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At least 1,423 records · Page 79Linked to original sources
PubMed1997

Modeling of anti-nucleosome immunoglobulin Fv domains: analysis of electrostatic interactions.

Three-dimensional structural models of six murine anti-(H2A-H2B) monoclonal autoantibody variable fragments were built by comparative molecular modeling using the COMPOSER software. Analysis of the antibody combining sites is based on the hypothesis that ionic and/or electrostatic interactions predominate in antigen antibody binding, as suggested by the cationic nature of histones and the amino acid sequences of the antibody hypervariable regions. The study of the electrostatic potentials of their combining site surfaces, computed with the MOLCAD software, and the comparison with the electrostatic potentials of 13 selected control mAbs show the lack of a unique electrostatic pattern. One group of three mAbs expresses a strong and large electronegative area, supporting the hypothesis that ionic interactions predominate in antigen recognition. The second group, containing the other three mAbs, exhibits an alternation of electropositive and electronegative areas. All, however, present a localized electronegative area in the vicinity of H-CDR1 and H-CDR2 loops that is generated by the presence of at least one acidic residue. The model suggesting that the binding activity may depend on charged residues at the same site is reminiscent of what was previously reported in anti-DNA mAbs. In addition, the alternation of electropositive areas and electronegative areas in second group mAbs is also frequently observed in certain anti-DNA mAbs. These data argue for the existence of relationships between these two autoantibody populations and suggest that they share a common immunogenic particle formed by anionic and cationic components, such as a nucleosome.

Amino Acid Sequence↗
PubMed1997

Fluorescence polarization study of a salt bridge between a single-chain Fv and its antigen ribonuclease A.

The interaction between a single-chain Fv (sFv) of the monoclonal antibody 3A21 and its antigen, bovine pancreatic ribonuclease A (RNase A), was studied by site-directed mutagenesis of the hypervariable regions and fluorescence polarization analysis. The affinity constants of wild-type sFv and a mutant sFv D31A (Asp31 of heavy chain was replaced by Ala) for RNase A were found to be 2.7 x 10(7) and 4.7 x 10(6) M-1 in PBS at pH 7.2 and 37 degrees C, respectively. While the affinity constant of D31A is not affected by NaCl concentration, that of wild-type sFv is almost the same as that of D31A in the presence of more than 1 M NaCl. These results demonstrate that Asp31 of the heavy chain interacts electrostatically with a positively charged amino acid residue of RNase A.

Alanine↗
PubMedDate not supplied

Fluorescence polarization study of a salt bridge between a single chain Fv and its antigen ribonuclease A.

The interaction between a single chain Fv (sFv) of the monoclonal antibody 3A21 and its antigen, bovine pancreatic ribonuclease A (RNase A), was studied by site-directed mutagenesis of the hypervariable regions and fluorescence polarization analysis. The affinity constants of wild-type sFv and a mutant sFv D31A (Asp31 of heavy chain was replaced by Ala) for RNase A were found to be 2.7 x 10(7) and 4.7 x 10(6) M(-1) in PBS at pH 7.2 and 37 degrees C, respectively. Whereas the affinity constant of D31A is not affected by NaCl concentration, that of wild-type sFv is almost the same as that of D31A in the presence of more than 1 M NaCl. These results demonstrate that Asp31 of the heavy chain interacts electrostatically with a positively charged amino acid residue of RNase A.

Animals↗
PubMedDate not supplied

Comparison of llama VH sequences from conventional and heavy chain antibodies.

Forty different PCR clones encoding a llama variable heavy chain domain were analysed. The majority of these clones are derived from heavy-chain antibody cDNA in which the entire CH1 exon is absent. It appears from the amino acid within the VHH framework 1 and 3 that all the llama clones belong to the VH III family. However, the individual llama VHH sequences differ more substantially from each other than expected for members of the same family. Several remarkable amino acid substitutions in the framework 2 hinder the proper association of the VL. However, they lay the foundation for the secretion from the endoplasmic reticulum and good solubility behaviour of llama H2 antibodies. The repertoire of the llama VHHs may be extensive due to the presence of a long CDR3-loop, often constrained by a disulfide bridge and the occurrence of H1 and H2 loop conformations not yet encountered in mice or human VHs. The variability plot of the amino acids in the VHH shows that the first hypervariable region coincides with the structural H1 loop in contrast to the situation found in mice and man where the CDR1 and H1 are slightly offset. We propose that the amino acids of the llama H1 loop participate actively in the antigen binding. All these observations are characteristic for the llama VHHs of the homodimeric heavy-chain H2 antibodies, but are not maintained in the llama clones from conventional heterotetrameric H2L2 immunoglobulins.

Amino Acid Sequence↗
PubMed1996

Attempts to demonstrate indirect T cell allorecognition of donor MHC peptides in transplant patients.

Indirect T cell allorecognition has been shown to play an important role in the rejection of allografts in experimental animals. Although there has been much speculation as to its role in clinical transplantation, especially with regard to chronic rejection, indirect T cell allorecognition has been difficult to demonstrate in transplant patients. In this paper, we looked for in vitro T cell proliferation to synthetic peptides corresponding to donor HLA-A and HLA-B incompatible antigens. Twelve 15 amino acid peptides corresponding to the hypervariable regions of six of the most common HLA class I alleles in Caucasian populations (A1, A2, A3, B7, B8 and B44) were studied. Blood was taken from 12 adult patients following one or more episodes of acute kidney graft rejection, and from three pediatric patients undergoing chronic rejection of heart/lung transplants. The donor-recipient combinations were selected such that at least one of the six HLA antigens above was present in the donor and absent in the recipient. Peripheral blood mononuclear cells from these patients responded strongly in proliferation assays to phytohemagglutinin. However, none responded to the incompatible donor HLA peptides. Compartmentalization of responding T cells, the effects of immunosuppression, and assay sensitivity are discussed as possible explanations for the negative results.

Adult↗
PubMed2003

Functional properties of myelin oligodendrocyte glycoprotein-reactive T cells in multiple sclerosis patients and controls.

Autoimmune T-cell reactivity to myelin components may be implicated in the initiation or maintenance of the inflammation leading to myelin destruction in multiple sclerosis (MS). Myelin oligodendrocyte glycoprotein (MOG), a quantitatively minor myelin protein, is an important candidate autoantigen in MS. We studied T-cell responses to recombinant MOG (extracellular domain, rMOG) and a panel of four peptides within this domain (amino acids 1-22, 34-56, 64-86 and 74-96) in MS patients and healthy controls (NS). Frequency analysis of T cells reactive to rMOG as measured by IFN-gamma ELISPOT did not reveal significant differences between MS patients and controls. MOG-reactive T-cell lines and clones (TCL/TCC) were generated by stimulating PBMC of four MS patients and three healthy subjects with a cocktail of the four MOG peptides. The functional properties of 50 MOG peptide-reactive TCL/TCC obtained were studied. All TCL were TCR alpha beta+CD4+ and 20 TCL showed reactivity to MOG peptides 1-22, 13 to 34-56, 1 to 64-86 and 16 to 74-96. No significant differences in peptide recognition were observed between MS patients and controls. The T-cell receptor (TCR) hypervariable regions of MOG-reactive TCL/TCC showed a heterogeneous usage of various TCR V(-D)-J elements. The data provide no evidence for clonal expansions within the MOG-reactive T-cell repertoire of the two study groups. Intracellular cytokine analysis demonstrated predominantly Th1-TCC (IFN-gamma+/IL-4-) in MS patients, while most MOG-reactive TCC of control subjects had a mixed Th0/Th1 phenotype. Furthermore, the MS-derived MOG-reactive TCC produced increased levels of TNF-alpha upon antigen stimulation as compared to controls. Most of the MS-derived MOG-TCC induced specific cytolysis of autologous MOG-pulsed PBMC (9/11) while none of the MOG-TCC isolated from control subjects showed this cytotoxicity (0/8). In conclusion, although the frequency of anti-MOG T cells was similar in MS patients and controls, our data indicate potential differences in the functional properties of MOG TCL in MS patients versus healthy controls which may relate to their role in the disease process.

Adult↗
PubMed1999

Structure-function studies of an anti-asialo GM1 antibody obtained from a phage display library.

Although gangliosides elicit human autoantibodies, they are extremely weak immunogens in mice. We obtained a monoclonal antibody Fab fragment (clone 10) that is specific for asialo GM1 (GA1), from a phage display library. The Vkappa domain of clone 10 could be replaced by two different Vkappa domains without changing the specificity of the antibody. Mutagenesis of the third hypervariable regions of the heavy and light chains of clone 10 yielded three mutants that exhibited a 3 to 4 times increase in avidity for GA1. A molecular model of clone 10 indicated that the putative antigen-binding site contained a shallow surface pocket. These data illustrate the use of recombinant DNA techniques to obtain anti-ganglioside antibodies, and to explore the molecular basis of their antigen-binding activity.

Amino Acid Sequence↗
PubMed2001

Optimisation and sensitivity of single-stranded conformation polymorphism for the detection of hepatitis C virus quasi-species.

Single-stranded conformation polymorphism (SSCP) is a technique used widely for the detection of differences in DNA sequence based on PCR technology. Developed by geneticists for the detection of mutations causing disease, it has been adopted more recently for the analysis of the quasi-species of viral genomes, such as hepatitis C virus (HCV). The rigorous standardisation and determination of the limit of detection of the technique has rarely been shown. Variants within the quasi-species of the hypervariable region of HCV were cloned into pUC119 and the resulting plasmids quantitated and used as templates to optimise SSCP. Variables studied included the number of variants detected, the sensitivity of detection of variants in the minority, the electrophoresis temperature, methods of generation of single-stranded DNA, effect of numbers of cycles of PCR and use of DNA polymerase with proof-reading ability. It was demonstrated that the optimised method could detect at least five variants within a quasi-species and that variants could be detected down to a level of 2% of the quasi-species. Electrophoresis at room temperature for 18 h was highly reproducible. Generation of single-stranded DNA using a single primer with Taq polymerase for 20 cycles gave an accurate reflection of the quasi-species make-up and use of Pfu polymerase reduced sensitivity of detection of minor bands. This SSCP method provides an accurate tool to evaluate HCV quasi-species.

Base Sequence↗
PubMed1997

Quasispecies analysis in hepatitis C virus infection by fluorescent single strand conformation polymorphism.

Hepatitis C virus (HCV) results frequently in chronic hepatitis and its sequelae liver cirrhosis and hepatocellular carcinoma. Interferon-alpha is at present the most effective treatment, resulting in a sustained response in about 20-25% of patients. HCV genotype, titer and quasispecies determine the success of treatment. In this study, fluorescent single strand conformation polymorphism (f-SSCP) was evaluated for the analysis of HCV quasispecies. Two sera from a chronically HCV-infected patient, obtained 6 years apart, were examined. The hypervariable region I (HVRI) of the HCV genome was amplified by reverse transcription and PCR. The PCR products were cloned and sequenced or fluorescein-labeled and subjected to f-SSCP. Both methods demonstrated a single HCV species in the early serum and multiple quasispecies in the late serum. Single clones of the heterogeneous virus population were used to optimize conditions for f-SSCP. The most important factors were the gel temperature and virus titer. At the optimal running temperature one base exchange in 218 bases was detectable. Repeat extractions and amplifications gave identical results. Dilution of the serum containing multiple quasispecies resulted in a 'loss' of species. Provided the running temperature is optimal and virus titer is sufficient, f-SSCP is shown to be fast and reliable for HCV quasispecies analysis.

Amino Acid Sequence↗
PubMedDate not supplied

Lack of association between polymorphisms of the coagulation factor VII and myocardial infarction in middle-aged Spanish men.

In order to determine the role of two polymorphisms in the factor VII gene (R353Q and intron 7 hypervariable region) in the susceptibility to develop early myocardial infarction, a total of 175 patients with acute myocardial infarction aged 50 years or less (mean age 41+/-7 years) and 200 controls (average age 42+/-6) without cardiovascular disease were genotyped for these polymorphisms. Gene and genotype frequencies did not differ between patients and controls. Although the 353-QQ genotype was at a higher frequency among controls (4%) compared to patients (1%), the difference did not reach statistical significance. Carriers of the H7-allele (intron 7 polymorphism) were at a slightly higher frequency among patients (51 vs. 43%; P=0.19; OR=1.36; 95% CI=1.09-1.70). Our data suggest a lack of association between both polymorphisms in the factor VII gene and early myocardial infarction in our population.

Adult↗
PubMed2002

Use of both 16S rRNA and engineered functional genes with real-time PCR to quantify an engineered, PCB-degrading Rhodococcus in soil.

A real-time PCR (RTm-PCR) assay using fluorescently labeled oligonucleotides (TaqMan probes) was used to detect and quantify the recombinant Rhodococcus sp. strain RHA1(fcb) in soil. One primer and probe set targeted a hypervariable region of the 16S rRNA gene unique to strain RHA1(fcb) and its phylogenetic relatives, and the other set targeted the recombinant 4-chlorobenzoate (4-CBA) degradation operon (fcb) and was strain-specific. The method had a 6-log dynamic range of detection (10(2)-10(7) cells ml(-1)) for both probes when DNA from pure cultures was used. Although the method was less sensitive in soil, the estimated number of cells in soil by real-time PCR corresponded to the measured number of RHA1(fcb) cells determined by colony-forming units.

Biodegradation, Environmental↗
PubMed2003

A comparison of DNA profiling techniques for monitoring nutrient impact on microbial community composition during bioremediation of petroleum-contaminated soils.

Amplicon length heterogeneity PCR (LH-PCR) and terminal restriction fragment length polymorphisms (TRFLP) were used to monitor the impact that nutrient amendments had on microbial community dynamics and structural diversity during bioremediation of petroleum-contaminated soils. Slurried soils contaminated with petroleum hydrocarbons were treated in airlift bench-scale bioreactors and were either amended with optimal inorganic nutrients or left unamended. Direct DNA extraction and PCR amplification of whole eubacterial community DNA were performed with universal primers that bracketed the first two or three hypervariable regions of the 16S rDNA gene sequences. The LH-PCR method profiled a more diverse microbial community than did the TRFLP method. The LH-PCR method also tracked differences between the communities due to nutrient amendments. An in silico database search for bacterial genera with amplicon lengths represented in the community fingerprints was performed. It was possible to qualitatively identify different groups in the microbial community based on the amplicon length variations. A similar "virtual" search was performed for the TRFLP fragments using the web-based TAP-TRFLP program. Cloning and sequencing of the PCR products confirmed the in silico database matches. The application of the LH-PCR method as a monitoring tool for bioremediation could greatly enhance and extend the current understanding of the microbial community dynamics during the biodegradation of environmental contaminants.

Bacteria↗
PubMed2000

Mutations occurring during serial passage of Japanese equine infectious anemia virus in primary horse macrophages.

An attenuated equine infectious anemia virus (EIAV), named V26, was previously obtained after 50 passages of the Japanese virulent strain V70 in primary macrophage culture. To clarify the differences between both viruses, their full-length sequences were determined. There were higher mutations in S2 (6.15% amino acid difference) and LTR (10.7% nucleotide difference). The presumed initiation codon of the S2 gene was absent from the sequence of V26. There was a large insertion within the long-terminal repeat (LTR) U3 hypervariable region of V26. In addition, there were minor mutations in gag (1.22% amino acid difference), pol (1.05% amino acid difference) and env (1. 65% amino acid difference) regions, but no mutation in tat region. No mutations were observed in the principal neutralizing domain in the gp90. Thus, the mutations in the S2 and LTR might be the major target sites of mutation in EIAV during serial passages in vitro.

Amino Acid Sequence↗
PubMed2001

Origin and evolution of Georgia 98 (GA98), a new serotype of avian infectious bronchitis virus.

We previously identified GA98, a new serotype of infectious bronchitis virus (IBV), which is closely related to the DE072 serotype of IBV genetically, but not antigenically. Herein, we analyzed the 421bp sequence of a hypervariable region (HVR) (position 114-534, counting from the ATG start site) of the S1 subunit of GA98 IBVs to further examine the evolution of these viruses. These viruses were isolated between the years 1997 and 2000. Phylogenetic analysis of the deduced amino acid sequence on that region indicated that GA98 isolates from different regions of Georgia were the result of a single introduction of the S1 gene of the DE072 serotype progenitor. Most of the mutations were nonsynonymous and had become fixed in a progressive manner. The evolutionary and mutation rates in the HVR was calculated as 2.5 and 1.5% per year, respectively. This new serotype of IBV appears to be evolving very fast compared with other serotypes of IBV. We further determined the complete coding sequence of the S1 gene of seven isolates obtained from one selected region in North Georgia. Together with virus neutralization data, it appears that GA98 arose from immune selection caused by DE072 vaccine use. Reasons for this conclusion are discussed.

Amino Acid Sequence↗
PubMed1998

Hepatitis E virus: complete genome sequence and phylogenetic analysis of a Nepali isolate.

The complete nucleotide sequence of the Nepali strain TK15/92 of hepatitis E (HEV) was determined. It showed the highest sequence homology with the Burmese B1 strain, but closer evolutionary relatedness to the Indian strains. Difficulties in reverse-transcribing and amplifying the hypervariable region in ORF1 suggested that strong secondary structures might be intrinsically responsible for the high mutational rate observed in this region of the HEV genome.

Base Sequence↗
PubMed1997

Evolution of hepatitis C virus quasispecies in mothers and infants infected through mother-to-infant transmission.

BACKGROUND/AIMS: Two mother-infant pairs (Pair H and P) were studied to determine the evolution of hepatitis C virus (HCV) quasispecies. METHODS: Eight clones of the hypervariable region of HCV cDNA from the infants' sera sampled at the age of 3 months, 1, 2, and 3 years and the time-corresponding maternal sample were also sequenced. The sequences were analyzed by the nucleotide diversity, substitution rate, and phylogenetic studies. RESULTS: HCV quasispecies of the infants were more homogeneous than those of their mothers, particularly at the age of 3 months (nucleotide diversity, pi = 0.18 x 10(-2)/site in infant H, and 0.22 x 10(-2)/site in infant P). The nucleotide substitution rate in infants also increased as they aged, from 1.2 x 10(-2) to 4.46 x 10(-2)/site/year in infant H, and from 0.21 x 10(-2) to 4.88 x 10(-2)/site/year in infant P respectively. The nucleotide sequence differences between infants and mothers increased from 2.63 x 10(-2) to 9.06 x 10(-2)/site in Pair H, and from 1.85 x 10(-2) to 5.33 x 10(-2)/site in Pair P within 3 years. Phylogenetic studies suggest the infants' initial quasispecies were closely related to their mothers', while they evolved differently. HCV RNA titer was stable during follow-up and the infants' titer was similar to their mothers'. The fluctuations in titer did not correlate with nucleotide diversity. CONCLUSIONS: HCV quasispecies evolved differently in each individual, even though they were genetically linked. The sequence in infants was not a complex as in their mothers.

Adult↗
PubMed1997

Effect of immunosuppression on composition of quasispecies population of hepatitis C virus in patients with chronic hepatitis C coinfected with human immunodeficiency virus.

BACKGROUND/AIMS: To study the effects of the immunosuppression caused by the reduction of CD4 activity on the composition of hepatitis C virus (HCV) populations, we analyzed the number of HCV quasispecies clones and the nucleotide diversity of the hypervariable region 1 (HVR1) of HCV in 37 patients with hemophilia with persistent HCV infection, with or without human immunodeficiency virus (HIV). METHODS: The numbers of HCV quasispecies clones were measured by fluorescence single-strand conformation polymorphism analysis. Direct sequencing was used to analyze the degree of diversity of HVR1. We compared these values according to coinfection with HIV, and CD4 counts of patients. RESULTS: There were no differences in either the number of HCV clones or the diversity between patients with and without HIV coinfection. In HIV coinfected patients the diversity decreased in association with the decrease in CD4 count while the number of HCV clones did not. The diversity of HVR1 was 3.64 +/- 5.03% in patients with a CD4 count < 50/microliters and 14.92 +/- 6.03% in patients with a CD4 count > or = 50/microliters; it was significantly lower in the former (p = 0.0002). CONCLUSIONS: A severe reduction in the CD4 count, which is considered to cause a decline in the activity of helper T-lymphocytes, induced changes in the composition of HCV populations; one or a few quasispecies clones are predominant in the HCV population in the serum of individual patients.

Adult↗
PubMed1997

Analysis of the cell tropism of HCV by using in vitro HCV-infected human lymphocytes and hepatocytes.

BACKGROUND/METHODS: We recently established two hepatitis C virus (HCV) replication systems, using MT-2, a human T-cell leukemia virus type I-infected human T-cell line, and PH5CH, a non-neoplastic human hepatocyte line immortalized with simian virus 40 large T antigen. These HCV replication systems were used to assess the infective potencies of seven sera containing more than 10(6) HCV genomes per ml obtained from HCV-positive blood donors. RESULTS: The results showed that these sera had different infectivities for MT-2 and PH5CH cells. One of the seven sera, 1B-1, was more infective for MT-2 cells than PH5CH cells, whereas all the sera except serum 1B-1 were more infective for PH5CH cells than for MT-2 cells. Intracellular HCV RNA could be detected at least 30 days after inoculation with three of the sera. These findings suggested that the infective potency of each serum depends on the type of target cells. To further investigate HCV replication in these cells, we examined the hypervariable region 1 (HVR1) populations of HCV recovered from both MT-2 and PH5CH cells at 8 days postinoculation. The results revealed that the shift to limited HVR1 populations from the quasi-species of HVR1 populations in both cells usually occurred within 8 days after virus inoculation. Furthermore, in two of four sera, the predominant HVR1 populations in MT-2 and PH5CH cells appeared to be different. CONCLUSION: These results suggest that HCV exhibits cell tropism.

Amino Acid Sequence↗