Tag: pregnancy

After our study of the tolerogeneic role of HLA-G in maternal-fetal tolerance, we designed experiments to identify the structural and functional characteristics of proteins generated by the HLA-G*0105N allele. First, we used site-directed mutagenesis to construct the HLA-G*0105Nfrom the nucleotide sequence of HLA-G*010102 and transfected it into the M8 human cell line. Then we characterized the transcripts and proteins generated by the HLA-G*0105Ngene before analyzing the functional capacity of the isoforms produced.

Although the deletion of one base in exon 3 of HLA-G*0105N disrupts the reading frame, it should have no effect on transcription of the primary transcript or on its alternative splicing. Indeed, in our RT-PCR analysis, the HLA-G mRNA profile was the same in both M8-HLA-G*0105N- and M8-HLA-G*010102-transfected cells, indicating the presence of all HLA-G mRNA transcripts normally found. Thus, deletion of the cytosine in codon 130 did not block the transcription and splicing mechanisms in the null allele. Nevertheless, real-time and classical RT-PCR showed that HLA-G mRNA levels were lower in M8-HLA-G*0105Nthan in M8-HLA-G*010102cells, and classical PCR revealed that the HLA-G1/HLA-G2 mRNA ratio was lower in M8-HLA-G*0105N than in M8-HLA-G*010102. This discrepancy in the expression level of HLA-G genotypes and isoforms has already been reported for alleles other than the null allele in pathological pregnancies in which altered HLA-G transcription was associated with certain HLA-G genotypes as well as in a study of the role of HLA-G alleles in determining the soluble HLA-G protein plasma level. Another explanation is that nonsense-mediated decay, a eukaryotic regulatory process that degrades mRNA with premature termination codons, could be responsible for the higher degradation of HLA-G1 transcripts in cells expressing the null allele compared with other alleles.

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Construction of HLA-G*0105N Null Allele Genomic DNA

To study proteins encoded by the HLA-G null allele, we generated a DNA sequence identical to HLA-G*0105N in the pcDNA 3.1 expression vector. In vitro site-directed mutagenesis of HLA-G*010102 genomic DNA allowed us to delete the cytosine at position 815 from ATG (exon 1), yielding the genomic dNa of the HLA-G*0105Nallele. We used the HLA-G*010102 rather than the HLA-G*010101 reference sequence since HLA-G*010102 and -G*0105N have identical DNA sequences with the exception of the cytosine deletion at codon 130. The Xbal-EcoRV HLA-G*010102 fragment linked into the pcDNA3.1 vector used as the template is represented in Figure 1, and the electropherogram of DNA sequences before and after directed mutagenesis demonstrating deletion of the cytosine in the newly synthesized DNA is shown Figure 2, the remaining sequence being identical. The genomic HLA-G*0105NDNa presented a deletion at codon 130 (CTG ® TGC), which generated a stop codon 171 nucleotides further on at codon 189 (GTG ® TGA) in exon 4 or 495 nucleotides further on at codon 297 (GTA ® TAG) in exon 5. At this step, we had obtained a copy of the HLA-G*0105Nallele of 3932 nucleotides, beginning 460 bp upstream from exon 1 and ending 541 bp downstream from exon 8.

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in Vitro Site-Directed Mutagenesis

In vitro site-directed mutagenesis was carried out using the Stratagene QuickChange XL site-directed mutagenesis kit, which allows site-specific mutation in double-stranded plasmids. The plasmid DNA template (50 ng) HLA-G*010102-pcDNA3.1, isolated from the dam+ JM109 Escherichia coli strain, was replicated by PfuTurbo DNA polymerase (2.5 U) using two synthetic primers, G.Null S and G.Null AS (125 ng each), consisting of 15 bases on both sides of the AC mutation. The oligonucleotide primers, each complementary to opposite strands of the HLA-G*010102 insert, were extended during temperature cycling (95°C for 1 min; 18 cycles at 95°C for 50 sec, 60°C for 50 sec, 68°C for 15 min) and terminated by 7 min at 68°C. Following temperature cycling, amplification reactions were cooled and digested for 1 h at 37°C using Dpn I restriction enzyme to eliminate the nonmutated parental supercoiled double-stranded DNA. At this step, genomic HLA-G*010102-pcDNA3.1 was replaced by genomic HLA-G*0105N-pcDNA3.1. The mutated DNA was then transformed in XL10-Gold ultracompetent cells.

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Protection of the semiallogenic fetus against maternal immune recognition and attack has been attributed principally to the high and almost unique expression of human leukocyte antigen-G (HLA-G) on trophoblast cells at the fetal-maternal interface. The nonclassical HLA-G gene is characterized by alternative splicing that yields seven proteins, four membrane bound (HLA-G1 through -G4) and three soluble (HLA-G5 through -G7). Full-length HLA-G mRNA encodes the HLA-G1 protein, which has three extracellular globular domains, one membrane-anchored domain, and one intracytoplasmic domain. Exons 3 and/or 4 may be deleted from the primary transcript, yielding the alternative mRNA HLA-G2, HLA-G3, and HLA-G4 forms. In addition, the insertion of introns 2 or 4 may generate soluble isoforms, such as HLA-G5 (the soluble full-length HLA-G1 counterpart), HLA-G6 (the soluble HLA-G2 counterpart), and HLA-G7 (the soluble HLA-G3 counterpart) (reviewed in ). The structures of both membrane-bound HLA-G1 and soluble HLA-G5 proteins are similar to those of classical class I proteins, consisting of three extracellular domains linked to (32-microglobulin (p2m).

Functional assays have demonstrated that both membrane-bound and soluble HLA-G proteins are able to inhibit NK cell and antigen-specific T-cell cytotoxicity and the proliferation of allogeneic T cells. Moreover, soluble HLA-G is able to induce apoptosis of both activated CD8+ T and NK cells.

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DISCUSSION(6)

The authors recognize that proteins other than UCRP could potentially immunoreact with the anti-UCRP antibody. However, a recent search of the NCBI-BLAST database revealed no known proteins with significant amino acid sequence identity to the UCRP peptide immunogen other than ubiquitin and huISG15 (see Fig. 1). Although unlikely, the existence of proteins that share antigenic epitopes but are not conjugated to UCRP would be a novel finding because these proteins would be induced by pregnancy and IFN-т. buy asthma inhaler

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Release of UCRP into the medium in response to rbo-IFN-t occurred after accumulation of cytosolic UCRP and its conjugates. The extended time (24 h) that was required for release of UCRP might be related to a secondary secreted role. For example, the primary role for UCRP might be regulating cytosolic proteins. After UCRP and its conjugates have accumulated, secretion of UCRP may occur to fulfill a hormonal role. Evidence to support a secreted role for UCRP is provided by presence of UCRP in uterine flushings from Day 18 pregnant cows and the activation of monocytes and macrophages by rhuISG15. buy ortho tri-cyclen online

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DISCUSSION(4)

In the present Western blot experiments, second antibodies used for detection cross-reacted with uterine proteins in the absence of primary anti-ubiquitin or anti-UCRP antibody (Fig. 4). This nonspecific staining occurred mostly with four bands of protein ^ 30 kDa, but it did not interfere with detection of ubiquitin or UCRP.

Anti-UCRP antibody immunoreacts with UCRP, does not cross-react with ubiquitin, and detects an array of endometrial proteins > 30 kDa (conjugates) on Western blots. Coincubation of antibody against UCRP with the antigenic UCRP peptide blocks detection of UCRP and its conjugates. Preadsorption of UCRP antibody with ubiquitin has no effect on detection of UCRP or UCRP conjugates. It is concluded from these findings that detection of UCRP and its conjugates by the anti-UCRP antibody is immunospe-cific. flovent inhaler

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