1H NMR study of the interaction of bacteriophage λ Cro protein with the O R 3 operator

The 17 base pair operator O _R ³ oligonucleotide, which is the preferential binding site for the Cro repressor of phage , was studied by two-dimensional NMR spectroscopy. A sequential assignment procedure based on two-dimensional Nuclear Overhauser Effect (NOESY) and scalar coupling correlated (COSY) NMR spectroscopy, together with the knowledge of the oligodesoxynucleotide sequence, made it possible to assign the non-exhangeable base protons and the H1 and the H2-H2 sugar protons of the O _R ³ operator DNA. The pattern of the observed NOE connectivities is consistent with a right-handed helical DNA structure. The base and sugar proton assignments provide the necessary information for further studies of the O _R ³ operator — Cro repressor interaction.Abbreviations COSY correlated spectroscopy - FID free induction decay - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect spectroscopy - RD relaxation delay - TSP sodium 3-trimethylsilyl-(2,2,3,3-²H₄)propionate - EDTA sodium ethylendiamine tetraacetate     

Immunofluorescence staining of thin-filament sections not participating in actomyosin crossbridges: Studies by use of a monoclonal antibody specific to actin

Summary Monoclonal antibodies (mcab) were produced in vitro by fusing mouse X63-Ag8.653 plasmacytoma cells with spleen cells from a Balb/c mouse immunized with primary cultures of chick skeletal muscle (pmcc). After cloning on agar, stable clones were obtained, the antibodies of which stain specifically the I-band of myofibrils in the immunofluorescence (IF) procedure. For further characterization of these mcab their affinities to muscle proteins were tested by immunoblotting and by enzyme-linked immunosorbent assay (ELISA). Mcab specific for actin were revealed by these criteria. One of the anti-actin antibodies, mcab 647, reveals a variety of IF-staining patterns on myofibrils. On rest-length myofibrils the I-band is labeled only. However, at sarcomere lengths below 2 m, where the thin filaments meet in the middle of the A-band and form a region of double overlap, an additional fluorescent band appears in this position. The fluorescence intensity of this band is increased significantly in shorter sarcomeres. Finally, when the I-band has disappeared at a sarcomere length of 1.5 m, fluorescence is located exclusively in the middle of the A-band. These IF-staining patterns suggest that only those sections of the thin filament are stained that do not participate in actomyosin crossbridges.     

Microfibrils, elastic anchoring components of the extracellular matrix, are associated with fibronectin in the zonule of Zinn and aorta

Microfibrils are striated tubules that play a role in the formation of elastin fibers by providing a scaffold upon which newly synthesized elastin is deposited. Ultrastructural and staining studies also demonstrate microfibrils that terminate where elastin is sparse or absent in basal laminae, plasma membranes, and the collagenous matrix. The most striking accumulation of microfibrils is found in the zonule of Zinn, the transparent and elastic suspensory ligament of the lens, which contains no elastin. Application of immunocytochemical staining with a peroxidase-antiperoxidase (PAP) procedure demonstrates that fibronectin is associated with the microfibrils of the zonule and aorta. Aggregates of microfibrils are identical to oxytalan ('acid enduring') fibers that have been described in peridontal membranes and other sites subject to mechanical stress and they can be found in sites as disparate as the rabbit zonule, rat hepatic stroma and human cardiac papillary muscle, indicating that microfibrils are a widely distributed connective tissue element with a function that extends beyond elastogenesis; their association with fibronectin and localization suggests that they serve as an elastic anchoring component of the extracellular matrix.     

Degradation of extracellular matrix by the trophoblastic cells of first-trimester human placentas

First-trimester human placental villi were cultured on 3H-leucine-labeled extracellular matrices isolated from the PF HR9 and PYS-2 cell lines. Both cell lines produced an extracellular matrix that contained basement membrane-specific macromolecules, including type IV collagen, laminin and proteoglycan. Both matrices promoted outgrowth of cells from the villi which, according to morphological criteria, were identified as cytotrophoblastic cells. As the cells migrated from the attachment site, they caused a marked focal dissolution of the matrix which was accompanied by a concomitant release of 3H-labeled material into the media. Approximately half of this material chromatographed near the inclusion volume of Sephadex G-50, indicating that the labeled matrix components had been degraded. This phenomenon was dependent on the age of the placenta. Second-trimester placental villi also adhered to the matrix, but no areas of dissolution were formed and no significant amounts of radioactivity were released into the medium. These results suggest that culture of first-trimester human placental villi on extracellular matrices may be useful for the study of some of the early embryonic events leading to human implantation, during which the trophoblastic cells erode the uterine epithelium.     

Deposition of type X collagen in the cartilage extracellular matrix

In cultured chick embryo chondrocytes, type X collagen is preferentially deposited in the extracellular matrix, the ratio between type II and type X collagen being about 5 times higher in the culture medium than in the cell layer. When the newly synthesized collagens deposited in slices from the epiphyseal cartilage of 17-day-old embryo tibiae were isolated, type X collagen was always the major species. In agreement with this result the mRNA for type X collagen was the predominant mRNA species purified from the same tissue. When the total collagen (unlabeled) deposited in the epiphyseal cartilage was analyzed, it was observed that type X collagen represented only 1/15 of the type II collagen recovered in the same preparation. The possible explanations for these differences are discussed.     

Bufonid nucleocytoplasmic hybrids arrested at the early gastrula stage lack a fibronectin-containing fibrillar extracellular matrix

Summary Most hybrids betweenBufo bufo andB. calamita obtained by nuclear transplantation become arrested at the early gastrula stage. In both parental controls and the hybrid embryos, the presence and distribution of extracellular matrix was analysed with fluorescent wheat germ agglutinin and by immunolabelling with antibodies directed against fibronectin. InB. bufo andB. calamita gastrulae and in the few hybrids that complete gastrulation, the inner surface of the blastocoel roof is covered by a fibronectin-rich fibrillar matrix. In nucleocytoplasmic hybrids whose development was arrested at the gastrula stage, the fibronectin-containing extracellular matrix was either totally absent or poorly developed and disorganized.     

Some factors involved in the dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by digestive fluids of Trichoplusia ni larvae

Factors involved in the dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus (AcNPV) by digestive fluid collected from fifth stage Trichoplusia ni larvae were studied in vitro. Observations were made at timed intervals using phase contrast microscopy and transmission electron microscopy. When digestive fluid was heated at 50°C proteases retained activity. Exposure of polyhedra to digestive fluid previously heated to 50°C resulted in polyhedral matrix dissolution and envelope disruption in a manner similar to that of unheated digestive fluid, only delayed slightly. After exposure of polyhedra for 3 min, only enveloped virons were observed. Heating the digestive fluid to 60° or higher inactivated the proteases and altered the effect on polyhedra. Dissolution of the occlusion body matrix occurred but the polyhedral envelope remained and only a few weakened areas were observed in its structure. Within the polyhedral envelope, enveloped virons were not observed, only nucleocapsids and capsids. Exposure of polyhedra to 0.1 m sodium carbonate buffer at pHs of 9.5 or higher had effects similar to those of the digestive fluid with heat (60°C)-inactivated proteases. The addition of trypsin and chymotrypsin to the 0.1 m sodium carbonate buffer had no effect, while the addition of a bacterial protease (Streptomyces griseus) at pHs of 9.5 or higher resulted in dissolution of the matrix and disruption of the polyhedral envelope like the digestive fluid. Material infectious to TN-368 cells was obtained by exposure of AcNPV to T. ni digestive fluid. Maximum infectivity resulted from a 5-min exposure to unheated digestive fluid, with a dramatic decrease in infectivity with longer exposure. Exposure to digestive fluid with heat (60°C)-inactivated proteases resulted in a slower release of infectious material from the occlusion body, with a steady increase in the level of infectivity throughout the 30-min digestion period.     

Growth Kinetics, Cell Shape, and the Cytoskeleton of Primary Astrocyte Cultures

Abstract: We examined correlations among growth kinetics, cell shape, and cytoskeletal protein content in rat astrocytes grown in primary culture. Cell suspensions from brains of newborn rats were seeded at densities from 0.2 to 3 × 10⁵/cm². At initial densities above 1 × 10⁵ the population increased to reach confluency by 10–12 days, after which cell number remained stable for many weeks. At low initial densities, 0.2–0.4 × 10⁵/cm², cells did not increase in number. Final density increased with increasing plating densities. High-density cells had small perikarya and several long cytoplasmic processes; low-density cells appeared flat and polygonal. All cultures were almost entirely astrocytic, as judged by immunofluorescent staining with antiserum against glial fibrillary acidic protein (GFAP). Cytoskeletal proteins were analyzed by gel electrophoresis after extraction from cells with nonionic detergent. Relative amounts of the proteins differed, in that low-density cells contained large amounts of cytoskeletal actin relative to the intermediate filament (IF) proteins vimentin and GFAP, whereas high-density cells contained relatively less actin and more IF proteins. Such differences in cytoskeletal proteins between the high- and low-density cultures were mirrored in the relative rates of synthesis of the cytoskeletal proteins. In the low-density cells amino acid incorporation into cytoskeletal-associated actin was more active than that into the IFs, whereas in the high-density cells higher rates of IF protein synthesis were observed.     

Characterization, Localization, and Biosynthesis of an Interstitial Retinol-Binding Glycoprotein in the Human Eye

Abstract: Human eyes contain an M_r 135K retinol-binding protein that is analogous to interstitial retinol-binding protein (IRBP) in the subretinal space of bovine eyes. It is a glycoprotein, because it binds ¹²⁵I-concanavalin A, ¹²⁵I-wheat germ agglutinin and ¹²⁵I- Lens culinaris hemagglutinin. It does not bind Ricinus communis agglutinin I. After desialation, it binds Ricinus communis agglutinin I, but loses its capacity to bind wheat germ agglutinin. These observations, coupled with the known specificities of these lectins, suggest that at least one of the oligosaccharide chains is a sialated, biantennary complex type containing fucose. Both by direct analysis of dissected ocular tissues and by immunocytochemistry it was shown that human interstitial retinol binding protein is an extracellular protein that is confined predominantly to the subretinal space. Monkey retinas incubated in vitro in medium containing [³H]leucine were shown to synthesize and secrete this protein into the medium, a conclusion that was confirmed by immunoprecipitation with an immunoglobulin fraction prepared from rabbit antibovine IRBP serum. Virtually no other labeled proteins were detectable in the medium. It is concluded that interstitial retinol-binding protein meets many of the requirements for a putative transport protein implicated in the transfer of retinol between the pigment epithelium and retina during the visual cycle, and that the neural retina may play an important role in regulating its amount in the subretinal space.