Probe Mix Research Articles

1017-LB

Aim While the expressed extracellular domains of the HLA antigens are responsible for determining antigenicity, there are nucleotide polymorphisms outside of this region that affect expression levels. In particular, several of the common well defined (CWD) null mutations lie outside of those typically amplified exons in SSO products (exon 2 and 3 for Class1; exon2 for Class 2) and cause the loss of antigen expression. The LIFECODES Null Allele SSO kit was developed to detect A*24:09N, B*51:11N, C*04:09N, DRB4*01:03:01:02N and DRB5*01:08N. When running standard SSO based assays, reflex testing is required for resolution of these null alleles. However, there is currently no single product that can resolve all of these null alleles. Methods Primers were designed to amplify exon 4 for HLA-A & B, exon 7 for HLA-C, exon 3 for DRB5 and the intron 1 - exon 2 border for DRB4. Probes were developed that are specific for the null alleles along with the complementary probes that are specific for the non-null alleles. Amplification conditions and hybridization conditions are identical to those of the other LIFECODES SSO products. The Null kit contains two assays: Null Class 1 and Null Class 2, composed of two separate PCR master mixes and one common probe mix for hybridization. Results Using input DNA from different sources, such as UCLA reference samples and whole blood preserved either in EDTA or ACD, the Null Class 1 and Null Class 2 assays showed ⩾ 97% typing accuracy per locus in 139 and 137 genomic samples, respectively. Both assays were generated expected typings using DNA ranging from 25 to 400 ng. Reproducibility among three lots and three operators showed ⩾ 99% concordance per locus for 162 Null Class 1 assays and for 126 Null Class 2 assays. Conclusions In a single product, the LIFECODES Null Allele SSO kit provides a method to simultaneously detect multiple CWD null alleles that lie outside of exon 2 and 3 for Class I and exon 2 for Class II.

1012-LB Enhanced resolution of LIFECODES HLA-C eRES SSO typing kit by the addition of 22 new probes

LIFECODES SSO typing kits are based upon the Luminex platform to provide intermediate level of resolution mostly focused upon resolving common well documented (CWD) HLA alleles. Out of 81 CWD HLA-C alleles identified by Cano et al (Human Immonol. 68: 392-417, 2007), 71 CWD alleles are unique at the 4-digit level. An analysis of the CWD-CWD allelic resolution by LIFECODES HLA-C product revealed that 14 CWD-CWD alleles could not be resolved. The aim of the study was to develop probes to resolve these and other ambiguities. Herein, we demonstrate enhanced resolution of the kit by the addition of 22 new probes. Sequence specific probes, directed to nucleotide polymorphisms in exons 2 and 3 of HLA-C genes were designed to resolve various ambiguities of the CWD alleles. These probes were added to current HLA-C probe mix to evaluate the enhanced resolution of the revised kit. Addition of 22 new probes to the LIFECODES HLA-C probe mix resulted in the incremental resolution of 10 CWD-CWD, 12 CWD-rare and 2 rare-rare allelic ambiguities. For CWD-CWD resolution, Probe-276 resolved C*04:01 from C*04:07; Probe-280 resolved C*03:10 from C*03:09; Probe-373 resolved C*03:05 from C*03:04; Probe-376 resolved C*05:05 from C*05:01; Probe-378 resolved C*15:09 from C*15:04; Probe-380 resolved C*03:06 from C*0302; Probe-382 resolved C*12:13 from C*12:03; Probe-383 resolved C*07:12 from C*07:04; Probe-384 resolved C*01:08 from C*01:02; and Probe-385 resolved C*08:06 from C*08:03. For allelic combinations, Probe-276 resolved C*04:01 +C*07:02 pair from C*07:64 + C*18:01/02 pair; Probe 279 resolved C*05:20/32 + C*07:01 pair from C*07:01 + C*08:02 pair; Probe-381 resolved C*12:03 + C*07:01 pair from C*06:11 + C*07:92 pair, and C*12:03 + C*07:40 pair from C*07:01 + C*16:04 pair. Other probes resolved C*01:02/01:07, C*01:02/01:18, C*02:04/02:02, C*02:10/02:14, C*04:01/04:05, C*06:02/06:15, C*07:05/07:27, C*07:14/07:27, C*07:35/07:55N, and C*17:04/17:01 allelic ambiguities. Out of 178 samples typed, 123 (69%) were resolved to a single pair of CWD alleles at 2-field resolution over exons 2 and 3. Efforts are underway to resolve the remaining CWD ambiguities.

Isotachophoresis with ionic spacer and two-stage separation for high sensitivity DNA hybridization assay

We present an on-chip electrophoretic assay for rapid and high sensitivity nucleic acid (NA) detection. The assay uses isotachophoresis (ITP) to enhance NA hybridization and an ionic spacer molecule to subsequently separate reaction products. In the first stage, the probe and target focus and mix rapidly in free solution under ITP. The reaction mixture then enters a region containing a sieving matrix, which allows the spacer ion to overtake and separate the slower probe-target complex from free, unhybridized probes. This results in the formation of two focused ITP peaks corresponding to probe and probe-target complex signals. For a 149 nt DNA target, we achieve a 220 fM limit of detection (LOD) within 10 min, with a 3.5 decade dynamic range. This LOD constitutes a 12× improvement over previous ITP-based hybridization assays. The technique offers an alternative to traditional DNA hybridization assays, and can be multiplexed and extended to detect other biomolecules.

Effectiveness of multiplex ligation dependent probe amplification (MLPA) in prenatal diagnosis of common aneuploidies

To assess the effectiveness of the MLPA method (multiplex ligation dependent probe amplification) in prenatal diagnosis of common aneuploidies and compare its concordance with traditional karyotyping. From October 2008 until July 2012 we performed 195 MLPA (MRC-Holland) tests with the P095 probe mix on DNA extracted from chorionic villi or amniotic fluid from pregnant women with elevated risk for abnormal fetal karyotype and 5 tests on miscarriage DNA samples. Cell culture and traditional karyotyping were performed in parallel. Traditional karyotyping was successfully performed in 192 cases (98.5%; 192/195). In 52 cases the fetal karyotype was abnormal (26.8%). The most common findings included aneuploidies of the following chromosomes: 13, 18, 21, X, Y (86.5%, 45/52). There were 179 conclusive and 1 inconclusive MLPA result (92.3%; 180/195). The absolute specificity and sensitivity of the MLPA test were 100%. In 9 cases traditional karyotyping revealed aberrations impossible to detect with the MLPA P095 kit. The MLPA reaction was successfully performed on all miscarriage DNA samples. MLPA is an effective method for detecting common aneuploidies. Its effectiveness for miscarriage DNA samples remains to be elucidated in further studies.

Abstract P3-06-32: Topoisomerase 1 gene copy aberration is a frequent finding in clinical breast cancer samples

Abstract Background: The topoisomerase-1 (TOP1) targeting drug irinotecan may be a new effective drug in the treatment of metastatic breast cancer patients1,2. However, the use of irinotecan should be guided by predictive biomarkers to avoid drug-induced site effects in the non-responding subgroup of patients. A newly developed TOP1 fluorescence in situ hybridization (FISH)3 probe mix (Dako, Denmark) may represent such a novel predictive biomarker assay. Materials and Methods: The TOP1/CEN-20 FISH probe mix was applied to normal breast tissue (n = 100) and human breast cancer biopsy material (n = 42). TOP1 copy number variations (Affymetrix GeneChip 100k SNP array) and TOP1 mRNA levels (Affymetrix u133a GeneChip) were studied in 313 breast cancer samples4. Results: Employing FISH, the mean diploid TOP1 copy number in normal breast epithelium was 1.7 (range 1.45–1.90) and the diploid CEN-20 number was 1.6 (range 1.38–1.82). The normal diploid range was defined as the mean plus/minus 0.5 times the haploid gene copy number (Table 1 and 2). Using these values as cut-off, 13 (30.9%) of the 42 breast cancer samples had TOP1 gene copy number in the diploid range, 18 (42.9%) had 1 extra copy and 11 (26.2%) had ≥ 2 extra copies. CEN-20 counts showed that 19 (45.5%) had CEN-20 values within the diploid range, 21 (50%) had 1 extra copy and 2 (4.8%) had ≥ 2 extra copies. Calculating the TOP1/CEN-20 ratio showed that 6 (14.3%) of the patients samples had a ratio above 1.5, while none had a ratio < 0.8. The Pearson correlation between TOP1 and CEN-20 counts in the 42 breast cancer samples analyzed with FISH was 0.73. In the 313 breast cancer samples 34/313 (10.9%) had TOP1 copy gain and a significant association (p < 0.0001) was observed between TOP1 copy number and TOP1 mRNA. Conclusions: This study shows that about 25% of breast cancers have ≥2 extra copies of TOP1 as determined by FISH analyses. The high correlation between TOP1 and CEN20 in the 42 breast cancer samples challenges the use of the ratio. Moreover, the significant association between TOP1 gene copy number (SNP array) and TOP1 mRNA expression in breast cancer biopsies suggests that TOP1 FISH results will correlate with TOP1 protein amounts and may therefore be used to predict sensitivity to the TOP1 targeting drug irinotecan in breast cancer. However, a prospective clinical study is needed to prove or disprove this hypothesis.

A DNA probe mix for the multiplex detection of ten artichoke viruses

Artichoke Italian latent virus (AILV), Artichoke latent virus (ArLV), Artichoke mottled crinkle virus (AMCV), Bean yellow mosaic virus (BYMV), Cucumber mosaic virus (CMV), Pelargonium zonate spot virus (PZSV), Tomato infectious chlorosis virus (TICV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV) and Turnip mosaic virus (TuMV) are damaging to artichoke. We have developed a protocol enabling the simultaneous detection of these artichoke viruses by non-isotopic dot blot hybridisation with DNA probes. The probe mix detected all viruses with high specificity and identical to that obtained using individual probes. The approach is proposed for the routine assessment of phytosanitary status for certified nursery production of globe artichoke.

Recurrent Rearrangement of the PHF1 Gene in Ossifying Fibromyxoid Tumors

Ossifying fibromyxoid tumor (OFMT) is a soft tissue tumor of unknown lineage. Although most cases are histologically and clinically benign, some show malignant morphological features and local recurrences are not uncommon; a few may even metastasize. In the present study, cytogenetic analysis identified different structural rearrangements of chromosome band 6p21 in tumor cells from three cases of OFMT, including one with typical, one with atypical, and one with malignant morphological features. Mapping of the 6p21 breakpoint by fluorescence in situ hybridization (FISH) indicated that the PHF1 gene was rearranged in all three cases. Further FISH, 5'-rapid amplification of cDNA ends, and RT-PCR analyses disclosed an EP400/PHF1 fusion transcript in one of the cases. Interphase FISH on tumor sections from 13 additional cases of OFMT showed rearrangement of the PHF1 locus in four of four typical, two of three atypical, and one of six malignant lesions. Thus, the PHF1 gene, previously shown to be the 3'-partner of fusion genes in endometrial stromal tumors, is also recurrently involved in the pathogenesis of OFMTs, irrespective of whether they are diagnosed as typical, atypical, or malignant lesions. The PHF1 protein interacts with the polycomb-repressive complex 2 (PRC2), which, in turn, regulates the expression of a variety of developmental genes. Thus, the results indicate that deregulation of PRC2 target genes is crucial for OFMT development.

Pigment epithelial cells isolated from human peripheral iridectomies have limited properties of retinal stem cells

The identification of cells with properties of retinal progenitor cells (RPCs) in the adult human ciliary margin (CM) prompted a number of studies of their proliferative and differentiation potential. One of the remaining challenges is to find a feasible method of isolating RPCs from the patient's eye. In the human CM, only the iris pigment epithelium (IPE) is easily obtained by a minimally invasive procedure. In the light of recent studies questioning the existence of RPCs in the adult mammalian CM, we wanted to assess the potential of the adult human IPE as source of RPCs. The IPE were isolated from peripheral iridectomies during glaucoma surgery, and IPE and ciliary body (CB) epithelium were also isolated from post-mortem tissue. Cells were cultivated in sphere-promoting conditions or as monolayers. Whole-tissue samples, undifferentiated and differentiated cells were studied by immunocytochemistry, RT-PCR and transmission electron microscopy. The adult human IPE, like the CB, expressed markers of RPCs such as Pax6, Sox2 and Nestin in vivo. Both sphere-promoting and monolayer cultures preserved this phenotype. However, both IPE/CB cultures expressed markers of differentiated epithelial cells such as Claudin, microphtalmia-associated transcription factor (MITF) and Cytokeratin-19. Ultrastructurally, IPE spheres displayed epithelial-like junctions and contained mature melanosomes. After induced differentiation, IPE-derived cells showed only partial neuronal differentiation expressing β-III-tubulin, Map-2 and Rhodopsin, whereas no mature glial markers were found. Proliferative cells with some properties of RPCs can be isolated from the adult human IPE by peripheral iridectomies. Yet, many cells retain properties of differentiated epithelial cells and lack central properties of somatic stem cells.

Wear resistant coatings: Silica sol–gel reinforced with carbon nanotubes

Abstract Pin-on-disc wear experiments have been carried out on sol–gel silica coatings reinforced with 0.1 wt.% carbon nanotubes (CNTs) deposited on WE54 magnesium alloy substrates by the dip-coating technique. Sol–gel solutions were fabricated using two different procedures: mechanical mixing (MM) and ultrasonic probe mixing. Dry sliding wear tests have been carried out at load of 1 N, speed of 0.1 m/s and sliding distance of 60 m. Friction coefficients were obtained from the tests and the specific wear rates ( k ) were calculated. The fabrication procedure of the coating influences its morphology and wear resistance. Friction coefficient was found to vary slightly with the addition of the CNTs. The wear volume of the magnesium substrate coated decreased by 40% and 80%, in terms of k , by using unreinforced and CNT-reinforced MM coatings, respectively. In MM layers reinforced with CNT uniform dispersion of the nanotubes was reached and toughening of the ceramic coating by pull-out and crack bridging mechanisms was observed.

Karyotype and Identification of All Homoeologous Chromosomes of AllopolyploidBrassica napusand Its Diploid Progenitors

Investigating recombination of homoeologous chromosomes in allopolyploid species is central to understanding plant breeding and evolution. However, examining chromosome pairing in the allotetraploid Brassica napus has been hampered by the lack of chromosome-specific molecular probes. In this study, we establish the identification of all homoeologous chromosomes of allopolyploid B. napus by using robust molecular cytogenetic karyotypes developed for the progenitor species Brassica rapa (A genome) and Brassica oleracea (C genome). The identification of every chromosome among these three Brassica species utilized genetically mapped bacterial artificial chromosomes (BACs) from B. rapa as probes for fluorescent in situ hybridization (FISH). With this BAC-FISH data, a second karyotype was developed using two BACs that contained repetitive DNA sequences and the ubiquitous ribosomal and pericentromere repeats. Using this diagnostic probe mix and a BAC that contained a C-genome repeat in two successive hybridizations allowed for routine identification of the corresponding homoeologous chromosomes between the A and C genomes of B. napus. When applied to the B. napus cultivar Stellar, we detected one chromosomal rearrangement relative to the parental karyotypes. This robust novel chromosomal painting technique will have biological applications for the understanding of chromosome pairing, homoeologous recombination, and genome evolution in the genus Brassica and will facilitate new applied breeding technologies that rely upon identification of chromosomes.

Performance of MLPA as a screening method for aneuploidy in uncultured amniocytes

To test whether the Multiplex Ligation-dependent Probe Amplification (MLPA) technique can be used as a screening test for rapid diagnosis of aneuploidies in uncultured amniocentesis. In this prospective blind study, MLPA with chromosomes 13,18,21,X and Y specific probe mixes was performed in 500 amniotic fluid samples. Chromosome copy numbers were determined by analyzing size and peak area for each MLPA probe. Results were compared with those of karyotyping/FISH. Conclusive test results were obtained in 98% of the samples, whereas 10 were inconclusive. In all conclusive tests, the MLPA results were concordant with that of cytogenetic and/or FISH analyses. There were no false-positive results. A case with 69,XXX triploidy could not be diagnosed by MLPA. In total, 28 aneuploidies were diagnosed. There were no false-positive results. The performance of each probe was determined. MLPA is a rapid, simple and reliable assay for aneuploidy screening in uncultured amniocytes.

Simultaneous Detection of Genomic Rearrangements In Myelodysplastic Syndromes (MDS) with the Multiplex Ligation-Dependent Probe Amplification (MLPA) Assay

Abstract Abstract 1863 Background: Clonal chromosome abnormalities are present in the marrow cells in about 50% of patients with myelodysplastic syndromes (MDS) at the time of presentation. Cytogenetic analysis of the bone marrow is not only indicated in MDS for diagnostic purposes, but also to assess the individual prognosis according to IPSS scoring guidelines and plan tailored therapy. Conventional cytogenetics (CC) analysis is performed in clinical practice to detect chromosomal abnormalities. It has been reported that fluorescence in situ hybridization (FISH) is a more sensitive approach, but this analysis is limited to detection of the more frequent abnormalities on chromosomes 5, 7, 8, 11, and 20, and reports from the literature provide contradictory data. A new method has recently been described for the measurement of gene/chromosome copy number using genomic DNA: Multiplex Ligation-dependent Probe Amplification (MLPA). Aims: The purpose of this study was to perform the MLPA assay in a series of 29 MDS patients (M: 21, F: 8, median age 71 years, range 44–84), and to compare the results obtained with CC data. According to the WHO classification, 7 cases were classified as RA, 7 as refractory cytopenia with multilineage dysplasia, 2 as RAEB-1, 8 as RAEB-2, 1 as MDS_U, and 4 as CMML. According to the IPSS score, 8 were considered low risk, 11 intermediate-1 risk, 5 intermediate-2 risk, and 5 high risk. Methods: The MLPA assay was performed for all samples in two independent reactions, one for each probe mix (SALSA Probe-Mix P144 and P145) according to the manufacturer's recommendations (MRC-Holland). This mix contains 61 target sequences specific for different chromosome regions commonly involved in MDS: 5q (9 probes) + 5p (1 probe), 7q (8 probes) + 7p (2 probes), 8q (8 probes) + 8p (2 probes), 11q (8 probes), 12p (6 probes), 17q (2 probes) + 17p (4 probes), 20q (5 probes) + 20p (1 probe) and 21q (5 probes). The Probe mixes also include 21 reference probes selected from chromosomal regions that appear to be “quiet” in MDS. Data were analyzed with Coffalyser Software (MRC-Holland) using DNA from ten healthy donors as controls. The CC study was performed following standard protocols and at least 20 metaphases were analyzed. Results and Conclusions: Our study showed a good correlation between the MLPA and CC results, as shown in Table I, since most of the alterations were detected by both techniques. Discrepancies were found in 5 (17%) samples. MLPA analysis did not detect: in sample n°5 the presence of a chromosomal (chr.) translocation; in sample n°12 a chr. deletion and a chr. translocation; in sample n°17 a chr. deletion; in sample n°22 several chr. translocations and deletions; in sample n°28 a chr. gain. In fact, MLPA is not able to detect chr. translocations because it can reveal only chr. loss or gain; it can only analyse the chr. regions commonly involved in MDS (5, 7, 8, 11, 12, 17, 20 and 21); it can reveal chr. abnormalities only if the percentage of cells carrying the alterations is about 30–35% and do not show mosaicism. On the other hand, with CC we observed a karyotype failure (no metaphases) in 3 samples. MLPA proved to be rapid, cost effective, relatively easy to perform, had high throughput and enabled simultaneous analysis of many samples by automated data processing. MLPA and CC result complementary techniques, and MLPA is particularly useful in MDS cases with Karyotype failure. Disclosures: No relevant conflicts of interest to declare.

GeneFISH – an in situ technique for linking gene presence and cell identity in environmental microorganisms

Our knowledge concerning the metabolic potentials of as yet to be cultured microorganisms has increased tremendously with the advance of sequencing technologies and the consequent discoveries of novel genes. On the other hand, it is often difficult to reliably assign a particular gene to a phylogenetic clade, because these sequences are usually found on genomic fragments that carry no direct marker of cell identity, such as rRNA genes. Therefore, the aim of the present study was to develop geneFISH - a protocol for linking gene presence with cell identity in environmental samples, the signals of which can be visualized at a single cell level. This protocol combines rRNA-targeted catalysed reporter deposition - fluorescence in situ hybridization and in situ gene detection. To test the protocol, it was applied to seawater samples from the Benguela upwelling system. For gene detection, a polynucleotide probe mix was used, which was designed based on crenarchaeotal amoA clone libraries prepared from each seawater sample. Each probe in the mix was selected to bind to targets with up to 5% mismatches. To determine the hybridization parameters, the T(m) of probes, targets and hybrids was estimated based on theoretical calculations and in vitro measurements. It was shown that at least 30%, but potentially the majority of the Crenarchaeota present in these samples harboured the amoA gene and were therefore likely to be catalysing the oxidation of ammonia.

Real-Time PCR Reveals Endosymbiont Titer Fluctuations inMetaseiulus occidentalis(Acari: Phytoseiidae) Colonies Held at Different Temperatures

Real-time PCR (Polymerase Chain Reaction) can estimate Wolbachia endosymbiont population density in mosquitoes (Wiwatanaratanabutr & Kittayapong 2009). The COS (?arbaryl-OP-Sul fur resistant) colony of the phytoseiid Metaseiulus (=Typhlodromus or Gcdendromus) occidentalis (Nesbitt) harbors several endosymbionts, includ ing Cardinium, Wolbachia, Enterobacter, and Bacteroidetes in their gut and reproductive tis sues (Johanowicz & Hoy 1996; Hoy & Jeyaprakash 2005). Johanowicz & Hoy (1998) ob served mating incompatibility when females from a heat-treated (33?C) COS colony (a treatment presumed to eliminate Wolbachia) was crossed with males from a room-temperature (23?C) COS colony containing Wolbachia, resulting in the pro duction of shriveled eggs that did not hatch. It was not clear whether the Wolbachia population was reduced or completely eliminated from the heat-treated mites because a standard PCR pro tocol was used, which is relatively insensitive compared to high-fidelity PCR (Jeyaprakash & Hoy 2000). In this paper, we estimate the endo symbiont population density in COS colonies of M. occidentalis held under different tempera tures in the laboratory using real-time PCR. One colony of M. occidentalis was maintained at room-temperature (23-25?C) and 2 colonies ini tiated from this line were maintained at 32 and 34?C for More than 1 year in growth chambers prior to experiments. Female mites (100) were isolated from each colony and their genomic DNA extracted by Puregene and QIAGEN methods (Jeyaprakash & Hoy 2007), and resuspended in 50 pL of sterile water. Four different plasmids carrying endosymbiont 16S rRNA sequences; pAJ233 (Cardinium), pAJ234 (Bacteroidetes), pAJ235 (Wolbachia) and pAJ239 (Enterobacter), were extracted and their yield estimated with a BioPhotometer Plus (Eppendorf AG, Hamburg, Germany). Species-specific primers were de signed with Primer 3 software (http:// frodo.wi.mit.edu/primer3/) and a probe was de signed with Primer X software (Applied Biosys tems, Foster City, CA) from the variable regions for each species (Table 1). Real-time PCR was per formed in a 20-pL reaction volume containing ge nomic DNA from 2 females (1 pL) or serially-di luted plasmid DNA (100 pg to 1 fg) or a no DNA control (1 pL), forward and reverse primers (400 pM), probe (100 pM) and TaqMan master mix (10 pL). Two linked profiles: (1) one cycle of 50?C for 2 min and 95?C for 10 min, and (2) 60 cycles consist ing of denaturation at 95?C for 15 s, annealing at 59?C (Cardinium) or 57?C (Wolbachia or Entero bacter or Bacteroidetes) for 30 s and extension at 72?C for 30 s, were used. Each treatment was rep l cated 3 times. The starting copy number of all plasmid DNA dilutions used in the real-time PCR was obtained with an open-source software (http:/

Enzymatic activity of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 11: upregulated in breast and cervical cancers

The conversion of lysophosphatidic acid (LPA) to phosphatidic acid is carried out by the microsomal enzymes 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs). These enzymes are specific for acylating LPA at the sn-2 (carbon 2) position on the glycerol backbone and are important, because they provide substrates for the synthesis of phospholipids and triglycerides. At least, mutations in one isoform, AGPAT2, cause near complete loss of adipose tissue in humans. We cloned a cDNA predicted to be an AGPAT isoform, AGPAT11. This cDNA has been recently identified also as lysophosphatidylcholine acyltransferase 2 (LPCAT2) and lyso platelet-activating factor acetyltransferase. When AGPAT11/LPCAT2/lyso platelet-activating factor acetyltransferase cDNA was expressed in CHO and HeLa cells, the protein product localized to the endoplasmic reticulum. In vitro enzymatic activity using lysates of Human Embryonic Kidney-293 cells infected with recombinant AGPAT11/LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA adenovirus show that the protein has an AGPAT activity but lacks glycerol-3-phosphate acyltransferase enzymatic activity. The AGPAT11 efficiently uses C18:1 LPA as acyl acceptor and C18:1 fatty acid as an acyl donor. Thus, it has similar substrate specificities for LPA and acyl-CoA as shown for AGPAT9 and 10. Expression of AGPAT11 mRNA was significantly upregulated in human breast, cervical, and colorectal cancer tissues, indicating its adjuvant role in the progression of these cancers. Our enzymatic assays strongly suggest that the cDNA previously identified as LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA has AGPAT activity and thus we prefer to identify this clone as AGPAT11 as well.

Integration of Genetic, Physical, and Cytogenetic Maps for Brassica rapa Chromosome A7

Bacterial artificial chromosome (BAC) contigs have been genetically mapped to the 10 linkage groups of Brassica rapa by BAC end sequences (BES). To integrate the genetic, physical, and cytogenetic maps, fluorescence in situ hybridization (FISH) was used to anchor the assembly of BAC contigs onto Brassica chromosomes using representative BACs. This BAC-FISH approach can be used to identify chromosome arms on separate mitotic metaphase chromosomes or to map multiple BACs to single long pachytene chromosomes. As part of an international consortium that is sequencing the B. rapa genome, we integrated the linkage and physical maps with the B. rapa cytogenetic map for chromosome A7 by hybridizing BACs to mitotic chromosomes and along the length of pachytene chromosome spreads. A total of 31 BACs that were putatively located on A7 were used as probes for FISH analyses; however, only 19 BACs mapped unambiguously to A7 while the remaining BACs either mapped to other chromosomes or hybridized to multiple locations. We then created a multicolor FISH cocktail of 16 BAC probes to simultaneously hybridize the entire length of the A7 chromosome. We successfully applied the 16 A7 BAC probe mix to B. rapa, B. oleracea, and domesticated and resynthesized genotypes of B. napus to demonstrate that this approach can facilitate studies of genome evolution by integrating the genetic, physical, and cytogenetic maps among closely related species of Brassica.

Prenatal study of common submicroscopic “genomic disorders” using MLPA with subtelomeric/microdeletion syndrome probe mixes, among gestations with ultrasound abnormalities in the first trimester

The present study aims to investigate the presence of common submicroscopic chromosomal rearrangements in fetuses with ultrasound abnormalities or positive screening in the first trimester and normal karyotype. We used the multiplex ligation-dependent probe amplification (MLPA) technique with subtelomeric (SALSA P036B) and microdeletion syndrome (SALSA P064B/P096) probe mixes as a screening method to measure copy number changes on the tested probes in chorionic villus sampling. MLPA with P036B and P064/P096 probe mixes was performed on 49 chorionic villi DNA samples obtained between the 11th and 13th week of gestation. The MLPA analyses did not detect any diminished or increased intensity for all the tested probes in the samples. Our results suggest that the common submicroscopic "genomic disorders" (microdeletion and microduplication syndromes) would not be frequently detected in the first trimester anomalies screening.

Influence of class B scavenger receptors on cholesterol flux across the brush border membrane and intestinal absorption

To learn more about how the step of cholesterol uptake into the brush border membrane (BBM) of enterocytes influences overall cholesterol absorption, we measured cholesterol absorption 4 and 24 h after administration of an intragastric bolus of radioactive cholesterol in mice with scavenger receptor class B, type 1 (SR-BI) and/or cluster determinant 36 (CD36) deleted. The cholesterol absorption efficiency is unaltered by deletion of either one or both of the receptors. In vitro determinations of the cholesterol uptake specific activity of the BBM from the mice reveal that the scavenger receptors facilitate cholesterol uptake into the proximal BBM. It follows that cholesterol uptake into the BBM is not normally rate-limiting for the cholesterol absorption process in vivo; a subsequent step, such as NPC1L1-mediated transfer from the BBM into the interior of the enterocyte, is rate-limiting. The absorption of dietary cholesterol after 4 h in mice lacking SR-BI and/or CD36 and fed a high-fat/high-cholesterol diet is delayed to more distal regions of the small intestine. This effect probably arises because ATP binding cassette half transporters G5 and G8-mediated back flux of cholesterol from the BBM to the lumen of the small intestine limits absorption and causes the local cholesterol uptake facilitated by SR-BI and CD36 to become rate-limiting under this dietary condition.

Rapid and reliable determination of transgene zygosity in mice by multiplex ligation-dependent probe amplification

The ability to rapidly and unequivocally distinguish heterozygous from homozygous transgenic mice is an integral part of any breeding strategy. Here we describe a quick and simple protocol for determining the zygosity of transgenic mice at multiple loci in a single reaction. This involved the development of a multiplex ligation-dependent probe amplification (MLPA) probe mix to simultaneously measure common transgenic alleles such as Cre recombinase (Cre), neomycin (Neo), beta-galactosidase (LacZ) and enhanced green fluorescent protein (eGFP), as well as loci specific to the X and Y chromosome to allow sexing. Each reaction required as little as 100 ng of genomic DNA isolated from a tail biopsy using a simple procedure. Normalization against autosomal control loci resulted in 100% call accuracy, with no ambiguous results. This probe mix can be easily implemented in any laboratory with access to a PCR machine and a DNA sequencer, and can be rapidly adapted to genotype any additional loci of interest.

Lipid G Protein-coupled Receptor Ligand Identification Using β-Arrestin PathHunter™ Assay

A growing number of orphan G-protein-coupled receptors (GPCRs) have been reported to be activated by lipid ligands, such as lysophosphatidic acid, sphingosine 1-phosphate (S1P), and cannabinoids, for which there are already well established receptors. These new ligand claims are controversial due to either lack of independent confirmations or conflicting reports. We used the beta-arrestin PathHunter assay system, a newly developed, generic GPCR assay format that measures beta-arrestin binding to GPCRs, to evaluate lipid receptor and ligand pairing. This assay eliminates interference from endogenous receptors on the parental cells because it measures a signal that is specifically generated by the tagged receptor and is immediately downstream of receptor activation. We screened a large number of newly "deorphaned" receptors (GPR23, GPR92, GPR55, G2A, GPR18, GPR3, GPR6, GPR12, and GPR63) and control receptors against a collection of approximately 400 lipid molecules to try to identify the receptor ligand in an unbiased fashion. GPR92 was confirmed to be a lysophosphatidic acid receptor with weaker responses to farnesyl pyrophosphate and geranylgeranyl diphosphate. The putative cannabinoid receptor GPR55 responded strongly to AM251, rimonabant, and lysophosphatidylinositol but only very weakly to endocannabinoids. G2A receptor was confirmed to be an oxidized free fatty acid receptor. In addition, we discovered that 3,3'-diindolylmethane, a dietary molecule from cruciferous vegetables, which has known anti-cancer properties, to be a CB(2) receptor partial agonist, with binding affinity around 1 microm. The anti-inflammatory effect of 3,3'-diindolylmethane in RAW264.7 cells was shown to be partially mediated by CB(2).