Atherosclerosis represents the most significant risk factor for coronary artery disease (CAD), the leading cause of death in developed countries. To better understand the pathogenesis of atherosclerosis, we applied a likeli hoodbased model selection method to infer genedisease causality relationships for the aortic lesion trait in a segregating mouse population demonstrating a spectrum of susceptibility to developing atherosclerotic lesions. We identified 292 genes that tested causal for aortic lesions from liver and adipose tissues of these mice, and we experimentally validated one of these candidate causal genes, complement component 3a receptor 1 (C3ar1), using a knockout mouse model. We also found that genes identified by this method overlapped with genes progressively regulated in the aortic arches of 2 mouse models of atherosclerosis during atherosclerotic lesion development. By comparing our gene set with findings from public human genomewide association studies (GWAS) of CAD and related traits, we found that 5 genes identified by our study overlapped with published studies in humans in which they were identified as risk factors for multiple atherosclerosisrelated pathologies, including myocardial infarction, serum uric acid levels, mean platelet volume, aortic root size, and heart failure. Candidate causal genes were also found to be enriched with CAD risk polymorphisms identified by the Wellcome Trust Case Control Consortium (WTCCC). Our findings therefore validate the ability of cau sality testing procedures to provide insights into the mechanisms underlying atherosclerosis development.

Lotus japonicus har1 mutants respond to inoculation with Mesorhizobium loti by forming an excessive number of nodules due to genetic lesions in the HAR1 autoregulatory receptor kinase gene. In order to expand the repertoire of mutants available for the genetic dissection of the root nodule symbiosis (RNS), a screen for suppressors of the L. japonicus har1-1 hypernodulation phenotype was performed. Of 150,000 M2 plants analyzed, 61 stable L. japonicus double-mutant lines were isolated. In the context of the har1-1 mutation, 26 mutant lines were unable to form RNS, whereas the remaining 35 mutant lines carried more subtle symbiotic phenotypes, either forming white ineffective nodules or showing reduced nodulation capacity. When challenged with Glomus intraradices, 18 of the 61 suppressor lines were unable to establish a symbiosis with this arbuscular mycorrhiza fungus. Using a combined approach of genetic mapping, targeting induced local lesions in genomics, and sequencing, all non-nodulating mutant lines were characterized and shown to represent new alleles of at least nine independent symbiotic loci. The class of mutants with reduced nodulation capacity was of particular interest because some of them may specify novel plant functions that regulate nodule development in L. japonicus. To facilitate mapping of the latter class of mutants, an introgression line, in which the har1-1 allele was introduced into a polymorphic background of L. japonicus ecotype MG20, was constructed.

Substrates of human and bovine epidermal transglutaminase (glutaminyl-peptide yglutamyltransferase, R-glutaminyl-peptide:amine-y-glutamyl-yltransferase, EC 2.3.2.13) were isolated and purified by ion exchange chromatography and preparative zone electrophoresis. These substrates of Mr 36,000, which we propose to call keratolinin, incorporated dansylcadaverine and were precipitated by antibody. (8,9,(11)(12)(13). In this paper, we report further biochemical characterization of the bovine envelope precursor and the first biochemical characterization of the envelope precursor from human epidermis. We propose "keratolinin" (from Greek keratos, horny tissue; lininos, to cover the inner surface of) as a suitable name for this soluble precursor protein. Keratolinin is crosslinked by epidermal TGase and ultimately located along the inner leaflet of the keratinocyte membrane as the cornifled envelope. Its relationship to involucrin, a soluble envelope precursor from cell culture, (4-6) is also examined. MATERIALS AND METHODSPurification of Keratinolin. Human and bovine keratolinins and both epidermal TGases were isolated and purified as described (10).Briefly, 0.2-mm slices of bovine snout epidermis or heatseparated human epidermis were snap-frozen and pulverized in liquid nitrogen and homogenized in 25 mM ammonium acetate buffer, pH 8.5/1 mM EDTA. Supernatant proteins clarified by centrifugation were dialyzed against acetate/EDTA buffer and applied to a 2.5 x 90 cm column of DEAE-Sepharose CL-6B. The column was washed with 1 liter of acetate/EDTA buffer and developed with a linear gradient to 0.7 M NaCl in acetate/EDTA buffer. These and all subsequent steps were carried out at 4°C.Fractions eluting from the DEAE column that produced a line of identity with antibody to keratolinin were concentrated to 5 ml by ultrafiltration using a UM-2 membrane (Amicon), dialyzed against acetate buffer (pH 8.5), applied to the origin of a 20 x 20 x 2 cm Pevikon block (9), and electrophoresed at 10 V/cm for 20 hr. Proteins were eluted from 1-cm slices by aspiration.Immunoreactive fractions were concentrated to 1.2 ml, dialyzed, and applied to an ACA 54 gel filtration column (1.5 x 90 cm). Either human or bovine keratolinin yielded a symmetrical peak corresponding to a Mr of 36,000 when compared to standards. Pure keratolinin was stored at -20°C for 4-6 months.

No abstract

No abstract

No abstract

Transglutaminase is a calcium-dependent enzyme found widely in nature. It catalyzes the formation of epsilon-(gamma-glutamyl)lysine bonds that participate in processes varying from fibrin clot formation to epidermal cell envelope formation. Epidermal transglutaminase is localized to the granular layer of the epidermis. It catalyzes the covalent cross-linking of a soluble cytoplasmic substrate into large polymers to form the cornified envelope that lines the inner membrane of keratinocytes in the stratum corneum. The soluble precursor from epidermis has been named keratolinin, and from keratinocyte culture, it has been named involucrin. Hair follicle transglutaminase is biochemically and immunochemically distinct from its epidermal counterpart. It has been localized to the inner root sheath and medulla of the hair follicle. The substrate of hair follicle transglutaminase has been poorly defined but appears to be rich in the amino acid citrulline. Transglutaminase has been shown to be an important marker of normal differentiation. There is a rise in its activity at the time of keratinization, and transglutaminase activity has been shown to be greatly decreased in basal cell epithelioma and in psoriasis. Keratinocyte cell culture has proven most helpful in delineating the processes of normal differentiation and keratinization, since the formation of the cell envelope in culture appears to parallel the formation in vivo.

Abstract. Pemphigus vulgaris is an autoimmune disease associated with an autoantibody directed against a keratinocyte membrane antigen. The purpose ofthis study was to purify the human pemphigus vulgaris antigen, to produce an antibody to this antigen, and to use the antibody to induce pemphigus in newborn mice. Various techniques to extract the membrane-rich pellet from human epidermal homogenate were compared; 1% sodium dodecyl sulfate (SDS) and 1% dimethysulfoxide proved to be superior to extract the pemphigus vulgaris antigen. This antigen was identified by transfer blotting to nitrocellulose paper, incubated with pemphigus vulgaris serum, or 20 control sera, and detected with fluorescein labeled antisera to human IgG.Since concanavalin A inhibits the binding of pemphigus vulgaris antibody to tissue sections, we studied the binding of the extracted proteins to concanavalin A covalently coupled to Sepharose. Pemphigus vulgaris antigen bound to the concanavalin A column and was released by 0.02 M methyl a-D-mannopyranoside. The proteins thus recovered were subjected to AcA 54 gel permeation chromatography, and the pemphigus antigen was detected by the transfer blot assay. The antigen corresponded to a discrete peak at 66,000 D by gel permeation and gave one homogenous band at 33,000 D in urea-SDS-polyacrylamide gel electrophoresis. Monospecific antibody to the antigen raised in rabbits stained human epidermis in the same manner as the pemphigus vulgaris autoantibody and induced pemphigus vulgaris in newborn