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PubMed2006

Detection of feline calicivirus (FCV) from vaccinated cats and phylogenetic analysis of its capsid genes.

We analysed genogroups of four feline calcivirus (FCV) isolates (FCV-S, H10, Ao198-1 and ML89) obtained from cats that experienced FCV infection after having been vaccinated against FCV. New PCR primer sets (8F/8R, Ao-S/Ao-A, cp-S/cp-A) were also designed, since the conventional Seal primer failed to amplify the target sequences in two samples. The genogroups of the four isolates as well as eight global and 17 domestic strains were determined by phylogenetic analysis of their amino acid sequences. One out of the four strains (25%) isolated in this study, H10, was grouped into genogroup I, along with the vaccine strains F9 and FCV-255. The other three isolates (75%) belonged to genogroup II. Thus, there were more isolates in genogroup II than in genogroup I. However, the antibody values of the four isolates against cat anti-F9 antisera were significantly decreased. There may be no relationship between the neutralizing antibody titre and genogroup. Amino acid sequence alignment of the four isolates showed that only a single amino acid in region C, which is involved in neutralization epitopes, was different in ML89 strain from that of F9. The other three strains, H10, Ao198-1 and FCV-B, shared the same amino acid sequence with F9. Alignment of amino acids for linear epitopes in the F9 strain, which are located at regions D and E, showed variations in 5' hypervariable region (HVR) of E, whereas D and conE had only synonymous substitutions i.e. no change in the amino acid sequence. This mutation in 5' HVR of region E suggested a vaccine breakdown, as the region is known to be essential for antigenicity. The genogroup II FCV is likely to be the cause of the FCV infection in this study, while the vaccine strains belong to genogroup I. Thus, the existing vaccine may need reevaluation for its effectiveness.

Amino Acid Sequence↗
PubMed1985

Primary structure of porcine Cu,Zn superoxide dismutase.

The complete amino acid sequence of Cu,Zn superoxide dismutase from porcine erythrocytes has been determined. Comparison of the sequence with that of the bovine enzyme shows an overall high degree of homology with conservation of the crucial residues and the presence of two regions prone to variation. In one of these hypervariable regions the insertion of one residue with respect to the bovine enzyme and evidence of structural microheterogeneity has been observed. On the basis of the three-dimensional structure of the bovine enzyme no obvious relationship is apparent between a specific amino acid replacement and the unique pH-dependence pattern of the activity of the porcine enzyme.

Amino Acid Sequence↗
PubMed1986

Optimal strategies for developing human-human monoclonal antibodies.

Human monoclonal antibodies are desirable, especially as therapeutic agents, but the best means of producing them is still a matter of investigation. It is clear that human antibodies of predicted specificity from patients with autoimmune disease can be derived, and this may help unlock some of the mysteries of these illnesses. Human monoclonal antibodies against tumor-specific antigens for use in in vivo diagnosis and therapy remain desirable goals. Problems involved in their routine development include the lack of available, adequately immunized, and differentiated lymphocytes and the nature and paucity of the available human "myeloma" cell lines. These lines have been compared now by a number of authors who have reached similar conclusions. Our study directly compared the greatest number of cell lines and found UC729-6 and HF2 to be the best; on the other hand, our success in developing IgG-secreting hybridomas from U-266, using hyperimmunized lymphocytes, suggests that this line may only be capable of secretion with the more differentiated cell, the human equivalent of those hyperimmunized murine spleens. Hence both sides of the fusion equation must be made optimal. Two new approaches to circumvent this problem involve the use of either a human-murine myeloma chimera as the parental myeloma line or, more recently, genetic engineering techniques to substitute human constant regions for the murine while retaining the murine hypervariable region, preserving the binding specificity of the murine antibody.

Antibodies, Monoclonal↗
PubMedDate not supplied

Mitogens, superantigens, and nominal antigens elicit distinctive patterns of TCRB CDR3 diversity.

The third complementarity-determining region (CDR3) is the only nongermline-encoded hypervariable region of the T cell receptor beta (TCRB) chain, and it is the region that has been predicted to confer fine specificity of the TCR for peptide-MHC complexes. For this reason analysis of TCRB CDR3 heterogeneity may provide insight into immune mechanisms operative in infectious and autoimmune diseases. PBMC stimulated with either mitogen (PHA), superantigen (TSST-1), or nominal antigen (tetanus toxoid) have been compared with unstimulated PBMC using a two-dimensional approach. Analysis of the expressed TCRBV gene repertoire CDR3 length profile coupled with SSCP methodology enabled the discrimination of sequences with the same CDR3 length. For both freshly isolated and PHA stimulated PBMC, a normally distributed spectrum of CDR3 lengths (five or more products) was observed. These products differed by 3 bp (1 amino acid) due to the strict requirement for in-frame rearrangements in the CDR3 region of TCR. By contrast, tetanus toxoid stimulated PBMC had restricted profiles for most TCRBV families after as few as 7 days of incubation. The oligoclonal nature of samples showing CDR3 length restriction was revealed by SSCP analysis and confirmed by sequence determination. Superantigen stimulation resulted in unique patterns of diversity, which included polyclonal expansion of specific TCRBV families as well as oligoclonal expansion of most other TCRBV families. These data reveal complex yet distinct patterns of TCR diversity in response to different T cell activation stimuli.

Adult↗
PubMed1982

Mouse immunoglobulin coding sequences for the heavy-chain variable region arose as repeats of the two short building blocks.

The coding sequence for the 97-amino-acid-residue-long immunoglobulin heavy-chain variable (VH) regions of the mouse apparently arose as repeats of the two short building blocks. Three of the recognizable copies of the one 21-base-long prototype sequence A-C-T-G-G-A-T-A-T-G-A-C-C-T-G-G-A-G-T-G-G are invariably found to occupy the fixed positions within the 5' half of each VH coding sequence. Interestingly, the first and third copies specify the relatively invariant regions represented by the 7th to 13th and 41st to 47th amino acid residues (the first and second framework regions), whereas the second copy specifies the first hypervariable region (31st to 35th amino acid residues). These copies maintain at least 57.2% (12 out of 21) base sequence homology to the above-noted prototype building block. Base sequences of the other 14- to 15-base-long prototype building block differ from each other by as much as 60% between individual VHS. Yet one of its copies invariably occupies the terminal region of each VH coding sequence, thus specifying the very invariant third framework region. Other copies occupy unfixed positions in the VH and its attendant hydrophobic leader coding sequence as well as in adjacent noncoding sequences. The homology thus revealed between the VH coding sequence and its adjacent noncoding sequences suggests their concordant evolution.

Amino Acid Sequence↗
PubMed2006

IWoCS: analyzing ribosomal intergenic transcribed spacers configuration and taxonomic relationships.

MOTIVATION: Lately the use of 16S-23S Intergenic Transcribed Spacer (ITS) sequences for bacterial typing purposes has increased. The presence of conserved regions like tRNA genes or boxes together with hypervariable regions allows performing intraspecific discrimination of very close bacterial strains. On the other hand this mosaic of variability makes the ITS a sequence difficult to analyze and compare. RESULTS: A software to study ITSs by a Word Count based System (IWoCS) is proposed. A large dataset of ITS was created (comprising 7355 sequences). A database indicating all the occurrences of possible n-mers (tags), describing each ITS sequence, was created (with n going from 5 to 13) including 32 061 819 entries. The database allows to analyze ITS sequences submitted by users using a web-based interface. The abundance in the database of each n-mer is given in a one-base sliding frame. A dominance plot reflects how common the tags are within different taxonomic levels. The obtained profile identifies highly repeated tags as evolutionarily conserved regions (like tRNA or boxes) or low frequency tags as regions specifically associated to taxonomic groups. The study of the dominance and abundance profiles combined with the taxonomy reports provides a novel tool for the use of the ITS in bacteria typing and identification. AVAILABILITY: The database is freely accessible at http://egg.umh.es/iwocs/.

Algorithms↗
PubMed1997

Role of basic residues in the subgroup-determining region of the subgroup A avian sarcoma and leukosis virus envelope in receptor binding and infection.

Receptor specificity in avian sarcoma and leukosis viruses (ASLV) maps to the central region of the envelope surface protein, SU. Two hypervariable regions, hr1 and hr2, within this region of SU are the principal determinants of receptor specificity. The cellular receptor for subgroup A ASLV, Tva, utilizes a 40-residue, acidic, cysteine-rich sequence for viral binding and entry. This domain in Tva is closely related to the ligand-binding domain of the low-density lipoprotein receptor (LDLR). Ligands bind to LDLR via the interaction of clustered basic residues in the ligand with the acidic cysteine-rich domains of the receptor. Analysis of the ASLV envelope sequences revealed a cluster of basic residues within hr2 that is unique to the subgroup A viruses, suggesting a possible role for these residues in receptor recognition. Therefore, the effects of altering these basic residues on subgroup A envelope expression, receptor binding, and infectivity were examined. Most of the mutant proteins were transported to the cell surface and processed normally. Receptor binding was diminished approximately 50% by alanine substitution at amino acid R213 or K227, whereas substitution by alanine at R210, R223, or R224 had no effect. However, when coupled with mutations at R213 or K227, changes at R223,R224 reduced envelope binding by 90%. Mutation of all five basic residues abrogated receptor binding. The effect of the hr2 mutations on ASLV envelope-mediated infection did not parallel the effect on receptor binding. Residues 210, 213, 223, and 224 were important for efficient infection, while mutations at residue 227 had little effect on infectivity. These results demonstrate that the basic residues in the ASLV envelope have roles in both receptor recognition and post-receptor binding events during viral entry.

Alpharetrovirus↗
PubMed1999

Antigen-driven selection of TCR In vivo: related TCR alpha-chains pair with diverse TCR beta-chains.

Ag-driven selection mediates effective T cell help and the development of Th cell memory in vivo. To analyze the dynamics of interclonal competition during the selection process in vivo, we use the I-Ek-restricted murine response to pigeon cytochrome c (PCC). The dominant PCC-specific clonotype expresses Valpha11Vbeta3 V regions with preferred sequence features in the third hypervariable regions (CDR3). In the current study we define and quantitatively monitor four subdominant PCC-specific clonotypes that express Valpha11 paired with non-Vbeta3 TCR beta-chains (Vbeta6, Vbeta8.1/8. 2, Vbeta8.3, and Vbeta14). The subdominant clonotypes emerge with similar dynamics to the dominant clonotype and together amount to similar numbers as the dominant clonotype in vivo. These subdominant clonotypes do not efficiently enter germinal centers, although they enter the memory compartment and rapidly re-emerge upon secondary challenge. Analysis of CDR3 diversity in the TCR alpha-chains identifies many preferred sequence features expressed by the dominant clonotype. These studies quantitatively demonstrate selection for diverse Th cells in vivo and highlight TCR alpha-chain dominance in Ag-driven selection for best fit.

Amino Acid Sequence↗
PubMed2001

Predominance of antibodies to hepatitis C virus envelope proteins in various disease statuses of hepatitis C.

The antibody profile to various proteins of hepatitis C virus (HCV) was studied in 113 patients positive for HCV RNA in various disease statuses of hepatitis C (HC). A single peptide (E2/NS1, aa 413-436 of HCV polyprotein) chosen from a conserved region at the C-terminus of the hypervariable region (HVR) HVR1 of HCV was found to be sufficient for reliable diagnosis of the infection, even in the acute phase. Six hundred and one suspected HC cases and 200 voluntary blood donors were tested by this peptide. The sensitivity of detection of HCV antibodies by this peptide did not increase with addition of peptides from other HCV proteins. Our results clearly demonstrate that antibodies to HCV envelope proteins occur in a higher percentage of the infected population than those to other proteins. This emphasizes the necessity of using representative sequences from HCV envelope proteins in diagnostic immunoassays of this viral infection.

Acute Disease↗
PubMed2001

[Cloning and sequence analysis of light chain gene of human antibody against HBV pre-S2].

OBJECTIVE: To obtain the gene of light chain of human antibody against HBV pre-S2. METHODS: A human antibody against HBV pre-S2 displayed on phage was obtained from the constructed human immunoglobulin phage displaying library through four rounds panning of "adherence-elution-amplification" with Pre-S2 (120aa-145aa) short peptide synthesized and help phage. The positive clone of anti-pre-s2 was characterized by ELISA competitive inhibition assay. The plasmid from the positive clone was amplified by PCR with designed oligonucleotide primers of kaph light chain. The light chain fragment was amplified with PCR and plasmid pUC18 digested by restriction endonuclease SacI & XbaI, respectively, then it was subcloned into the plasmid pUC18 and was transformed into E.coli XL1-blue sensitized by CaCl2. The recombinant plasmid pUC18-kaph was sequenced. RESULTS: The competitive inhibition rate of the positive clone of anti-pre-S2 was 65% at 1:50 dilution. Sequence was compared with data in gene bank through internet and analyzed with Blast. According to the sequence and ELISA competitive inhibition assay the integral gene of kaph light chain of human antibody against HBV-PreS2 was obtained. Its variable region located on 1-328bp including three hypervariable regions (82-105bp, 145~177bp, 274~309bp) and four frame regions (1-81bp, 106-144bp, 178-273bp, 310-328bp). Its constant region was located on 329-645bp. CONCLUSIONS: The HBV pre-S2 kaph light chain is screened from phage antibody displaying library by antigen-antibody special response.

Base Sequence↗
PubMed1993

Broadly neutralizing monoclonal antibodies to the V3 region of HIV-1 can be elicited by peptide immunization.

The third hypervariable region (V3) of the HIV-1 envelope gp120 protein contains the principal neutralizing domain. Most neutralizing antibodies directed toward this region are very type-specific. Conserved sequences do exist within this region, however, and may prove useful in developing vaccines and therapeutic monoclonal antibodies (MABs) capable of targeting diverse HIV-1 isolates. We have used synthetic peptides containing conserved V3 sequences as immunogens to produce a panel of neutralizing MABs. The characterization of these MABs is described here. In addition, a series of in vitro assays has been developed that may be useful in predicting the neutralization potential of individual antibodies.

Amino Acid Sequence↗
PubMed1995

Human immunodeficiency virus envelope V1 and V2 regions influence replication efficiency in macrophages by affecting virus spread.

The V3 hypervariable region of the HIV-1 envelope protein is a major determinant of viral tropism for macrophages. However, the replication of macrophage-tropic HIV-1 strains varies considerably in macrophages, and this variability has been linked to the V1 and V2 envelope regions. In the present study, recombinant HIV clones were generated by inserting V1 and V2 sequences from the Ba-L HIV isolate, which has a high macrophage replication level, into the genomic background of a macrophage-tropic clone with a low macrophage replication level. Infection of macrophages with varying multiplicities of infection and direct detection of the number of infected macrophages per culture showed that the Ba-L V1 and V2 envelope sequences enhanced the ability of virus to spread in the cultures. In contrast, macrophage-tropic clones with low replication efficiency infected macrophages initially but showed no evidence of spread to additional cells during the culture period. This effect on virus spread appeared to be macrophage-specific as it was not observed in cultures of T lymphocytes. Comparison of recombinant clones containing V1, V2, and V3 envelope sequences from high-efficiency Ba-L and JR-FL strains indicated that markedly different V1 and V2 sequences could impart the same rapidly spreading phenotype in macrophages.

Amino Acid Sequence↗
PubMed2001

Prevalence of an ulcerative colitis-associated CD8+ T cell receptor beta-chain CDR3-region motif and its association with disease activity.

The normal human intestinal mucosa contains clonal T cell expansions. Clonal populations of T cells can be determined through evaluation of the idiotypic, hypervariable region of their T cell receptor (TCR). We have previously reported that there exists a highly conserved TCR pattern among intestinal CD8+ T cells in the majority of ulcerative colitis (UC) patients undergoing colectomy that was not present in normal control individuals. This TCR pattern, or motif, was characterized by particular beta-chain usage (TCRBV3 and TCRBJ1S6) and a defined length in the hypervariable third complementarity determining region (CDR3). The aim of this study was to assess the motif's relationship to disease activity. Subjects were 66 with UC, 19 with Crohn's disease, 14 inflammatory controls, and 6 normal controls. cDNA and gDNA were prepared from colonic biopsies and paraffin blocks, respectively, obtained from study subjects and used to assess TCRBV CDR3 region length and usage, as well as for cloning and sequencing of TCRs. The TCRBV CDR3 region was present in 25 of a series of 48 UC subjects but only 3 of 19 Crohn's disease patients and 3 of 14 inflammatory controls. The motif was more common in UC than either Crohn' s disease or inflammatory controls (chi2 = 7.5, P = 0.006, and chi2 = 4.1, P = 0.04, respectively). The motifs presence was not dependent upon histologic disease activity (either active or inactive UC). Clinical UC disease activity was also not significantly associated with an increased presence of the motif in 14 paired biopsies, which were taken during times of clinical activity or inactivity. There was a trend toward persistence of the motif, as it was present in 6 of 14 subjects over a 3- to 6-month time period. The previously described UC-associated TCRBV CDR3 region motif located in the intestinal CD8+ T-cell subset is found in a significant proportion of UC subjects. The TCR motif does not significantly discriminate active from inactive disease states. The persistent and diffuse nature of this TCR-associated motif in UC suggests that an ongoing T-cell response to a particular antigen(s) is occuring in this disorder.

Amino Acid Sequence↗
PubMed1994

Sequence variation in the gp135 gene of Maedi visna virus strain EV1.

We have used the PCR and HIRT DNA obtained from MVV-infected tissue culture cells as a template to generate a number of independently derived clones representing overlapping fragments of the gp135 region (env) of Maedi visna virus (MVV) strain EV1. Sequencing these clones revealed that homology between selected regions of gp135 ranged from 93.2% to 99.8%. Four hypervariable regions and one large highly conserved region have been identified. These data provide information on the variability of EV1 env, which extends and complements the data previously available on env variability between geographically distinct isolates of MVV.

Amino Acid Sequence↗
PubMed1994

A research on DLA-DRB1 genotyping by PCR-RFLP. I. To select a appropriate oligonucleotide primer pair.

In order to study the DLA (Dog Leucocyte Antigen) class II region we utilized the polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) method, which has been reported previously as an efficient and simple technique for accurate definition of the HLA class II alleles. To search for a appropriate primer pair a series of amplifications with 4 different primer pairs DLA-DR-SP/Stop, DLA-DR-SP/P3, HLA-DRB-GH46/50 and HLA-DRB-AMP-A/B were provided. Only one satisfactory amplification was obtained with the primer pair HLA-DRB-AMP-A/B. The analogous sequences of the primer pair are found in the sequence of HLA-DRB-cDNA. The amplification region of the primer pair includes also the three hypervariable regions (HVR) in the sequence of DLA-DRB cDNA. Southern blot hybridization analysis confirmed the specificity of the primer pair HLA-DRB-AMP-A/B. The results of Hae III and Hinfl digestion show high polymorphism in DLA-D region and allele specific polymorphic patterns. Therefore, it is suggested that the primer pair HLA-DRB-AMP-A/B is at present the only available and usefull primer pair in PCR-RFLP study of DLA-DRB1 gene.

Alleles↗
PubMed1999

Analysis of sheep T-cell receptor beta-chain heterogeneity.

We analyzed nucleotide and deduced amino acid sequence heterogeneity of sheep T-cell receptor beta-chain cDNAs isolated from an anchored-polymerase chain reaction library. Evaluation of 34 individual rearrangements has defined 18 new beta-chain variable region sequences which have been clustered into 13 families. Presumptive allelic polymorphisms of four of these variable regions have been defined, as well as ten distinct beta-chain joining region sequences. The present analysis indicates that sheep T-cell receptor beta-chains are composed of characteristic leader, variable, joining, and constant region sequences, and that imprecise joining and N-region addition contribute significantly to diversity in the third hypervariable region. Thus, it appears that sheep, like all other mammals studied to date, employ somatic rearrangement of multiple germline genes to create beta-chain heterogeneity. These findings have allowed us to estimate the diversity of the sheep T-cell receptor beta-chain variable region repertoire, and they provide information that will permit the evaluation of the role that specific T-cell populations play in naturally occurring and experimental diseases of sheep.

Alleles↗
PubMed1991

Techniques used to define human MHC antigens: polymerase chain reaction and oligonucleotide probes.

The various HLA class II specificities, identifiable by serological or cellular typing reagents, reflect underlying polymorphisms of the constituent alpha and beta chains, encoded at several loci within the HLA-D region. Variation in these genes is concentrated in allelic hypervariable regions but can also be found elsewhere. Sequence-specific oligonucleotides (SSO) can be used to detect individual alleles with a high degree of accuracy by probing dot-blotted DNA, amplified to high copy number by the polymerase chain reaction (PCR). The classic DR specificities (DR1-DRw14) can be identified with certainty by sequential hybridization to a series of 14 SSO probes. In addition, fine resolution of specificities such as the subtypes of DR4 is possible using a combination of SSO probes and group-specific amplification in which PCR primers designed to amplify only DR4 alleles are employed. Similar methods can be applied to the typing of DQ and DP alleles even on whole blood samples which have been stored at -20 degrees C for up to 8 years. A full HLA class II type may be obtained on a large number of samples simultaneously without the need to separate and store viable lymphocytes. Thus, the sensitivity and robustiness of this technique give it major applications in the analysis of histocompatibility and disease associations, particularly in circumstances where facilities for the initial preparation and storage of samples may be limited.

Alleles↗
PubMed1992

Genomic typing of hepatitis C viruses present in China.

Hepatitis C virus (HCV) genomic clones were obtained from the serum of Chinese HCV carriers using a polymerase chain reaction-based approach. Consensus sequences were derived from (1) the structural region (nt 1-1543) for one carrier, (2) the hypervariable region V (nt 1156-1233) from four carriers and (3) region V3 from four carriers. Region V3, located in the nonstructural domain NS5 (nt 7066-7137), has been previously shown to be a particularly good marker for the genomic typing of HCV isolates [Inchauspe et al., Proc. Natl. Acad. Sci. USA 88 (1991) 10292-10296]. Comparison of these sequences with sequences from geographically distinct HCV isolates indicates that Chinese HCV strains are closely related to, though distinguishable from, Japanese prototype strains. One amino acid motif, GGAA, located in region V, was found to be conserved only among Chinese isolates. This may define a new subgroup among HCV isolates.

Alanine Transaminase↗