Immunocytochemical localization of two retinoid-binding proteins in vertebrate retina

The recent discovery and characterization of several proteins that purify with endogenous, bound retinoid have given rise to the suggestion that these proteins, which are abundant in retina, perform a role in transport and function of vitamin A. Immunocytochemical techniques were used to localize two retinoid-binding proteins in the retina of four species. Antisera to cellular retinal-binding protein (CRALBP) and an interphotoreceptor retinoid-binding protein (IRBP) were obtained from rabbits immunized with antigens purified from bovine retina. Antibodies from each antiserum reacted with a single component in retinal homogenates and supernatants which corresponded to the molecular weight and charge of the respective antigen (non-SDS and SDS PAGE, electrophoretic transfer to nitrocellulose, immunochemical staining). Immunocytochemistry controls were antibodies from nonimmune serum and antibodies absorbed with purified antigen. Antigens were localized on frozen-sectioned bovine, rat, monkey, and human retina using immunofluorescence and the peroxidase-antiperoxidase technique. Specific staining with anti-IRBP was found in the space that surrounds photoreceptor outer segments, with heaviest labeling in a line corresponding to the retinal pigment epithelium (RPE) apical surface. Cone outer segments were positive. Staining with anti-CRALBP was found in two cell types in all species: the RPE and the Muller glial cell. Within the RPE, labeling filled the cytoplasm and was heaviest apically, with negative nuclei. Labeling of Muller cells produced Golgi- like silhouettes with intense staining of all cytoplasmic compartments. Staining of the external limiting membrane was heavy, with labeled microvilli projecting into the interphotoreceptor space. Localization of IRBP to this space bordered by three cell types (RPE, photoreceptor, and Muller) is consistent with its proposed role in transport of retinoids among cells. Localization of CRALBP in RPE corroborates previous biochemical studies; its presence in the Muller cell suggests that this glial cell may play a hitherto unsuspected role in vitamin A metabolism in retina.     

Vascular remnants of pupillary membrane in the albino rat eye.

As remnants of pupillary membrane, some albino rat eyes revealed vascular loops starting from arteriovenous bridges near the pupillary margin and crossing the pupil. These vascular loops bend in miosis and straighten in mydriasis, which prevents them from being broken during pupillary movement. The vessel wall reveals endothelial cells and pericytes. The lumen contains red blood cells, which means that they are functional vessels. They may function as vascular shunts between opposite sides of the albino rat iris.     

Interaction of Ca2+ and H+ with heme A in cytochrome oxidase

Ca²⁺ ions shift the absorption spectrum of reduced cytochromea in mitochondria by acting from the outside of the membrane. In isolated cytochrome oxidase the shift may be induced by either Ca²⁺ or H⁺, the apparent pK varying between 6.20 and 5.75 depending on the state of cytochromea ₃. Studies of the Soret band show that Ca²⁺ also shifts the spectrum of ferrocytochromea ₃ in isolated oxidase in contrast to the situation in mitochondria or isolated oxidase reconstituted into liposomes. Model studies with reduced bis-imidazole heme A reveals an analogous spectral shift induced by Ca²⁺. Esterification of the propionate carboxyls of heme A abolishes the spectral shift, suggesting that it is due to interaction of Ca²⁺ with these groups. When taken together with the data with intact mitochondria, this suggests that the propionate side chains of cytochromea are accessible to Ca²⁺ and H⁺ from the outside of the mitochondrial membrane. In the soluble enzyme both hemesa anda ₃ are accessible. Thus hemea may be located near the outside of the inner membrane whereas hemea ₃ experiences a different environment in which no Ca²⁺ shift occurs.     

Effect of cis-diamminedichloroplatinum (II) on metallothionein induction and trace element metabolism in rats fed different amounts of dietary zinc

Recent studies have suggested that the induction of metallothionein synthesis in kidneys of mice by the acute administration of bismuth and other trace elements might protect against cis-diamminedichloroplatinum (II) nephrotoxicity. The present study was designed to determine the effects of dietary zinc and cis-diamminedichloroplatinum (II) on the induction of liver and kidney metallothionein and its subsequent effect on nephrotoxicity and trace element metabolism in rats. Male rats were fed diets containing 5, 20, 80, or 320 mg zinc/kg diet for 3 weeks. Each dietary group was subdivided into 3 groups. In one group, each rat received an i.p. injection of 7.5 mg cis-diamminedichloroplatinum (II)/kg b.w. All other rats received saline. During the next three days a second group of rats was pair-fed to the cis-diamminedichloroplatinum (II) injected group. A third group received no treatment and was allowed to eat ad libitum. Results showed that when dietary zinc was increased from 5 mg/kg diet to higher amounts, kidney metallothionein concentration increased twofold. cis-diamminedichloroplatinum (II) treatment increased kidney metallothionein even further, but elevated metallothionein gave no protection from the toxic effects of the drug. Serum copper concentration and ceruloplasmin activity were significantly lower with higher concentrations of dietary zinc, which indicated that these rats were mildly copper-deficient. There was a small but significant depression of superoxide dismutase activity and a highly significant increase in thiobarbituric acid reactive substances in kidneys of rats treated with cis-diamminedichloroplatinum (II) compared to either pair-fed or ad libitum controls. This supports the hypothesis that part of the mechanism for cis-diamminedichloroplatinum (II)-induced toxicity might be caused by free-radical generation. However, the data do not support the hypothesis that metallothionein induction protects the kidney from cis-diamminedichloroplatinum (II) toxicity.     

Repeated Home-Applied Dual-Light Antibacterial Photodynamic Therapy Can Reduce Plaque Burden,Inflammation, and aMMP-8 in Peri-Implant Disease—A Pilot Study

Until now, in clinical dentistry, antibacterial photodynamic therapy (aPDT) has been restricted to in-office treatments, which hampers repeated applications. This pilot study tested the benefit of a commercially available Lumoral^® device designed for regular periodontal dual-light aPDT treatment at home. Seven patients with peri-implant disease applied dual-light aPDT daily in addition to their normal dental hygiene for four weeks. A single Lumoral^® treatment includes an indocyanine green mouth rinse followed by 40 J/cm² radiant exposure to a combination of 810 nm and 405 nm light. A point-of-care analysis of active-matrix metalloproteinase (aMMP-8), visible plaque index (VPI), bleeding on probing (BOP), and peri-implant pocket depth (PPD) measurements was performed on day 0, day 15, and day 30. Reductions in aMMP-8 (p = 0.047), VPI (p = 0.03), and BOP (p = 0.03) were observed, and PPD was measured as being 1 mm lower in the implant (p = ns). These results suggest a benefit of regular application of dual-light aPDT in peri-implantitis. Frequently repeated application can be a promising approach to diminishing the microbial burden and to lowering the tissue destructive proteolytic and inflammatory load around dental implants. Further studies in larger populations are warranted to show the long-term benefits.     

Chinese hamster ovary cell mutants deficient in an anion exchanger functionally similar to the erythroid band 3

Studies in Chinese hamster ovary cells demonstrate the presence of an anion exchanger, which has some of the properties of the band 3 transporter in erythrocytes. 1) Extracellular chloride is a competitive inhibitor of sulfate influx and stimulates sulfate efflux, suggesting that the mechanism of uptake is SO2-(4)/Cl- exchange. 2) The anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits sulfate uptake in a dose-dependent manner. Half-maximal inhibition is achieved at 0.06 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. 3) Low extracellular pH markedly stimulates sulfate uptake. A 6-fold decrease in the apparent Km is observed at pHout 5.5 as compared to pHout 7.5. However, studies carried out over a broad range of extracellular SO2-(4) concentrations indicate the presence of three components of this transport activity in Chinese hamster ovary cells: two high affinity low capacity systems, one in the range 0.5 microM less than [SO2-(4)]out less than 50 microM and one in the range 50 microM less than [SO2-(4)]out less than 150 microM, and a low affinity, high capacity system (at [SO2-(4)]out greater than 150 microM). These properties have not been previously reported for the erythroid band 3 transporter. The availability of mutants deficient in these activities has enabled us to carry out studies which suggest that the high affinity systems are functionally independent of the low affinity system, but that all systems are dependent on the same anion exchange protein. Studies in a mutant which lacks all components of the transport activity indicates that the anion exchanger may be instrumental in the regulation of the intracellular pH in Chinese hamster ovary cells.     

Ethane production in copper-deficient rats

Evidence is accumulating which indicates that copper-deficient animals are prone to oxidative damage. To investigate this possibility further, we measured the production of breath ethane, a hydrocarbon by-product of lipid peroxidation, in copper-deficient rats. Male, weanling Sprague-Dawley rats were fed either a purified diet which was deficient in copper (CuD) or the same diet made sufficient with 5 ppm of copper (CuS). After 33 to 34 days the rats were placed individually in gastight metabolic cages through which ethane-free air or 100% O2 was passed. Expired ethane was absorbed onto cold, activated charcoal, liberated by heating, and measured by gas chromatography. Ethane production rates (pmoles/min/100 g +/- SD) were 3.3 +/- 0.8 (CuS-air), 4.3 +/- 1.4 (CuD-air), 8.3 +/- 2.5 (CuS-O2), and 12.2 +/- 4.3 (CuD-O2). Repeated measures analysis of variance indicated that both copper deficiency (P less than 0.01) and breathing 100% O2 (P less than 0.0001) enhanced ethane production, with no interaction between treatments. This finding complements previous evidence that increased lipid peroxidation occurs in copper-deficient rats.     

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial–stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpin^cpdm), and mice with a stromal (S100a4‐Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin^cpdm mammary epithelial cells transplanted in vivo into wild‐type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin^cpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpin^cpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro. Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.