction who may have concurrent gingival enlargement. Subgingival paper point samples taken from the deepest probing sites of 38 subjects (one per patient) were examined using direct microscopy and culture techniques. A complex microflora comprising gram‐positive and gram‐negative cocci, rods and filaments, fusiforms, curved rods and spirochetes was observed using microscopy. Yeasts were occasionally detected. Significantly higher proportions of gram‐positive morphotypes, including gram‐positive cocci, were observed in samples from periodontally healthy patients. The predominant cultivable microflora from anaerobic culture comprised several species of facultative and obligate anaerobes. Colonization of the subgingival sites by ‘foreign’ microbes that are normally dermal, intestinal or vaginal flora was detected in up to 50% of the samples. High mean proportions of lost or unidentified species were also occasionally noted. The results showed that the subgingival biofilm of renal transplant recipients with chronic periodontitis comprised mainly gram‐negative rods and spirochetes. Besides the usual predominant cultivable subgingival microbiota associated with periodontitis, the high prevalence of unidentified and ‘foreign’ microbes indicates the possibility of subgingival microbial alteration in renal transplant patients.