bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a novel O1variant allele at the ABO blood group locus
Authors: Bugert P. , Rütten L. , Goerg S. , Klüter H.
Published: March 2003

Abstract:We describe the identification and molecular characterization of a novel variant O1allele of the ABO blood group locus. The allele was found in a young child and by analyzing the maternal DNA we were able to show that a meiotic recombination event between the maternal O1v‐3and B1‐1alleles recreated a O1/B hybrid allele. Further characterization of intron 6 sequences delineated the putative recombination breakpoint between nucleotide position 42 and 163 of the intron. We propose that the novel O1variantallele should be named O1v‐7and is a combination of exon 6 from a O1v‐3allele and exon 7 from a B1‐1allele.
Cellular, serological, and molecular polymorphism of the class I and class II loci of the canine Major Histocompatibility Complex
Authors: Wagner J.L. , Sarmiento U.M. , Storb R.
Published: March 2003

Abstract:This study was undertaken to determine the relationships between canine cellular and serological determinants and more recently described genes. Such relationships might reveal information about immunological reactivity or function of various proteins. To do this we studied the haplotypic associations of dog leukocyte antigen (DLA) class I and class II alleles determined from a panel of 14 DLA‐D homozygous dogs. This panel of dogs was typed for the serological determinants DLA‐A, DLA‐B and DLA‐C. Polymorphisms for DLA‐DQA1, DLA‐DQB1, DLA‐DRB1 and DLA‐88 were also determined. The number of alleles (one or two) for two microsatellite markers in the DLA region were also determined. Analyses of the nucleotide sequences and of the serological and cellular typing data revealed that phenotypic homozygosity, as defined by the DLA‐D type in mixed leukocyte culture (MLC), tended to correlate with homozygosity at the DLA‐DRB1 locus but not necessarily at the DLA‐DQB1 locus. Furthermore, MLC specificity was determined by other loci besides DLA‐DRB1 and DLA‐DQB1. The amino acid at position 63 of the DR β chain could contribute to the DLA‐B serological specificity. DLA‐88, the most polymorphic class I gene characterized to date, did not have an easily identifiable association with either the DLA‐A or DLA‐C class I serological specificities. Homozygosity or heterozygosity of each of two microsatellite markers, FH 2200 and FH 2202, located in the class I or class II region, respectively, did not correlate with homozygosity or heterozygosity of the most polymorphic known class I (DLA‐88) or class II (DLA‐DRB1) genes.
Tumor escape from killing: Role of killer inhibitory receptors and acquisition of tumor resistance to cell death
Authors: Chouaib S. , Thiery J. , Gati A. , Guerra N. , El Behi M. , Dorothée G. , Mami‐Chouaib F. , Bellet D. , Caignard A.
Published: March 2003

Abstract: Immunotherapy of cancer has always been a very attractive fourth‐modality therapeutic approach. Over the past few years, advances in the identification of tumor antigens have offered new perspectives and provided new opportunities for more accurate immunotherapy for cancer. However, when applied to patients with established tumors, it rarely leads to an objective response. This is partly due to the fact that tumors evade host immunity at both the induction and effector phases. Thus, understanding tumor escape mechanisms may be the key to successful immunotherapy for cancer. In the present review, we will focus on how the expression of killer Ig receptors (KIR) on tumor infiltrating lymphocytes can compromise their function and how tumors evade apoptotic death – two additional mechanisms of tumor escape.
Class II transactivator‐induced expression of HLA‐DOβ in HeLa cells
Authors: Khalil H. , Deshaies F. , Bellemare‐Pelletier A. , Brunet A. , Faubert A. , Azar G.A. , Thibodeau J.
Published: March 2003

Abstract: HLA‐DO is an intracellular nonclassical MHC class II molecule expressed in the endocytic pathway of B lymphocytes. It shapes the repertoire of peptides bound to classical class II molecules such as HLA‐DR by regulating the activity of HLA‐DM. Using a peptide corresponding to the cytoplasmic tail of HLA‐DOβ, we have developed a mouse monoclonal antibody, HKC5. Immunofluorescence microscopy revealed that HKC5 recognizes HLA‐DO molecules present in the endoplasmic reticulum as well as those in vesicular compartments of the endocytic pathway. In addition, the antibody detects the isolated β chain on Western blots. Using mutants of the DOβ cytoplasmic tail fused to a reporter molecule and expressed in epithelial cells, we showed by flow cytometry that the antibody epitope includes one or both of the leucine residues forming the lysosomal sorting signal. Finally, we have used HKC5 to evaluate the presence of the HLA‐DOβ chain in HeLa cells expressing the class II transactivator protein CIITA. Our flow cytometry and confocal microscopy analyses showed a marked expression of DOβ suggesting that HLA‐DO could accumulate under the influence of CIITA in non‐B cells.
The twists and turns of β‐peptides
Authors: DeGrado W.F. , Schneider J.P. , Hamuro Y.
Published: March 2003

Abstract: Recently, it has been discovered that peptides composed of β‐amino acids are capable of adopting novel secondary structures demonstrating that peptides composed of α‐amino acids are not unique in their ability to fold into well‐defined structures. Cyclic as well as acyclic peptides composed of β‐amino acid residues adopt turn, helical, and sheet‐like conformations. Here, we discuss the synthesis and conformational preferences of individual, substituted β‐amino acids as well as the structures that peptides composed of these residues, β‐peptides, may adopt.
Parallel synthesis and pharmacological screening of nonpeptide ligands of the neuropeptide Y receptor subtype Y5
Authors: Henlin J.M. , Duchêne‐Roger F. , Desmet‐Beaufort C. , Levens N. , Fauchère J.L. , Boutin J.A. , Nicolas J.P.
Published: March 2003

Abstract: Several series of low‐molecular‐mass ligands of the neuropeptide receptor subtype Y5 were prepared using a mixed strategy of synthesis on solid phase and in solution. Collections of single compounds were obtained by an automated parallel procedure which allowed quick variation and investigation of the central spacer moiety, as well as of the aromatic substituents on each side. The strategy of parallel synthesis and screening of partially purified analogs helped to select rapidly potent and selective leads which displayed comparable antagonistic potency against neuropeptide Y activity on the Y5 receptor and better receptor selectivity than the original reference compounds.
Studies on a growth‐inhibitory peptide derived from alpha‐fetoprotein and some analogs
Authors: Eisele L.E. , Soldwedel H. , MacColl R. , Mizejewski G.J. , Mesfin F.B. , Bennett J.A. , Andersen T.T. , Jacobson H.I.
Published: March 2003

Abstract: A 34‐amino acid synthetic peptide was derived from the third domain of human alpha‐fetoprotein, and the peptide was shown to inhibit estrogen‐stimulated growth. Under certain conditions, however, the peptide lost growth‐inhibitory activity. A biophysical study of the peptide was undertaken with a goal of obtaining completely reliable preparations. The peptide was studied using gel‐filtration column chromatography as a function of peptide concentration and age of solution, and was found to exhibit complex aggregation behaviors. During the early period (0–3 h) after dissolving lyophilized peptide into pH 7.4 buffer, solutions were composed mostly of trimers. At higher peptide concentrations (≥ 3.0 g/L), the trimers aggregated extensively to a large aggregate (minimum size ≈ 102 peptides). At 5.0–8.0 g/L, these large aggregates increased in size (up to ≈ 146 peptides) until trimers were largely exhausted from solution. During the later times (> 3 h) after sample preparation, the trimeric oligomer of the peptide dissociated slowly to form dimers for samples at 0.10–3.0 g/L. After their build‐up, a very small number of dimers associated to form hexamers. Disulfide bonds stabilized the dimers as indicated by the conversion of dimers to trimers upon the addition of a reducing agent, and the failure of dimers to form in the presence of reducing agent. Reducing agent did not affect trimer or large aggregate formation. Trimers were found to be active in an assay monitoring inhibition of estrogen‐stimulated growth, whereas dimers and large aggregates were inactive. The two cysteines in the peptide were modified to either S‐methylcysteine or S‐(2‐aminoethyl)cysteine, and both derivatives showed significant growth‐inhibition activity. A serine analog in which both cysteines were replaced had very different aggregation behavior than the cysteine peptide and lacked its growth inhibitory ability. Peptide aggregation is critically important in establishing the ability of the peptide to inhibit growth and have anticancer activity, but the state of its two cysteines is of little influence.
Synthesis, activity on NK‐3 tachykinin receptor and conformational solution studies of scyliorhinin II analogs modified at position 16
Authors: Rodziewicz‐Motowidło S. , Lesner A. , Łęgowska A. , Czaplewski C. , Liwo A. , Rolka K. , Patacchini R. , Quartara L.
Published: March 2003

Abstract: Two analogs of a tachykinin family peptides – scyliorhinin II (ScyII): [Aib16]ScyII and [Sar16]ScyII were synthesized by the solid‐phase method using Fmoc chemistry. Conformational studies in water and DMSO‐d6 on these peptides were performed using a combination of two‐dimensional NMR and theoretical conformational analysis. The solution structure of the peptides studied is interpreted as an equilibrium of several conformers with different statistical weights. The structure of [Sar16]ScyII in water appeared to be more flexible, especially in the C‐terminal fragment. A better defined structure for this analog was obtained in DMSO‐d6, in which the analysis resulted in a family of conformers with similar shapes. Some of these conformers were characterized by the presence of a 310‐helix in the N‐terminal fragment and middle part of the molecule. The introduction of the Aib residue in position 16 significantly rigidifies the structure. For [Aib16]ScyII in both solvent systems very similar populations of conformations were obtained which are characterized by the presence of a 310‐helix in the 13–18 fragment. A common structural motif was found in conformationally constrained Cys7−Cys13 fragment, which resembles the Greek letter ‘ω’. The differences in the solution structure of the C‐terminal fragment of the peptides studied are responsible for their specificity. [Aib16]ScyII showed 25% the agonistic activity of selective NK‐3 agonist – senktide, but it also showed antagonist effect vs. this peptide, whereas [Sar16]ScyII appeared to be a full agonist of NK‐3 tachykinin receptor.
Development of a synthetic cyclized peptide derived from α‐fetoprotein that prevents the growth of human breast cancer
Authors: Mesfin F.B. , Zhu S. , Bennett J.A. , Andersen T.T. , Jacobson H.I.
Published: March 2003

Abstract: The peptide, EMTPVNPG, derived from alpha‐fetoprotein, inhibits estrogen‐stimulated growth of immature mouse uterus and estrogen‐dependent proliferation of human breast cancer cells. However, the biological activities of the peptide diminish over time in storage, even when in the lyophilized state, probably because of peptide aggregation through hydrophobic interaction among monomers. Two analogs of EMTPVNPG were designed with the intent of minimizing aggregation and retaining biological activity during prolonged storage. EMTOVNOG, where O is 4‐hydroxyproline, is a linear peptide generated by substituting 4‐hydroxyproline for the two prolines, thereby increasing peptide hydrophilicity. This analog exhibited a dose‐dependent inhibition of estrogen‐stimulated growth of immature mouse uterus similar to that of EMTPVNPG (maximal activity at 1 µg/mouse). A second analog, cyclo‐(EMTOVNOGQ), a hydrophilic, cyclic analog with increased conformational constraint, was as potent as the other peptides in its inhibition of estrogen‐dependent growth of immature mouse uterus, and had an expanded effective dose range. Both linear and cyclized hydroxyproline‐substituted analogs exhibited indefinite shelf‐life. Furthermore, both analogs inhibited the estrogen‐dependent growth of MCF‐7 human breast cancer growing as a xenograft in SCID mice. These analogs may become significant, novel agents for the treatment of breast cancer.
The influence of steric interactions on the conformation and biology of oxytocin. Synthesis and analysis of penicillamine6‐oxytocin and penicillamine6‐5‐tert‐butylproline7‐oxytocin analogs
Authors: Bélec L. , Lubell W.D. , Maletinska L. , Slaninová J.
Published: March 2003

Abstract: Six [Pen6]oxytocin analogs were synthesized by substituting penicillamine for cysteine in oxytocin, [Mpa1]oxytocin, [dPen1]oxytocin, [5‐t‐BuPro7]oxytocin, [Mpa1, 5‐t‐BuPro7]oxytocin and [dPen1, 5‐t‐BuPro7]oxytocin. When tested in the uterotonic test in vitro[Pen6]oxytocin, [Pen6, 5‐t‐BuPro7]oxytocin, [Mpa1, Pen6]oxytocin and [Mpa1, Pen6, 5‐t‐BuPro7]oxytocin, all were found to possess both agonistic and antagonistic properties. Their agonistic potency ranged from negligible (0.08 IU/mg) to low (5.85 IU/mg) and their antagonistic potency (pA2) was estimated to range from 6.6 to 7.9. [dPen1, Pen6]Oxytocin and [dPen1, Pen6, 5‐t‐BuPro7]oxytocin were found to be pure antagonists with similarly high pA2 values of ≈ 8.2. Replacement of proline by 5‐tert‐butylproline increased binding affinity by a factor of two in [Pen6]oxytocin and had no influence on the binding affinity of [Mpa1, Pen6]oxytocin and [dPen1, Pen6]oxytocin. Assignment of the proton signals for prolyl amide cis‐ and trans‐isomers by NMR experiments in water indicated that the Pen6−5‐tert‐BuPro7 peptide bond cis‐isomer population was augmented relative to the prolyl peptides and measured, respectively, at 20, 35 and 35% in the 5‐tert‐butylproline7 analogs of [Pen6]oxytocin, [Mpa1, Pen6]oxytocin and [dPen1, Pen6]oxytocin. This augmentation in cis‐isomer population was correlated with a 21‐fold reduction in the agonistic potency and 2‐fold augmentation in antagonistic potency for [Pen6, 5‐t‐BuPro7]oxytocin relative to [Pen6]oxytocin. Augmentation of cis‐isomer population was also correlated to reduced agonist potency without effect on antagonism on conversion of [Mpa1, Pen6]oxytocin to [Mpa1, Pen6, 5‐t‐BuPro7]oxytocin. In the potent oxytocin antagonist, [dPen1, Pen6]oxytocin, substitution of 5‐tert‐butylproline for proline augmented the cis‐isomer population without affecting antagonistic potency. The synthesis and evaluation of [Pen6]oxytocin and [Pen6, 5‐t‐BuPro7]oxytocin analogs 1–6 indicated that steric interactions influenced agonist and antagonist activity by modifying peptide conformation. Augmentations in the prolyl cis‐isomer population caused by 5‐tert‐butylproline occurred concurrently with enhanced or maintained antagonistic potency and binding affinity and reduced agonistic potency.
Helix‐stabilizing effects of the pentapeptide KIFMK and its related peptides on the sodium channel inactivation gate peptides
Authors: Maeda Y. , Nakagawa T. , Kuroda Y.
Published: March 2003

Abstract: We have previously found by NMR and CD spectroscopic studies that the helical content of the sodium channel inactivation gate‐related peptide (Ac‐GGQDIFMTEEQK‐NH2; MP‐1A) in 80% trifluoroethanol solutions was increased by adding a pentapeptide, KIFMK. In order to study in further detail whether the presence of the IFM motif and the two lysine residues is a prerequisite for stabilizing the helical conformation, we examined interactions between various oligopeptides (RIFMR, KIFMTK, KIQMK, KAFAK, KIIIK) and MP‐1A and its related peptides; that is, MP‐2A in which Phe was replaced by Gln, MP‐1MMA in which Thr was replaced by Met, MP‐1TA in which Thr was removed from MP‐1A, and MP‐1A′ in which l‐Phe was replaced by d‐Phe. It was found that the IFM motif was absolutely necessary in both the oligopeptide and the inactivation gate peptide. This finding means that hydrophobic interactions are operative between KIFMK and MP‐1A. In contrast, KIFMK destabilized the helical structure of MP‐1MMA, MP‐1TA, and MP‐1A′, showing that the conformation around the IFM motif in the inactivation gate peptides is an important factor. It was concluded that the IFM motif and the two Lys residues are a prerequisite for effectively stabilizing the α‐helix of MP‐1A.
Purification and characterization of charantin, a napin‐like ribosome‐inactivating peptide from bitter gourd (Momordica charantia) seeds
Authors: Parkash A. , Ng T.B. , Tso W.W.
Published: March 2003

Abstract:  A peptide designated charantin, with a molecular mass of 9.7 kDa, was isolated from bitter gourd seeds. The procedure comprised affinity chromatography on Affi‐gel blue gel, ion‐exchange chromatography on Mono S and gel filtration on Superdex 75. The N‐terminal sequence of charantin exhibited marked similarity to that of the 7.8‐kDa napin‐like peptide previously isolated from bitter gourd seeds. Charantin inhibited cell‐free translation in a rabbit reticulocyte lysate system with an IC50 of 400 nm, a potency lower than that of the previously reported small ribosome‐inactivating protein γ‐momorcharin (IC50 = 55 nm) which also exhibited an abundance of arginine and glutamate/glutamine residues. Charantin reacted positively in the N‐glycosidase assay, yielding a band similar to that formed by the small ribosome‐inactivating proteins γ‐momorcharin and luffin S.
Design and characterization of a membrane permeable N‐methyl amino acid‐containing peptide that inhibits Aβ1–40 fibrillogenesis
Authors: Gordon D.J. , Tappe R. , Meredith S.C.
Published: March 2003

Abstract: Alzheimer's disease, Huntington's disease and prion diseases are part of a growing list of diseases associated with formation of β‐sheet containing fibrils. In a previous publication, we demonstrated that the self‐association of the Alzheimer's β‐amyloid (Aβ) peptide is inhibited by peptides homologous to the central core domain of Aβ, but containing N‐methyl amino acids at alternate positions. When these inhibitor peptides are arrayed in an extended, β‐strand conformation, the alternating position of N‐methyl amino acids gives the peptide two distinct faces, one exhibiting a normal pattern of peptide backbone hydrogen bonds, but the other face having limited hydrogen‐bonding capabilities due to the replacement of the amide protons by N‐methyl groups. Here, we demonstrate, through two‐dimensional NMR and circular dichroic spectroscopy, that a pentapeptide with two N‐methyl amino acids, Aβ16–20m or Ac‐K(Me)LV(Me)FF‐NH2, does indeed have the intended structure of an extended β‐strand. This structure is remarkably stable to changes in solvent conditions and resists denaturation by heating, changes in pH (from 2.5 to 10.5), and addition of denaturants such as urea and guanindine‐HCl. We also show that this peptide, despite its hydrophobic composition, is highly water soluble, to concentrations > 30 mm, in contrast to the nonmethylated congener, Aβ16–20 (Ac‐KLVFF‐NH2). The striking water solubility, in combination with the hydrophobic composition of the peptide, suggested that the peptide might be able to pass spontaneously through cell membranes and model phospholipid bilayers such as unilamellar vesicles. Thus, we also demonstrate that this peptide is indeed able to pass spontaneously through both synthetic phospholipid bilayer vesicles and cell membranes. Characterization of the biophysical properties of the Aβ16–20m peptide may facilitate the application of this strategy to other systems as diverse as the HIV protease and chemokines, in which there is dimerization through β‐strand domains.
Study of the binding environment of α‐factor in its G protein‐coupled receptor using fluorescence spectroscopy
Authors: Ding F.‐X. , Patri R. , Arshava B. , Naider F. , Lee B.‐K. , Hauser M. , Becker J.M.
Published: March 2003

Abstract: Mating in Saccharomyces cerevisiae is induced by the interaction of α‐factor (W1H2W3L4Q5L6K7P8G9Q10P11M12Y13) with its cognate G protein‐coupled receptor (Ste2p). Fifteen fluorescently labeled analogs of α‐factor in which the 7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl (NBD) group was placed at the αN‐terminus and in side‐chains at positions 1, 3, 4, 6, 7, 12 and 13 were synthesized and assayed for biological activity and receptor affinity. Eleven of the analogs retained 6−60% of the biological activity of the α‐factor, as judged using a growth arrest assay. The binding affinities depended on the position of NBD attachment in the peptide and the distance of the tag from the backbone. Derivatization of the positions 3 and 7 side‐chains with the NBD group resulted in analogs with affinities of 17−35% compared with that of α‐factor. None of the other NBD‐containing agonists had sufficient receptor affinity or strong enough emission for fluorescence analysis. The position 3 and 7 analogs were investigated using fluorescence spectroscopy and collisional quenching by KI in the presence of Ste2p in yeast membranes. The results showed that the λmax of NBD in the position 7 side‐chain shifted markedly to the blue (510 nm) when separated by 4 or 6 bonds from the peptide backbone and that this probe was shielded from quenching by KI. In contrast, separation by 3, 5, 10 or more bonds resulted in λmax (≈ 540 nm) and collisional quenching constants consistent with increasing degrees of exposure. The NBD group in the position 3 side‐chain was also found to be blue shifted (λmax=520 nm) and shielded from solvent. These results indicate that the position 7 side‐chain is likely interacting with a pocket formed by extracellular domains of Ste2p, whereas the side‐chain of Trp3 is in a hydrophobic pocket possibly within the transmembrane region of the receptor.
Characterization of the solution conformations of leuprolide acetate
Authors: Meyer J. D. , Manning M. C. , Vander Velde D. G.
Published: March 2003

Abstract: Leuprolide acetate (pGlu‐His‐Trp‐Ser‐Tyr‐d‐Leu‐Leu‐Arg‐Pro‐NHEt), a potent LHRH agonist in wide clinical use, was characterized conformationally by NMR and circular dichroism. It displayed quite different preferred conformations under different solution conditions: two low population β‐turns in water, a nascent helix in TFE/water at low pH, and a high population β‐turn in TFE/water at slightly acidic pH. The pH‐related conformational change in TFE/water is attributed to the pKa of the acetate counterion, not to ionizable groups on the peptide. None of these conformations are in exact agreement with previous computational predictions.
Fast and efficient peptide bond formation using bis‐[α,α‐bis(trifluoromethyl)‐benzyloxy]diphenylsulfur. Part I
Authors: Slaitas A. , Yeheskiely E.
Published: March 2003

Abstract: This study towards the development of sulfurane‐based coupling agents shows that bis‐[α,α‐bis(trifluoromethyl)‐benzyloxy]diphenylsulfur (BTBDS) can facilitate rapid amide bond formation between Nα‐urethane‐protected l‐amino acids and l‐phenylalanine ethyl ester in the absence of an external base. The corresponding dipeptide esters were obtained in excellent yields and with no detectable racemization, as judged by analysis of the formed dipeptides by chiral‐phase HPLC. In addition, BTBDS‐mediated condensation of benzoyl‐l‐phenylalanine with l‐phenylalanine ethyl ester was also investigated. The results indicate that sulfuranes can be useful for application in racemization‐sensitive systems, such as segment condensation.
Multiple anchoring of myelopeptides on sequential oligopeptide carriers (SOCn): synthesis, conformation and studies in human leukemia cells
Authors: Keramisanou D. , Tsikaris V. , Sakarellos‐Daitsiotis M. , Sakarellos C. , Mikhailova A. A. , Strelkov L. A.
Published: March 2003

Abstract: Myelopeptides, MP‐6 (Val‐Asp‐Pro‐Pro) and MP‐4 (Phe‐Arg‐Pro‐Arg‐Ile‐Met‐Thr‐Pro), induce metabolic changes in human leukemia cells, HL‐60, characteristic of the differentiation process, which should be regarded as a promising therapeutic approach in cancer and related diseases. With the aim to optimize the differentiation effect of MPs, they were coupled to the Lys‐NεH2 groups of a sequential oligopeptide carrier Ac‐(Lys‐Aib‐Gly)4, SOC4, and the constructs obtained were studied. The rigid 310 secondary structure of the carrier is preserved even after linkage of the MPs, which also maintain their initial conformations without interacting either with each other or with the carrier, as demonstrated by 1H nuclear magnetic resonance (NMR) spectroscopy. It is concluded that the carrier accommodates the presentation of MPs, thus improving their differentiation effect on human leukemia cells.
Utility of peptide–protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide*
Authors: Gururaja T.L. , Li W. , Payan D.G. , Anderson D.C.
Published: March 2003

Abstract:We used aN‐biotinylated peptide analog of the C‐terminal domain of the tumor suppressor protein, p21cip1/waf1to elucidate peptide/protein interacting partners. The C‐terminal domain of p21cip1/waf1protein spanning 141–160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell‐cycle control. This C‐terminal 20‐mer efficiently extracts PCNA in the presence of a variety of N‐ or C‐terminally attached affinity tags. Using difference silver stained 2D gels combined with in‐gel tryptic digests, we identified the difference spots using MALDI‐TOF mass spectrometry‐based peptide mass fingerprinting followed by a database search usingprofoundagainst NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor‐1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion‐trap LC‐MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches usingsequestconfirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90α, HSP40 and T‐complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21cip1/waf1protein. The use ofN‐biotinylated peptide derived from the C‐terminal domain of p21cip1/waf1protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.
Acid‐induced acid tolerance and acidogenicity of non‐mutans streptococci
Authors: Takahashi N. , Yamada T.
Published: March 2003

Acid tolerance and acidogenicity of non‐mutans streptococci and their capacity of acid adaptation were studied. The cells of non‐mutans streptococci (Streptococcus sanguis[Streptococcus sanguinis], Streptococcus gordonii, Streptococcus oralis and Streptococcus mitis) grown at pH 7.0 showed 0.0088% to 71% viability after acidification at pH 4.0 for 60 min, whereas the cells of mutans streptococci (Streptococcus mutans) were not killed by the acidification. Washed cells of non‐mutans streptococci lowered pH to 4.04–4.33 in the presence of glucose, while mutans streptococci cells lowered pH to 3.70. When the growth pH was shifted to 5.5 for 30–90 min, the viability of non‐mutans streptococci after the acidification at pH 4.0 for 60 min increased (0.25% to 91%) and the minimum pH values of the cells in the presence of glucose decreased (3.90–4.19). Along with the increase in acid tolerance and acidogenicity, non‐mutans streptococci increased activities of H+‐ATPase and arginine deiminase and amounts of stress proteins cross‐reacting with 60 kDa and 70 kDa heat shock proteins (Hsp60 and Hsp70). These results indicate that non‐mutans streptococci were capable of increasing their acid tolerance and acidogenicity in response to environmental acidification. Furthermore, it is suggested that the acid adaptation observed in non‐mutans streptococci cells could involve the induction of H+‐ATPase, arginine deiminase and stress protein syntheses. The strains of non‐mutans streptococci, which are pioneer bacteria for dental plaque formation and predominant in plaque microbial flora, may play a significant role in shifting the dental plaque environment toward acidic and consequently promoting the colonization of more acid‐tolerant and acidogenic bacteria such as mutans streptococci and lactobacilli.
Age‐related acquisition of oral and nasopharyngeal yeast species and stability of colonization in young children
Authors: Hannula J. , Saarela M. , Jousimies‐Somer H. , Takala A. , Syrjänen R. , Könönen E. , Asikainen S.
Published: March 2003

The occurrence and stability of colonization of oral yeast species and strains was determined from 40 healthy children during a 22‐month follow‐up at the ages 2, 6, 12, 18 and 24 months. In addition, salivary samples were obtained from the mothers at baseline (2 months) to study the role of the mother as the source of yeasts for the child. Yeasts were recovered at least once from 17/40 (43%) children by the age of 2 years. Of the 40 children, 11 (28%) were yeast‐positive at multiple sampling occasions. No significant differences were found in recovery frequency of yeasts at different ages. Candida parapsilosis was isolated in 18/33 (55%) yeast‐positive samples, and it predominated (share of positive findings 76%) at ages 12 to 24 months. The same yeast species was rarely detected in successive follow‐up samples and thus on species level yeasts were transient colonizers in the developing oral flora of the children. Of the mothers 20/40 (50%) harbored yeasts. Candida albicans was recovered from 19/20 (95%) of the yeast‐positive mothers and C. parapsilosis from none. Only 7/20 (35%) of the mothers with a yeast‐positive finding had a yeast‐positive child. In 5/7 (71%) of these mother‐child pairs, both harbored the same yeast species (C. albicans) and in 3/5 (60%) of the pairs the AP‐PCR profiles of the yeast isolates were identical suggesting possible transmission. In children, significant relationships (Fisher's exact‐test, P<0.05) were found between recovery of yeasts and use of pacifier at age over 12 months, eruption of first teeth at age over 6 months, mother cooling the child's food by blowing and mother cleaning the child's pacifier in her own mouth. In mothers, a significant relationship existed between recovery of yeasts and use of antibiotics.
Taxonomy and virulence of oral spirochetes
Authors: Chan E. C. S. , McLaughlin R.
Published: March 2003

All oral spirochetes are classified in the genus Treponema. This genus is in the family Spirochaetaceae as in Bergey’s manual of systematic bacteriology. Other generic members of the family include Spirochaeta, Cristispira and Borrelia. This conventional classification is in accord with phylogenetic analysis of the spirochetes based on 16S rRNA cataloguing. The oral spirochetes fall naturally within the grouping of Treponema. Only four species of Treponema have been cultivated and maintained reliably: Treponema denticola, Treponema pectinovorum, Treponema socranskii and Treponema vincentii. These species have valid names according to the rules of nomenclature except for Treponema vincentii, which only has had effective publication. The virulence factors of the oral spirochetes updated in this mini‐review have been discussed within the following broad confines: adherence, cytotoxic effects, iron sequestration and locomotion. T. denticola has been shown to attach to human gingival fibroblasts, basement membrane proteins, as well as other substrates by specific attachment mechanisms. The binding of the spirochete to human gingival fibroblasts resulted in cytotoxicity and cell death due to enzymes and other proteins. Binding of the spirochete to erythrocytes was accompanied by agglutination and lysis. Hemolysis releases hemin, which is sequestered by an outer membrane sheath receptor protein of the spirochete. The ability to locomote through viscous environments enables spirochetes to migrate within gingival crevicular fluid and to penetrate sulcular epithelial linings and gingival connective tissue. The virulence factors of the oral spirochetes proven in vitro underscore the important role they play in the periodontal disease process. This role has been evaluated in vivo by use of a murine model.
Pathways for amino acid metabolism by Prevotella intermedia and Prevotella nigrescens
Authors: Takahashi N. , Yamada T.
Published: March 2003

Pathways for amino acid metabolism by Prevotella intermedia and Prevotella nigrescens were investigated. Prevotella strains grew anaerobically in tryptone‐based medium and their growth increased upon the addition of aspartate to the medium. Washed cells of tryptone‐grown strains metabolized aspartate to succinate, acetate, fumarate, malate, formate and ammonia, while from tryptone they produced isobutyrate and isovalerate in addition to the end products from aspartate. Cell extracts obtained from the tryptone‐grown cells had aspartate ammonia‐lyase for the conversion of aspartate to fumarate. Methylviologen‐dependent fumarate reductase was found to reduce fumarate to succinate. A series of enzymatic activities, including fumarase, NAD‐dependent malate dehydrogenase, oxaloacetate decarboxylase, methylviologen‐dependent pyruvate oxidoreductase, phosphotransacetylase and acetate kinase, was detected for the oxidative conversion of fumarate to acetate. Pyruvate formate‐lyase and NAD‐dependent formate dehydrogenase were also found for the production and consumption of formate, respectively. Methylviologen: NAD(P) oxidoreductase was found to be responsible for linkage between these reductive and oxidative pathways. Furthermore, the cell extracts had branched‐chain amino acid aminotransferase and methylviologen‐dependent branched‐chain 2‐oxoacid oxidoreductase, concomitantly with NAD‐dependent glutamate dehydrogenase. Valine and leucine could be converted to isobutyryl CoA and isovaleryl CoA, respectively, through the sequential catalyses of these enzymes, and consequently to isobutyrate and isovalerate, respectively.
Profile of subgingival microbiota in children with mixed dentition
Authors: Kamma J. J. , Diamanti‐Kipioti A. , Nakou M. , Mitsis F. J.
Published: March 2003

A diversity of microbial species has been detected in children’s oral flora at an early age. To investigate the composition of the subgingival microbiota of different groups of teeth in children with mixed dentition, 40 systemically healthy children, aged 7–8 years, randomly chosen, were examined. Subgingival plaque samples were taken from the mesiobuccal sites of 21, 41, 16 and 36 permanent teeth and 53, 73, 64 and 84 deciduous teeth. The samples were cultured for bacterial isolation anaerobically and in 10% CO2 plus air using selective and nonselective media. Forty‐five different microbial species were isolated from both permanent and deciduous teeth. Streptococcus sanguis (79–70%), Streptococcus mitis (66–65%), Prevotella melaninogenica (51–57%), Eikenella corrodens (51–52%), Capnocytophaga gingivalis (46–34%), Capnocytophaga ochracea (45–45%), Actinomyces naeslundii (39–60%) and Prevotella intermedia (42–35%) were among the most frequently detected species in permanent and deciduous teeth respectively. Several suspected periodontal pathogens, such as Porphyromonas gingivalis, Prevotella loescheii, Campylobacter gracilis, Bacteroides forsythus, Campylobacter concisus, Peptostreptococcus micros and Selenomonas sputigena, albeit less frequently detected, were present in the microbiota of these children. The bacterial species Streptococcus constellatus, Peptostreptococcus micros, Pseudoramibacter alactolyticus, E. corrodens and Fusobacterium nucleatum were associated with non‐bleeding permanent and deciduous teeth whereas Streptococcus intermedius, C. concisus, P. intermedia and P. loescheii were associated with bleeding.
Suppression of urease levels in Streptococcus salivarius by cysteine, related compounds and by sulfide
Authors: Sissons C. H. , Yakub S.
Published: March 2003

Urease synthesis in Streptococcus salivarius is induced by an acid environment, carbohydrate and a high growth rate. We now report that both cysteine and sulfide above 1 mM strongly suppress S. salivarius urease levels. Close structural relatives of cysteine (cysteamine, ethanedithiol and penicillamine) at 5 mM buffered to pH 7.0 also caused urease suppression, but thiols in general (2‐mercaptoethanol, dithiothreitol and glutathione) did not. In cultures buffered below pH 5.9, the cysteine‐induced urease suppression was lifted substantially, but the sulfide suppression increased, suggesting involvement of different processes. Urease activity was inhibited 50% by 5 mM mercaptoethanol but unaffected by 5 mM cysteine or sulfide, hence modification of enzyme activity by thiols is not directly related to suppression of their levels in culture. Cysteine, arising primarily through protein hydrolysis which also raises the pH, could be a surrogate pH feedback signal for nearby alkaline conditions, and sulfide may reflect activity of periodontopathic plaques.
Inhibitory effect of sorbitol on sugar metabolism of Streptococcus mutans in vitro and on acid production in dental plaque in vivo
Authors: Takahashi‐Abbe S. , Abbe K. , Takahashi N. , Tamazawa Y. , Yamada T.
Published: March 2003

This study was conducted to find out whether sorbitol inhibits the sugar metabolism of Streptococcus mutansin vitro and the acid production in dental plaque in vivo. S. mutans NCIB 11723 was anaerobically grown in sorbitol‐containing medium. The rate of acid production from sugars was estimated with a pH stat. The rate of acid production from glucose or sucrose was not changed at various concentrations of oxygen. By the addition of sorbitol to sugar, however, the acid production was decreased with increasing levels of oxygen. Intracellular NADH/NAD+ ratio and (dihydroxyacetone‐phosphate+glyceraldehyde‐phosphate)/3‐phosphoglycerate ratio were high whenever the acid production was inhibited by sorbitol. Sorbitol also inhibited the acid production in dental plaque in vivo. These results suggest that the increased NADH/NAD+ ratio during sorbitol metabolism through the inactivation of pyruvate formate‐lyase by oxygen inhibited glyceraldehyde‐phosphate dehydrogenase and then the acid production of S. mutans and the one in dental plaque.
Genetic variation in the recognition of Porphyromonas gingivalis antigens in mice
Authors: Gemmell E. , Sernia C. , Grieco D. A. , Bird P. S. , Allen C. J. , Seymour G. J.
Published: March 2003

T‐cell cytokine profiles, anti–Porphyromonas gingivalis antibodies and Western blot analysis of antibody responses were examined in BALB/c, CBA/CaH, C57BL6 and DBA/2J mice immunized intraperitoneally with different doses of P. gingivalis outer membrane antigens. Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)‐4, interferon (IFN)‐gamma and IL‐10 by FACS analysis and levels of anti–P. gingivalis antibodies in the serum samples determined by enzyme‐linked immunosorbent assay. Western blot analysis was performed on the sera from mice immunized with 100 μg of P. gingivalis antigens. The four strains of mice demonstrated varying degrees of T‐cell immunity, although the T‐cell cytokine profiles exhibited by each strain were not affected by different immunizing doses. While BALB/c and DBA/2J mice exhibited responses that peaked at immunizing doses of 100–200 μg of P. gingivalis antigens, CBA/CaH and C57BL6 demonstrated weak T‐cell responsiveness compared with control mice. Like the T‐cell responses, serum antibody levels were not dose dependent. DBA/2J exhibited the lowest levels of anti–P. gingivalis antibodies followed by BALB/c with CBA/CaH and C57BL6 mice demonstrating the highest levels. Western blot analysis showed that there were differences in reactivity between the strains to a group of 13 antigens ranging in molecular weight from 15 to 43 kDa. Antibody responses to a number of these bands in BALB/c mice were of low density, whereas CBA/CaH and C57BL6 mice demonstrated high‐density bands and DBA/2J mice showed medium to high responses. In conclusion, different immunizing doses of P. gingivalis outer membrane antigens had little effect on the T‐cell cytokine responses and serum anti–P. gingivalis antibody levels. Western blot analysis, however, indicated that the four strains of mice exhibited different reactivity to some lower‐molecular‐weight antigens. Future studies are required to determine the significance of these differences, which may affect the outcome of P. gingivalis infection.
Subgingival occurrence and antimicrobial susceptibility of enteric rods and pseudomonads from Brazilian periodontitis patients
Authors: Barbosa F. C. B. , Mayer M. P. A. , Saba‐Chujfi E. , Cai S.
Published: March 2003

The occurrence and in vitro antimicrobial sensitivity of isolates of enteric rods and pseudomonads were examined in 80 periodontitis patients, 17 to 58 years of age, in São Paulo, Brazil. Speciation and in vitro antimicrobial susceptibility testing were performed using the BBL Crystal enteric/nonfermenter system and the Etest for amoxicillin/clavulanic acid, ciprofloxacin and doxycycline. A total of 30 strains were isolated from 25 (31.2%) of the study subjects. Pseudomonas aeruginosa occurred in nine patients, Serratia marcescens in seven, and five other species were recovered in lower prevalence. All study organisms demonstrated high susceptibility to ciprofloxacin but exhibited variable susceptibility patterns to the other antimicrobial agents tested. In conclusion, the high occurrence of enteric rods and pseudomonads in these subjects may be important in the pathogenesis of periodontitis, and ciprofloxacin might be the antibiotic of choice to eradicate these pathogens from periodontal pockets.
Differentiation of Streptococcus mutans and Streptococcus sobrinus via genotypic and phenotypic profiles from three different populations
Authors: Li Y. , Caufield P. W. , Emanuelsson I. Redmo , Thornqvist E.
Published: March 2003

Routine identification of Streptococcus mutans and Streptococcus sobrinus is generally based upon growth on various selective media, colony morphology and biochemical characteristics. We examined various approaches of differentiating these two species through a combination of the conventional phenotypic methodology with chromosomal DNA fingerprint (CDF) and arbitrarily primed polymerase chain reaction (AP‐PCR) methods. Initially, ten ATCC type strains and 20 randomly selected clinical isolates of mutans streptococci (MS) were characterized and grouped into two major types based on patterns generated by the CDF using HaeIII digestion. The CDF’s patterns with restriction fragments equal to or greater than 6.6 kb were defined as the CDF‐1 group. The CDF’s patterns with restriction fragments less than 6.6 kb were defined as the CDF‐2 group. Both groups were then examined for biotype, serotype, and composition of DNA via thermal denaturation. AP‐PCR was applied and evaluated for the capability of delineating S. mutans from S. sobrinus strains. Results of this study showed that all CDF‐1 strains fit within a G+C range of 36.2% to 42.2%, whereas the CDF‐2 strains had a G+C range of 45.8% to 47.0%. The serotyping assay exhibited 100% sensitivity, 90% specificity and 86.7% agreement with the CDF. The biotyping assay presented the poorest specificity (38.5%), indicating the highest variability. The capability of AP‐PCR in differentiation of S. mutans from S. sobrinus was comparable to the CDF method, suggesting that either of these two approaches can and may serve as a viable alternative method to serotyping or biotyping of MS.
Selective sensitization of bacteria to peroxide damage associated with fluoride inhibition of catalase and pseudocatalase
Authors: Phan T.‐N. , Kirsch A. M. , Marquis R. E.
Published: March 2003

Fluoride and sulfide are known inhibitors of heme catalases in acid environments. Staphylococcus aureus H cells were found to be sensitized by fluoride or sulfide to H2O2 killing at acid pH values in the range of 3.5 to 4.0, and catalase activity was reduced concomitantly. In contrast, fluoride had little effect on H2O2 killing of Streptococcus mutans GS‐5, which has fluoride‐insensitive peroxidase activity, but still is more sensitive to H2O2 than is S. aureus in the absence of fluoride. Fluoride but not sulfide was inhibitory also for the Mn‐containing, non‐heme pseudocatalase of Lactobacillus plantarum ATCC 14431 over a wide pH range, and this inhibitory effect was reflected in enhanced H2O2 killing in the presence of fluoride. In addition, we found that catalase‐positive S. aureus or Neisseria sicca could protect catalase‐negative S. mutans against killing by H2O2 in mixed suspensions, but protection was compromised by fluoride or sulfide under acid conditions. Thus, catalase‐positive organisms could protect a catalase‐negative organism against peroxide damage, but inhibition of catalase reduced protection. These findings are pertinent to the widespread use of fluoride and peroxide in oral health care products.
HLA‐DR4 and salivary immunoglobulin A reactions to oral streptococci
Authors: Wallengren M. L. L. , Ericson D. , Hamberg K. , Johnson U.
Published: March 2003

The aim of this study was to describe and compare salivary immunoglobulin A (IgA) antibody reactions to extracts of strains of three oral streptococci in human leukocyte antigen (HLA)‐DR4‐positive and ‐DR4‐negative subjects. Whole paraffin‐stimulated saliva samples were collected from 27 apparently healthy subjects. Previous HLA typing showed that 20 subjects were DR4 positive and 7 were DR4 negative. HLA‐DRB1*04 subtyping was performed among the DR4‐positive subjects. Whole‐cell antigen extracts from Streptococcus mutans (KPSK 2), Streptococcus sobrinus (OMZ 65) and Streptococcus parasanguis (Nt 62) were separated in SDS‐PAGE. The antigens were immunoblotted with diluted saliva (Western blot), scanned and analyzed in a computer system. All immunoblot bands were recorded in DR4‐positive and DR4‐negative saliva pools, and bands with an optical density ≥0.1 were selected for analysis in individual salivas. The DR4‐negative subjects in general had more immunoblot bands and more distinct bands than did the DR4‐positive subjects. A higher concentration of total IgA in saliva was correlated with more bands, especially to antigens separated from S. mutans. When the number of bands was calculated per IgA unit, significant differences were observed between DR4‐positive and DR4‐negative salivas. This was particularly seen for S. mutans and S. parasanguis. As the number of bands was analyzed in relation to DR4 subgroups, DRB1*04, there was a lower salivary IgA activity to S. mutans in the DRB1*0401 and *0404. The variable level of correlation previously demonstrated for S. mutans colonisation and serologically defined DR4 positive subjects might be explained by the heterogeneity in this group, and the relation should be sought on a subgroup level.