Ultrastructural Localization of Rhodopsin in the Vertebrate Retina

Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care

Biochemical and immunochemical analysis of the arrangement of connexin43 in rat heart gap junction membranes

A 43 × 10^3 M_r protein (designated connexin43 or Cx43) is a major constituent of heart gap junctions. The understanding of its arrangement in junctional membranes has been extended by means of site-directed antibodies raised against synthetic peptides of Cx43. These represent part of the first extracellular loop (EL-46), the cytoplasmic loop (CL-100), the second extracellular loop (EL-186) and carboxy-terminal sequences (CT-237 and CT-360). All of the antibodies raised reacted with their respective peptides and the Cx43 protein on Western blots. By immunoelectron microscopy two of the antibodies (CL-100 and CT-360) were shown to label the cytoplasmic surface of isolated gap junction membranes. Immunofluorescent labeling at locations of neonatal cardiac myocyte-myocyte apposition required an alkali/urea treatment when the EL-46 and EL-186 antibodies were used. Immunoblot analysis of endoproteinase Lys-C-digested gap junctions revealed that the Cx43 protein passed through the lipid bilayer four times. Alkaline phosphatase digestion of isolated junctions was used to show that the CT-360 antibody recognized many phosphorylated forms of Cx43. Our results unequivocally confirm models of the organization of Cx43 that were based on a more limited set of data and a priori considerations of the sequence

Reversibility of cell surface label rearrangement

Cell surface labeling can cause rearrangements of randomly distributed membrane components. Removal of the label bound to the cell surface allows the membrane components to return to their original random distribution, demonstrating that label is necessary to maintain as well as to induce rearrangements. With scanning electron microscopy, the rearrangement of concanavalin A (con A) and ricin binding sites on LA-9 cells has been followed by means of hemocyanin, a visual label. The removal of con A from its binding sites at the cell surface with alpha- methyl mannoside, and the return of these sites to their original distribution are also followed in this manner. There are labeling differences with con A and ricin. Under some conditions, however, the same rearrangements are seen with both lectins. The disappearance of labeled sites from areas of ruffling activity is a major feature of the rearrangements seen. Both this ruffling activity and the rearrangement of label are sensitive to cytochalasin B, and ruffling activity, perhaps along with other cytochalasin-sensitive structure, may play a role in the rearrangements of labeled sites

Loss and reappearance of gap junctions in regenerating liver

Changes in intercellular junctional morphology associated with rat liver regeneration were examined in a freeze-fracture study. After a two-thirds partial hepatectomy, both gap junctions and zonulae occludentes were drastically altered. Between 0 and 20 h after partial hepatectomy, the junctions appeared virtually unchanged. 28 h after partial hepatectomy, however, the large gap junctions usually located close to the bile canaliculi and the small gap junctions enmeshed within the strands of the zonulae occudentes completely disappeared. Although the zonulae occludentes bordering the bile canaliculi apparently remained intact, numerous strands could now be found oriented perpendicular to the canaliculi. In some instances, the membrane outside the canaliculi was extensively filled with isolated junctional strands, often forming very complex configurations. About 40 h after partial hepatectomy, very many small gap junctions reappeared in close association with the zonulae occludentes. Subsequently, gap junctions increased in size and decreased in number until about 48 h after partial hepatectomy when gap junctions were indistinguishable in size and number from those of control animals. The zonulae occludentes were again predominantly located around the canalicular margins. These studies provide further evidence for the growth of gap junctions by the accretion of particles and of small gap junctions to form large maculae

Spatial and temporal patterns of distribution of the gap junction protein connexin43 during mouse gastrulation and organogenesis

Connexin43 (Cx43) is a member of the family of channel-forming proteins that make up the gap junction and are believed to provide pathways for cell-cell exchange of developmental signals. We have used immunofluorescence and confocal microscopy to characterize the patterns of distribution of Cx43 in postimplantation mouse embryos representing stages of development extending through gastrulation and the major period of organogenesis [through 13.5 days post coitum (dpc)]. We find that Cx43 is expressed early after implantation by the undifferentiated, pluripotent cells of the primitive embryonic ectoderm from which all tissues of the fetus are believed to be derived. As cells become committed to particular developmental pathways, there is a progressive restriction of Cx43 to specific areas and organ systems. The patterns are complex and not limited by germ layer of origin, although there is a clear preference for expression in ectodermal and, to a lesser extent, mesodermal derivatives. Expression in lens, retina, kidney, brain, pineal and pituitary glands is initiated early in organogenesis. In heart, the first clear signal for Cx43 appears in the ventricle at about 10 dpc and is only subsequently detected in the atrium at about 13-13.5 dpc. Particularly intriguing with regard to functional implications is the high level expression observed at sites of inductive interaction; the eye lens and optic cup, the infundibulum and the apical ectodermal ridge of the limb bud

Inertia-Constrained Pixel-by-Pixel Nonnegative Matrix Factorisation: a Hyperspectral Unmixing Method Dealing with Intra-class Variability

Blind source separation is a common processing tool to analyse the constitution of pixels of hyperspectral images. Such methods usually suppose that pure pixel spectra (endmembers) are the same in all the image for each class of materials. In the framework of remote sensing, such an assumption is no more valid in the presence of intra-class variabilities due to illumination conditions, weathering, slight variations of the pure materials, etc... In this paper, we first describe the results of investigations highlighting intra-class variability measured in real images. Considering these results, a new formulation of the linear mixing model is presented leading to two new methods. Unconstrained Pixel-by-pixel NMF (UP-NMF) is a new blind source separation method based on the assumption of a linear mixing model, which can deal with intra-class variability. To overcome UP-NMF limitations an extended method is proposed, named Inertia-constrained Pixel-by-pixel NMF (IP-NMF). For each sensed spectrum, these extended versions of NMF extract a corresponding set of source spectra. A constraint is set to limit the spreading of each source's estimates in IP-NMF. The methods are tested on a semi-synthetic data set built with spectra extracted from a real hyperspectral image and then numerically mixed. We thus demonstrate the interest of our methods for realistic source variabilities. Finally, IP-NMF is tested on a real data set and it is shown to yield better performance than state of the art methods

Structural changes in lipid-free humic acids during composting of sewage sludge

Structural changes in humic acids (HAs), extracted after lipid removal from sewage sludge during composting, were investigated using various chemical methods (elemental analysis, Fourier transform infrared spectroscopy and 13C-nuclear magnetic resonance (NMR) spectroscopy). Compared to non-purified HAs, lipid-free HAs (LFHAs) exhibit higher C and N contents and high absorbance around 1652, 1540 and 1230 cm1, which indicates the intensity of the etherified aromatic structures and nitrogencontaining components. Less absorbance around 2920, 1600, 1414 and 1100 cm1 could be assigned to their low level of aliphatic compounds, mainly those with a carboxyl group. According to 13C-NMR spectroscopy, almost 45% of aliphatic structures are removed by lipid extraction and these correspond mainly to long-chain fatty acids. During composting, significant decomposition of non-substituted alkyl structures and N-containing components occurred, increasing the relative intensity of etherified aromatic structures

Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31

A new member of the connexin gene family has been identified and designated rat connexin-31 (Cx31) based on its predicted molecular mass of 30,960 daltons. Cx31 is 270 amino acids long and is coded for by a single copy gene. It is expressed as a 1.7-kilobase mRNA that is detected in placenta, Harderian gland, skin, and eye. Cx31 is highly conserved and can be detected in species as distantly related to rat as Xenopus laevis. It exhibits extensive sequence similarity to the previously identified connexins, 58, 50, and 40% amino acid identity to Cx26, Cx32, and Cx43, respectively. When conservation of predicted phosphorylation sites is used to adjust the alignment of Cx31 to other connexins, a unique alignment of three predicted protein kinase C phosphorylation sites near the carboxyl terminus of Cx31 with three sites at the carboxyl terminus of Cx43 is revealed

Tip-sample interactions in atomic force microscopy: I. Modulating adhesion between silicon nitride and glass

An adhesive interaction between a silicon nitride AFM tip and glass substrate in water is described. This adhesion is in the range 5-40 nN, of which a large component is likely to be due to hydrogen bonding between the silanol groups on both surfaces. The interaction can be modulated by a variety of buffers commonly used in biochemical and biological research, including sodium phosphate, tris(hydroxymethyl)aminomethane, glycine, and N-2-hydroxyethyl-piperazine N'-2-ethanesulfonic acid. Using these buffers it appears that there are effects of ion concentration, ion type and pH on the measured adhesion. Of the conditions examined, phosphate was most effective at reducing adhesion and could be used at concentrations as low as 10 mM at neutral pH. The results demonstrate that the chemical interactions between tip and sample can be modulated, and provide a basis for designing conditions for imaging and manipulating biological molecules and structures

Expression of the gene for main intrinsic polypeptide (MIP): separate spatial distributions of MIP and beta-crystallin gene transcripts in rat lens development

The main intrinsic polypeptide (MIP) is the major protein present in the lens fiber cell membrane and is the product of a gene which, as far as is known, is expressed only in the lens. We have used in situ hybridization and immunofluorescence microscopy to characterize the expression of this gene during the course of development in the rat. At progressive stages of lens morphogenesis, we find that synthesis of the protein is closely tied to the accumulation of MIP mRNA in cells that are committed to terminal differentiation, first in the elongating presumptive primary lens fibers and later in the secondary fibers as they differentiate from the anterior epithelial cells. The transcripts accumulate in the basal cytoplasm of the primary fibers and in the cytoplasm which surrounds the cell nucleus in the secondary fibers. We have compared this pattern of expression with that of a gene for a cytoplasmic protein, beta-crystallin beta-A1/A3. In sharp contrast to the localized concentrations seen for the MIP mRNA, beta-A1/A3 transcripts are relatively uniformly distributed throughout the cytoplasm. Neither MIP nor crystallin gene appears to be transcriptionally active in the undifferentiated epithelial cell, but transcripts from the beta-A1/A3 gene appear earlier in fiber cell differentiation than do those from the gene for MIP