bluuebunny/crossref_metadata_2025 huggingface dataset:
Polarity of the P1 anchor residue determines peptide binding specificity between HLA‐A*3101 and HLA‐A*3303
Authors: Takiguchi M. , Matsuda T. , Tomiyama H.
Published: March 2003

A previous pool sequence analysis showed that HLA‐A*3101 and HLA‐A*3303 binding peptides have the same anchor residues at P2 and the C‐terminus, the only difference being that HLA‐A*3303 binding peptides have two additional P2 anchor residues. Using a stabilization assay with RMA‐S transfectants expressing HLA‐A*3101 and human β2‐microglobulin, we tested the binding of 232 8‐ to 11‐mer peptides carrying HLA‐A*3303 anchor residues to HLA‐A*3101. One hundred of these peptides (43.1%) bound to HLA‐A*3101, confirming that these residues are also anchors for HLA‐A*3101. Although aromatic hydrophobic P2 residues were previously shown to be stronger anchors than aliphatic hydrophobic P2 residues in HLA‐A*3303 binding peptides, we detected no significant difference in HLA‐A*3101 binding affinity between peptides carrying aromatic or aliphatic hydrophobic P2 residues. Statistical analysis previously showed a positive effect of negatively charged P1 residues and a negative effect of positively charged P1 residues for peptide binding to HLA‐A*3303. In contrast such analysis demonstrated a positive effect of positively charged P1 residues and a negative effect of negatively charged P1 residues for peptide binding to HLA‐A*3101. Analysis using mutated peptides confirmed these results. The present study therefore demonstrates that peptide binding specificity between HLA‐A*3101 and HLA‐A*3303 is determined by the polarity of the P1 anchor residue.
Molecular diversity of HLA‐A*02 in Asian Indians: predominance of A*0211
Authors: Mehra N.K. , Jaini R. , Rajalingam R. , Balamurugan A. , Kaur G.
Published: March 2003

Abstract: The North Indians are considered predominantly Caucasoid with an admixture of genes from the Mongoloid and Aryan races. The present study was undertaken to investigate the genetic diversity of HLA‐A*02 in the North Indian population and determine the frequency distribution of its molecular subtypes at the population level. The study revealed a high occurrence of A*0211 (33.8%) in this population along with increased frequencies of the common Oriental alleles, A*0206 (7.5%) and A*0207 (32.5%) and also of HLA‐A*0205 (15%) commonly observed in negroid populations. HLA‐A*0211 has only been reported with very low frequencies among the Ticuna Jews, Thai population, and Colombian Blacks in the malaria endemic areas of Africa. Significantly, we observed an unexpectedly low frequency of A*0201 (3.8%) in contrast to its distribution in Western Caucasians in whom it constitutes 95% of the HLA‐A2 repertoire. Prevalence of HLA‐A*0211 at very high frequencies among North Indians may be a consequence of the founder effect, racial admixture or selection pressure due to environmental factors in this population.
Sequencing based typing for HLA‐C. Identification of three new alleles: Cw*0307, Cw*0502 and Cw*0504
Authors: Santos S. , Vicario J.L. , Garcia‐Sanchez F. , Aviles M.J. , Balas A.
Published: March 2003

Abstract: HLA‐C has been described as a transplantation locus in the unrelated bone marrow transplantation setting, and noticeably the number of mismatches between HLA‐A,‐B,‐DRB1 compatible pairs is considerably high. Sequencing based typing (SBT) is an accurate and efficient methodology utilised in the HLA class I and II allele level of resolution. SBT for HLA‐C locus was applied on a sample of 40 HLA‐A,B,DRB1,DRB3/4/5,DQB1‐compatible bone marrow recipient‐donor pairs, and 3 new HLA‐C alleles have been found. Cw*0307, well defined by serology as Cw3, showed two amino acid changes at the NK motif 77–80 regarding all described Cw*03 alleles, N77K80 instead of S77N80. Two new Cw*05 alleles were described, Cw*0502 properly typed by serology, and Cw*0504 that behaves as a short antigen. Cw*0502 differed from Cw*0501 by only one nucleotide at exon 3, that generated an amino acid replacement at codon 177, K to E. Cw*0504 differs from Cw*0501 by two clustered amino acid positions (114 and 116) placed at the peptide binding site. The rate of new HLA‐C alleles found in this small series evidences a high grade of hidden HLA‐C diversity in the Spanish population, particularly in the well‐defined serologic specificities.
Diversity of MICA (PERB11.1) and HLA haplotypes in Northeastern Thais
Authors: Romphruk A.V. , Naruse T.K. , Romphruk A. , Kawata H. , Puapairoj C. , Kulski J.K. , Leelayuwat C. , Inoko H.
Published: March 2003

Abstract: MICA or PERB11.1 is a polymorphic major histocompatibility complex (MHC) class I‐related gene located 46 kb centromeric of the HLA‐B gene in the HLA class I region. It is expressed mainly in gut epithelial cells, keratinocytes, endothelial cells, fibroblasts and monocytes, and is upregulated by heat stress. MICA has been found to interact with γδ T cells, αβ CD8+ and natural killer (NK) cells bearing the NKG2D/DAP10 receptor. The MICA gene displays a high degree of polymorphism with at least 54 alleles. In the present study, polymorphic exons 2, 3 and 4 of the MICA gene were analyzed using sequencing based typing (SBT) in 255 unrelated healthy northeastern Thais. Thirteen previously reported MICA alleles were detected. MICA*008, *010, *002 and *019 were highly predominant with the allele frequencies of 21.4%, 18.2%, 17.6% and 15.3%, respectively. Five of these 13 MICA alleles show significantly different frequencies from those of the Japanese and Caucasian populations. Interestingly, MICA052, which is a very rare allele in other populations, was prevalent with the allele frequency of 8.2%, mainly on the HLA haplotype carrying HLA‐B*13 in this population. Strong linkage disequilibria were observed between MICA and HLA‐B, as similarly observed in other populations, namely MICA*010‐B*4601, MICA052‐B*13, MICA*002‐B*5801, and MICA*019‐B*15 (1502, 1508, 1511, 1515, 1528, 1530). A large variety of three‐locus (MICA – HLA‐B – HLA‐Cw) and six‐locus (HLA‐DQB1 – HLA‐DRB1 – MICA – HLA‐B – HLA‐Cw – HLA‐A) haplotypes were recognized in the northeastern Thai population. This is the first report on MICA allelic distribution in Southeast Asian populations. These data will provide the important basis for future analyses on the potential role of the MICA gene in disease susceptibility and transplantation matching in Southeast Asian populations.
Prenatal HLA typing of uncultured amniocytes prior to the collection of related allogeneic cord blood
Authors: Bugert P. , Zieger W. , Klüter H. , Eichler H.
Published: March 2003

Abstract: DNA samples isolated from corresponding uncultured amniotic fluid, cord blood and maternal blood (n=5) were subjected to low resolution typing of the HLA‐A, ‐B and ‐DRB loci by the polymerase chain reaction using sequence‐specific primers (PCR‐SSP). Furthermore, the effect of ethylene diamine tetraacetate disodium salt (EDTA) on the quality of genomic DNA isolated from amniotic fluid samples after long‐term storage was evaluated. Unambiguous results of HLA typing could be achieved from all amniotic fluid samples stabilized with EDTA. PCR‐SSP typing failed in DNA samples from amniotic fluid without the addition of EDTA. In all cases the fetal HLA type could be confirmed by the result from the corresponding cord blood typing. Contamination with maternal DNA led to additional weak PCR‐SSP bands in one case, but data interpretation was still unambiguous. Reliable fetal HLA typing can be achieved directly from amniotic fluid and culturing of amniocytes is not required.
Cloning of rhesus monkey killer‐cell Ig‐like receptors (KIRs) from early pregnancy decidua
Authors: Grendell R.L. , Hughes A.L. , Golos T.G.
Published: March 2003

Abstract: To define the killer‐cell Ig‐like receptors (KIR) molecules expressed within the decidua in the rhesus monkey, an early pregnancy rhesus decidual library was screened with an oligonucleotide located within the KIR D2 domain. We obtained 26 full‐length clones which represented 11 mRNAs. The cDNAs encode proteins containing 2 or 3 Ig domains, and short or long cytoplasmic domains. The long domains contained immunotyrosine inhibitory motifs (ITIM) motifs indicating inhibitory function. Three clones contained single or dinucleotide deletions within the D1 domain, which would result in proteins truncated within this domain. Phylogenetic analysis of the Ig domains supports a D0 – D2 organization of rhesus KIR2DL4.1, similar to human KIR2DL4 and KIR2DL5. The rhesus KIR2DL4.1 transcript contains a long cytoplasmic tail homologous with human KIR2DL4.
MOST PATIENTS WHO REJECT A KIDNEY TRANSPLANT HAVE ANTI‐HLA‐ANTIBODIES
Authors: El‐Awar N , Terasaki PI , Lazda V , Nikaein A , Arnold AN
Published: March 2003

To test the humoral theory of allograft rejection, we investigated whether all patients who reject a kidney have HLA‐antibodies. Sera from 1363 patients at four transplant centers who had rejected a kidney graft and transplant or who had rejected a graft and were waiting for a regraft were first tested by cytotoxicity AHG. Sera that were negative were tested by solid phase assays (ELISA or flow cytometry). Among the 1363 patients who had rejected a kidney, 90% were found to have HLA‐antibodies. Eighty‐four per cent tested positive for the CDC‐AHG assay and an additional 6% of sera, when tested by solid‐phase assays for IgG, IgM and IgA antibodies, was found to be positive for HLA. Among the four different centers the range of positivity was 89–91%. We conclude that 90% of patients who reject a kidney transplant have detectable HLA‐antibodies.
HLA‐MATCHING BY DNA METHODS: IMPACT ON A LIVING‐RELATED RENAL TRANSPLANTATION PROGRAMME
Authors: Zafar MN , Ahmed N , Abbas Y , Abbas K , Naqvi SAA , Rizvi SAH
Published: March 2003

DNA methods have resulted in improved renal allograft survival rates in cadaveric renal transplantation. This paper describes the impact of DNA typing by PCR‐SSP on a living‐related renal transplant (LRRT) programme. It evaluates error rates in serology, acute rejections, graft function and survival rates between the two typing methods. Serological typing was carried out on CTS 120 antisera Class 1, 60 antisera Class 2, 72 antisera Terasaki Class 1 and 72 antisera Class 2 antigens. Low‐resolution PCR‐SSP typing was carried out by 24 primers for HLA‐A, 48 for HLA‐B and 24 for HLA‐DR. Of the 585 transplants: 159 (Group I) were serology based; 172 were serology and PCR‐SSP based for HLA‐DR (Group II); and 254 were serology and PCR‐SSP based for HLA‐A and ‐B, and only PCR‐SSP based for HLA‐DR (Group III). Error rates in serology compared with PCR‐SSP were 24% for HLA‐A, 16% for HLA‐B and 35% for HLA‐DR. Acute rejections were 39%, 30% 26% in Groups I, II and III, respectively (P=0.02). Graft function of serum creatinine <1.5 mg/dl at 1 year was found in 26% of the Group I patients compared with 48% of those in Group III (P<0.0001). One‐ and 3‐year graft survival was 93% and 87% for Group II compared with 81% and 69% for Group I, respectively (P=0.0001). Matching by this combination of serology and PCR‐SSP is not only economical for a developing country but also improves graft survival by 12% and 18% at 1 and 3 years, respectively.
OUR EARLY EXPERIENCE OF TACROLIMUS CONVERSION IN THE KIDNEY TRANSPLANTS
Authors: Moray G , Yağmurdur MC , Emiroğlu R , Başaran Ö , Haberal M
Published: March 2003

Tacrolimus has been known as an effective immunosuppressive drug since 1987, and in our clinic we have used this agent since November 1999.

Our transplantation team has performed 1359 kidney transplantations since 1975. Until November 1999, all of these recipients received a standard low‐dose triple‐drug immunosuppressive regimen of prednisolone, azathioprine, and cyclosporine A. Since November 1999, cyclosporine A was replaced by tacrolimus in 95 kidney recipients. In this study, our early results are analyzed.

The mean follow‐up period after alteration of the immunosuppressive protocol was 12 (1–24) months. The mean time interval between the transplantation and the tacrolimus replacement was 34±11.4 months (1–158 months). Chronic allograft nephropathy in 16 (17%) patients, nephrotoxicity related to cyclosporine A in 22 (23%) patients and steroid‐resistant acute rejection in 57 (58%) were the indications of tacrolimus conversion. One acute‐rejection episode in 30 patients, two in 17, and three in 10 patients were observed before starting the tacrolimus. After the drug conversion, one acute rejection in 17 and two acute rejections in four patients were observed. Graft loss was seen in 15 (15%) patients after the drug conversion. Tacrolimus was withdrawn because of diabetes mellitus in seven patients, epilepsy in four, and severe Nocardia sepsis, lymphoma and Kaposi sarcoma in one patient each. Decreases of serum creatinin levels and also increases in blood glucose levels showed a statistically significant association with the tacrolimus doses (P=0.0004, P=0.04, respectively). The decrease in creatinin clearance values was closely related to the increase in tacrolimus levels. The target range with maximum efficacy and minimum toxicity seems to be 10–15ng/ml.

Tacrolimus conversion can be successful in cases of rejection and nephrotoxicity, but dose‐dependent blood glucose elevations must be followed closely in these patients.
GENERICS IN TRANSPLANTATION, AND THE INTERNATIONAL RULES AND REGULATIONS ON THEIR USEMA
Authors: Masri MA
Published: March 2003

By definition a product identified by its official chemical name rather than an advertised brand name is called a generic. If a drug exerts its pharmacological effects at the same site, and has the same potency, dosage form and bioavailability as a brand name reference‐listed drug (RLD), it is consider as a generic. However, inactive ingredients can differ between a brand name and a generic. Generics gained ground in the drug market through the regulations of the FDA; they currently account for more than 42% of the total prescription rate in the USA. These regulations include the abbreviated new drug application (ANDA) for the registration process and drug substitution at the pharmacy level without patient or physician consent. This coupled with a keen interest of third‐party payers and the health authorities to reduce the high transplant health budget (>2 billion US$) made it necessary to introduce generics into the field of transplantation. Using the above‐mentioned definition we can theoretically say that all antilymphocytes produced in the same animal species are generic of each other. Moreover monoclonal antibodies that are directed against the same target and have the same bioavailability are also considered generics of each other. Of all the immunosuppressive drugs that have been introduced into the field of transplantation, none has been as dominant as Cyclosporine. Cyclosporine became and remains the backbone for any immunosuppressive protocol. In 1992, Consupren, the first non‐FDA approved generic to Sandimmun, was introduced. Although Consupren was not bioequivalent to Neoral, the long‐term results in kidney transplantation have been similar for both drugs. The introduction of Consupren resulted in a nearly 40% reduction in the total cost of immunosuppressive therapy. Interestingly, the cost of the brand name drug Neoral was also reduced by 20%. The cost reduction allowed the introduction of the new immunosuppressive agents MMF and Rapamycin. Currently there are five FDA‐approved Cyclosporine generics with a 20% market share in the USA and 0% in Europe. Alternative formulations to both Rapa and MMF should be available soon. These forms are not by definition generics and are considered by the FDA to be new brand names acting on the same site as Cell Cept and Rapaimmune. Their introduction would be a great welcome and would definitely result in cost saving in transplantation.
Sequence‐based typing techniques for rhesus macaque MhcMamu‐DQB1 allow the identification of more than 35 alleles
Authors: Vigon N. , Sauermann U.
Published: March 2003

Abstract: In the recent years, substantial progress has been made in the characterization of the rhesus macaque MHC region, which is more complex than in humans. To cope with the increasing knowledge, improved typing techniques for MHC genes are required. Including the DNA‐sequences in this report, 39 rhesus macaque (Mamu)‐DQB1 alleles, corresponding to 38 deduced protein sequences, are known. Here, we present a typing technique for Mamu‐DQB1 alleles and the DNA‐sequences of 9 novel DQB1 alleles. The technique consists of one or two rounds of screening followed by DNA‐sequence determination. The first round represents a low‐resolution screening sufficiently insensitive to identify novel alleles, which also allows sequencing of both alleles in most heterozygous individuals. The second round consists of a high‐resolution screening. This is only necessary for animals in which one PCR‐product was generated by the initial screening. The technique was validated by analyzing samples of more than 200 rhesus macaques of different origin, and DNA‐sequence determination of more than 230 PCR‐products.
Description of six new HLA‐B alleles in the 5C CREG including a B*58 intron two sequence
Authors: Gans C.P. , Mitton W. , Ng J. , Hartzman R.J. , Hurley C.K.
Published: March 2003

Abstract: Five of the six novel HLA‐B alleles described in this report contain polymorphic motifs previously reported in HLA‐B alleles, suggesting an origin resultant from a gene conversion mechanism. The sixth allele, B*35012, contains a polymorphism previously unreported in HLA‐B molecules. In addition to previously reported polymorphisms, B*5806 carries a previously unreported intronic substitution in a sequencing primer annealing site.
Seven new HLA‐B alleles associated with antigens in the B7 CREG
Authors: Gans C.P. , Mitton W. , Ng J. , Hartzman R.J. , Hurley C.K.
Published: March 2003

Abstract: This paper describes seven novel HLA‐B alleles. Five of these new alleles contain polymorphic motifs previously reported in HLA‐B alleles, suggesting an origin resultant from a gene conversion mechanism. B*0723 contains a polymorphism previously unreported in class I HLA molecules. B*4105 contains a nucleotide substitution previously unreported in class I HLA molecules, which encodes a protein sequence previously reported only in HLA‐C locus alleles.
Second HLA‐A*68 null allele, A*6818 N, identified
Authors: Dormoy A. , Froelich N. , Parissiadis A. , Cazenave J.‐P. , Tongio M.M.
Published: March 2003

Abstract: A second HLA‐A*68 null allele, HLA‐A*6818 N, was identified in our laboratory after discrepant results were obtained between class I serological and molecular typing in a male patient suffering from narcolepsy. HLA‐A*6818 N displays a sequence identical to that of the HLA‐A*6802 allele, except in exon 2 where 20 nucleotides inserted at codon position 48 are a repeat of the 20 preceding nucleotides. This duplication creates a shift of the reading frame, which leads to a premature non‐sense codon at position 59 of the null allele.
New HLA‐B alleles identified and sequences extended in potential bone marrow donors: B*5804, B*4418, B*1558, and B*4805
Authors: Williams T.M. , Wu J. , Bassinger S. , Feldman D. , Birkos S. , Kearns J. , Kamoun M.
Published: March 2003

Abstract: Nucleic acid‐based methods for allele identification have revealed more than 470 polymorphic variants at the HLA‐B locus. Screening of potential bone marrow donors with sequence specific primer polymerase chain reactions and sequence specific oligonucleotide probe hybridization assays revealed apparent variants within the B*58, *44, *15, and *48 allele groups. DNA sequencing of cloned DNA identified the new alleles B*5804, B*4418, and B*1558 within these groups and observed new sequence information for the previously reported allele B*4805. These findings further extend our knowledge of the substantial genetic variation present at the HLA‐B locus within human populations.
The role of CD55 in protecting the tumour environment from complement attack
Authors: Morgan J. , Spendlove I. , Durrant L.G.
Published: March 2003

Abstract: CD55 is a complement regulatory protein expressed by cells to protect them from bystander killing by complement. CD55 is over‐expressed 2–100‐fold on tumour cells and is deposited in large amounts within tumour matrix. Vascular endothelial growth factor (VEGF) produced by tumours to stimulate angiogenesis, also up‐regulates endothelial cell surface expression of CD55 and stimulates the release of matrix degrading metalloproteinases. This study investigated the effects of VEGF on CD55 deposition into matrix and the release of CD55 by metalloproteinases. In contrast to inflammatory cytokines, CD55 was up‐regulated by VEGF at the cell surface and within the extracellular matrix (ECM). Interestingly, human umbilical vein endothelial cells (HUVEC) exposed to VEGF released similar amounts of CD55 into the ECM as a tumour cell line expressing 50‐fold higher level of CD55 on its cell surface. Furthermore, in contrast to earlier studies, both tumour and HUVEC‐derived CD55 was functionally active. However, in contrast to papain that degrades CD55, and collagenase that fails to release CD55, MMP‐7 released intact CD55 from ECM. This suggests that it may have a further role to play in protecting cells during inflammation and invasion.
A common KIR2DS4 deletion variant in the human that predicts a soluble KIR molecule analogous to the KIR1D molecule observed in the rhesus monkey
Authors: Maxwell L.D. , Wallace A. , Middleton D. , Curran M.D.
Published: March 2003

Abstract: A KIR2DS4 deletion variant allele, previously identified through KIR PCR‐SSOP typing studies, was characterized, alongside a normal KIR2DS4 allele, by cDNA cloning and sequencing and its prevalence in the population determined using a deletion specific probe. The KIR2DS4 deletion variant was found in 72 of the 90 individuals screened and differed from the normal KIR2DS4 sequence by a single 22 bp deletion in exon 5. The deletion causes a frameshift predicting a truncated KIR2DS4 protein with a significantly altered D2 domain that would be secreted due to the loss of the transmembrane/cytoplasmic domains. Parallels with a recent study in the rhesus monkey highlighting access to the same open reading frame as the deletion variant, also predicting a soluble KIR molecule, are drawn.
HLA class II sequence‐based typing in normal Saudi individuals
Authors: Al‐Hussein K.A. , Rama N.R. , Butt A.I. , Meyer B. , Rozemuller E. , Tilanus M.G.J.
Published: March 2003

Abstract: We have adopted a system that combines low resolution PCR‐SSP followed by sequence‐based typing (SBT) to analyze HLA‐DRB1, ‐DPB1 and ‐DQB1 alleles in the Saudi population. The SBT method was used to identify HLA class II alleles in Saudis for the first time. Nineteen HLA‐DRB1 alleles in currently recognized subtypes of the DRB locus were detected. DR1 and DR9 were not encountered. SBT did not detect diversity within the DR7 and DR10 alleles. Sixteen HLA‐DQB1 and 10 HLA‐DPB1 alleles were identified. This study represents the first molecular report on the HLA class II allele frequency in the population of Saudi Arabia.
Effect of the three‐dimensional structure on the deamidation reaction of ribonuclease A
Authors: Capasso S. , Salvadori S.
Published: March 2003

Abstract: Kinetic data on the deamidation reaction of Asn67 in RNase A and of Asn3 in the two peptides Ac‐Cys‐Lys‐Asn‐Gly‐Gln‐Thr‐Asn‐Cys‐NH2 and Ac‐Cys(Me)‐Lys‐Asn‐Gly‐Gln‐Thr‐Asn‐Cys(Me)‐NH2, whose sequences are similar to that of the deamidation site in the enzyme, have been determined in a wide range of pH and buffer concentrations. The values of the observed rate constant (k) for the enzyme are markedly lower than those for the peptides. However, the k dependence on pH and buffers is similar for all three substrates, indicating a similar reaction mechanism. The lower k‐values for the enzyme have been quantitatively related to the thermal stability and the three‐dimensional structure of the enzyme.
Modeling of α‐MSH conformations with implicit solvent
Authors: Prabhu N.V. , Perkyns J.S. , Pettitt B.M.
Published: March 2003

Abstract: A conformational search for the most probable structures of the hormone α‐MSH in aqueous solution was performed in order to help determine the structural features necessary for biological activity. The free‐energy surface was modeled using methods from integral equation theory, and high‐temperature molecular dynamics was used to enhance conformational sampling. Families of low free‐energy structures have been found. The minimum energy structure shows a stable β‐turn conformation in the putative message region that is stabilized by a salt bridge between Glu5 and Lys11. The orientation of the side chains reflects the amphiphilic nature of the peptide, and a close interaction between the side chains of the His6, Phe7 and Trp9 was observed. Several structural features observed in the minimum energy structure agree well with experimental results. The conformational features led to a hypothesis of a receptor–hormone interaction model in which the hydrophobic side chains of Phe7 and Trp9 interact with the transmembrane portion of the human melanocortin (MC1) receptor. Also, the positively charged side chain of Arg8 and the imidazole side chain of His6 may interact with the negatively charged portions of the receptor which may even be on the receptor's extracellular loops.
Selective isolation and identification of N‐terminal blocked peptides from tryptic protein digests
Authors: Betancourt L. , Besada V. , González L.J. , Morera V. , Padrón G. , Takao T. , Shimonishi Y.
Published: March 2003

Abstract: A method for the easy isolation and direct sequencing of N‐terminally blocked peptide in proteins refractory to N‐terminal sequencing was developed. It is based essentially on tandem enzymatic treatments of the protein with trypsin and carboxypeptidase B, and selective isolation of the Nα‐blocked peptide using ion‐exchange chromatography. The chromatographic step was optimized for picomole amounts of sample and very short elution times by placing a thin layer of the resin over the membrane of an ultrafiltration tube. The isolated fraction can be analyzed directly using MALDI or ESI mass spectrometry. The method was applied to several recombinant and natural N‐terminal acetylated proteins. A critical discussion on the intrinsic limitations of the method is also given.
DNA binding properties of basic helix‐loop‐helix fusion proteins of Tal and E47
Authors: Ghosh I. , Bishop P. , Chmielewski J.
Published: March 2003

Abstract: The basic helix‐loop‐helix (bHLH) transcription factor Tal has been shown to form heterodimers with the ubiquitously expressed bHLH transcription factor E47 and thereby modulate gene expression. The absence of homodimeric Tal−DNA complexes had been attributed to the inability of Tal to homodimerize, but subsequent studies have shown that the bHLH region of Tal does homodimerize. In order to correlate the contributions of both the basic region and the helix‐loop‐helix (HLH) domain to the lack of DNA binding by Tal homodimers, mutant and fusion proteins based on Tal and E47 were designed and synthesized. Size‐exclusion chromatography established that all mutant and fusion proteins were dimeric. Point mutations were made within the basic region of Tal based on residues within E47 that are essential for DNA binding, but an affinity for DNA was not observed. Even complete replacement of the basic region in Tal with the basic region of E47, in an E47−Tal fusion protein, did not confer DNA binding upon the protein. However, when the dimerization domain in Tal was replaced with its E47 counterpart, in a Tal−E47 fusion protein, sequence specific DNA binding was observed with an apparent dissociation constant of 3.6 × 10−9 m2. Furthermore, circular dichroism studies showed that the basic region of Tal in the Tal−E47 fusion protein underwent a random coil to helix transition in the presence of a specific DNA probe. These experimental observations indicate that the inability of Tal homodimers to recognize DNA stems from a misalignment of its basic region with respect to the HLH domain, rather than an intrinsic inability of the Tal basic region to bind DNA.
Anin vivostudy of novel bioactive peptides that inhibit the growth ofEscherichia coli
Authors: Walker J.R. , Altman E. , Roth J.R.
Published: March 2003

Abstract:We have created a system in which synthetically produced novel bioactive peptides can be expressedin vivoinEscherichia coli. Twenty thousand of these peptides were screened and 21 inhibitors were found that could inhibit the growth ofE. colion minimal media. The inhibitors could be placed into one of two groups, 1‐day inhibitors, which were partially inhibitory, and 2‐day inhibitors, which were completely inhibitory. Sequence analysis showed that two of the most potent inhibitors were actually peptide−protein chimeras in which the peptides had become fused to the 63 amino acid Rop protein which was also contained in the expression vector used in this study. Given that Rop is known to form an incredibly stable structure, it could be serving as a stabilizing motif for these peptides. Sequence analysis of the predicted coding regions from the next 10 most inhibitory peptides showed that four of the 10 peptides contained one or more proline residues either at or very near the C‐terminal end of the peptide which could act to prevent degradation by peptidases. Collectively, based on what we observed in our screen of synthetic bioactive peptides that could prevent the growth ofE. coliand what has been learned from structural studies of naturally occurring bioactive peptides, the presence of a stabilizing motif seems to be important for small peptides, if they are to be biologically active.
O‐Linked glycopeptides retain helicity in water
Authors: Palian M.M. , Jacobsen N.E. , Polt R.
Published: March 2003

Abstract: A 17‐residue O‐linked glycopeptide model incorporating a central α‐mannosyl serine residue, and its unglycosylated analog both demonstrate substantial helicity in water. The peptide sequence was derived from previous studies in which differences in overall helicity as a function of single amino acid substitutions were measured by circular dichroism (CD). The helical content was predicted by molecular modeling, and confirmed by CD and NMR. Moreover, the glycopeptide retained its helicity in the presence of SDS micelles, whereas the native peptide lost secondary structure in the presence of micelles. The inference is that the peptide sequence is a more important helix determinant than glycosylation per se.
Ligand based structural studies of the CB1 cannabinoid receptor
Authors: Picone R. P. , Fournier D. J. , Makriyannis A.
Published: March 2003

Abstract: The structural characterization of G‐protein coupled receptors (GPCRs) is quite important as these proteins represent a vast number of therapeutic targets involved in drug discovery. However, solving the three‐dimensional structure of GPCR has been a significant obstacle in structural biology. A variety of reasons, including their large molecular weight, intricate interhelical packing, as well as their membrane‐associated topology, has hindered efforts aimed at their purification. In the absence of pure protein, available in the native conformation, classical methods of structural analysis such as X‐ray crystallography and nuclear magnetic resonance spectroscopy cannot be utilized successfully. Alternative methods must therefore be explored to facilitate the structural features involved in drug–receptor interactions. The methods described herein detail the use of covalent probes, or affinity labels, capable of binding covalently to a target GPCR at its binding site(s). Our approach involves the incorporation of a number of reactive moieties in different regions of the ligand molecule each of which is expected to react with different amino acid residues. Information obtained from such work coupled with computer modeling and validated by the use of site‐directed mutagenesis of GPCRs allows for three‐dimensional mapping of the receptor binding site. It also sheds light on the different possible binding motifs for the various classes of agonists and antagonists and identifies amino acid residues involved with GPCR activation or inactivation.
Biophysical studies and anti‐growth activities of a peptide, a certain analog and a fragment peptide derived from α‐fetoprotein
Authors: Butterstein G. , MacColl R. , Mizejewski G. J. , Eisele L. E. , Meservey M.
Published: March 2003

Abstract: A chemically synthesized 34‐amino acid peptide, an analog, and a fragment of the peptide have been purified and studied. Biophysical studies were carried out to determine some of the metal ion binding properties of the original peptide and an analog of this parent peptide, in which the two histidine residues were replaced by alanines. As shown by visible absorption spectroscopy, Co (II) forms a complex with the parent peptide, but not with the analog peptide, and one or two histidines in the parent peptide are ligands for Co (II) ion binding. The effects on disulfide bond formation in the peptide by Zn (II) and Co (II) ions were also examined for this analog. Anti‐growth assays were performed using the original cysteine‐containing peptide with Zn (II) ion complexed to the peptide through the two cysteine residues. These rat uterine growth assays showed that the complexing of Zn (II) ion to the peptide maintained the anti‐growth activity of the peptide, while gel‐filtration experiments showed the zinc ions maintained the peptide in its anti‐growth form indefinitely in solution. A saliently important part of this research was the discovery that a fragment of the peptide consisting of a middle sequence of 14 amino acids was found to have significant anti‐growth activity in the rat uterine assay. Its activity suggested that this fragment might be considered a viable candidate for testing in anti‐cancer protocols.
Synthesis and immunogenic properties of phosphopeptides related to the human insulin receptor
Authors: Coba M. P. , Turyn D. , Peña C.
Published: March 2003

Abstract:  Two phosphoserine tetradecapeptides corresponding to sequences 987–1000 (peptide pSer994) and 1017–1030 (peptide pSer1023/1025) from the human insulin receptor involved in the regulation of its activity were successfully synthesized using Fmoc‐based chemistry. Phosphorylation was performed by post‐assembly phosphitylation followed by oxidation. The selective phosphorylation of Ser residues was achieved incorporating into the peptide chain the Ser (Trt) derivative and t‐Bu blocking groups at sites other than those intended to be phosphorylated. The Trt group was selectively removed with dichloroacetic acid while under this condition t‐Bu protecting groups remained unaltered. Following conjugation to keyhole limpet hemocyanin phosphopeptides were used as immunogens to generate sequence‐specific phosphoserine antibodies. Peptide pSer994 induced antibodies in New Zealand white rabbits which discriminated between the phosphorylated and nonphosphorylated forms of the peptide, thus representing promising candidates to recognize signaling pathways associated to the regulation of the human insulin receptor.
Effects of pH on the glucose and lactate metabolisms by the washed cells of Actinomyces naeslundii under anaerobic and aerobic conditions
Authors: Takahashi N. , Yamada T.
Published: March 2003

Effects of pH on the glucose and lactate metabolism by the washed cells of Actinomyces naeslundii genospecies 1 and 2 under anaerobic and aerobic conditions were studied. The rate of acid production from glucose was the highest at pH 7.0 and decreased as the pH lowered to 4.5, irrespective of atmospheric conditions. The anaerobic end‐product in the absence of bicarbonate was mainly lactate, while in the presence of bicarbonate the rate of acid production increased 1.8–2.5 times with the production of formate, acetate and succinate in addition to lactate. Under aerobic conditions, the cells produced acids from glucose along with oxygen consumption and the end‐product was mainly acetate. In contrast to the glucose metabilism, the cells produced base from lactate along with oxygen consumption. The rates of base production and oxygen consumption were the highest at pH 5.5. The end‐products from lactate were acetate and pyruvate. These results indicate that oral actinomyces has a various activity of glucose and lactate metabolism at a wide range of environmental pH and suggest its flexibility in surviving in dental plaque, where the environmental factors fluctuate.
Attachment of Fusobacterium nucleatum PK1594 to mammalian cells and its coaggregation with periodontopathogenic bacteria are mediated by the same galactose‐binding adhesin
Authors: Weiss E. I. , Shaniztki B. , Dotan M. , Ganeshkumar N. , Kolenbrander P. E. , Metzger Z.
Published: March 2003

It has been shown that Fusobacterium nucleatum PK1594 coaggregates with Prophyromonas gingivalis PK1924 through a galactose‐binding adhesin. In the present study, attachment of F. nucleatum PK1594 to a variety of mammalian cells was characterized. F. nucleatum PK1594 attached to all eukaryotic cells tested, including human buccal epithelial cells, gingival and periodontal ligament fibroblasts, HeLa cells and murine lymphocytes, macrophages, and polymorphonuclear leukocytes. These attachments were (i) inhibited by galactose, lactose and N‐acetylgalactosamine and (ii) inhibited by monoclonal antibody specific for the galactose‐binding adhesin of F. nucleatum PK1594. In addition, a coaggregation‐defective mutant of F. nucleatum PK1594 (PK2172), which does not exhibit galactose binding activity, did not attach to the mammalian cells. Coaggregation of F. nucleatum PK1594 with P. gingivalis PK 1924 and Actinobacillus actinomycetemcomitans JP2, but not with other bacteria, showed a similar pattern with sugars, monoclonal antibody, and the adhesin‐deficient mutant. The results suggest that the attachment of F. nucleatum PK1594 to mammalian cells and its coaggregation with periodontal pathogens are mediated by the same galactose‐binding adhesin.
Actinobacillus actinomycetemcomitans induces lethal effects on the macrophage‐like human cell line U937
Authors: Fukunaga M. , Tsuruda K.
Published: March 2003

We examined the cytotoxicity in culture medium of Actinobacillus actinomycetemcomitans against the human monocyte‐macrophage‐like cell line U937 using the trypan blue exclusion test and WST‐1 test. We found that A. actinomycetemcomitans Y4 showed the highest cytotoxic activity among the three different serotype strains and the cytotoxic effects of both bacterial cells and culture supernatants in A. actinomycetemcomitans Y4 were stronger on phorbol‐12‐myristate 13‐acetate (PMA)‐induced U937 cells than uninduced U937 cells. Morphological changes in PMA‐induced U937 cells treated with culture supernatants differed from those treated with leukotoxin, and a difference in the susceptibility to 56 °C heat treatment was found between culture supernatants and leukotoxin. The cytotoxic activity by WST‐1 was determined more rapidly and strongly than that by trypan blue assay. These findings suggested that the cytotoxic effect of A. actinomycetemcomitans was influenced by the differentiation of U937 cells and may be more potent on the respiratory chain than the cell membrane.
Immune response to Bacteroides forsythus in a murine model
Authors: Bird P. S. , Shakibaie F. , Gemmell E. , Polak B. , Seymour G. J.
Published: March 2003

A murine skin abscess model was used to study the immune response to an acute infection with Bacteroides forsythus. BALB/c mice were given subcutaneous injections of either viable or heat‐killed B. forsythus, while a third sham‐immunized control group received phosphate‐buffered saline. Weights and lesion sizes were measured. Blood was collected from the heart and specific antibodies to B. forsythus measured by an ELISA. Swabs taken from the lesions and also from pooled blood were cultured anaerobically for viable B. forsythus. Viable B. forsythus–induced lesions reached maximum size at day 7. B. forsythus cells were recovered from lesions up to day 4 although none were cultured from blood samples. Heat‐killed bacteria induced much smaller lesions. Serum antibody levels increased during the 9‐day study period, being significantly higher in mice injected with viable compared with heat‐killed B. forsythus. Antibody levels in sham control mice were significantly lower than those seen in the other two groups. These results showed that a subcutaneous injection of viable cells of B. forsythus elicited a pronounced abscess formation and induce higher levels of specific antibodies compared with that produced by an injection of dead bacteria. This suggests that, as with other periodontopathic organisms, this mouse model can be used to study the immune response to B. forsythus.
Actinomyces naeslundii fimbrial protein Orf977shows similarity to a streptococcal adhesin
Authors: Hoflack L. , Yeung M. K.
Published: March 2003

The nucleotide sequence of the chromosomal DNA, upstream of Actinomyces naeslundii T14V fimbrial gene fimA, was determined. One open reading frame (orf977) encoding 977 amino acids was found, preceded by a gene homologous to elongation factor TU. Database searches revealed that Orf977 was homologous to CshA, a Streptococcus gordonii protein involved in cell adhesion. Previous studies had already determined two genes in the type 2 fimbrial gene cluster of A. naeslundii T14V: the structural subunit fimA, and orf365 with unknown function, followed by ribosomal genes. This study completes the type 2 fimbrial gene cluster sequence.
Microbial flora of root canal–treated teeth associated with asymptomatic periapical radiolucent lesions
Authors: Cheung G. S. P. , Ho M. W. M.
Published: March 2003

This study aimed to investigate the composition of microflora in endodontically treated teeth associated with asymptomatic periapical lesions in southern Chinese patients. Twenty‐four teeth which had received nonsurgical root canal treatment more than 4 years previously, and which presents an acceptable coronal restoration with a periapical radiolucent area, were re‐treated nonsurgically. Bacteriological samples were obtained after removal of the old root canal filling. The samples were inoculated on enriched trypticase soy agar and four selective media for incubation at 37°C in both a carbon dioxide‐enriched atmosphere and anaerobically. Eighteen teeth that had received gutta‐percha root canal fillings were grouped for analysis, 12 (66.7%) of which contained cultivable microorganisms. The total colony forming units per ml of transport medium ranged from 0 to 2.3×105. The number of bacterial genera recovered ranged between 0 and 6, with facultative gram‐positive cocci being the most prevalent group of bacteria isolated. Facultative anaerobic bacteria were present in all, whereas strict anaerobic bacteria were found in 3 out of the 12 teeth with positive growth. The size of the periapical rarefaction did not show any relationship with the quantity of microorganisms recovered. Coagulase‐negative staphylococci, streptococci and Pseudomonas aeruginosa were most frequently isolated in this group of patients. The possible origin of these organisms is discussed.
Fetal immune responsiveness and routes of allergic sensitization
Authors: Jones Catherine A. , Holloway Judith A. , Warner John O.
Published: March 2003

There is much interest in the role of early‐life events in the subsequent development of atopy and/or atopic disease. Despite the ongoing debate about the intrauterine exposure of the fetus to environmental allergens and the establishment of T‐cell memory, it is clear that the immunological response of the neonate at risk of atopy is more immature than that of the neonate likely to be non‐atopic. The reasons for this remain unknown, but might reflect maternally transmitted signals that adapt the neonatal immune response. An inadvertent consequence of this might be an inappropriate host response to environmental signals such as those from microbial products during early post‐natal life that result in an inability to dampen neonatal T helper 2‐skewed responses. The developing gastrointestinal tract and the exogenous factors that impact on this, such as microbial flora and breast milk, should therefore be a focus of investigation.
Evaluation of the risk of anaphylactic reactions by wasp venom‐extract challenges in children
Authors: Schultze‐Werninghaus Claudia , Wahn Ulrich , Niggemann Bodo
Published: March 2003

Diagnostic sting challenges have been shown to provide information on the risk of further anaphylactic reactions to bee stings. We present a follow‐up study in wasp venom‐hypersensitive children after diagnostic venom extract challenges to analyze their risk of further anaphylactic reactions. Responses were obtained from 104 patients with wasp venom hypersensitivity out of 115 former patients. Only one of the 104 patients showed more than a severe local reaction to the sting challenge irrespective of the performance of a single or sequential challenge; therefore, only one patient received venom immunotherapy. The performance of a diagnostic sting challenge with wasp venom extract in children had a high negative predictive value of 94.6% for the risk of further systemic reactions. This was shown by analysis of later field stings, since 37 children experienced further field stings and only two of these children(5.4%) developed a mild systemic reaction (urticaria) equal to or less severe than the index sting. The value of the venom extract challenge can be interpreted in two ways: either it is less sensitive than a native sting challenge since the rate of systemic reactions to the challenge was very low, or the prognosis of wasp venom hypersensitivity in children is extremely favorable. Since the latter hypothesis is supported by the low incidence of systemic field sting reactions, we postulate that venom immunotherapy is necessary only in a minority of children with wasp venom hypersensitivity with an index sting reaction of Mueller grade I or II. However, the value of venom extract challenges as a general diagnostic instrument in children with Mueller I and II reactions due to wasp venom hypersensitivity may be questioned. It may have a place as a safe procedure in demonstrating to parents and physicians the often self‐limiting natural course in most of these children.
The relationship among markers of allergy, asthma, allergic rhinitis, and eczema in Costa Rica
Authors: Celedón Juan C. , Soto‐Quiros Manuel E. , Hanson Lars Å. , Weiss Scott T.
Published: March 2003

The association between allergy markers and asthma and allergic rhinitis is stronger in countries with a Western lifestyle than in rural areas of Africa and Asia. We examined the relationship among allergy markers, asthma, rhinitis, and eczema in a case‐control study of 198 schoolchildren, 10–13 years of age, living in Costa Rica, a Latin American country. The geometric mean total serum immunoglobulin E (IgE) level in subjects with and without asthma was 465.0 and 143.0 IU/ml, respectively (difference = 322 IU/ml, 95% CI = 141.8–616.1 IU/ml, p < 0.001), and that in subjects with and without allergic rhinitis was 442.5 and 144.3 IU/ml, respectively (difference = 298.2 IU/ml, 95% CI = 125.7–581.0 IU/ml, p < 0.001). After adjusting for age, gender, and skin test reactivity to allergens, we found a linear relationship between serum total IgE level and the log odds ratio (OR) of having asthma. In a multivariate analysis, there was a linear relationship between skin test reactivity to allergens and the log OR of having allergic rhinitis. The OR of having allergic rhinitis was almost three times higher in children who had four positive skin tests than in non‐reactors. Skin test reactivity to greater than five aeroallergens was an independent predictor of eczema in a multivariate analysis (OR = 3.1, 95% CI = 1.1–8.4). Although the geometric mean total serum IgE levels of Costa Rican children with either asthma or allergic rhinitis are higher than those of children with asthma or allergic rhinitis in most industrialized countries, the relationship among markers of allergy, asthma, rhinitis, and eczema in Costa Rica is similar to that found in countries with a Western lifestyle and different from that found in rural areas of Asia and Africa.