Crystal structure of a chimeric Fab' fragment of an antibody binding tumour cells.

The crystal structure of a chimeric Fab' fragment of a monoclonal antibody is presented. The Fab' comprises the murine light chain and heavy chain variable domains of the carcinoma-binding antibody B72.3 fused to the constant domain of human kappa, and the first constant domain and hinge domain of human gamma 4, respectively. A model for the Fab' has been determined by molecular replacement and refined to a resolution of 3.1 A with an R-factor of 17.6%. The additional residues that distinguish a Fab' from a Fab fragment are seen to be disordered in the crystals. The H3 hypervariable loop is short and adopts a sharp hairpin turn in a conformation that results from an interaction between the lysine side-chain of H93 and the main-chain carbonyl group of H96. The remaining hypervariable loops display conformations similar to those predicted from the canonical structures approach, although loop H2 is apparently displaced by a salt-bridge formed between H55 Asp and the neighbouring H73 Lys. These and other features of the structure likely to be important in grafting the hypervariable loops to an otherwise human framework are discussed.     

Concentration and detection of hepatitis A virus and rotavirus from shellfish by hybridization tests.

A modified polyethylene glycol precipitation method for concentration of virus followed by a new method to recover nucleic acid was used to detect hepatitis A virus (HAV) and rotavirus (SA11) in shellfish (oysters and hard-shell clams) by hybridization tests. Infectious virus, seeded into relatively large quantities of shellfish, was recovered consistently, with greater than 90% efficiency as measured by either in situ hybridization (HAV) or plaque assay (rotavirus SA11). Viral nucleic acid for dot blot hybridization assays was extracted and purified from virus-containing polyethylene glycol concentrates. Separation of shellfish polysaccharides from nucleic acid was necessary before viral RNA could be detected by dot blot hybridization. Removal of shellfish polysaccharides was accomplished by using the cationic detergent cetyltrimethylammonium bromide (CTAB). Use of CTAB reduced background interference with hybridization signals, which resulted in increased hybridization test sensitivity. After polysaccharide removal, dot blot hybridization assays could detect approximately 10(6) physical particles (corresponding to approximately 10(3) infectious particles) of HAV and 10(4) PFU of SA11 rotavirus present in 20-g samples of oyster and clam meats. These studies show continuing promise for the development of uniform methods to directly detect human viral pathogens in different types of shellfish. However, practical applications of such methods to detect noncultivatable human viral pathogens of public health interest will require additional improvements in test sensitivity.     

Biological activities of melanotropic peptide fatty acid conjugates.

Four fatty acids (FA, palmitic, myristic, decanoic, hexanoic) were individually conjugated to the N-terminus of the alpha-MSH fragment analog, H-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10-NH2. This resulted in enhanced potency of the conjugates (compared to the unconjugated melanotropin analog) as determined in the lizard skin bioassay and in the mouse melanoma cell tyrosinase bioassay. The shorter conjugates of hexanoic and decanoic acid were at least equipotent to alpha-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogs were 100 times less active. The myristoyl and palmitoyl conjugates exhibited a "creeping" potency in the lizard skin bioassay-that is, potency of the peptides increased with time in contact with the skins. These observations may be related to the more lipid nature of these FA-conjugates. In the tyrosinase assay, the conjugates were 10-100 times more active than alpha-MSH or the unconjugated analog. Each of the FA-melanotropic peptide conjugates exhibited prolonged (residual) melanotropic activity in both the lizard skin and melanoma cell bioassays. In other words, after removal of the melanotropin conjugates from contact with the skins or cells, responses were still manifested for hours or days thereafter. As little as 1 hr of contact with melanoma cells resulted in enhanced enzyme activity as measured 48 hr later. Since the conjugates, but not H-[Asp5, D-Phe7, Lys10]alpha-MSH5-10-NH2, exhibited prolonged activity, the conversion of reversible agonists to irreversible agonists was demonstrated.     

Post-translational modification of a monocyte-specific chemoattractant synthesized by glioma, osteosarcoma, and vascular smooth muscle cells

Chemotaxis is an important step in monocyte recruitment in inflammation, wound healing, and tumor growth. We reported previously that monocyte chemotactic activity secreted by malignant cells and normal smooth muscle cells is associated with a protein or family of proteins that are related to the monocyte-specific smooth muscle cell-derived chemotactic factor (SMC-CF) (Graves, D. T., Jiang, Y. L., Williamson, M. J., and Valente, A. J. (1989) Science 245, 1490-1493). Similar monocyte chemotactic proteins (MCP-1) produced by U-105MG human glioma cells have also been identified (Yoshimura, T., Robinson, E. A., Tanaka, S., Appella, E., Kuratsu, J., and Leonard, E. J. (1989) J. Exp. Med. 169, 1449-1459). We now report that the MCP-1 gene is expressed in MG-63 human osteosarcoma and vascular smooth muscle cells and that SMC-CF antiserum specifically immunoprecipitates proteins synthesized by U-105MG glioma cells. Experiments were undertaken to elucidate the processing pathway of MCP-1/SMC-CF-like proteins in each of these cell types. These experiments demonstrate that larger MCP-1/SMC-CF-like proteins are derived from a Mr = 9000 precursor. Post-translational modification involves the addition of O-linked carbohydrates and sialic acid residues. Differences in carbohydrate processing account for the heterogeneity in MCP-1/SMC-CF-like proteins produced by different cell types. Secretion of these proteins occurs rapidly following processing events in the endoplasmic reticulum-Golgi compartment.     

Immuno-electron microscopical studies on the retinal basement membrane of chick embryo

Retinal basement membrane (RBM), also called inner limiting membrane of retina, is constituted by extracellular matrix. It was reported that neurite outgrowth of a neuron was closely related to extracellular matrix, particularly the laminin. In this laboratory RBM was used as the optimal substrate for retinal cells in culture. We have studied the surface of RBM and its relation to neurite outgrowth by scanning electronmicroscopy and immunogold transmission electronmicroscopy. RBM could be separated by mechanical disruption of the retina mounted between 2 adhesive substrata (membrane filter and poly-L-lysine coated glass). The surface of RBM studied was the side of RBM facing the optic fiber layer and ganglion cell layer. Small particles densely distributed on surface of RBM (Plate I, Fig. 1 and 2) were shown to be chrysanthemum-like structures with radiative arms under the scanning electronmicroscopy (Plate I, Fig. 3 and 4). The radiative arms of RBM of 12-day old chick embryo (E 12) were more in number and longer in length than that of the 6-day old chick embryo (E 6). The axons of ganglion cell from E 6 retinal strip extended out very well on RBM (Plate I, Fig. 5). Growth cone was active with filopodia. The chrysanthemum-like structures changed to ball-particles when the RBM was cultured for 24 hr. Some of ball-particles lay over the growth cone, and some beside it. Over and beside the nerve fiber could also be seen some ball-particles. When many neurites grew on RBM, a lot of ball-particles were shown to be displaced and piled up (Plate I, Fig. 6). The whole amount RBM labeled by indirect immunogold staining of Müller glial cell could be observed by transmission electronmicroscopy. The gold particles wer located at the chrysanthemum-like structure of E 6 RBM (Plate II, Fig. 7) and E 12 RBM (Plate II, Fig. 8). It was suggested that those structures were the end foot of Müller glial cells. Staining of PBS control or mouse serum control was negative (Plate II, Fig. 9 and 10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Long noncoding RNA lncMREF promotes myogenic differentiation and muscle regeneration by interacting with the Smarca5/p300 complex

Long noncoding RNAs (lncRNAs) play important roles in the spatial and temporal regulation of muscle development and regeneration. Nevertheless, the determination of their biological functions and mechanisms underlying muscle regeneration remains challenging. Here, we identified a lncRNA named lncMREF (lncRNA muscle regeneration enhancement factor) as a conserved positive regulator of muscle regeneration among mice, pigs and humans. Functional studies demonstrated that lncMREF, which is mainly expressed in differentiated muscle satellite cells, promotes myogenic differentiation and muscle regeneration. Mechanistically, lncMREF interacts with Smarca5 to promote chromatin accessibility when muscle satellite cells are activated and start to differentiate, thereby facilitating genomic binding of p300/CBP/H3K27ac to upregulate the expression of myogenic regulators, such as MyoD and cell differentiation. Our results unravel a novel temporal-specific epigenetic regulation during muscle regeneration and reveal that lncMREF/Smarca5-mediated epigenetic programming is responsible for muscle cell differentiation, which provides new insights into the regulatory mechanism of muscle regeneration.