Evidence of heterogeneity of protein-turnover states in cultured cells.

Previous studies on L-cell cultures [Amenta & Sargus (1979) Biochem. J. 182, 847--859] have suggested: (a) that degradation of slow-turnover proteins occurs in a distinct cell state (D-state); (b) that cells randomly enter the D-state with a first-order transition constant, rapidly degrade cell protein, and return to a quiescent G0-state. In the present study we have tested the hypothesis that the putative D-state exists as a substate within A-state (non-replicating) fibroblasts. Rat-embryo fibroblasts were prelabelled with [14C]leucine and [3H]thymidine, 'chased' for 24 h, and then placed in fresh growth medium containing either vinblastine (10 microM) or colchicine (25 microM) for three successive 24 h periods. Cells trapped in mitosis were separated from the residual non-replicating cells and rates of protein synthesis, degradation and net accumulation were measured in both populations. We observed that significant protein degradation occurred only in the non-replicating population, although both populations showed equally high rates of protein synthesis induced by fresh growth medium. These data support the hypothesis that degradation of slow-turnover protein is heterogeneous, occurring only in A-state cells. A model that proposes a separate D-state within G0-phase successfully accounts for these observations and previous reports on this cell line [Amenta, Sargus & Baccino (1978) J. Cell. Physiol. 97, 267--283] showing no differences in degradation of the slow-turnover protein pool in growth-stimulated and stationary-phase fibroblast cultures.     

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.     

In the mammalian eye type VI collagen tetramers form three morphologically different aggregates.

The organization of the aggregates occurring in the stroma: (1) of the murine and human cornea after incubation in an ATP acidic solution; (2) of surgically excised epiretinal membranes (ERM); and (3) of the trabecular meshwork of monkey eyes was investigated morphologically and immunocytochemically on thin section electron microscopy. Morphology. The aggregates in the cornea appeared as cross-banded fibrils. The bands were uniformly electron dense (single banded form); they were separated from each other by interbands consisting of a bundle of filaments emerging in cross section as small areas of randomly assembled dot-like structures. In the ERM, most of the aggregates stood out as heteromorphic cross-banded bodies showing dense bands with electron denser borders (double banded form) and interbands composed of longitudinally oriented, parallel sheets or laminae of amorphous material enclosing thin, similarly oriented filaments. These extended, thinner and double in number (since interlacing with similar components of the opposite sheet), into the pale central zone of the dense band. The aggregates of the trabecular meshwork were heteromorphic, had uniformly dense bands (single banded form as in the cornea), but their interbands displayed longitudinal sheets (as the ERM aggregates). Immunocytochemistry revealed type VI collagen in the three eye aggregates with gold particles preferentially localized at the interbands. The specificity of the antibodies used was tested by Western blot analysis of type VI collagen samples extracted from human placenta and on homogenates of human cornea. In conclusion, the results indicate that the tetramers of type VI collagen may aggregate differently into structures with distinct supramolecular arrangements. These are illustrated in schematic drawings.     

Evidence for highly stable nuclear poly(A) in cultured mammalian cells.

The kinetics of turnover of nuclear poly(A) were determined under conditions which facilitated the detection of relatively stable classes of the molecule. Growing 3T6 or HeLa cells were labeled with [3H]adenosine for several hours. The turnover of nuclear poly(A) was then followed over long time intervals using a variety of chase conditions. When a cordycepin chase was employed, a class of nuclear poly(A) with a half life of 2.5 h was observed. When the chase was effected by allowing the intracellular ATP pool specific activity to decay as a result of normal metabolic processes, a more stable class of nuclear poly(A) was detected (half life = 8--12 h). These results indicate that a significant portion of poly(A)-hnRNA has a long half-life.     

Isolation from bovine elastic tissues of collagen type VI and characterization of its form in vivo.

Foetal-bovine nuchal ligament and aorta, together with adult-bovine aorta and pregnant uterus, were extracted under dissociative conditions in the absence and in the presence of a reducing agent. A collagenous glycoprotein of Mr 140000 [designated component 140K(VI)], identified in these extracts as the major periodate/Schiff-positive component, was shown to be related to collagen type VI. Digestion of non-reduced extracts with pepsin yielded periodate/Schiff-positive peptides that, on the basis of their electrophoretic mobilities, amino acid analyses and peptide 'maps', were identical with type VI collagen fragments prepared by standard procedures. It is concluded that collagen type VI occurs in vivo as molecule comprising three chains of Mr 140000 in which the helical domains account for about one-third of each polypeptide. Biosynthetic experiments with nuchal-ligament fibroblasts in culture demonstrated that a bacterial-collagenase-sensitive [3H]fucose-labelled glycoprotein, Mr 140000, was immunoprecipitated from culture medium by a specific antibody to the pepsin-derived form of collagen type VI. This result suggests that the collagenous polypeptides [140K(VI) components] represent the biosynthetic precursors of type VI collagen that do not undergo processing to smaller species before deposition in the extracellular matrix. Analyses of 5M-guanidinium chloride extracts of tissues with markedly different elastin contents and at different stages of development suggested that there was no relationship between collagen type VI and elastic-fibre microfibrils, a conclusion supported by the observation that the immunoprecipitated glycoprotein, Mr 140000, was distinct from the glycoprotein MFPI, Mr 150000, believed to be a constituent of these microfibrils [Sear, Grant & Jackson (1981) Biochem. J. 194, 587-598].     

Molecular assembly, secretion, and matrix deposition of type VI collagen

Monoclonal antibodies reactive with the tissue form of type VI collagen were used to isolate the type VI collagen polypeptides from cultured fibroblasts and muscle cells. Two [35S]methionine-labeled polypeptides of 260 and 140 kD were found intracellularly, in the medium, and in the extracellular matrix of metabolically labeled cells. These polypeptides were disulfide cross-linked into very large complexes. The 260- and 140-kD polypeptides were intimately associated and could not be separated from each other by reduction without denaturation. In the absence of ascorbic acid, both polypeptides accumulated inside the cell, and their amounts in the medium and in the matrix were decreased. These results suggest that both the 260- and the 140-kD polypeptides are integral parts of the type VI collagen molecule. Examination of type VI collagen isolated from the intracellular pool by electron microscopy after rotary shadowing revealed structures corresponding to different stages of assembly of type VI collagen. Based on these images, a sequence for the intracellular assembly of type VI collagen could be discerned. Type VI collagen monomers are approximately 125 nm long and are composed of two globules separated by a thin strand. The monomers assemble into dimers and tetramers by lateral association. Only tetramers were present in culture media, whereas both tetramers and multimers were found in extracellular matrix extracts. The multimers appeared to have assembled from tetramers by end-to-end association into filaments that had prominent knobs and a periodicity of approximately 110 nm. These results show that, unlike other collagens, type VI collagen is assembled into tetramers before it is secreted from the cells, and they also suggest an extracellular aggregation mechanism that appears to be unique to this collagen.     

Anticlastogenicity in cultured mammalian cells.

Basic and applied research on anticlastogenicity has not only revealed valuable evidence on the mechanisms governing the induction of chromosomal aberrations by environmental mutagens, but also contributed effective ideas on a practical employment of this knowledge for the protection of individuals at risk. Considering the basic role played by chromosomal anomalies in oncogenesis, additional weight must be attributed to studies on anticlastogenicity. The employment of human cells in this kind of study dates back to 1969/70, while classical mammalian cell systems were used only later on. Various modes of application of both clastogens and anticlastogens (AC) were examined, but simultaneous addition to the cultures of both reagents was the most favored way. A wide spectrum of cytogenetic endpoints can be studied, but differences can be demonstrated with regard to efficacy of inhibitors on different types of cytogenetic changes, e.g., open breaks vs. rearrangements, but also vs. SCEs. Depending on their mode of influence on this spectrum, ACs can be arranged in various categories which are of practical importance, for instance, with regard to their oncogenic potential. A wide variety of factors was shown to influence AC action, e.g., time and mode of application of the test substances, physiologic and metabolic features of the cell types studied, type and mechanism of the clastogen used, etc. The addition of S9 mix can drastically change the patterns of efficacy of the ACs. The combined application of two or more ACs, as far as investigated, apparently neither potentiates nor even merely adds their effects.     

Isolation of biogenetically competent mitochondria from mammalian tissues and cultured cells

This article describes a quick basic method adapted for the purification of mammalian mitochondria from different sources. The organelles obtained using this protocol are suitable for the investigation of biogenetic activities such as enzyme activity, mtDNA, mtRNA, mitochondrial protein synthesis, and mitochondrial tRNA aminoacylation. In addition, these mitochondria are capable of efficient protein import and the investigation of mtDNA/protein interactions by DNA footprinting is also possible.     

Basement membrane zone type XV collagen is a disulfide-bonded chondroitin sulfate proteoglycan in human tissues and cultured cells

Type XV collagen has a widespread distribution in human tissues, but a nearly restricted localization in basement membrane zones. The alpha1(XV) chain contains a highly interrupted collagenous region of 577 residues, and noncollagenous amino- and carboxyl-terminal domains of 530 and 256 residues, respectively. Cysteines are present in each domain and consensus sequences for O-linked glycosaminoglycans are situated in the amino terminus and in two large, noncollagenous interruptions. We now report that type XV collagen is a chondroitin sulfate proteoglycan in human tissues and cultured cells, and that the alpha chains are covalently linked by interchain disulfide bonds only between the two cysteines in the collagenous region. Western blotting of tissue extracts revealed a diffuse smear with a mean size >/=400 kDa, which after chondroitinase digestion resolved into a 250-kDa band in umbilical cord, and 250- and 225-kDa bands in placenta, lung, colon, and skeletal muscle. The latter two bands were also directly visualized by alcian blue/silver staining of a purified placenta extract. In a human rhabdomyosarcoma cell line, almost all of the newly synthesized type XV collagen was secreted into the medium and upon chondroitinase digestion just the 250-kDa alpha chain was generated. Chondroitinase plus collagenase digestion of tissue and medium proteins followed by Western blotting using domain-specific antibodies revealed a 135-kDa amino-terminal fragment containing glycosaminoglycan chains and a 27-kDa fragment representing the intact carboxyl terminus. However, a truncated carboxyl peptide of approximately 8-kDa was also evident in tissue extracts containing the 225-kDa form. Our data suggest that the 225-kDa form arises from differential carboxyl cleavage of the 250-kDa form, and could explain the approximately 19-kDa endostatin-related fragments (John, H., Preissner, K. T., Forssmann, W.-G., and St?ndker, L. (1999) Biochemistry 38, 10217-10224), which may be liberated from the alpha1(XV) chain.     

Molecular complementation of a collagen mutation in mammalian cells using yeast artificial chromosomes.

The cloning of large contiguous segments of mammalian DNA in Saccharomyces cerevisiae has become possible with the advent of Yeast Artificial Chromosomes (YACs). We are interested in extending the technique of genetic complementation analysis to the molecular level through the introduction of YACs into mammalian cells and the mammalian germline. We report the successful introduction of homogeneous DNA derived from a 150 kbp YAC spanning the murine Col1a1 locus into murine fibroblasts carrying a mutation at this locus. The YAC DNA was fractionated by pulse field electrophoresis, condensed with polyamines, and introduced into mutant fibroblasts via DNA-lipid micelles. The DNA was integrated as a stable intact unit in 10% of the transfected clones conferring collagen RNA expression to the mutant cells.     

Evidence for highly stable nuclear poly(A) in cultured mammalian cells

The kinetics of turnover of nuclear poly(A) were determined under conditions which facilitated the detection of relatively stable classes of the molecule. Growing 3T6 or HeLa cells were labeled with [³H]adenosine for several hours. The turnover of nuclear poly(A) was then followed over long time intervals using a variety of chase conditions. When a cordycepin chase was employed, a class of nuclear poly(A) with a half life of 2.5 h was observed. When the chase was effected by allowing the intracellular ATP pool specific activity to decay as a result of normal metabolic processes, a more stable class of nuclear poly(A) was detected (half life = 8–12 h). These results indicate that a significant portion of poly(A)-hnRNA has a long half-life.     

Molecular diversity in the adenylylcyclase family. Evidence for eight forms of the enzyme and cloning of type VI.

The conservation of amino acid sequence among types I-IV adenylylcyclase has made it possible to apply the polymerase chain reaction to examine the extent of the molecular diversity within this family of enzymes. cDNA templates from rat heart, liver, kidney, guinea pig brain and testes, and mouse skeletal muscle were amplified with primers specific to adenylylcyclase sequences. Evidence was obtained for a total of eight distinct gene products divisible into five subfamilies. Five of the products correspond to regions from cloned forms of adenylylcyclase, while three are previously unidentified. As many as seven different adenylylcyclases are expressed in rat heart, liver, and kidney based on this analysis. Two newly identified polymerase chain reaction (PCR) products were utilized to screen a rat cDNA library from H35 Reuber hepatoma cells. A 6080-nucleotide cDNA contains an open reading frame encoding the 1166-amino acid type VI protein which has a predicted topography similar to that of other adenylylcyclases. The type VI message is abundantly expressed in rat heart, kidney, and brain. Human embryonal kidney cells stably expressing the cDNA showed an enhanced response to isoproterenol that could be inhibited by carbachol in intact cells. Increases in intracellular Ca2+ contribute to the inhibitory effect of carbachol.     

Base composition heterogeneity of mammalian DNAs in CsCl-netropsin density gradient.

DNAs of 15 mammals and some lower organisms were analysed by CsCl-netropsin density gradient centrifugation. Increased resolving power of this method enabled to detect many new components in mammalian DNAs. Distinct components were detected in the density range of the main band. These components found in different mammalian DNAs have probably limited variation in the G+C content. Most of other components seems to be species specific. The DNAs of lower organisms form homogeneous band even in the presence of netropsin. The relation between densities in CsCl-netropsin and CsCl density gradient is nonlinear. This result supports a hypothesis that in high ionic strength netropsin is preferentially bound to (dA.dT) clusters.     

The distribution of type VI collagen in the developing tissues of the bovine femoral head

Type VI collagen appears central to the maintenance of tissue integrity. In adult articular cartilage, type VI collagen is preferentially localised in the chondron where it may be involved in cell attachment. In actively remodelling developing cartilage, the distribution is less certain. We have used confocal immunohistochemistry and in situ hybridisation to investigate type VI collagen distribution in third trimester bovine proximal femoral epiphyses. In general, type VI collagen immunofluorescence was concentrated in the chondrocyte pericellular matrix, with staining intensity strongest in regions which persist to maturity and weakest in regions that remodel during development. Type VI collagen was also present in cartilage canals. In the growth plate and around the secondary centre of ossification, the intensity of type VI collagen stain rapidly decreased with chondrocyte maturation and was absent at hypertrophy, except where canal branches penetrated the growth plate and stain was retained around the adjacent chondrocytes. In situ hybridisation confirmed the presence of type VI collagen mRNA in cartilage canal mesenchymal cells but the signal was low in chondrocytes, suggesting minimal levels of synthesis and turnover. The results are consistent with a role for type VI collagen in stabilising the extracellular matrix during development.     

胶原蛋白与Ⅵ型胶原蛋白的结构概述

描述了胶原蛋白的结构和Ⅵ型胶原蛋白的结构以及胶原蛋白的应用 ,并对人类胶原蛋白的生产进行了展望。     

Evidence for proteins specific for vascular elements in intact and cultured tissues and cells of maize

A group of antigenically distinct proteins characteristic for the tissue complex of the vascular cylinders was found in maize (Zea mays L.) seedlings using an immunofiltration technique. Specific stelar antigens present in the fully developed stele (vascular cylinder) of the primary root were also found in steles extracted from adventitious roots and from the mesocotyl but were absent, within the limits of sensitivity of the immunodiffusion tests employed, in root cortex and epidermis. Some of the stelar antigens were also evident in the meristem of the primary root and were present in traces in the scutellum, the mesocotyl node, and the primary leaves plus coleoptile. The specific stelar antigens could be traced in 13- and 15-day-old developing embryos and were definitely expressed by the 21 st day after pollination. Several stelar-specific antigens were found in embryo-derived callus tissues and in stem-derived cells maintained in serial suspension culture. Higher resolution of the stelar antigens by a modified technique of crossed immunoelectrophoresis was used to demonstrate several minor stelar antigens that were presumably characteristic exclusively of the completely differentiated stele. This technique along with sequential immunoprecipitation of labelled proteins provided a semiquantitative estimate of the specific stelar antigens in the meristem and the stele of the primary root, and in suspension-cultured cells which were devoid of noticeable signs of vascular differentiation.     

Interaction of intact type VI collagen with hyaluronan.

The capacity of non-pepsinyzed type VI collagen to bind to hyaluronan was investigated. Type VI collagen was extracted from bovine meniscal cartilage with 6 M GuHCl and purified by extraction of PEG precipitates and dissociative Sephacryl S-500 HR chromatography. Type VI collagen, detected with a monoclonal antibody, bound in 0.5 M NaCl to hyaluronan-coated micro-wells, the degree of binding being higher at 37 degrees C than 23 degrees C and 4 degrees C. Incubation of type VI collagen in competitive inhibition assays with testicular hyaluronidase digests of hyaluronan in liquid phase, reduced binding of the protein to hyaluronan-coated microwells to background levels. Thus, non-pepsinyzed type VI collagen binds to hyaluronan in vitro.     

Production of type VI collagen by human macrophages: a new dimension in macrophage functional heterogeneity

Macrophages derived from human blood monocytes perform many tasks related to tissue injury and repair. The main effect of macrophages on the extracellular matrix is considered to be destructive in nature, because macrophages secrete metalloproteinases and ingest foreign material as part of the remodeling process that occurs in wound healing and other pathological conditions. However, macrophages also contribute to the extracellular matrix and hence to tissue stabilization both indirectly, by inducing other cells to proliferate and to release matrix components, and directly, by secreting components of the extracellular matrix such as fibronectin and type VIII collagen, as we have recently shown. We now report that monocytes and macrophages express virtually all known collagen and collagen-related mRNAs. Furthermore, macrophages secrete type VI collagen protein abundantly, depending upon their mode of activation, stage of differentiation, and cell density. The primary function of type VI collagen secreted by macrophages appears to be modulation of cell-cell and cell-matrix interactions. We suggest that the production of type VI collagen is a marker for a nondestructive, matrix-conserving macrophage phenotype that could profoundly influence physiological and pathophysiological conditions in vivo.