Identification and phylogeny of novel human endogenous retroviral sequences belonging to the HERV-W family on the human X chromosome

Summary.  A new family (HERV-W) ofhuman endogenous retroviruses (HERVs) has recently been described that is related to MSRV (multiple sclerosis related retrovirus) sequences that have been identified in particles recovered from monocyte cultures from patients with multiple sclerosis. We investigated the pol fragment of HERV-W on the human X chromosome by the polymerase chain reaction (PCR) using a monochromosomal somatic cell hybrid DNA panel and compared these with related sequences in the genome database. We identified three novel sequences on the human X chromosome, HWXI, HWX3, and HWX5, which have a high degree of homology (88–98%) with the MSRV pol fragment and with seven other sequences. HWX5, like MSRV, has an uninterrupted open reading frame. Phylogenetic analysis showed HWX5 to be related to the HWX1, HWX3, and BAC clone B353C18 sequences with 91–93% homology. The ratio of synonymous to nonsynonymous substitutions indicated that negative selective pressure is acting on HWX5. Further studies on the structure and expression of this sequence are indicated. Received February 8, 1999/Accepted July 13, 1999     

Increased DNA damage sensitivity of Cornelia de Lange syndrome cells: evidence for impaired recombinational repair

Cornelia de Lange syndrome (CdLS) is a rare dominantly inherited multisystem disorder affecting both physical and mental development. Heterozygous mutations in the NIPBL gene were found in about half of CdLS cases. Scc2, the fungal ortholog of the NIPBL gene product, is essential for establishing sister chromatid cohesion. In yeast, the absence of cohesion leads to chromosome mis-segregation and defective repair of DNA double-strand breaks. To evaluate possible DNA repair defects in CdLS cells, we characterized the cellular responses to DNA-damaging agents. We show that cells derived from CdLS patients, both with and without detectable NIPBL mutations, have an increased sensitivity for mitomycin C (MMC). Exposure of CdLS fibroblast and B-lymphoblastoid cells to MMC leads to enhanced cell killing and reduced proliferation and, in the case of primary fibroblasts, an increased number of chromosomal aberrations. After X-ray exposure increased numbers of chromosomal aberrations were also detected, but only in cells irradiated in the G(2)-phase of the cell cycle when repair of double-strand breaks is dependent on the establishment of sister chromatid cohesion. Repair at the G(1) stage is not affected in CdLS cells. Our studies indicate that CdLS cells have a reduced capacity to tolerate DNA damage, presumably as a result of reduced DNA repair through homologous recombination.     

Baton pass hypothesis: successive incorporation of unconserved endogenous retroviral genes for placentation during mammalian evolution

It is well accepted that numerous RNAs derived from endogenous retroviruses (ERVs) are expressed in mammalian reproductive structures, particularly in the uterus, trophoblast, and placenta. Syncytin 1 and syncytin 2 in humans and syncytin A and syncytin B in mice are membrane proteins originating from Env genes of ERVs. These ERVs are involved in the fusion of trophoblast cells, resulting in multinucleated syncytiotrophoblast formation. Evidence accumulated indicates that syncytin‐like fusogenic proteins are expressed in the placenta of rabbits, dogs/cats, ruminant ungulates, tenrecs, and opossums. The syncytin genes so far characterized are known to be endogenized to the host genome only within the past 12–80 million years, more recently than the appearance of mammalian placentas, estimated to be 160–180 million years ago. We speculate that ERVs including syncytin‐like gene variants integrated into mammalian genomes in a locus‐specific manner have replaced the genes previously responsible for cell fusion. We therefore propose the ‘baton pass’ hypothesis, in which multiple successive ERV variants ‘take over’ cell‐fusion roles, resulting in increased trophoblast cell fusion, morphological variations in placental structures, and enhanced reproductive success in placental mammals.     

Embryo donation for medical research: attitudes and concerns of potential donors

BACKGROUND: The recent derivation of embryonic stem cell lines from human blastocysts and related implications for regenerative medicine has intensified a longstanding debate about the use of human embryos for research purposes. However, studies have shown that few couples with stored embryos opt to donate them for research. Herein, the attitudes and concerns of potential embryo donors to donation of surplus embryos for medical research were examined. METHODS: From a total of 509 couples who had stored frozen embryos and who had received a questionnaire about embryo donations for medical research, 152 women (30%) and 123 male partners (24%) responded. Embryos had been stored for a mean of 2.25 years (range 3 months to 12 years). RESULTS: Some 10% of respondents indicated it probable, and 34% possible, that they would donate their surplus embryos for research in the future. Women respondents whose embryos had been stored longer, and those committed to the practice of a religion, were more worried about their embryos. Respondents positively disposed to donation commented on their desire not to waste embryos, a desire to help infertile couples, and/or to advance scientific knowledge. Those with negative views commented on the embryo as a potential child and expressed concerns about a perceived lack of control over the type of research to be carried out. CONCLUSIONS: Findings indicate a need for tailored education and counselling about embryo donation for medical research.     

Identification of altered dipeptidyl-peptidase activities as potential biomarkers for unipolar depression

Background

Changes in circulatory aminopeptidases [dipeptidyl-peptidase-IV (DPP-IV), Prolyl-oligopeptidase (POP) and Leucine aminopeptidase (LAP)] activities have been found to be associated with psychiatric illnesses and inflammatory diseases.

Methods

The discriminatory indices of aminopeptidases activities were assessed by enzymatic assays in plasma samples from 240 unipolar depression (UD) patients and 264 matched controls. In addition the relationship between soluble and cellular DPP-IV activity was determined in plasma and blood cells from healthy subjects.

Results

Greater than 95% of the plasma DPP-IV activity could be blocked by inhibitors, demonstrating the specificity of the assay. Also, DPP-IV protein and activity levels were strongly correlated. In contrast, only 50% of the membrane-bound activity in blood cells was inhibited, which suggested that other similar peptidases may be present in these cells. UD patients had decreased plasma levels of DPP-IV and POP activities compared to healthy controls with a concomitant increase in LAP activity. Finally, testing of the LAP/DPP-IV ratio resulted in good discrimination of UD patients from controls with an area under the curve—receiver operating characteristic of 0.70.

Limitations

Further biological validation studies using different cohorts are warranted.

Conclusions

The finding that plasma DPP-IV activity was decreased and LAP activity was increased in UD patients suggests the potential value for testing the levels of these enzymes for improved classification of patients. In addition, the changes in these enzymes, suggests that the proteolytic maturation of their proneuropeptide and prohormone subtrates may also be affected in UD, resulting in altered production of the associated bioactive peptides.     

Identification of a group of Mus dunni endogenous virus-like endogenous retroviruses from the C57BL/6J mouse genome: proviral genomes, strain distribution, expression characteristics, and genomic integration profile

About 10 % of the mouse genome is occupied by sequences associated with endogenous retroviruses (ERVs). However, a comprehensive profile of the mouse ERVs and related elements has not been established yet. In this study, we identified a group of ERVs from the mouse genome and characterized their biological properties. Using a custom ERV mining protocol, 191 ERVs (159 loci reported previously and 32 new loci), tentatively named Mus dunni endogenous virus (MDEV)-like ERVs (MDL-ERVs), were mapped on the C57BL/6J mouse genome. Seven of them retained putative full coding potentials for three retroviral polypeptides (gag, pol, and env). Among the 57 mouse strains examined, all but the Mus pahari/Ei strain had PCR amplicons corresponding to a conserved MDL-ERV region. Interestingly, the Mus caroli/EiJ??s amplicon was somewhat larger than the others, coinciding with a substantial phylogenetic distance between the MDL-ERV populations of M. caroli/EiJ and C57BL/6J strains. MDL-ERVs were highly expressed in the lung, spleen, and thymus of C57BL/6J mice compared to the brain, heart, kidney, and liver. Seven MDL-ERVs were mapped in the introns of six annotated genes. Of interest, some MDL-ERVs were mapped periodically on three clusters in chromosome X. The finding that these MDL-ERVs were one of several types of retroelements, which form mosaic-repeat units of tandem arrays, suggests that the formation of the mosaic-repeat unit preceded the tandem arrangement event. Further studies are warranted to understand the biological roles of MDL-ERVs in both normal and pathologic conditions.     

Identification of Pru du 6 as a potential marker allergen for almond allergy

Background

Oral food challenges have demonstrated that diagnosis of almond allergy based on extract-sIgE tests displays low specificity. Molecular allergy diagnosis is expected to improve accuracy, but its value in diagnosing almond allergy remains unknown. The aim of this study was to identify relevant almond allergens and examine their ability to improve almond allergy diagnosis.

Methods

IgE-reactive proteins were purified from almond kernels. IgE binding to almond extract and the allergens was analyzed by quantitative ELISA using sera from 18 subjects with a proven almond allergy. The control group consisted of sera from 18 subjects allergic to peanut and/or tree nuts but tolerant to almond.

Results

Three IgE-binding proteins were identified: legumin (Pru du 6), alpha-hairpinin (Pru du 8), and mandelonitrile lyase (Pru du 10). Positive IgE (≥0.35 kU/L) to almond extract showed 94% sensitivity but only 33% specificity. IgE to Pru du 6 maintained high sensitivity (83%) and provided superior specificity (78%). Sera from almond-allergic subjects had significantly higher IgE levels to almond extract (P < .0001) and Pru du 6 (P < .0001) than sera from tolerant donors. Sensitization to Pru du 6 was highly specific for almond allergy, while frequencies of sensitization to legumins from peanut, walnut, hazelnut, and cashew were similar in both groups. IgE to Pru du 8 and Pru du 10 was less sensitive (41% and 67%), but showed specificities of 100% and 61%.

Conclusion

The use of almond allergens markedly increases the diagnostic specificity compared to the extract. Pru du 6 is a potential new molecular marker for almond allergy.

     

Identification of circulating miR-663a as a potential biomarker for diagnosing osteosarcoma

PurposeOsteosarcoma (OS) is the most common malignant bone tumor in children and young adults. The overall survival rate of OS patients has not been improved during the last 25 years, in part due to the lack of sensitive and specific biomarkers. This study aimed to investigate miRNAs existing in the peripheral blood as diagnostic biomarkers of OS.Patients and methodsOS patients and healthy controls (HCs) in this study were enrolled from Nanjing First Hospital. Candidate miRNA was selected by comprehensive analysis of GEO database. The expressions of candidate miRNA in tissues and plasma samples were subsequently examined through qRT-PCR. The diagnostic utility of candidate miRNA was examined by using receiver operating characteristic (ROC) curve analysis. Results: After analysis of GEO database and clinical plasma samples, miR-663a was selected as a candidate miRNA to further investigate its value for diagnosing OS due to the highest differential fold change. We discovered that miR-663a expressions were remarkably elevated in both tissues and plasmas of OS patients. In addition, upregulated miR-663a in peripheral blood of OS patients was proved to be tumor-derived. The area under the ROC curve (AUC) was 0.86 (95%CI: 0.78 to 0.93) for miR-663a with 67.35% sensitivity and 89.8% specificity.ConclusionPlasma miR-663a was identified as a novel potential biomarker for the diagnosis of OS.     

Initial repertoire of anti-(4-hydroxy-3-nitrophenylacetyl) antibodies as potential donors for effective affinity maturation

We previously found that there are two distinct antibody (Ab) maturation pathways for the immune response of C57BL/6 mice to 4-hydroxy-3-nitrophenylacetyl (NP), one involving Abs with high evolvability (group-H) and the other involving Abs with low evolvability (group-L). Commitment to whichever pathway is followed pre-determined in B cells at an early developmental stage. Candidates for the group-L or -H pathway are thus expected to pre-exist in the initial repertoire of the immune response. In the present study, we examined the initial Ab repertoire from the viewpoint of the latent potential of these Abs for effective affinity maturation. At first, we prepared anti-NP B cell hybridomas at 1 week postimmunization. Although the diversity of the obtained repertoire was maintained mainly by the third complementarity determining region of the heavy chain (CDR-H3), their changes in the near UV circular dichroism resulting from NP-binding allowed for classification into three groups according to the same rules applied in the pathway classification of the maturated Abs. This suggested that the innate structural properties of CDR-H3 were conserved throughout maturation. In other words, in exploring the structure of CDR-H3, it is possible to distinguish the latent potentials of Abs in effective affinity maturation even those making up the initial Ab repertoire. We then examined an artificially designed group-H Ab prototype and found its NP-binding ability sufficient for engagement in the initial repertoire. The question arose here as to why the majority of the actual initial repertoire consisted of the group-L ancestors regardless of their middling NP-binding affinity, which called for further discussion from the viewpoint of the dynamics possibly shaping the repertoire.     

Regional multilineage differentiation potential of meniscal fibrochondrocytes: Implications for meniscus repair

The knee menisci are wedge‐shaped semilunar fibrocartilaginous structures that reside between the femur and tibia and function to transmit and distribute load. These structures have characteristics of both fibrous and cartilaginous tissues. The cartilage‐like inner region and the fibrous vascularized outer region each has a distinct extracellular matrix, and resident meniscal fibrochondrocytes (MFCs) with distinct morphologies dependent on their location. Damage to the meniscus is common, and disruption of tissue structure and function result in erosion of the underlying articular cartilage. It has been observed that damage in the vascular periphery undergoes spontaneous repair, whereas damage of the inner region does not heal. While vascularity of the peripheral region plays a role in healing, recent findings have also suggested that local cellular composition influences local healing capacity. This study examined the variation in multipotential characteristics of cell populations isolated from different regions of the bovine meniscus. MFCs were isolated from the outer (vascular), inner (avascular), and horn (mixed) regions and induced toward chondrogenic, adipogenic, and osteogenic lineages. The results of this study suggest that MFCs from all regions of the meniscus possess a multilineage differentiation capability, particularly toward chondrogenesis and adipogenesis. MFCs from the outer region were most plastic, differentiating along all three mesenchymal lineages. These findings may underlie the experimental observation of improved integration of meniscus grafts from the outer zone and may have implications for developing strategies of cell‐based meniscus repair. Anat Rec, 290:48–58, 2007. © 2006 Wiley‐Liss, Inc.     

Strict conservation of the retroviral nucleocapsid protein zinc finger is strongly influenced by its role in viral infection processes: characterization of HIV-1 particles containing mutant nucleocapsid zinc-coordinating sequences

The retroviral nucleocapsid (NC) protein contains highly conserved amino acid sequences (-Cys-X2-Cys-X4-His-X4-Cys-) designated retroviral (CCHC) Zn2+ fingers. The NC protein of murine leukemia viruses contains one NC Zn2+ finger and mutants that were competent in metal binding (CCCC and CCHH) packaged wild-type levels of full-length viral RNA but were not infectious. These studies were extended to human immunodeficiency virus type 1 (HIV-1), a virus with two NC Zn2+ fingers. Viruses with combinations of CCHC, CCCC, and CCHH Zn2+ fingers in each position of HIV-1 NC were characterized. Mutant particles contained the normal complement of processed viral proteins. Four mutants packaged roughly wild-type levels of genomic RNA, whereas the remaining mutants packaged reduced levels. Virions with mutated C-terminal position NC fingers were replication competent. One interesting mutant, containing a CCCC Zn2+ finger in the N-terminal position of NC, packaged wild-type levels of viral RNA and showed approximately 5% wild-type levels of infectivity when examined in CD4-expressing HeLa cells containing an HIV-1 LTR/beta-galactosidase construct. However, this particular mutant was replication defective in H9 cells; all other mutants were replication defective over the 8-week course of the assay. Two long terminal repeat viral DNA species could be detected in the CCCC mutant but not in any of the other replication-defective mutants. These studies show that the N-terminal Zn2+ finger position is more sensitive to alterations than the C-terminal position with respect to replication. Additionally, the retroviral (CCHC) NC Zn2+ finger is required for early infection processes. The evolutionary pressure to maintain CCHC NC Zn2+ fingers depends mainly on its function in infection processes, in addition to its function in genome packaging.     

Identification and sequencing of HLA-B*0714 and B*2718 alleles and novel exon 1 sequences of B*0709 and B*2714 alleles in potential bone marrow donors

Sequence specific oligonucleotide probe hybridization and sequence specific primer PCR typing of volunteer bone marrow donors suggested the presence of variants of known HLA-B alleles in two individuals. PCR products encompassing HLA-B locus exons 1, 2, and 3 were prepared, subcloned and sequenced. A Hispanic individual had a novel B*07 allele (B*0714) and a Chinese individual had a novel B*27 allele (B*2718). In two other individuals, a previously unknown sequence of exon 1 was determined for HLA-B*0709 (African American) and B*2714 (Native American). These findings further illustrate the substantial genetic variation present at the HLA-B locus within human populations. We discuss the structural variation in the protein sequence for these HLA-B alleles and its potential functional effects.     

运用亚细胞蛋白质组学方法发现胃腺癌潜在标志物UCHL1

目的:通过对胃腺癌细胞系SGC7901和永生化胃正常上皮细胞系GES胞浆组分蛋白进行差异蛋白质组学分析,为胃腺癌诊断提供候选生物标志物,进而为阐明胃腺癌发病机理提供新的线索和思路。方法:运用亚细胞蛋白组份分离结合双向凝胶电泳和基质辅助激光解吸电离飞行时间质谱(MALD I-TOF-MS)技术筛选鉴定出SGC7901和GES细胞系胞浆蛋白组分间的差异蛋白质,并且利用免疫印迹和半定量RT-PCR方法对得到的差异蛋白进行验证。结果:筛选得到10个差异蛋白,进一步证实,差异蛋白泛素羧基末端水解酶-L1(UCHL1)在蛋白质和mRNA水平上,在胃癌细胞系SGC7901、AGS、BGC823和MKN45中的表达水平均低于胃正常上皮细胞系GES;其在胃癌组织中的表达水平也远远低于癌旁正常胃组织中的水平。结论:UCHL1蛋白质分子具有作为胃腺癌诊断检测标志物的潜在应用价值,为开发胃腺癌诊断标志物提供了新的候选蛋白。     

Rescue of the genetically engineered Cul4b mutant mouse as a potential model for human X-linked mental retardation

Mutation in CUL4B, which encodes a scaffold protein of the E3 ubiquitin ligase complex, has been found in patients with X-linked mental retardation (XLMR). However, early deletion of Cul4b in mice causes prenatal lethality, which has frustrated attempts to characterize the phenotypes in vivo. In this report, we successfully rescued Cul4b mutant mice by crossing female mice in which exons 4-5 of Cul4b were flanked by loxP sequences with Sox2-Cre male mice. In Cul4b-deficient (Cul4b(Δ)/Y) mice, no CUL4B protein was detected in any of the major organs, including the brain. In the hippocampus, the levels of CUL4A, CUL4B substrates (TOP1, β-catenin, cyclin E and WDR5) and neuronal markers (MAP2, tau-1, GAP-43, PSD95 and syn-1) were not sensitive to Cul4b deletion, whereas the number of parvalbumin (PV)-positive GABAergic interneurons was decreased in Cul4b(Δ)/Y mice, especially in the dentate gyrus (DG). Some dendritic features, including the complexity, diameter and spine density in the CA1 and DG hippocampal neurons, were also affected by Cul4b deletion. Together, the decrease in the number of PV-positive neurons and altered dendritic properties in Cul4b(Δ)/Y mice imply a reduction in inhibitory regulation and dendritic integration in the hippocampal neural circuit, which lead to increased epileptic susceptibility and spatial learning deficits. Our results identify Cul4b(Δ)/Y mice as a potential model for the non-syndromic model of XLMR that replicates the CUL4B-associated MR and is valuable for the development of a therapeutic strategy for treating MR.     

Homozygous and frequent deletion of proximal 8p sequences in human prostate cancers: Identification of a potential tumor suppressor gene site

By using tissue microdissection and polymerase chain reaction (PCR) techniques, we examined 85 prostate tumors that were paired with normal tissues from the same patients for allelic loss at 26 highly polymorphic microsatellite sequences, 21 spanning 8p and 5 localized to 8q. Sixty-four tumors (75%) demonstrated loss of at least one 8p locus. Separate distal and proximal regions of deletion were observed as well as an intervening, staggered breakpoint. A novel region of homozygous deletion of sequences at the D8S87 locus was detected both by multiplex PCR and by fluorescence in situ hybridization within this breakpoint region. These data suggest that a tumor-suppressor gene mapping to proximal 8p is deleted frequently and is likely to be important for tumorigenesis in prostate tumors. Genes Chromosomes Cancer 23:255–262,1998. © 1998 Wiley-Liss, Inc.