bluuebunny/crossref_metadata_2025 huggingface dataset:
Validation of DNA‐based HLA‐A and HLA‐B testing of volunteers for a bone marrow registry through parallel testing with serology
Authors: Noreen H.J. , Yu N. , Setterholm M. , Ohashi M. , Baisch J. , Endres R. , Fernandez‐Vina M. , Heine U. , Hsu S. , Kamoun M. , Mitsuishi Y. , Monos D. , Perlee L. , Rodriguez‐Marino S. , Smith A. , Yang S.Y. , Shipp K. , Hegland J. , Hurley C.K.
Published: March 2003

Abstract: A total of 42,160 individuals were typed for HLA‐A and HLA‐B by both serology and PCR‐based typing. The HLA assignments included all of the known serological equivalents. The majority of the individuals (99.9%) were from U.S. minority population groups. The serologic typing was performed between 1993 and 1997 at the time of recruitment for the National Bone Marrow Program (NMDP) registry. The polymerase chain reaction (PCR)‐based typing was carried out in two phases. In phase I, DNA typing was performed by PCR using sequence‐specific oligonucleotide probes (PCR‐SSOP) or PCR using sequence‐specific primers (PCR‐SSP) without knowledge of the serologic assignments. Discrepancies were identified between the serologic and DNA assignments in 24% of the volunteers (8% of volunteers differed for only HLA‐A assignments, 13% for HLA‐B, and 3% for both HLA‐A and ‐B) and a potential explanation was assigned each discrepant serology/DNA pair. In phase II, a random sampling scheme was used to select a statistically significant number of individuals for repeat DNA typing from each of these categories. The categories included antigens missed by serology, nonexpressed (null) alleles, PCR amplification failures, misassignment of antigens and nomenclature issues. Only a single individual was found to carry a null allele. DNA‐based testing correctly typed nearly 99% of the donors at HLA‐A, more than 98% at HLA‐B, and more than 97% at both HLA‐A and ‐B validating this methodology for registry typing.
New polymorphic microsatellite markers in the human MHC class III region
Authors: Matsuzaka Y. , Makino S. , Nakajima K. , Tomizawa M. , Oka A. , Bahram S. , Kulski J.K. , Tamiya G. , Inoko H.
Published: March 2003

Abstract: The human major histocompatibility complex (MHC) class III region spanning approximately 760 kb is characterized by a remarkably high gene density with 59 expressed genes (one gene every 12.9 kb). Recently, susceptibility loci to numerous diseases, such as Graves disease, Crohn disease, and SLE have been suggested to be localized to this region, as assessed by associations mainly with genetic polymorphisms of TNF and TNF‐linked microsatellite loci. However, it has been difficult to precisely localize these susceptibility loci to a single gene due to a paucity to date of polymorphic markers in the HLA class III region. To facilitate disease mapping within this region, we have analyzed 2∼5 bases short tandem repeats (microsatellites) in this region. A total of 297 microsatellites were identified from the genomic sequence, consisting of 69 di‐, 62 tri‐, 107 tetra‐, and 59 penta‐nucleotide repeats. It was noted that among them as many as 17 microsatellites were located within the coding sequence of expressed genes (NOTCH4, PBX2, RAGE, G16, LPAAT, PPT2, TNXB, P450‐CYP21B, G9a, HSP70‐2, HSP70‐1, HSP‐hom, MuTSH5 and BAT2). Eight microsatellite repeats were collected as polymorphic markers due to their high number of alleles (11.9 on average) as well as their high polymorphic content value (PIC) (0.63). By combining the 38 and the 22 polymorphic microsatellites we have previously collected in the HLA class I and class II regions, respectively, we have now established a total of 68 novel genetic markers which are uniformly interspersed with a high density of one every 63.3 kb throughout the HLA region. This collection of polymorphic microsatellites will enable us to search for the location of any disease susceptible loci within the HLA region by association analysis.
Twenty‐nine new HLA‐B alleles associated with antigens in the 5C CREG
Authors: Steiner N.K. , Kosman C. , Jones P.F. , Gans C.P. , Rodriguez‐Marino S.G. , Rizzuto G. , Baldassarre L.A. , Edson S. , Koester R. , Sese D. , Mitton W. , Ng J. , Hartzman R.J. , Hurley C.K.
Published: March 2003

Abstract: This paper describes 29 novel HLA‐B locus alleles identified during low‐resolution typing. The majority of the novel alleles carry new patterns of previously known polymorphic motifs or codons. Three alleles carry alterations in the Bw4/Bw6 epitope. Five alleles carry novel substitutions.
Report of a new DRB1*13 allele: DRB1*1336
Authors: Jezek D.A. , Lower F.E. , Wagenknecht D.R. , Britton R.M. , Getty R.R. , Pulliam J.F. , McIntyre J.A. , Jennings C.D.
Published: March 2003

Abstract: This brief communication describes the characterization of a new allele, DRB1*1336.
HLA‐DR and ‐DQ polymorphism in Cameroon
Authors: Pimtanothai N. , Hurley C.K. , Leke R. , Klitz W. , Johnson A.H.
Published: March 2003

Abstract: HLA‐DRB1, ‐DQA1 and ‐DQB1 allele frequencies were determined by high‐resolution polymerase chain reaction using sequence‐specific oligonucleotide probes (PCR‐SSOP) and/or DNA sequencing in 126 healthy individuals in Cameroon. Eighteen DRB1, 11 DQA1, and 18 DQB1 alleles were observed. The most common alleles at each locus were DRB1*1503 (29%), DRB1*1301 (13%); DQA1*0102 (38%), DQA1*0302 (11%), DQA1*0401 (11%); and DQB1*0602 (31%), DQB1*0301 (13%), DQB1*0501 (12%). Forty‐four different haplotypes were identified including 12 novel haplotypes demonstrating the HLA class II diversity resulting from allele combinations in this population. A single predominant DRB1*1503‐DQA1*0102‐DQB1*0602 haplotype was observed with a frequency of 27%. In summary, this study of HLA class II polymorphism in Cameroon demonstrates the extent of diversity in this population.
Polymorphism of the human retinoid X receptor β and linkage disequilibrium with HLA‐DPB1
Authors: Rajsbaum R. , Fici D. , Fraser P.A. , Flores‐Villanueva P.O. , Awdeh Z.L.
Published: March 2003

Abstract: The human retinoid X receptor β (RXRB) gene is localized in the major histocompatibility complex (MHC) region between DPB1 and RING2. The RXRB gene sequence reported by different investigators suggests that the gene may be polymorphic. In this study, we confirmed one polymorphism by sequencing genomic DNA from four Caucasian individuals. We also developed a restriction fragment length polymorphism (RFLP) analysis to detect this specific polymorphism. Linkage analysis studies between RXRB alleles and a number of HLA markers showed significant linkage disequilibrium between RXRB*T and HLA‐DPB1*0401.
Identification of the novel allele HLA‐DRB1*1137 which probably originated from DRB1*11011: implications for mismatch with its ancestor allele at bone marrow transplantation
Authors: Elsner H.‐A. , Kotsch K. , Blasczyk R.
Published: March 2003

Abstract: The identification of the new allele HLA‐DRB1*1137, which was found in a Caucasian individual, is described. In the sequence analysis the new allele differs from DRB1*11011 by position 227 (T>A) which is located in exon 2. At the protein level, the new allele has one amino acid difference compared to DRB1*1101 (Phe47Tyr). Residue 47 is likely to contribute to the peptide binding site of HLA‐DR11 and thus to be important for peptide binding. However, as phenylalanine and tyrosine have very similar physical and chemical features allogenicity in case of mismatch at bone marrow transplantation may be weak.
The absence of disease‐specific polymorphisms within the HLA‐B51 gene that is the susceptible locus for Behçet’s disease
Authors: Sano K. , Yabuki K. , Imagawa Y. , Shiina T. , Mizuki N. , Ohno S. , Kulski J.K. , Inoko H.
Published: March 2003

Abstract: Behçet’s disease is known to be associated with HLA‐B51 in many different populations. Genetic evidence supports that the susceptible gene for Behçet’s disease is the HLA‐B51 allele at the HLA‐B locus. This study was aimed to determine the HLA‐B51 nucleotide sequence variation in three Behçet’s disease patients and three healthy controls in order to elucidate if any disease specific mutations or polymorphisms may exist in the HLA‐B51 gene of patients. Long‐range polymerase chain reaction (PCR) was first carried out to give a PCR‐amplified product of 9.5 kb which was then used as a template for nested PCR to give a final amplified product of 4.2 kb. This final product containing the 1.3‐kb promoter/enhancer region and the entire HLA‐B gene except for a 363‐bp 3′ terminal end segment encoding the 3′ untranslated region was subcloned by the BP cloning technique and sequenced. The sequencing results showed that all the patients possessed the HLA‐B*51011 allele, and there were no differences in the exonic nucleotide sequences between the three Behçet’s disease patients and the three healthy controls. The HLA‐B*51011 intronic and promoter/enhancer nucleotide sequences from the three patients had 22 single nucleotide polymorphisms (SNPs), a single insertion of 6 bp and a single deletion of 2 bp. On the other hand, the three healthy controls had 24 SNPs in their intronic and promoter/enhancer regions. However, none of these polymorphisms in the patients were specific for the disease. Therefore, these results clearly demonstrate that the HLA‐B exonic sequence that encodes the HLA‐B51 allele is the real pathogenic factor in Behçet’s disease.
New insights regarding HLA‐B27 diversity in the Asian population
Authors: García‐Fernández S. , Gonzalez S. , Miña Blanco A. , Martinez‐Borra J. , Blanco‐ Gelaz M. , López‐Vazquez A. , López‐Larrea C.
Published: March 2003

Abstract: A polymerase chain reaction‐sequence‐specific primer (PCR‐SSP) method which distinguishes all B27 alleles described at present (B*2701–23) has been developed. The distribution of B27 alleles was characterised in six different Asian populations. HLA‐B*2705, 02, 04, 07, 22 (formerly B*2706) subtypes found in Asian populations differ in their ethnic distribution, which may be the result of different genetic and geographic origins. Furthermore, two novel B27 alleles were found in this study. B*2714 was identified in two Siberians, one of whom was a patient with ankylosing spondylitis. B*2715 was found in two patients with ankylosing spondylitis in Thais. These associations have not previously been reported in either ethnic group.
Identification of a novel HLA‐DQB1*06 allele, DQB1*0618
Authors: Casamitjana N. , Gil J. , Campos E. , Santos M. , Nogués N. , Ribera A. , Palou E.
Published: March 2003

Abstract: We report here the identification of a novel DQB1*06 allele, DQB1*0618, found in a bone marrow donor. The new allele was detected during routine DNA‐based HLA typing by an ambiguous pattern of probe hybridization, obtained by polymerase chain reaction using sequence‐specific oligonucleotides (PCR‐SSO). Molecular cloning and sequencing confirmed that the new allele is identical to DQB1*0609 at exon 2 except for 3 nucleotide substitutions at positions 353, 356 and 367, also found in other alleles. These nucleotide changes may explain its anomalous reactivity.
Three new DP alleles identified in a study of 800 unrelated bone marrow donor‐recipient pairs
Authors: Grams S.E. , Wu J. , Noreen H.J. , Mangaccat J. , Cognato M.A. , Johnson S. , Segall M. , Williams T.M. , Begovich A.B.
Published: March 2003

Abstract: HLA‐DP genotyping of 800 unrelated donor‐recipient pairs in phase 5 of a retrospective analysis of unrelated bone marrow transplantation, sponsored by the National Marrow Donor Program (NMDP®), has identified two new DPB1 alleles (DPB1*8701 and DBP1*8801) and one new DPA1 (DPA1*0108) allele. Sequencing confirmed that all three of these new alleles represent novel combinations of previously described sequence motifs, reinforcing the notion that “gene conversion‐like” events play an important role in generating HLA allelic diversity. The identification of these new alleles brings the total number of DPA1 alleles to 20 and the total number of DPB1 alleles to 94.
Identification of a novel allele HLA‐B*7805 in a Japanese female
Authors: Sekimoto T. , Araseki M. , Sato S. , Sakaya S. , Kato T. , Ikeda H.
Published: March 2003

Abstract: A new HLA‐B*78 allele, B*7805, was identified in a healthy Japanese female. The results of her serological HLA class I typing showed an unusual Bw4/Bw6 pattern with strongly positive reactivity to anti‐Bw6, i.e. A24, ‐, B52, ‐, Bw4, Bw6. In DNA typing, she was typed as A*24, ‐, B*52, B*78‐like, Cw1202, ‐, (Bw4, Bw6). Cloning and sequencing of exon 2 and exon 3 of her B locus genes revealed a new allele B*7805. The cloned B*7805 differed from B*78021 by three nucleotide substitutions in exon 2 at position 259 (A to G), 261 (C to G) and 272 (A to C), and contained sequences defining Bw6 motif in the region of codon 77 to 83.
Sequence of two new HLA‐Cw alleles: HLA‐Cw*0313 and HLA‐Cw*1208
Authors: Cox S.T. , Ogilvie H. , Bohan E.M. , Holman R. , Prentice H.G. , Potter M. , Madrigal J.A. , Little A.‐M.
Published: March 2003

Abstract: Two new HLA‐C alleles have been identified by reverse line blot assay and direct sequencing of polymerase chain reaction (PCR) products. The first polymorphism encodes an amino acid change from lysine in Cw*12022 to asparagine in Cw*1208 at residue 66. The second polymorphism encodes two amino acid changes from isoleucine in Cw*03031 to threonine in Cw*0313 at residue 93 and isoleucine to leucine at residue 94. The functional significance of these polymorphisms on peptide‐binding and/or T‐cell recognition is unknown.
Non‐expression of HLA‐A*2901102 N is caused by a nucleotide exchange in the mRNA splicing site at the beginning of intron 4
Authors: Elsner H.‐A. , Bernard G. , Eiz‐Vesper B. , De Matteis M. , Bernard A. , Blasczyk R.
Published: March 2003

Abstract: We describe the identification of the novel human leukocyte antigen (HLA) blank allele A*2901102 N which was detected in an individual of mixed race. The serological HLA class I typing was A1; B7,44 whereas PCR‐SSP indicated the presence of an additional A*29 allele. The pedigree analysis demonstrated that the new blank allele segregated with the haplotype A*29null B*07, inherited from the individual's Vietnamese father. A single G→Τ transversion was detected at position 1 of intron 4, which is a highly conserved nucleotide position in vertebrate splice donor sites. Accordingly, it is very likely that this nucleotide exchange inhibits the splicing of intron 4, resulting in a premature stop codon further downstream. Despite this alteration, transcription into mRNA was demonstrated.
Allele specific PCR for the minor Histocompatibility antigen HA‐2
Authors: Wilke M. , Pool J. , Goulmy E.
Published: March 2003

Abstract: The hematopoietic system restricted minor Histocompatibility antigen (mHag), HA‐2, is encoded by the novel human class I Myosin gene, MYO1G, located on the short arm of chromosome 7. The HA‐2 encoding region is di‐allelic and comprises the HLA‐A2 restricted T cell epitope YIGEVLVSV (HA‐2V) and its allelic counterpart YIGEVLVSM (HA‐2M). We designed a sequence specific PCR (SSP) for both HA‐2 alleles. The HA‐2 genomic typing results were compared with the HA‐2 CTL phenotyping in three families and revealed exact correlation. The mHag HA‐2 SSP can be incorporated in DNA based typing protocols.
DRB1*14 diversity and DRB3 associations in four major population groups in the United States
Authors: Gans C.P. , Tang T.F. , Slack R. , Ng J. , Hartzman R.J. , Hurley C.K.
Published: March 2003

Abstract: At least 59 DRB1*14 positive individuals from each of four U.S. population groups, Caucasoids, African Americans, Asians/Pacific Islanders, and Hispanics, were randomly selected from a database of 82,979 individuals. DRB1*14 alleles were identified by DNA sequence analysis using intron‐specific primers to obtain complete exon 2 sequences. Only 23% of the known DRB1*14 alleles were detected. DRB1*14011 was the predominant DRB1*14 allele in three populations while Hispanics carried DRB1*1402 and DRB1*1406 more frequently. Asians/Pacific Islanders were the most diversified carrying seven alleles. DRB3*0101, DRB3*02021 and DRB3*0210 were detected in a subset of individuals typed for this locus and 15 DRB1‐DRB3 haplotypes were defined. This study completes the exon 2 sequences of previously identified alleles, DRB1*1405‐*1408, including the identification of two silent codon 90 variants of DRB1*1407. In addition, two new DRB1*14 alleles, DRB1*1441 and DRB1*1442, are described.
Two novel CD1 E alleles identified in black African individuals
Authors: Tamouza R. , Sghiri R. , Ramasawmy R. , Neonato M.G. , Mombo L.E. , Poirier J. C. , Schaeffer V. , Fortier C. , Labie D. , Girot R. , Toubert A. , Krishnamoorthy R. , Charron D.
Published: March 2003

Abstract: CD1 gene (CD1A to CD1E) products are involved in non‐peptide antigen presentation, such as lipids and glycolipids, to T cells. With a similar function to MHC, namely antigen presentation, these genes nevertheless displayed a much lower level of polymorphism as compared to MHC. We report here two additional CD1E variants identified in black African individuals, designated herein CD1E*05 and CD1E*06. While the former differs from the common (wild type) allele sequence by two substitutions at nucleotide positions 217 and 229 of exon 2, the latter only by a single base change at position 91 of exon 3. These substitutions lead to amino acid changes at position 73 and 77 of the α1 domain in the former and at position 30 of the α2 domain in the latter. Identification of these additional variants suggests that the CD1 locus, especially the CD1E gene, is much more polymorphic than previously assumed1.
The HLA‐DRA*0102 allele: Correct nucleotide sequence and associated HLA haplotypes
Authors: Kralovicova J. , Marsh S.G.E. , Waller M.J. , Hammarstrom L. , Vorechovsky I.
Published: March 2003

Abstract: Here we correct the nucleotide sequence of a single known variant of the HLA‐DRA gene. We show that the coding regions of the HLA‐DRA*0101 and HLA‐DRA*0102 alleles do not differ at two codons as reported previously, but only in codon 217. Using nucleotide sequencing and DNA samples from individuals homozygous in the major histocompatibility complex, we found that the variant, leucine 217‐encoding HLA‐DRA*0102 allele was present on the haplotypes HLA‐B*0801, DRB1*03011, DQB1*0201 (ancestral haplotype AH8.1), HLA‐B*07021, DRB1*15011, DQB1*0602 (AH7.1), HLA‐B*1501, DRB1*15011, DQB1*0602, HLA‐B*1501, DRB1*1402, DQB1*03011 and HLA‐A3, B*07021, DRB1*1301, DQB1*0603. The HLA‐DRA*0101 allele coding for valine 217 was observed on the haplotypes HLA‐B*5701, DRB1*0701, DQB1*03032 (AH57.1), HLA‐DRB1*04011, DQB1*0302, HLA‐DRB1*0701, DQB1*0202, and HLA‐DRB1*0101, DQB1*05011.
Identification of a new HLA‐DRB1 allele, DRB1*0321
Authors: Gagne K. , Bonneville F. , Cheneau M.‐L. , Herry P. , Pinson M.‐J. , Herfray P. , Cesbron‐Gautier A. , Folléa G. , Bignon J.‐D.
Published: March 2003

Abstract: This communication reports the identification of a new HLA‐DRB1*03 allele identified in three members of a Caucasian French family. This new allele has been officially named HLA‐DRB1*0321 by the World Health Organization Nomenclature Committee. The complete exon 2 sequence of DRB1*0321 is identical to that of DRB1*0307 except for the first and second nucleotides of codon 37 (TT replacing AA), which lead to the substitution of a tyrosine for a phenylalanine (AAC→TTC at position 37). The family study showed that this new allele was transmitted into the HLA‐A*0101/09, ‐B*0801/14, ‐Cw*0701, ‐DRB1*0321, ‐DRB3*0101, ‐DQB1*0503 and ‐DPB1*0401 haplotype. The complete exon 2 sequence of this new allele has been previously deposited in the EMBL Sequence Database under accession number AF297266.
Tissue typing in support of unrelated hematopoietic cell transplantation
Authors: Petersdorf E.W. , Anasetti C. , Martin P.J. , Hansen J.A.
Published: March 2003

Abstract: The success of unrelated hematopoietic cell transplantation (HCT) for the treatment of hematologic malignancies has closely paralleled development of robust typing methods for comprehensive and precise donor–recipient matching. The application of molecular methods in clinical research has led to a more complete understanding of the immunogenetic barriers involving host‐vs‐graft (HVG) and graft‐vs‐host (GVH) reactions. Along with the development of less toxic transplant regimens, advances in the prevention and treatment of graft‐vs‐host disease (GVHD) and in the supportive care of the transplant recipient, improved HLA matching of potential unrelated donors has led to clinical results that begin to compare favorably with that of HLA‐identical sibling transplants.
HLA‐B phenotype modifies the course of Behçet's disease in Moroccan patients
Authors: Choukri F. , Chakib A. , Himmich H. , Marih L. , Caillat‐Zucman S.
Published: March 2003

Abstract: In Moroccan patients, predisposition to Behçet's disease is associated with HLA‐B*51, mostly in males with young age at disease onset. In addition, the disease is associated with B*15 both in females and in males with late disease onset. We analyzed the clinical presentation, the severity and the course of the disease in 86 Moroccan patients according to their HLA‐B phenotype. The presence of the B*51 or B*15 did not predispose to a particular clinical manifestation, nor to a more severe presentation of the disease. By contrast, outcome of the disease significantly differed depending on HLA‐B phenotype, with an increase of symptoms in most B*51+ patients and in half of B*15 patients, and a remission or a decrease of symptoms in all B*51‐B*15‐ patients. This variable course was mostly observed for ocular lesions, skin lesions, articular symptoms, and neurological symptoms. These data may suggest that treatment should be given early in the course of the disease in B*51 or B*15‐positive patients in order to stabilize the inflammatory process.
Synthesis and effect of shortened oostatic decapeptide (TMOF) analogs with isosteric structures on reproduction of Neobellieria bullata
Authors: Mařík J. , Tykva R. , Buděšínský M. , Hlaváček J. , Bennettová B.
Published: March 2003

Abstract: Oligopeptides 2a−2d derived from the oostatic decapeptide (TMOF) sequence, H‐Tyr‐Asp‐Pro‐Ala‐Pro‐Pro‐Pro‐Pro‐Pro‐Pro‐OH (1a) and containing isosteric structures were synthesized and assayed to determine their effect during reproduction in the flesh fly Neobellieria bullata. The N‐terminal linear tetra‐ and pentapeptides 2a, 2b containing the Pro‐ψ[CH2O]Ala isosteric linkage affect egg development in 80–90% of ovarioles resulting in some resorbed egg chambers, abnormal yolk deposition, the formation of large eggs with irregular yolk granules and proliferation of follicular epithelium. In comparison with their nonisosteric precursors 1b, 1c they exhibit even more accelerated oostatic activity. However, peptides 2c, 2d containing a Pro‐ψ[CH2S]Ala isosteric linkage are less active.
Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its β‐subunit: possible involvement of the disulfide bonds Cys9−Cys57 and Cys23−Cys72 in receptor binding of the hormone
Authors: Mishra A.K. , Mahale S.D. , Iyer K.S.
Published: March 2003

Abstract: Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The α‐ and β‐subunits of hCG are highly cross‐linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGβ in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGβ were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [125I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9−57) was found to be ≈ 4‐fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23−72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys9−Cys57 and Cys23−Cys72 of the β‐subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.
Development of potent glucagon antagonists: structure−activity relationship study of glycine at position 4
Authors: Ahn J.M. , Medeiros M. , Trivedi D. , Hruby V.J.
Published: March 2003

Abstract: We examined the functional role of glycine at position 4 in the potent glucagon antagonist [desHis1, Glu9]glucagon amide, by substituting the l‐ and d‐enantiomers of alanine and leucine for Gly4 in this antagonist. The methyl and isobutyl side‐chain substituents were introduced to evaluate the preference shown by the glucagon receptor, if any, for the orientation of the N‐terminal residues. The l‐amino acids demonstrated only slightly better receptor recognition than the d‐enantiomers. These results suggest that the Gly4 residue in glucagon antagonists may be exposed to the outside of the receptor. The enhanced binding affinities of analogs 1 and 3 compared with the parent antagonist, [desHis1, Glu9]glucagon amide, may have resulted from the strengthened hydrophobic patch in the N‐terminal region and/or the increased propensity for a helical conformation due to the replacement of alanine and leucine for glycine. Thus, as a result of the increased receptor binding affinities, antagonist activities of analogs 1–4 were increased 10‐fold compared with the parent antagonist, [desHis1, Glu9]glucagon amide. These potent glucagon antagonists have among the highest pA2 values of any glucagon analogs reported to date.
Glutaraldehyde cross‐linking alters the environment around Trp29 of cobrotoxin and the pathway for regaining its fine structure during refolding
Authors: Chang L‐s. , Lin S‐r. , Yang C‐c.
Published: March 2003

Abstract: Cobrotoxin, purified from the venom of Naja naja atra (Taiwan cobra), was subjected to modification with glutaraldehyde in order to prepare intra‐ and intermolecule cross‐linked derivatives. Monomeric and dimeric derivatives were separated from polymeric derivatives by gel filtration. The results of amino acid analysis and sequence determination revealed that only Lys residues were selectively modified by glutaraldehyde. Glutaraldehyde cross‐linking was accompanied by a change in the gross conformation of cobrotoxin as revealed by circular dichroism spectra of the modified derivatives. Compared with cobrotoxin, Trp29 of monomeric and dimeric derivatives was in an apolar microenvironment. This was in agreement with acrylamide quenching studies showing that the spatial position of the Trp indole ring became buried in the interior of the molecule after glutaraldehyde cross‐linking. Moreover, the Trp of modified derivatives was less accessible for iodide than that observed with cobrotoxin. Notably, disulfide reduction could not completely unfold the structure of glutaraldehyde‐modified derivatives as evidenced by the results of acrylamide quenching studies and enzyme‐linked immunoassay. Study of the characteristic changes in Trp fluorescence after the initiation of refolding suggested that the fine structure around Trp29 of cobrotoxin and glutaraldehyde‐modified derivatives was formed differently. These results suggest that glutaraldehyde cross‐linking leads to a change in the microenvironment of cobrotoxin Trp29 and alters the pathway of its fine structure formation during the refolding of cobrotoxin.
Solution structures of the N‐terminal domain of histone H4
Authors: Bang E. , Lee C.‐H. , Yoon J.‐B. , Lee W. , Lee D.W.
Published: March 2003

Abstract: Histones, nuclear proteins that interact with DNA to form nucleosomes, are essential for both the regulation of transcription and the packaging of DNA within chromosomes. The N‐terminal domain of histone H4 contains four acetylation sites at lysine residues and may play a separate role in chromatin structure from the remainder of the H4 chain. We performed circular dichroism and NMR characterization of both native (H4NTP) and acetylated (Ace‐H4NTP) peptides containing N‐terminal acetylation domain of histone H4 for various pH environments. Data from CD and NMR suggested that H4NTP exhibited a pH‐dependent conformational change, whereas the Ace‐H4NTP is insensitive to pH change. However, both peptides showed a defined structural form at acidic pH environments. The solution structure for Ace‐H4NTP shows two structurally independent regions comprising residues of Leu10‐Gly13 and Arg19‐Leu22, demonstrating relatively well‐defined turn‐type structures. Our results suggest that N‐terminal acetylated region of H4 prefers an extended backbone conformation at neutral pH, however, upon acetylation, the regions containing lysine residues induce structural transition, having defined structural form for its optimum function.
Effects of Na2SO4 on hydrophobic and electrostatic interactions between amphipathic α‐helices
Authors: Lazo N.D. , Downing Donald T.
Published: March 2003

Abstract: The effects of 2 molal Na2SO4 at neutral pH on hydrophobic and electrostatic interactions between amphipathic α‐helices were investigated by circular dichroism spectroscopy. The amphipathic peptides that were studied included LEK (acetyl‐LEELKKKLEELKKKLEEL‐NH2) and LEE (acetyl‐LEELEEELEELEEELEEL‐NH2). In phosphate buffer at neutral pH, only LEK adopted a predominantly α‐helical conformation, attributable to glu––lys+ interactions where a major contribution is evidently a hydrogen bond (Biochemistry32: 9668–9676). Despite the presence of lys+ in the e and g′ positions of the abcdefg heptad repeat, LEK exhibited mean‐residue ellipticities at 222 nm ([θ]222) which were dependent on peptide concentration, indicating the presence of a coiled coil. In the presence of 2 molal Na2SO4 at 25–75°C, the helical content of LEK increased, with the greatest increase observed at 75°C. The value of the ellipticity ratio R ([θ]222/[θ]208) of LEK in 2 molal Na2SO4 also increased, indicating a stronger interhelical association. At 50°C and 75°C, LEK remained predominantly α‐helical. In phosphate buffer at neutral pH, LEE was mainly random coil. In the presence of 2 molal Na2SO4, however, the peptide formed α‐helices that associated to form a coiled coil. At 50°C and 75°C, LEE became predominantly random coil but the remaining α‐helices were still associating. These results are consistent with the strengthening of interhelical hydrophobic interactions and the absence of screening of helix‐stabilizing and helix‐destabilizing electrostatic interactions in amphipathic α‐helices by Na2SO4.
The use of coiled‐coil interactions for the analysis and micropreparative isolation of adenomatous polyposis coli protein complexes
Authors: Catimel B. , Faux M.C. , Nerrie M. , Rothacker J. , Nice E.C. , Burgess A.W. , Otvos L. , Wade J.D.
Published: March 2003

Abstract: The coiled coil is a common structural motif found both as the dominant structure in fibrous proteins and as an oligomerization domain in a variety of cytoskeletal and extracellular matrix proteins. Coiled‐coils typically consist of two to four helices that are supercoiled around one another in either parallel or antiparallel orientations. In the past few years our knowledge of the structure and specificity of coiled coil interactions has increased, allowing the de novo design and preparation of coiled‐coils with well‐defined structure and specificity. Indeed, the design and synthesis of a peptide that binds specifically to a single coiled‐coil‐containing protein, adenomatous polyposis coli (APC) has been reported. We have optimized solid‐phase synthesis techniques to produce a modified form of the anti‐APC peptide that contains a biotin moiety specifically placed so as to allow selective orientation onto the surface of a biosensor or affinity support. These peptide surfaces have been used to both monitor and purify APC and APC complexes from cellular extracts.
Isolation and characterization of a novel postsynaptic/cytotoxic neurotoxin from Daboia russelli russelli venom
Authors: Shelke R. R. J. , Sathish S. , Gowda T.V.
Published: March 2003

Abstract: A postsynaptic neurotoxin was purified from Daboia russelli russelli venom using gel filtration, ion‐exchange chromatography and reverse‐phase high‐performance liquid chromatography. The N‐terminal sequence, molecular mass and pharmacological activities of the neurotoxin/cytotoxin indicate that it is a short‐chain neurotoxin like that found in Elapid venom. This is the first report on the presence of such a postsynaptic neurotoxin from D. r. russelli venom.
The CEC behaviour of several synthetic peptides related to the activin βA–βD subunits
Authors: Walhagen K. , Boysen R.I. , Hearn M.T.W. , Unger K.K.
Published: March 2003

Abstract:  The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin βA–βD subunits, has been studied using capillary electrochromatography (CEC) with Hypersil n‐octadecylsilica as the sorbent. The results confirm that the CEC migration of these peptides can be varied in a charge‐state‐specific manner as the properties of the background electrolyte, such as pH, salt concentration and content of organic modifier, or temperature are systematically changed. Acidic peptides followed similar trends in retention behaviour, which was distinctly different to that shown by more basic peptides. The CEC separation of these peptides with the Hypersil n‐octadecyl‐silica involved distinguishable contributions from both electrophoretic mobility and chromatographic retention. Temperature effects were reflected as variations in both the electro‐osmotic flow and the electrophoretic mobility of the peptides. When the separation forces acting on the peptides were synergistic with the electro‐osmotic flow, as, for example, with the positively charged peptides at a particular pH and buffer electrolyte composition, their retention coefficient, κcec, decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their κcec values increased slightly with increasing temperature. Moreover, when the content of organic modifier, acetonitrile, was sufficiently high, e.g. > 40% (v/v) and nonpolar interactions with the Hypersil n‐octadecyl‐silica sorbent were suppressed, mixtures of both the basic and acidic synthetic peptides could be baseline resolved under isocratic conditions by exploiting the mutual processes of electrophoretic mobility and electrostatic interaction. A linear relationship between the ln κcec values and the volume fractions, ψ, of the organic modifier over a limited range of ψ‐values, was established for the negatively charged peptides under these isocratic conditions. These findings thus provide useful guidelines in a more general context for the resolution and analysis of structurally related synthetic peptides using CEC methods.
A convenient incorporation of conformationally constrained 5,5‐dimethylproline into the ribonuclease A 89–124 sequence by condensation of synthetic peptide fragments
Authors: Čeřovský V. , Welker E. , Scheraga H. A.
Published: March 2003

Abstract:  The presence of l‐5,5‐dimethylproline (dmP) within an amino acid sequence results in the formation of an X‐dmP peptide bond predominantly locked in a cis conformation. However, the common use of this unnatural amino acid has been hampered by the difficulty of the economical incorporation of the dmP residue into longer peptide segments due to the steric hindrance imposed by the dimethyl moieties. Here, we describe synthesis of the C‐terminal 36‐residue peptide, corresponding to the 89–124 sequence of bovine pancreatic ribonuclease A (RNase A), in which dmP is incorporated as a substitute for Pro93. The peptide was assembled by condensation of protected 5‐ and 31‐residue peptide fragments, which were synthesized by solid‐phase peptide methodology using fluorenylmethyloxycarbonyl chemistry. We focused on optimizing the synthesis of the Fmoc‐Ser(tBu)‐Ser(tBu)‐Lys(Boc)‐Tyr(tBu)‐dmP‐OH pentapeptide (residues 89–93) with efficient acylation of the sterically hindered dmP residue. In a comparative study, the reagent O‐(7‐azabenzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate was found to be superior to bromo‐tris‐pyrrolidino‐phosphonium hexafluorophosphate and tetramethylfluoroformamidinium hexafluorophosphate for the synthesis of the ‐Tyr(tBu)‐dmP‐ peptide bond in solution as well as on a resin.
Characterization of a Porphyromonas gingivalis gene prtK that encodes a lysine‐specific cysteine proteinase and three sequence‐related adhesins
Authors: Slakeski N. , Cleal S. M. , Bhogal P. S. , Reynolds E. C.
Published: March 2003

Porphyromonas gingivalis extracellular arginine‐ and lysine‐specific proteinases have been implicated as major virulence factors in the development of adult periodontitis. We have previously purified a 48‐kDa lysine‐specific cysteine proteinase, designated PrtK48, from a P.gingivalis W50 cell–associated multiprotein complex. PrtK48 was non‐covalently associated with three sequence‐related adhesins designated PrtK39, PrtK15 and PrtK44 in the multiprotein complex. In this study we cloned and characterized the gene, designated prtK, that encodes a polyprotein that is post‐translationally processed to yield the Lys‐specific proteinase PrtK48 and the three sequence‐related adhesins PrtK39, PrtK15 and PrtK44.
The proportion of interleukin‐4, interferon‐gamma and interleukin‐10‐positive cells in Porphyromonas gingivalis–specific T‐cell lines established from P. gingivalis–positive subjects
Authors: Gemmell E. , Grieco D. A. , Cullinan M. P. , Westerman B. , Seymour G. J.
Published: March 2003

T‐cell cytokine profiles in ten adult periodontitis and seven age‐matched healthy or gingivitis subjects were determined. Porphyromonas gingivalis–specific T‐cell lines were established from the peripheral blood of these individuals all of whom had past or present evidence of P. gingivalis infection. FACS analysis was used to determine the percentage of CD4‐ and CD8‐positive cells in each line staining for cytoplasmic interleukin (IL)‐4, interferon‐gamma and IL‐10. There were no differences in the mean percentage of IL‐4‐, interferon‐gamma‐ or IL‐10‐positive T cells between the two groups. However, the individual profiles showed that the CD4 cells in five of the seven healthy or gingivitis lines had a higher proportion of interferon‐gamma‐positive cells, with two lines demonstrating higher percentages of IL‐10‐ and/or IL‐4‐positive CD4 cells. Five of the ten adult periodontitis lines demonstrated either equal or higher percentages of IL‐4‐positive and/or IL‐10‐positive CD4 cells. With respect to the CD8 cells, two of the seven lines established from the healthy or gingivitis subjects and six of the ten adult periodontitis lines showed profiles with a higher percentage IL‐4‐ and/or IL‐10‐positive cells. When the total T‐cell contribution (CD4 plus CD8) for each T‐cell line was determined from the individual CD4:CD8 ratios, only one of the healthy or gingivitis lines showed a profile with a higher proportion of IL‐10‐positive cells, while the results for the adult periodontitis lines were the same as indicated for the CD4 cell profiles, with five lines showing a higher percentage of IL‐4‐ and/or IL‐10‐positive cells. In conclusion, this study has shown that in P. gingivalis–responsive T‐cell lines established from adult periodontitis and healthy or gingivitis subjects, there was a predominant trend towards a higher percentage of interferon‐gamma positive cells than either IL‐4‐ or IL‐10‐positive cells. However, there were variations from this trend, although whether these variations indicate true susceptibility to progressive disease has yet to be determined.
Isolation and characterization of the cell‐surface polysaccharides of Porphyromonas gingivalis ATCC 53978
Authors: Farquharson S. I. , Germaine G. R. , Gray G. R.
Published: March 2003

The cell‐surface carbohydrates of Porphyromonas gingivalis strain ATCC 53978 were isolated and partially characterized. Three separate polysaccharides were found to be present: an extracellular polysaccharide, capsular polysaccharide and lipopolysaccharide. The capsular polysaccharide, which had peculiar, gel‐like viscoelastic properties, was found to be comprised of mannuronic acid, glucuronic acid, galacturonic acid, galactose, and 2‐acetamido‐2‐deoxy‐
d‐glucose in relative molar ratios of 0.6:0.9:0.5:0.5:1.0, respectively. The extracellular polysaccharide was found to contain mannose, rhamnose, glucose, galactose, and 2‐acetamido‐2‐deoxy‐
d‐glucose in relative molar ratios of 13.5:1.4:1.0:2.0:1.0, respectively. The lipopolysaccharide was found to contain an O‐antigen with a regular tetrasaccharide repeat unit comprised of 4‐linked α‐
l‐rhamnopyranosyl, 6‐linked α‐
d‐glucopyranosyl, 3‐linked α‐
d‐galactopyranosyl, and 4‐linked 2‐acetamido‐2‐deoxy‐β‐
d‐glucopyranosyl residues in equimolar proportions.
The diversity of periodontal spirochetes by 16S rRNA analysis
Authors: Dewhirst F. E. , Tamer M. A. , Ericson R. E. , Lau C. N. , Levanos V. A. , Boches S. K. , Galvin J. L. , Paster B. J.
Published: March 2003

The purpose of this study was to examine the diversity of spirochetes in the subgingival pocket of multiple subjects with a range of periodontal conditions, including two healthy, one adult periodontitis, three acute necrotizing ulcerative gingivitis, eight refractory periodontitis, and one human immunodeficiency virus (HIV) periodontitis. The 16S rRNA genes of spirochetes in plaque were amplified by polymerase chain reaction using spirochete selective primers. Over 500 clones were sequenced and subjected to phylogenetic analysis. The sequences clustered into the 10 known cultivated Treponema species and into 47 as‐yet‐uncultivated Treponema species. Most of these Treponema species were identified from multiple clones and subjects. The human periodontal pocket harbors a highly diverse treponeme population. Of the cultivated species, Treponema denticola, Treponema maltophilum and Treponema sp. Smibert‐3 were most commonly encountered in diseased subjects but rarely in healthy subjects. Molecular tools based on the sequence data from this study will allow determination of the prevalence of cultivable and uncultivable treponemes in oral diseases.
The oral microbiota of children undergoing liver transplantation
Authors: Sheehy E. C. , Beighton D. , Roberts G. J.
Published: March 2003

This study investigated the oral microbiota of children undergoing liver transplantation. Oral swabs were taken using a standardized procedure from 27 children before liver transplantation and at 3 and 100 days post‐transplantation and from 27 healthy controls at baseline and 90 days. Viridans streptococci, yeasts, staphylococci, enterococci and Enterobacteriaceae were enumerated and identified using conventional techniques. The oral microbiota of the patients changed significantly immediately post‐transplantation, but by the final examination, it had returned to baseline levels. The oral microbiota of the controls did not change significantly. The numbers and proportions of Streptococcus salivarius, Streptococcus sanguis and Streptococcus gordonii as percentages of the total streptococcal counts and of the total anaerobic counts decreased significantly 3 days post‐transplantation (P≤0.006). There were no significant changes in the numbers and proportions of Streptococcus oralis and Streptococcus mitis isolated pre‐ and post‐transplantation. The isolation frequencies and numbers of yeasts were significantly higher in patients than controls. Staphylococci were isolated in low numbers from all children. Enterococci and Enterobacteriaceae were isolated infrequently from patients.
Abiotrophia species in early dental plaque
Authors: Mikkelsen L. , Theilade E. , Poulsen K.
Published: March 2003

The most characteristic traits that distinguish Abiotrophia strains from other streptococci are nutrient requirements, satellitism and pyrrolidonyl arylamidase activity. The presence of Abiotrophia strains was studied in early dental plaque formed on sucrose‐containing and sucrose‐free (glucose‐containing) diets. The isolates were from 0‐ to 3‐day dental plaque formed on the buccal surface of a lower premolar in six subjects. Identification of Abiotrophia strains was based on their pyrrolidonyl arylamidase activity. They were among the predominant cultivable microorganisms isolated from diluted suspensions of early dental plaque formed on both diets. Using biochemical tests without testing for pyrrolidonyl arylamidase activity, the Abiotrophia strains would be biochemically identified as Streptococcus mitis or unidentified streptococci, and many isolates first classified as unidentified streptococci were Abiotrophia strains. Generally, identifications using partial 16S rRNA sequences confirmed the identifications obtained biochemically. However, Abiotrophia elegans and Gemella strains were biochemically identified as Abiotrophia adiacens. Abiotrophia strains produce hydrogen sulfide, which may influence the metabolism and ecology of dental plaque and act as a virulence factor in periodontal disease. They are not able to grow on tryptic soy agar. In the present study, some S. mitis strains were not able to grow on mitis salivarius agar, and some Abiotrophia strains were able to grow on this medium. These observations indicate that the use of these media for estimation of total viable counts and number of streptococci introduces systematic error in studies of microorganisms in dental plaque.
Acquisition of iron from human transferrin by Porphyromonas gingivalis: a role for Arg‐ and Lys‐gingipain activities
Authors: Brochu V. , Grenier D. , Nakayama K. , Mayrand D.
Published: March 2003

Porphyromonas gingivalis, a key causative agent of adult periodontitis, is known to produce a variety of virulence factors including proteases. The aim of this study was to evaluate the participation of Arg‐ and Lys‐gingipain activities of P. gingivalis in the acquisition of iron from human transferrin and its subsequent utilization in growth. Iron‐saturated transferrin was found to support the long‐term growth of P. gingivalis. Our results indicated that P. gingivalis does not produce siderophore and does not possess ferric reductase and transferrin‐binding activities. Incubating transferrin with P. gingivalis resulted in degradation of the protein, a step that may be critical for the acquisition of iron from transferrin. Spontaneous and site‐directed mutants of P. gingivalis deficient in one or several proteases were used to demonstrate the key role of specific enzymes in degradation of transferrin and subsequent utilization for growth. The lack of both Arg‐ and Lys‐gingipain activities (mutants M1 and KDP128) was associated with an absence of degradation of transferrin and the incapacity of bacteria to grow in the presence of transferrin as the sole source of iron. It was also found that the Lys‐gingipain activity is more critical than the Arg‐gingipain activity since the mutant KDP112 (deficient in Arg‐gingipain A and B) could grow whereas the mutant KDP129 (deficient in Lys‐gingipain) could not. The fact that growth of mutant KDP112 was associated with a lower final optical density and a generation time much longer compared with the parent strain suggests that the Arg‐gingipain activity also participates in the acquisition of iron from transferrin. Selected inhibitors of cysteine proteases (TLCK, leupeptin and cathepsin B inhibitor II) were tested for their capacity to reduce or inhibit the growth of P. gingivalis under different iron conditions. All three inhibitors were found to completely inhibit growth of strain ATCC 33277 in a medium supplemented with transferrin as the source of iron. The inhibitors had no effects when the bacteria were grown in a medium containing hemin instead of transferrin. The ability of P. gingivalis to cleave transferrin may be an important mechanism for the acquisition of iron from this protein during periodontitis.