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.

A fine structural analysis of intercellular junctions in the mouse liver.

Zonulae occludentes and gap junctions were examined both in the intact mouse liver and in a junction-rich membrane fraction from homogenized mouse liver. These preparations were visualized with the techniques of uranyl acetate staining en bloc, staining with colloidal lanthanum, negative staining with phosphotungstate, and freeze-cleaving. The zonula occludens is arranged as a meshwork of branching and anastomosing threadlike contacts sealing the lumen of the bile canaliculus from the liver intercellular space. The gap junction is characterized in section by a 20 A gap between the apposed junctional membrane outer leaflets, and permeation of this space with lanthanum or phosphotungstate reveals a polygonal lattice of subunits with a center-to-center spacing of 90-100 A. Freeze-cleaved gap junctions show a similar lattice. Extraction of junction-rich fractions with 60% aqueous acetone results in a disappearance of the 20 A gap in sectioned pellets and an inability to demonstrate the polygonal lattice with either the freeze-cleave or negative staining techniques. Extraction of the membranes with 50% acetone does not produce this effect. Thin-layer chromatography of the acetone extracts reveals a group of phospholipids in the 60% extract that are not detectable in the 50% extract. Acetone does not cause any detectable change in the structure of the zonula occludens, but the occluding junction becomes leaky to lanthanum following acetone treatment. The effects of other reagents on the junctions are reported.

The permeability of isolated and in situ mouse hepatic gap junctions studied with enzymatic tracers.

We have studied the effects of phospholipase C from Clostridium welchii on gap junctions in the intact mouse liver and in a junction-rich fraction prepared from mouse liver. Treatment of the isolated junctions results in the disappearance of both the 20 A gap and of the polygonal lattice visible with lanthanum. The junctions are morphologically unaltered, however, when whole livers are perfused with phospholipase via the portal vein. These results suggest that extracellular phospholipase cannot diffuse into the junctional area, but that the enzyme may affect structures within the gap from its cytoplasmic surfaces which become exposed in the isolated preparations. Horseradish peroxidase, which has physical dimensions similar to those of Clostridium phospholipase is also denied access to the 20 A gap in whole liver, while peroxidase reaction product can be seen in the gap in isolated preparations. Beef liver catalase, however, a tracer molecule much larger than peroxidase, cannot penetrate even in isolated fractions. If the cytoplasmic approaches to the gap junction used by peroxidase and phospholipase are available in vivo, and have not been created during the process of mechanical isolation, they may play a role in cell-to-cell passage of molecules larger than ions.

Coexistence of gap and sseptate junctions in an invertebrate epithelium.

The intercellular junctions of the epithelium lining the hepatic caecum of Daphnia were examined. Electron microscope investigations involved both conventionally fixed material and tissue exposed to a lanthanum tracer of the extracellular space. Both septate junctions and gap junctions occur between the cells studied. The septate junctions lie apically and resemble those commonly discerned between cells of other invertebrates. They are atypical in that the high electron opacity of the extracellular space obscures septa in routine preparations. The gap junctions are characterized by a uniform 30 A space between apposed cell membranes. Lanthanum treatment of gap junctions reveals an array of particles of 95 A diameter and 120 A separation lying in the plane of the junction. As this pattern closely resembles that described previously in vertebrates, it appears that the gap junction is phylogenetically widespread. In view of evidence that the gap junction mediates intercellular electrotonic coupling, the assignment of a coupling role to other junctions, notably the septate junction, must be questioned wherever these junctions coexist.

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.

Endothelial cell junctions.

In the course of a freeze-cleave study on intercellular junctions in the regenerating rat liver, we observed an unusual array of intramembranous particles located in regions of contact between endothelial cells lining the hepatic sinusoids. These arrays were characterized by an accumulation of particles which resembled a zonula occludens in their linear deployment but differed in that the contact regions were composed of individual particles which remained separated from each other by regular particle-free intervals.

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.

Demonstration of the transmembrane nature of the acetylcholine receptor by labeling with anti-receptor antibodies.

Antibodies raised in rabbits to Triton-solubilized, purified acetylcholine receptor from Torpedo californica were used to immunospecifically label intact T. californica electroplaque membrane vesicles attached to cover slips and oriented with the extracellular face of the synaptic membrane facing outward. Hemocyanin conjugated to Protein A was then used as a marker, making the antibody binding visible at the electron microscope level. Parallel labeling experiments were performed on vesicles attached to cover slips and sheared by sonication, leaving their cytoplasmic faces fully exposed to the labeling solution. While differences in antibody populations among different rabbits were observed, antigenic determinants of the receptor were present on both faces of the membrane, with those on the extracellular side more numerous than those on the cytoplasmic side, demonstrating the transmembrane nature of the receptor molecule.

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.

Intercellular communication in normal and regenerating rat liver: a quantitative analysis.

We have compared intercellular communication in the regenerating and normal livers of weanling rats. The electrophysiological studies were conducted at the edge of the liver, and we have found that here as elsewhere in the liver there is a dramatic decrease in the number and size of gap junctions during regeneration. The area of hepatocyte membrane occupied by gap junctions is reduced 100-fold 29-35 h after hepatectomy. By combining observations made with the scanning electron microscope with our freeze fracture data we have estimated the number of "communicating interfaces" (areas of contact between hepatocytes that include at least one gap junction) formed by hepatocytes in normal and regenerating liver. In normal liver a hepatocyte forms gap junctions with every hepatocyte it contacts (approximately 6). In regenerating liver a hepatocyte forms detectable gap junctions with, on average, only one other hepatocyte. Intercellular spread of fluorescent dye and electric current is reduced in regenerating as compared with normal liver. The incidence of electric coupling is reduced from 100% of hepatocyte pairs tested in control liver to 92% in regenerating liver. Analysis of the spatial dependence of electronic potentials indicates a substantial increase in intercellular resistance in regenerating liver. A quantitative comparison of our morphological and physiological data is complicated by tortuous pattern of current flow and by inhomogeneities in the liver during regeneration. Nevertheless we believe that our results are consistent with the hypothesis that gap junctions are aggregates of channels between cell interiors.

Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies.

We examined the roles of the extracellular domains of a gap junction protein and a cell adhesion molecule in gap junction and adherens junction formation by altering cell interactions with antibody Fab fragments. Using immunoblotting and immunocytochemistry we demonstrated that Novikoff cells contained the gap junction protein, connexin43 (Cx43), and the cell adhesion molecule, A-CAM (N-cadherin). Cells were dissociated in EDTA, allowed to recover, and reaggregated for 60 min in media containing Fab fragments prepared from a number of antibodies. We observed no cell-cell dye transfer 4 min after microinjection in 90% of the cell pairs treated with Fab fragments of antibodies for the first or second extracellular domain of Cx43, the second extracellular domain of connexin32 (Cx32) or A-CAM. Cell-cell dye transfer was detected within 30 s in cell pairs treated with control Fab fragments (pre-immune serum, antibodies to the rat major histocompatibility complex or the amino or carboxyl termii of Cx43). We observed no gap junctions by freeze-fracture EM and no adherens junctions by thin section EM between cells treated with the Fab fragments that blocked cell-cell dye transfer. Gap junctions were found on approximately 50% of the cells in control samples using freeze-fracture EM. We demonstrated with reaggregated Novikoff cells that: (a) functional interactions of the extracellular domains of the connexins were necessary for the formation of gap junction channels; (b) cell interactions mediated by A-CAM were required for gap junction assembly; and (c) Fab fragments of antibodies for A-CAM or connexin extracellular domains blocked adherens junction formation.

Major loss of the 28-kD protein of gap junction in proliferating hepatocytes.

There is a reduction in the 28-kD gap junction protein detectable by immunofluorescence in livers of partially hepatectomized rats and in cultured hepatocytes stimulated to proliferate. By the coordinate use of antibodies directed to the hepatic junction protein (HJP28) and the use of a monoclonal antibody that recognizes bromodeoxyuridine (BrdU) incorporated into DNA, we have been able to study the relationship between detectable gap junction protein and cell division. Hepatocytes that label with BrdU in the regenerating liver and in cell culture show a significant reduction of HJP28. Cells that do not synthesize DNA, on the other hand, show normal levels and distribution of immunoreactive gap junction protein. We postulate that the quantitative changes in gap junction expression might play an important role in the control of proliferation in the liver.

The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains.

Analysis by SDS-PAGE of gap junction fractions isolated from heart suggests that the junctions are comprised of a protein with an Mr 43,000. Antibodies against the electroeluted protein and a peptide representing the 20 amino terminal residues bind specifically on immunoblots to the 43-kD protein and to the major products arising from proteolysis during isolation. By immunocytochemistry, the protein is found in ventricle and atrium in patterns consistent with the known distribution of gap junctions. Both antibodies bind exclusively to gap junctions in fractions from heart examined by EM after gold labeling. Since only domains of the protein exposed at the cytoplasmic surface should be accessible to antibody, we conclude that the 43-kD protein is assembled in gap junctions with the amino terminus of the molecule exposed on the cytoplasmic side of the bilayer, that is, on the same side as the carboxy terminus as determined previously. By combining proteolysis experiments with data from immunoblotting, we can identify a third cytoplasmic region, a loop of some 4 kD between membrane protected domains. This loop carries an antibody binding site. The protein, if transmembrane, is therefore likely to cross the membrane four times. We have used the same antisera to ascertain if the 43-kD protein is involved in cell-cell communication. The antiserum against the amino terminus blocked dye coupling in 90% of cell pairs tested; the antiserum recognizing epitopes in the cytoplasmic loop and cytoplasmic tail blocked coupling in 75% of cell pairs tested. Preimmune serum and control antibodies (one against MIP and another binding to a cardiac G protein) had no or little effect on dye transfer. Our experimental evidence thus indicates that, in spite of the differences in amino acid sequence, the gap junction proteins in heart and liver share a general organizational plan and that there may be several domains (including the amino terminus) of the molecule that are involved in the control of junctional permeability.

Vascular system defects and impaired cell chemokinesis as a result of Galpha13 deficiency.

Heterotrimeric GTP-binding proteins (G proteins) participate in cellular signaling and regulate a variety of physiological processes. Disruption of the gene encoding the G protein subunit alpha13 (Galpha13) in mice impaired the ability of endothelial cells to develop into an organized vascular system, resulting in intrauterine death. In addition, Galpha13 (-/-) embryonic fibroblasts showed greatly impaired migratory responses to thrombin. These results demonstrate that Galpha13 participates in the regulation of cell movement in response to specific ligands, as well as in developmental angiogenesis.

Atomic force microscopy and dissection of gap junctions.

An atomic force microscope (AFM) was used to study the structure of isolated hepatic gap junctions in phosphate-buffered saline (PBS). The thickness of these gap junctions appears to be 14.4 nanometers, close to the dimensions reported by electron microscopy (EM). When an increasing force is applied to the microscope tip, the top membrane of the gap junction can be "dissected" away, leaving the extracellular domains of the bottom membrane exposed. When such "force dissection" is performed on samples both trypsinized and fixed with glutaraldehyde, the hexagonal array of gap junction hemichannels is revealed, with a center-to-center spacing of 9.1 nanometers.

Chemical and spectroscopic analysis of olive mill waste water during a biological treatment.

The treatment of olive mill waste water was studied on the laboratory scale. Physico-chemical analyses showed the final products had a mean pH of 5.4 without neutralisation and 5.7 when lime was added to the process. Raising the pH by adding lime had a positive outcome on the degradation of phenols, whose levels were reduced by over 76%. The lime also changed the structure of the organic matter, as seen in the infra-red spectra. Combining the FT-IR and 13C NMR data showed that with addition of lime, the density of aliphatic groups decreased to the benefit of aromatic groups, indicating that polymerisation of the organic matter occurred during the bioprocess. Under our experimental conditions, the biotransformation of olive mill waste water appears to favour the stabilisation of the organic matter through mechanisms analogous to those that lead to the formation of humus in the soil.

Structural study of the fulvic fraction during composting of activated sludge-plant matter: elemental analysis, FTIR and 13C NMR.

The starting fulvic structures isolated from an initial mixture of activated sludge and plant matter presented abundant peptide structures and hydrocarbons that absorb in FTIR spectra around (1650 and 1560 cm(-1)) and 1072 cm(-1), respectively. They also present a high resonance signal in the O- and N-alkyl areas of (13)C NMR spectra. As composting proceeded, some changes led to the formation of the molecular structures of fulvic fraction as demonstrated by a decrease of intensity of compounds absorbing around 1072 cm(-1) and an increase of those absorbing around 1140 cm(-1). The resonance of O- and N-substituted alkyl carbon also decreased from 55.7% to 33.8%, with an increase in the intensity of aromatic carbons, alkyls and carboxyls. These data indicate that the microbial community that developed during composting used polysaccharides as an energy source, structures which are supplied in abundance in the initial material. The fulvic fraction of the final compost is much richer in aromatic structures and aliphatic ethers/esters, which are most likely preserved from the original material but probably also synthesized through the microbial activities. The occurrence of alkyl ethers/esters at the end of composting is demonstrated by strong absorbance around 1140 cm(-1) in the FTIR spectra and large peaks at 32 and 174 ppm in the NMR spectra. These structures could also be produced following the creation of ether/ester bonds during the humification process.

Optimisation of biodegradation conditions for the treatment of olive mill wastewater.

The aim of the present paper was to optimise the conditions of aerobic treatment of olive mill wastewater. To do so, the waste was treated following the experimental optimal design methodology studying the set of factors susceptible to influence the treatment (pH, C/N ratio, aeration and temperature). The results of a first series of experiments showed a strong correlation between the reduction in the levels of polyphenols and three of the parameters studied, i.e. the C/N ratio, aeration and temperature. Optimised conditions led to a 94% drop in polyphenols. Then, for a finer study of the conditions, just two parameters were varied, the pH and the C/N ratio. The results showed that the conditions of pH modification (addition of lime or sodium hydroxide) and the C/N ratio (urea or ammonium nitrate) allowed the microbiological activity to be very significantly improved. This led to polyphenol reductions of 51% and 76%.

Photochemical UV/TiO2 treatment of olive mill wastewater (OMW).

Olive mill wastewater (OMW) was treated by photocatalysis using TiO2 under UV irradiation on the laboratory scale. The chemical oxygen demand, the coloration at 330nm, and the level of phenols all showed decreases which, after a 24-h treatment, reached 22%, 57% and 94%, respectively. The differences between these three values indicate the persistence of colourless, non-phenolic compounds. Application of the novel Fictitious Atomic-Group Separation method showed an increase in carbon oxidation state and confirmed that the attack primarily concerns, aromatic moieties. A fine spectroscopic study revealed the occurrence of three successive phases during the degradation process, thought to correspond to three different categories of molecules in the OMW and the presence of pectin compounds.

Fate of polycyclic aromatic hydrocarbons during composting of activated sewage sludge with green waste.

The level and fate of 16 polycyclic aromatic hydrocarbons (PAHs), targeted by the US Environmental Protection Agency (USEPA), has been studied over 90 days of composting of activated sludge with green waste, under a semi-arid climate. The total PAH calculated from the sum of the amounts of the 16 PAHs in the initial mixture of activated sludge and green waste, was lower than accepted European Union cut-off limits by about 0.48mgkg(-1). The treatment by composting led to a decrease of all PAHs mainly in the stabilization phase, but some differences could be observed between PAHs with three or fewer aromatic rings (N< or = 3) and those with four or more (N> or = 4). The former (except phenanthrene) exhibited a continuous decrease, while the latter PAHs with N of four or more and phenanthrene showed increases in the intermediate stages (30-60 days). This indicates the high potential sorption mainly of PAH with high molecular weight (> or = N4) plus phenanthrene, their tight adsorption makes them inaccessible for microbial attack. The high molecular weight PAHs showed a greater reduction of their bioavailability than those of low molecular weight. Naphthalene, with the lowest molecular weight, showed the smallest decrease (about 67.8%) compared to other PAHs of higher molecular weight (decrease reaching 100%). This is in agreement with the fact that the adsorption is less reversible with increased numbers of fused aromatic rings or an increase of their hydrophobicity.