Enzyme-linked immunoassay for anti-HLA antibodies--an alternative to panel studies by lymphocytotoxicity.

In order to provide a simple and specific assay for the detection and quantitation of IgG and IgM anti-HLA antibodies in sera, HLA antigens purified from a pool of 240 random donor platelets were used to develop a solid-phase enzyme-linked immunoassay (EIA). The reference values for identifying the presence of IgG or IgM anti-HLA antibodies were determined by assaying sera from 39 healthy individuals without prior HLA alloimmunization. The assay was evaluated by studying sera from 122 patients who had been characterized previously for panel reactive antibodies by the lymphocytotoxicity assay (LCA). A significant linear correlation between two assays was noted (r = 0.8, P = 0.0001). Further analyses of the data demonstrated that the newly developed EIA has 100% specificity and 95.3% sensitivity as compared with the LCA. Additional studies revealed that patients whose PRA increased or decreased over time were in parallel with antibody levels measured by EIA. When the EIA was used to measure anti-HLA antibody titers, it was more sensitive than the LCA. Since the EIA is sensitive, specific, and technically less demanding, it should provide an useful alternative to reduce the number of the more laborious panel studies for monitoring anti-HLA antibody status in candidates for organ transplantation and recipients of blood transfusions.

The bicoid and dorsal morphogens use a similar strategy to make stripes in the Drosophila embryo.

The anterior-posterior (A-P) and dorsal-ventral (D-V) axes of the early Drosophila embryo are established by two key maternal morphogens: bicoid (bcd) and dorsal (dl), respectively. The bcd protein is expressed in a broad concentration gradient along the A-P axis, with peak levels present at the anterior pole, while dl is expressed in a gradient along the D-V axis with peak levels along the ventral surface. The two morphogens are unrelated and their gradients are formed by distinct processes. Nonetheless, we have obtained evidence that they generate sharp on/off stripes of target gene expression through a similar mechanism. Both morphogens establish overlapping patterns of transcriptional activators and repressors in the early embryo. The activators and repressors bind to closely linked sites within short (300 to 500 bp) target promoter elements that have the properties of on/off switches. The activators act in concert with the morphogen to define a broad region where target genes can be initiated. Borders of target gene expression are established by the repressors, resulting in the formation of stripes.

At least 27 alternatively spliced forms of the neural cell adhesion molecule mRNA are expressed during rat heart development.

The major membrane-associated or transmembrane isoforms of the neural cell adhesion molecule (NCAM) are generated by alternative splicing at the 3' end of the mRNA. Further diversity in NCAM structure is observed in the extracellular region of the polypeptide, where the insertion of additional amino acid residues can result from alternative splicing events occurring at the exon 7-exon 8 and exon 12-exon 13 junctions. Here we report the characterization of tissue-specific patterns of alternative splicing at the exon 12-exon 13 junction by using the polymerase chain reaction. Nine alternatively spliced sequences in rat heart between exon 12 and exon 13 were identified. Each sequence consisted of different combinations of the three small exons (15, 48, and 42 bp in length) and the AAG triplet that make up MSD1, the 108-bp muscle-specific sequence found in human skeletal muscle NCAM (G. Dickson, H.J. Gower, C. H. Barton, H. M. Prentice, V. L. Elsom, S. E. Moore, R. D. Cox, C. Quinn, W. Putt, and F. S. Walsh, Cell 50:1119-1130, 1987). Although the rat equivalent of MSD1 (designated 15+ 48+ 42+ 3+) was detected in all ages of heart examined, it was only one of four or five major splice combinations at any given age. The only alternatively spliced sequence found in the exon 7-exon 8 junction of heart NCAM mRNA was the 30-bp variable alternatively spliced exon previously identified in rat brain. Twenty-seven NCAM forms with distinct sequences were found by analysis of individual NCAM transcripts from postnatal day 1 heart tissue for alternative splicing at the exon 7-exon 8 junction, the exon 12-exon 13 junction and the 3' end. Several combinations of splicing patterns in these three different regions of the gene appeared to be preferentially expressed. The observation that the expression of alternatively spliced forms of NCAM is developmentally regulated suggests a role for NCAM diversity in cardiac development.

Expression of the unique NCAM VASE exon is independently regulated in distinct tissues during development.

During development of the rat central nervous system, neural cell adhesion molecule (NCAM) mRNAs containing in the extracellular domain a 30-bp alternative exon, here named VASE, replace RNAs that lack this exon. The presence of this alternative exon between previously described exons 7 and 8 changes the predicted loop structure of the derived polypeptide from one resembling an immunoglobulin constant region domain to one resembling an immunoglobulin variable domain. This change could have significant effects on NCAM polypeptide function and cell-cell interaction. In this report we test multiple rat tissues for the presence of additional alternative exons at this position and also examine the regulation of splicing of the previously described exon. To sensitively examine alternative splicing, polymerase chain reactions (PCRs) with primers flanking the exon 7/exon 8 alternative splicing site were performed. Four categories of RNA samples were tested for new exons: whole brain from embryonic day 11 to adult, specific brain regions dissected from adult brain, clonal lines of neural cells in vitro, and muscle cells and tissues cultured in vitro and obtained by dissection. Within the limits of the PCR methodology, no evidence for any alternative exon other than the previously identified VASE was obtained. The regulation of expression of this exon was found to be complex and tissue specific. Expression of the 30-bp exon in the heart and nervous system was found to be regulated independently; a significant proportion of embryonic day 15 heart NCAM mRNAs contain VASE while only a very small amount of day 15 nervous system mRNAs contain VASE. Some adult central nervous system regions, notably the olfactory bulb and the peripheral nervous system structures adrenal gland and dorsal root ganglia, express NCAM which contains very little VASE. VASE is undetectable in NCAM PCR products from the olfactory epithelium. Other nervous system regions express significant quantities of NCAM both with and without VASE. Clonal cell lines in culture generally expressed very little VASE. These results indicate that a single alternative exon, VASE, is found in NCAM immunoglobulin-like loop 4 and that distinct tissues and nervous system regions regulate expression of VASE independently both during development and in adult animals.

Polypeptide variation in an N-CAM extracellular immunoglobulin-like fold is developmentally regulated through alternative splicing.

The alternative splicing of a previously undiscovered 30 base exon confers a new level of polypeptide diversity on the N-CAM family of cell-surface glycoproteins. It results in the insertion of 10 amino acids into the fourth of five extracellular immunoglobulin-like folds. Each major size class of rat brain N-CAM mRNAs consists of members that contain or lack the exon. Furthermore, this splicing event is developmentally controlled: RNAs containing the inserted exon are expressed at extremely low levels (less than 3%) in embryonic brain but increase postnatally to 40%-45% of all N-CAM mRNAs in adult brain. Antibodies that recognize the alternative 10 amino acid segment react with a subset of N-CAM-expressing neurons in cultures of embryonic rat cells.

Smooth muscle cells transiently express NCAM.

NCAM (neural cell adhesion molecule) polypeptides were first detected on neuronal cells and were subsequently found to be expressed at least transiently by a number of other cell types including skeletal and cardiac but not smooth muscle. We report here that rat smooth muscle expresses NCAM in vitro and transiently in vivo. Using a monoclonal antibody 3F4 which reacts with most rat NCAM polypeptides, NCAM was found on the surface of cultured rat aortic smooth muscle lines A10 and A7r5 and mouse smooth muscle like line BC3H1 in abundances equal to or greater than those of cardiac muscle, skeletal muscle, and neuronal cell lines. The major NCAM polypeptide of muscle cells was Mr = 140 kDa with lesser amounts of the 120 kDa form. Consistent with these results, a major NCAM mRNA of 6.7 kb was detected in Northern analyses with lesser amounts of the 4.3 and 2.9 kb mRNA size classes. The relative abundance of NCAM mRNA was similar in RNA prepared from smooth muscle A7r5 cells, L6 skeletal muscle cells, and 9-day-old rat brain. NCAM was distributed across the entire surface of cultured smooth muscle cells in a highly punctate manner. Cryostat sections of rat aorta, intestine and bladder were examined by immunofluorescence to determine if NCAM is also expressed on smooth muscle in vivo. In each case NCAM was found to be transiently expressed by the smooth muscle cells of these tissues. Highest NCAM levels were observed at embryonic day 17 which then declined to undetectable levels in tissues from adults. These results extend previous observations to indicate all muscle types transiently express NCAM in development.

Identification of a cDNA clone that contains the complete coding sequence for a 140-kD rat NCAM polypeptide.

Neural cell adhesion molecules (NCAMs) are cell surface glycoproteins that appear to mediate cell-cell adhesion. In vertebrates NCAMs exist in at least three different polypeptide forms of apparent molecular masses 180, 140, and 120 kD. The 180- and 140-kD forms span the plasma membrane whereas the 120-kD form lacks a transmembrane region. In this study, we report the isolation of NCAM clones from an adult rat brain cDNA library. Sequence analysis indicated that the longest isolate, pR18, contains a 2,574 nucleotide open reading frame flanked by 208 bases of 5' and 409 bases of 3' untranslated sequence. The predicted polypeptide encoded by clone pR18 contains a single membrane-spanning region and a small cytoplasmic domain (120 amino acids), suggesting that it codes for a full-length 140-kD NCAM form. In Northern analysis, probes derived from 5' sequences of pR18, which presumably code for extracellular portions of the molecule hybridized to five discrete mRNA size classes (7.4, 6.7, 5.2, 4.3, and 2.9 kb) in adult rat brain but not to liver or muscle RNA. However, the 5.2- and 2.9-kb mRNA size classes did not hybridize to either a large restriction fragment or three oligonucleotides derived from the putative transmembrane coding region and regions that lie 3' to it. The 3' probes did hybridize to the 7.4-, 6.7-, and 4.3-kb message size classes. These combined results indicate that clone pR18 is derived from either the 7.4-, 6.7-, or 4.3-kb adult rat brain RNA size class. Comparison with chicken and mouse NCAM cDNA sequences suggests that pR18 represents the amino acid coding region of the 6.7- or 4.3-kb mRNA. The isolation of pR18, the first cDNA that contains the complete coding sequence of an NCAM polypeptide, unambiguously demonstrates the predicted linear amino acid sequence of this probable rat 140-kD polypeptide. This cDNA also contains a 30-base pair segment not found in NCAM cDNAs isolated from other species. The significance of this segment and other structural features of the 140-kD form of NCAM can now be studied.