Tissue form of type VII collagen from human skin and dermal fibroblasts in culture

The triple-helical domain of type VII collagen was isolated from human placental membranes by mild digestion with pepsin, and polyclonal antibodies were raised in rabbits against this protein. After affinity purification the antibodies specifically recognized type VII collagen in both the triple-helical and the unfolded state. They also reacted with the fragments P1 and P2, derived from the triple-helical domain by further proteolysis with pepsin, but did not crossreact with other biochemical components of the dermal connective tissue. In skin the presence of a fragment of type VII collagen, similar to that isolated from placenta, was demonstrated by SDS-PAGE and immunoblotting. Type VII collagen represented less than 0.001% of the total collagen extracted by pepsin digestion from newborn or adult skin. The tissue form of type VII collagen was obtained from dermis after artificial epidermolysis with strongly denaturing buffers under conditions reducing disulfide bonds. The protein was identified by immunoblotting with the antibodies. The molecule was composed of three polypeptides with an apparent molecular mass of about 250 kDa, each. Similar large-molecular-mass chains could be identified by immunoblotting in extracts of human fibroblasts in culture.     

Structural and electronic properties of the liver fluke hemoglobin heme cavity by nuclear magnetic resonance: hemin isotope labeling

Reconstitution of liver fluke (Dicrocoelium dendriticum) apo-hemoglobin with hemins selectively deuterated at specific positions has permitted the assignment of several heme resonances in the proton nuclear magnetic resonance spectrum of the Met-aquo and Met-cyano forms of the holoprotein. It was established that in the Met-aquo form the meso protons resonate at positions characteristic of a six-co-ordinated in-plane iron. From this, we deduced that the Met-aquo species retains a bound water molecule at pH values as low as 4.5. The orientation of the proximal histidine imidazole ring with respect to the heme group in the cavity was determined through the identification of the heme methyl signals and the analysis of the hyperfine shift pattern in the Met-cyano hemoglobin proton nuclear magnetic resonance spectrum. Compared to sperm whale myoglobin, the heme appears to be rotated by 180 degrees about the alpha, gamma meso-axis. Protein isomers with the heme group in a reversed orientation were not detected, even shortly after reconstitution. In the Met-cyano form, the resonances most affected by the Bohr transition were shown to arise from the heme propionates.     

The globular domains of type VI collagen are related to the collagen-binding domains of cartilage matrix protein and von Willebrand factor.

Type VI collagen is a transformation-sensitive glycoprotein of the extracellular matrix of fibroblasts. We have isolated and sequenced several overlapping cDNA clones (4153 bp) which encode the entire alpha 2 subunit of chicken type VI collagen. The deduced amino acid sequence predicts that the alpha 2(VI) polypeptide consists of 1015 amino acid residues that are arranged in four domains: a hydrophobic signal peptide of 20 residues, an amino-terminal globular domain of 228 residues, a collagenous segment of 335 residues and a carboxy-terminal globular domain of 432 residues. The collagenous domain contains seven Arg-Gly-Asp tripeptide units, some of which are likely to be used as cell-binding sites. The globular domains contain three homologous repeats with an average length of 180 amino acid residues. These repeats show a striking similarity to the collagen-binding motifs found in von Willebrand factor and cartilage matrix protein. We therefore speculate that the globular domains of the alpha 2(VI) polypeptide may interact with collagenous structures.     

广东哺乳类及两栖类寄生吸虫四新种的记述（吸虫纲：蹲茎科,蛙蠕科）

作者在广东省梅县市发现四个吸虫新种:1.鼠丽色吸虫,新种Reesella ratta sp. nov., 2.后囊蛙蠕吸虫,新种Batrachotrema optstosacca sp. nov., 3.梅县后平睾吸虫,新种Opisthioparorchis meixianensis sp. nov., 4.大卵后平睾吸虫,新种Opisthioparorchis megaloonos sp. nov.,它们分别归隶于蹲茎科Cathaemasiidae及蛙蠕科Bstrachotrematidae。模式标本保存于中山医科大学寄生虫学研究所。     

Regulation of type VI collagen synthesis in transformed mesenchymal cells.

We have analysed the effects of oncogenic transformation on the expression of type VI collagen in mesenchymal cells. Synthesis of type VI collagen was almost completely inhibited in fibroblasts transformed by DNA or RNA tumour viruses or in cells derived from spontaneous mesenchymal tumours. Inhibition of type VI collagen synthesis appears, therefore, to be a common phenomenon of transformed mesenchymal cells. When introduced into normal cells by viral vectors, the 'nuclear' oncogene v-myc had an inhibitory effect similar to that of the 'cytoplasmic' oncogene v-src. Fibroblasts infected with a temperature-sensitive strain of Rous sarcoma virus (NY68) produced type VI collagen at the restrictive, but not at the permissive temperature. If such cells were shifted from the permissive to the restrictive temperature, synthesis of the individual subunits of type VI collagen was co-ordinately induced. These results demonstrate that the activity of a single oncogene product is sufficient to inhibit type VI collagen expression.     

D-periodic distribution of collagen type IX along cartilage fibrils

It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril.     

Encapsulation of hemoglobin in phospholipid liposomes: characterization and stability

Hemoglobin is encapsulated in liposomes of different lipid composition. The resulting dispersion consists primarily of multilamellar liposomes (hemosomes) of a wide particle size distribution (diameter ranging mainly between 0.1 and 1 micron). The encapsulation efficiency is significantly larger with liposomes containing negatively charged lipids as compared to liposomes made of phosphatidylcholine. The integrity of the phospholipid bilayer is maintained in the presence of hemoglobin. The reaction rate of CO binding to encapsulated hemoglobin is reduced compared to that of free hemoglobin, but it is still greater than that observed in red blood cells. Hemoglobin encapsulated in liposomes made from negatively charged phospholipids is less stable than hemoglobin entrapped in isoelectric phosphatidylcholine. The instability of hemoglobin is due to the protein interacting with the negatively charged lipid bilayer. This interaction leads in turn to hemoglobin denaturation, possibly involving the dissociation of the heme group from the heme-globin complex. The nature of the negatively charged phospholipid is important in promoting the interaction with hemoglobin, the effect being in the order phosphatidic acid greater than phosphatidylinositol congruent to phosphatidylglycerol greater than phosphatidylserine. The presence of equimolar amounts of cholesterol in the phospholipid bilayer has a stabilizing effect on hemoglobin. This effect is pronounced with saturated phospholipids, but it is also observed, though to a lesser extent, with unsaturated ones, indicating that the bilayer fluidity has a modulating effect. The presence of cholesterol possibly interferes with secondary interactions following the binding of hemoglobin to the negatively charged lipid bilayer.     

Kinetics of oxygen and carbon monoxide binding to liver fluke (Dicrocoelium dendriticum) hemoglobin. An extreme case?

The kinetics of oxygen and carbon monoxide binding to the monomeric liver fluke (Dicrocoelium dendriticum) hemoglobin have been studied. The ligand association rates are approximately 1 X 10(8) and approximately 3 X 10(8) M-1 s-1, respectively, for CO and O2 and show no pH dependence. On the contrary the ligand dissociation rates decrease by lowering the pH below 7, the pK of the transition being around 5.5. These findings, together with spectroscopic properties of the protein, are discussed in relation to the fact that, in this hemoglobin, the distal histidine is replaced by a glycine.     

Cartilage contains mixed fibrils of collagen types II, IX, and XI

The distribution of collagen XI in fibril fragments from 17-d chick embryo sternal cartilage was determined by immunoelectron microscopy using specific polyclonal antibodies. The protein was distributed throughout the fibril fragments but was antigenically masked due to the tight packing of collagen molecules and could be identified only at sites where the fibril structure was partially disrupted. Collagens II and IX were also distributed uniformly along fibrils but, in contrast to collagen XI, were accessible to the antibodies in intact fibrils. Therefore, cartilage fibrils are heterotypically assembled from collagens II, IX, and XI. This implies that collagen XI is an integral component of the cartilage fibrillar network and homogeneously distributed throughout the tissue. This was confirmed by immunofluorescence.     

Induction and prevention of chondrocyte hypertrophy in culture

Primary chondrocytes from whole chick embryo sterna can be maintained in suspension culture stabilized with agarose for extended periods of time. In the absence of FBS, the cells remain viable only when seeded at high densities. They do not proliferate at a high rate but they deposit extracellular matrix with fibrils resembling those of authentic embryonic cartilage in their appearance and collagen composition. The cells exhibit many morphological and biochemical characteristics of resting chondrocytes and they do not produce collagen X, a marker for hypertrophic cartilage undergoing endochondral ossification. At low density, cells survive in culture without FBS when the media are conditioned by chondrocytes grown at high density. Thus, resting cartilage cells in agarose cultures can produce factors required for their own viability. Addition of FBS to the culture media leads to profound changes in the phenotype of chondrocytes seeded at low density. Cells form colonies at a high rate and assume properties of hypertrophic cells, including the synthesis of collagen X. They extensively deposit extracellular matrix resembling more closely that of adult rather than embryonic cartilage.