Expression, purification, and functional testing of recombinant CYR61/CCN1

The human cysteine-rich protein 61 (CYR61/CCN1) belongs to the CCN family of genes which plays an important role in cellular processes such as proliferation, migration, adhesion, and differentiation. These extracellular matrix signaling molecules consist of a modular structure and contain 38 conserved cysteine residues. Previously, we have shown that CYR61 is expressed in human osteoblasts and is regulated by bone-relevant growth factors. The protein also plays a role in angiogenesis. The open reading frame was cloned into a baculovirus expression vector and transfected into SF-21 insect cells. Recombinant protein was expressed as a fusion protein with the Fc-domain of human IgG and purified using affinity chromatography on protein G-Sepharose columns. The chorioallantoic membrane assay verified that blood vessel formation was stimulated by rCYR61. Additionally, human primary mesenchymal stem cells, osteoblasts, and endothelial cells responded to CYR61 treatment by a markedly stimulated proliferation. rCYR61-Fc represents a tool to elucidate its role in cells of the bone microenvironment.     

Genetic control and carrier and suppressor effects in the antibody response of mice to procollagen

The response potential of inbred mouse strains against bovine type I procollagen and its separated structural domains, i.e., the globular procollagen peptide and the triple-helical collagen moiety, was compared by passive hemagglutination and radioimmune assays. Studies with congenic and recombinant lines and with backcross populations showed that the antibody response to procollagen peptide is under the control of a gene located in theIA, IB subregion of theH-2 complex. The strain distribution pattern of high response to the procollagen peptide is not identical to that of the high response to collagen. Both the procollagen peptide and procollagen are thymus-dependent antigens, since no response was observed in nude mice. Low response to procollagen peptide and/or collagen could be corrected in some, but not all mouse strains by using procollagen as immunogen. Strains which show low response against collagen but high response against procollagen were injected with collagen prior to a challenge with procollagen. This treatment reduced the antibody response to the procollagen peptide but not to collagen. Carrier and suppressor effects in the response to procollagen are apparently more complex than those observed in the response to synthetic peptides.     

The major histocompatibility complex of the rat,RT1

The antigenic determinants expressed on RBC and lymphocytes and coded for by the MHC, RT1,of the MNR (RT1 ( m )) rat strain were compared to those of the BN.DA(RT1 ( a )), ALB (RT1 ( b )), and AUG (RT1 ( c )) strains by direct cytotoxicity and absorption analysis with RT1 typing sera, sera produced against MNR cells, and sera produced in MNR responders against cells carrying thea, b, andc haplotype determinants. The results indicate that MNR shares major class I (A) antigens with DA, and major class II (B) determinants with AUG, but that MNR differs from DA and AUG with respect to both classes of determinant. It appears, therefore, that the MNR haplotype does not represent a simple composite of the two other haplotypes,RT1 ( a ) andRT1 ( c ), as reported earlier.     

Palmitoylation of CD95 facilitates formation of SDS-stable receptor aggregates that initiate apoptosis signaling

Apoptosis signaling through CD95 (Fas/APO-1) involves aggregation and clustering of the receptor followed by its actin-dependent internalization. Internalization is required for efficient formation of the death-inducing signaling complex (DISC) with maximal recruitment of FADD, caspase-8/10 and c-FLIP occurring when the receptor has reached an endosomal compartment. The first detectable event during CD95 signaling is the formation of SDS-stable aggregates likely reflecting intense oligomerization of the receptor. We now demonstrate that these SDS-stable forms of CD95 correspond to very high molecular weight DISC complexes (hiDISC) and are the sites of caspase-8 activation. hiDISCs are found both inside and outside of detergent-resistant membranes. The formation of SDS-stable CD95 aggregates involves palmitoylation of the membrane proximal cysteine 199 in CD95. Cysteine 199 mutants no longer form SDS-stable aggregates, and inhibition of palmitoylation reduces internalization of CD95 and activation of caspase-8. Our data demonstrate that SDS-stable forms of CD95 are the sites of apoptosis initiation and represent an important early step in apoptosis signaling through CD95 before activation of caspases.     

Insights into polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium

A novel levansucrase was identified in the supernatant of a cell culture of Bacillus megaterium DSM319. In order to test for the contribution of specific amino acid residues to levansucrase catalysis, the wild-type enzyme along with 16 variants based on sequence alignments and structural information were heterologously produced in Escherichia coli. The purified enzymes were characterized kinetically and the product spectrum of each variant was determined. Comparison of the X-ray structures of the levansucrases from Gram-positive Bacillus subtilis and Gram-negative Gluconacetobacter diazotrophicus in conjunction with the corresponding product spectra identified crucial amino acid residues responsible for product specificity and catalysis. Highly conserved regions such as the previously described RDP and DXXER motifs were identified as being important. Two crucial structural differences localized at amino acid residues Arg370 and Asn252 were of high relevance in polymer compared with oligosaccharide synthesis.     

The IMiD target CRBN determines HSP90 activity toward transmembrane proteins essential in multiple myeloma

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