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Yaofeng Zhao

Publications and source records attributed to Yaofeng Zhao.

16 recordsLinked to original sources
PubMed2006

Lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced diarrhea.

BACKGROUND: Rotavirus-induced diarrhea poses a worldwide medical problem in causing substantial morbidity and mortality among children in developing countries. We therefore developed a system for passive immunotherapy in which recombinant lactobacilli constitutively express neutralizing variable domain of llama heavy-chain (VHH) antibody fragments against rotavirus. METHODS: VHH were expressed in Lactobacillus paracasei, in both secreted and cell surface-anchored forms. Electron microscopy was used to investigate the binding efficacy of VHH-expressing lactobacilli. To investigate the in vivo function of VHH-expressing lactobacilli, a mouse pup model of rotavirus infection was used. RESULTS: Efficient binding of the VHH antibody fragments to rotavirus was shown by enzyme-linked immunosorbent assay and scanning electron microscopy. VHH fragments expressed by lactobacilli conferred a significant reduction in infection in cell cultures. When administered orally, lactobacilli-producing surface-expressed VHH markedly shortened disease duration, severity, and viral load in a mouse model of rotavirus-induced diarrhea when administered both fresh and in a freeze-dried form. CONCLUSIONS: Transformed lactobacilli may form the basis of a novel form of prophylactic treatment against rotavirus infections and other diarrheal diseases.

Animals↗
PubMed2006

Identification of IgF, a hinge-region-containing Ig class, and IgD in Xenopus tropicalis.

Only three Ig isotypes, IgM, IgX, and IgY, were previously known in amphibians. Here, we describe a heavy-chain isotype in Xenopus tropicalis, IgF (encoded by C(phi)), with only two constant region domains. IgF is similar to amphibian IgY in sequence, but the gene contains a hinge exon, making it the earliest example, in evolution, of an Ig isotype with a separately encoded genetic hinge. We also characterized a gene for the heavy chain of IgD, located immediately 3' of C(mu), that shares features with the C(delta) gene in fish and mammals. The latter gene contains eight constant-region-encoding exons and, unlike the chimeric splicing of muC(H)1 onto the IgD heavy chain in teleost fish, it is expressed as a unique IgD heavy chain. The IgH locus of X. tropicalis shows a 5' V(H)-D(H)-J(H)-C(mu)-C(delta)-C(chi)-C(upsilon)-C(phi) 3' organization, suggesting that the mammalian and amphibian Ig heavy-chain loci share a common ancestor.

Animals↗
PubMed2006

Cloning and characterization of the dromedary (Camelus dromedarius) neonatal Fc receptor (drFcRn).

The full length cDNA of the dromedary neonatal Fc receptor (drFcRn) alpha chain was isolated and found that it is similar to the neonatal Fc receptor (FcRn) of other species with a high overall similarity to ruminant FcRn alpha chains. The drFcRn/Fc contact residues are highly conserved and predicted to bind both conventional (IgG1) and heavy chain (IgG2a, IgG3) antibodies. Using immunohistochemistry, we detected its expression in the hepatocytes and in epithelial cells of portal bile ductuli and also in the mammary gland acini and ducti. Remarkably, Ser313, that was identified to be crucial for apical to basolateral transcytosis, is substituted in the drFcRn alpha chain. The full length of the dog and orangutan FcRn alpha chains was also identified from databases. Analyzing the phylogenetic relatedness of this gene we found that dromedary clustered together with artiodactyls, dog is located between artiodactyls and primates, where the orangutan was branched, reflecting the accepted evolutionary relationships.

Amino Acid Sequence↗
PubMed2006

Disparate roles of ATR and ATM in immunoglobulin class switch recombination and somatic hypermutation.

Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes initiated by activation-induced cytidine deaminase. Here, we have studied the role of ataxia telangiectasia and Rad3-related protein (ATR) in CSR by analyzing the recombinational junctions, resulting from in vivo switching, in cells from patients with mutations in the ATR gene. The proportion of cells that have switched to immunoglobulin (Ig)A and IgG in the peripheral blood seems to be normal in ATR-deficient (ATRD) patients and the recombined S regions show a normal "blunt end-joining," but impaired end joining with partially complementary (1-3 bp) DNA ends. There was also an increased usage of microhomology at the mu-alpha switch junctions, but only up to 9 bp, suggesting that the end-joining pathway requiring longer microhomologies (> or =10 bp) may be ATR dependent. The SHM pattern in the Ig variable heavy chain genes is altered, with fewer mutations occurring at A and more mutations at T residues and thus a loss of strand bias in targeting A/T pairs within certain hotspots. These data suggest that the role of ATR is partially overlapping with that of ataxia telangiectasia-mutated protein, but that the former is also endowed with unique functional properties in the repair processes during CSR and SHM.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2006

The bovine antibody repertoire.

Cattle are able to produce a full range of Ig classes including the long-elusive IgD through rearrangement of their germline genes. Several IgL groupings have been reported but as in several other livestock species (e.g. sheep, rabbits, chickens), rearrangement per se fails to generate significant IgH diversity. This is largely because of the modest number of bovine VH segments that participate in rearrangement and their conserved sequences. Perhaps in compensation, bovine Ig heavy chains carry CDR3 sequences of exceptional length. Processes that operate post-rearrangement to generate diversity remain ill defined as are the location, timing and triggers to these events. Reagents are needed to understand better the maturation of B lymphocytes, their responses to antigens and cytokines, and to provide standards for the quantitation of Ig responses in cattle; recombinant methods may help meet this need as Ab engineering technologies become more widely used.

Amino Acid Sequence↗
PubMed2005

Isolation of the gene encoding the bovine neonatal Fc receptor.

The role of an IgG-Fc receptor (FcRn) that resembles a class I MHC Ag in transporting IgGs through epithelial cells was recently shown in selected species. Here we report our preliminary characterization of a clone encoding the alpha chain of the bovine FcRn from a BAC library. The recombinant BAC DNA was digested, analyzed by Southern blot hybridization and a bovine FcRn positive 9 kb long fragment was subcloned and partially sequenced. The exon/intron organization of the bovine FcRn alpha chain gene was deduced by comparison with its cDNA sequence. The sequence revealed a similar organization to the human and mouse FcRn alpha chain genes. The bovine FcRn alpha chain gene has acquired several repetitive sequences in its 5'-flanking region, including multiple SINE and LINE elements. Potential binding sites for transcription factors within the 5'-flanking sequence were identified using TESS and TFSEARCH programs. The 5'-flanking region of the bFcRn alpha chain gene was analyzed for its ability to directly express the luciferase reporter gene in bovine mammary gland epithelial cells. Transient transfection of a luciferase construct revealed that there was promoter activity in the region -1787 to +92 of the 5'-flanking sequence.

Animals↗
PubMed2005

AID from bony fish catalyzes class switch recombination.

Class switch recombination was the last of the lymphocyte-specific DNA modification reactions to appear in the evolution of the adaptive immune system. It is absent in cartilaginous and bony fish, and it is common to all tetrapods. Class switching is initiated by activation-induced cytidine deaminase (AID), an enzyme expressed in cartilaginous and bony fish that is also required for somatic hypermutation. Fish AID differs from orthologs found in tetrapods in several respects, including its catalytic domain and carboxy-terminal region, both of which are essential for the switching reaction. To determine whether evolution of class switch recombination required alterations in AID, we assayed AID from Japanese puffer and zebra fish for class-switching activity in mouse B cells. We find that fish AID catalyzes class switch recombination in mammalian B cells. Thus, AID had the potential to catalyze this reaction before the teleost and tetrapod lineages diverged, suggesting that the later appearance of a class-switching reaction was dependent on the evolution of switch regions and multiple constant regions in the IgH locus.

Animals↗
PubMed2005

Identification of the activation-induced cytidine deaminase gene from zebrafish: an evolutionary analysis.

In the present study, we report the identification of the activation-induced cytidine deaminase (AID) encoding gene in frog, dog and chimpanzee, where both somatic hypermutation and class switch recombination (CSR) occurs and in zebrafish and fugu, species lacking CSR. The cDNA sequence of the zebrafish AID reported here suggests both N and C ends of the previously predicted protein sequence are incorrect. A comparison of AID sequences among mammals, birds, amphibians and fish revealed conserved aa residues which may be essential for AID activity, although the cytidine deaminase active motif in the latter is nine amino acids longer. Furthermore, an aa deletion, and extensive substitutions in the C terminal end of AID from bony fish indicate that the molecule may not yet have developed a capacity to recruit the specific cofactor(s) needed to initiate CSR.

Amino Acid Sequence↗
PubMed2004

Selective IgG2 deficiency due to a point mutation causing abnormal splicing of the Cgamma2 gene.

The mechanism underlying selective IgG subclass deficiency is largely unknown in humans. We have previously reported the acquisition of a complete IgG2 deficiency in a leukemia patient after bone marrow transplantation. Southern blot analysis showed a deletion including the Cgamma2 and Cgamma4 genes on one chromosome in the donor, suggesting that the remaining Cgamma2 gene allele was silent. In the patient and his two IgG2 deficient brothers, the silent Cgamma2 gene showed both germ-line transcription and switch recombination and no structural defects were found in the intronic promoter or the switch region of the gene. However, an A-->G transition in the fourth nucleotide in the 5' portion of intron 1 was identified. Transfection of artificial constructs into the human B cell lines demonstrated that this A-->G transition inactivated the normal splice site, and instead, a cryptic splice site in the CH1 exon was used in RNA post-transcriptional processing, leading to a 16 bp deletion of the gamma2 CH1 exon. This aberrantly spliced RNA that is mostly derived from germ-line transcription in vivo was also detected in both homozygous and heterozygous individuals carrying this mutation. These findings suggest a novel genetic mechanism as the cause of IgG subclass deficiency in selected patients.

Adult↗
PubMed2003

Presence of the di-leucine motif in the cytoplasmic tail of the pig FcRn alpha chain.

The sequence of the pig FcRn alpha chain was recently published. The lack of a conserved di-leucine motif in the cytoplasmic tail suggests a rare polymorphism in the described animal, alternatively, a sequencing error. We therefore cloned and sequenced the pig FcRn alpha chain. Our sequence, along with a previous NCBI GenBank submission and five pig derived EST clones clearly demonstrate the presence of di-leucine motif in the cytoplasmic tail of the pig FcRn. No polymorphism in the cytoplasmic tail-encoding region was found in 25 animals from six pig breeds based on single-stranded conformation polymorphism and sequencing analysis, suggesting that the previously described pig FcRn alpha chain may represent a sequencing error in the 3' portion of the gene.

Amino Acid Motifs↗
PubMed2003

The porcine Ig delta gene: unique chimeric splicing of the first constant region domain in its heavy chain transcripts.

The pig delta gene is located approximately 3.4 kb downstream of the second transmembrane exon of the micro gene and shows a similar genomic structure to its counterpart in cow with three exons encoding the CH1, CH2, and CH3 domains. The porcine genomic deltaCH1 exon has been replaced by a recent duplication of the micro CH1 and its flanking sequences, a genetic event that also led to the formation of a short switch delta region, immediately upstream of the delta gene. The deltaCH1 exhibits a 98.7% similarity (314 of 318 bp) to the micro CH1 at the DNA level, whereas the homologies between the deltaCH2 and micro CH3, and the deltaCH3 and micro CH4 are only 33.3 and 35.8%, respectively. Either of the two CH1 exons ( micro and delta) could be observed in the expressed porcine IgD H chain cDNA sequences VDJ- micro CH1-H-deltaCH2-deltaCH3 or VDJ-deltaCH1-H-deltaCH2-deltaCH3, showing a pattern that has not been observed previously in vertebrates. In addition, transfection of a human B cell line, using artificial constructs resembling the porcine C micro -Cdelta locus, also generated both VDJ- micro CH1-deltaCH1-H1-deltaCH2 and VDJ -deltaCH1-H1-deltaCH2 transcripts. An examination of the pig delta genomic sequence shows a putative, second hinge region-encoding exon. Due to the lack of a normal branchpoint sequence for RNA splicing, this exon is not present in the normal pig delta cDNA. However, the exon could be spliced into most of the expressed transcripts in vitro in cell transfection experiments after introduction of a single T nucleotide to restore the branchpoint sequence upstream of the putative H2 exon.

Amino Acid Sequence↗
PubMed2003

Physical mapping of the bovine immunoglobulin heavy chain constant region gene locus.

Bovine antibodies have recently attracted increasing attention, as they have been shown to exhibit prophylactic and therapeutic properties in selected infectious diseases in humans. In the present study, we have isolated bacterial artificial chromosomes and cosmid clones containing the bovine JH, mu, delta, gamma 1, gamma 2, gamma 3, epsilon, and alpha genes, which allowed us to make a contig of the genes within the bovine IGHC locus. The genes are arranged in a 5'-JH-7 kb-mu-5 kb-delta-33 kb-gamma 3-20 kb-gamma 1-34 kb-gamma 2-20 kb-epsilon- 13 kb-alpha-3' order, spanning approximately 150 kb DNA. Examination of the bovine germline JH locus revealed six JH segments, two of which, JH1 and JH2, were shown to be functional although there was a strong preference for expression of the former. Sequence alignment of the bovine 5' E mu enhancer core region with those of other mammals, demonstrated an absence of the mu E3 motif and a shortened spacer between the mu A and mu B sites within the bovine E mu enhancer core region. Furthermore, the essential sequence element for class switching, switch mu, spanning approximately 3-kb repetitive sequence and abundant in the switch region motifs CTGGG (187 repeats) and CTGAG (127 repeats), was identified immediately upstream of the mu gene. A further sequence comparison revealed that the bovine IGHC genes display an extensive polymorphism leading to expression of multiple antibody allotypes.

Amino Acid Sequence↗
PubMed2003

ATM is not required in somatic hypermutation of VH, but is involved in the introduction of mutations in the switch mu region.

Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes that share common key factors such as activation-induced cytidine deaminase. We have previously shown a role for ATM (mutated in ataxia-telangiectasia) in CSR. In this paper we show that the frequency, distribution, and nature of base pair substitutions in the Ig variable (V) heavy chain genes in ataxia-telangiectasia patients are largely similar to those in normal donors, suggesting a normal SHM process. Characterization of the third complementarity-determining region in B cells from ataxia-telangiectasia patients also shows a normal V(D)J recombination process. SHM-like mutations could be identified in the switch (S) mu region (up to several hundred base pairs upstream of the S mu -S(alpha) breakpoints) in normal in vivo switched human B cells. In the absence of ATM, mutations can still be found in this region, but at less than half the frequency of that in normal donors. The latter mutations are mainly due to transitions (86% compared with 58% in controls) and are biased to A or T nucleotides. An ATM-dependent mechanism, different from that generating SHM in V genes, is therefore likely to be involved in introducing SHM-like mutations in the S region. ATM may thus be one of the factors that is not shared by the CSR and SHM processes.

Adolescent↗
PubMed2003

Cloning of the complete rat immunoglobulin delta gene: evolutionary implications.

The recent discovery of a Cdelta encoding gene in artiodactyls has raised questions regarding the evolution of the gene. In the present study, we have analysed the complete rat Cdelta gene both at the cDNA and genomic levels, showing that the rat Cdelta gene is structurally similar to the corresponding mouse gene. Analysis of the rat immunoglobulin D heavy chain cDNA tail sequences, revealed two transcripts for the secreted form with varying sizes of their 3' untranslated region (UTR), resulting from usage of two different poly(A) addition signals. Furthermore, a membrane-bound form encoding transcript, possessing a long 3' UTR, was also observed. Phylogenetic analysis supports that the Cdelta gene appeared early in the evolution of vertebrates, and it was probably duplicated from the C micro gene more than 400 million years ago.

3' Untranslated Regions↗
PubMed2002

Artiodactyl IgD: the missing link.

IgD has been suggested to be a recently developed Ig class, only present in rodents and primates. However, in this paper the cow, sheep, and pig Ig delta genes have been identified and shown to be transcriptionally active. The deduced amino acid sequences from their cDNAs show that artiodactyl IgD H chains are structurally similar to human IgD, where the cow, sheep, and pig IgD H chain constant regions all contain three domains and a hinge region, sharing homologies of 43.6, 44, and 46.8% with their human counterpart, respectively. According to a phylogenetic analysis, the Cdelta gene appears to have been duplicated from the Cmu gene >300 million yr ago. The ruminant mu CH1 exon and its upstream region was again duplicated before the speciation of the cow and sheep, approximately 20 million yr ago, inserted upstream of the delta gene hinge regions, and later modified by gene conversion. A short Sdelta (switch delta) sequence resulting from the second duplication, is located immediately upstream of the bovine Cdelta gene and directs regular mu-delta class switch recombination in the cow. The presence of Cdelta genes in artiodactyls, possibly in most mammals, suggests that IgD may have some as yet unknown biological properties, distinct from those of IgM, conferring a survival advantage.

Amino Acid Sequence↗