scholarly journals Fucosylated protein of retinal cone photoreceptor outer segments: morphological and biochemical analyses

1982 ◽  
Vol 92 (2) ◽  
pp. 269-276 ◽  
Author(s):  
AH Bunt ◽  
JC Saari
Keyword(s):  
Intravitreal Injection ◽  
Time Course ◽  
Apparent Molecular Weight ◽  
Sucrose Density Gradient ◽  
Neutral Sugar ◽  
Electron Microscopic ◽  
Outer Segments ◽  
Photoreceptor Membranes ◽  
Electron Microscopic Radioautography

Cone outer segments (OS) of the goldfish retina are diffusely labeled after intravitreal injection of [(3)H]fucose while rod OS remain unlabeled. By electron microscopic radioautography, the OS of red- and blue-sensitive cones are heavily labeled while green- sensitive cone OS are lightly labeled. The time-course and pattern of OS labeling in all cone types from 30 min to 24 h resemble that of incorporation of other sugars into rhodopsin in rod OS. The nature of the cone OS-specific fucosylated component(s) was examined using biochemical techniques. Cone OS were prelabeled by intravitreal injection of [(3)H]fucose 24 h before sacrifice. Photoreceptor OS were isolated using a discontinuous sucrose density gradient and it was verified by electron microscopic radioautography that the only source of radioactivity in the preparations was cone OS. The different cone types could be recognized by the heaviness of labeling, characteristic membrane spacing, and 'staining' of green cone OS in vitro with horseradish peroxidase. After acid hydrolysis of prelabeled photoreceptor membranes, 90 percent of the counts were in the neutral sugar fraction which was analyzed by thin-layer chromatography. Approximately 70 percent of the radioactivity co-chromatographed with authentic fucose. SDS-PAGE/fluorography of prelabeled photoreceptor membranes revealed a single radioactive component that was lightly stained with coomassie blue and showed an apparent molecular weight of 33,000. This cone-derived band was separated from unlabeled rod opsin which was well stained and showed an apparent mol wt of 38,000. Isoelectric focusing under denaturing conditions produced two major and one minor band of radioactivity with isoelectric points of 8.2, 8.6, and 8.8 respectively. No radioactivity was found in association with a stained band corresponding in isoelectric point to that of bovine opsin (pl, 6.2). The fucosylated component was readily digested by pronase, indicating its protein nature. Washing of the isolated OS with isotonic and hypotonic buffers failed to extract major amounts of the radioactivity, suggesting that the fucosylated component is an integral membrane protein. The presence of a fucosylated protein thus represents a major difference between cone and rod OS in the goldfish and has enabled us to identify cone OS in preparations of isolated photoreceptor membranes and to demonstrate the separation of a cone-derived glycoprotein from rod opsin.

Copper transport in rats involving a new plasma protein

1985 ◽  
Vol 249 (1) ◽  
pp. E77-E88 ◽  
Author(s):  
K. C. Weiss ◽  
M. C. Linder
Keyword(s):  
Time Course ◽  
Specific Activity ◽  
High Specific Activity ◽  
Apparent Molecular Weight ◽  
Female Rats ◽  
Whole Body ◽  
Peripheral Tissues ◽  
Chelex 100

The time course of distribution of high-specific activity 67CuCl2 to tissues and plasma components was followed in adult, female rats. Immediately after intubation or injection, tracer 67Cu associated with two components of the blood plasma separable on columns of Sephadex G-150: albumin and another (larger) component, which was not ceruloplasmin. The latter, tentatively named transcuprein, had an apparent molecular weight of 270,000 and a high affinity for Cu2+, as judged by processing through Chelex-100, dilution, and exchange with albumin copper, in vitro and in vivo. It was capable of donating copper to tumor cells in serum-free medium. Analysis of "cold" plasma by furnace atomic absorption confirmed the presence of 10-15% of plasma copper in this peak. Plots of percent dose and 67Cu specific activity against time showed that copper followed a very specific pathway after binding to albumin and transcuprein, entering mainly the liver, then reappearing in the plasma on ceruloplasmin, and then achieving peak distribution in peripheral tissues (muscles, brain, etc.). 67Cu disappeared from liver and kidney with an apparent half-life of 4.5 days, the same exponential rate found for whole body turnover. Apparent turnover of ceruloplasmin copper was more rapid. Even after 7-12 days, tracer copper in plasma was still found exclusively with ceruloplasmin. The results indicate that copper follows a carefully prescribed path, on entering the blood and binding to a new transport protein.

scholarly journals Receptor-mediated entry of beta-glucuronidase into the parasitophorous vacuoles of macrophages infected with Leishmania mexicana amazonensis.

1983 ◽  
Vol 157 (5) ◽  
pp. 1471-1482 ◽  
Author(s):  
V L Shepherd ◽  
P D Stahl ◽  
P Bernd ◽  
M Rabinovitch
Keyword(s):  
Bone Marrow ◽  
Half Life ◽  
Mannose Receptor ◽  
Leishmania Mexicana ◽  
Preputial Gland ◽  
Electron Microscopic ◽  
Infected Cells ◽  
Secondary Lysosomes ◽  
Electron Microscopic Radioautography

125I-labeled rat preputial gland beta-glucuronidase was shown by light and electron microscopic radioautography to accumulate within the parasitophorous vacuoles of in vitro derived bone marrow macrophages infected with Leishmania mexicana amazonensis. beta-glucuronidase uptake was mediated by the mannose receptor, since the penetration of the ligand was inhibited by mannan. Uptake was detected as soon as 4 h after incubation of infected cells with the ligand, and increased at 24 and 48 h. The label persisted in the vacuoles for at least 24 h after a 24-h pulse with the ligand, a finding compatible with the relatively long half-life of labeled beta-glucuronidase in normal macrophages. Parasitophorous vacuoles were also labeled in macrophages exposed to the ligand only before infection, indicating that secondary lysosomes containing the ligand fused with the parasitophorous vacuoles. Another mannosylated ligand, mannose-BSA, which, in contrast to beta-glucuronidase, is rapidly degraded in macrophage lysosomes, did not detectably accumulate in the vacuoles. The results support and extend information previously obtained with electron opaque tracers that emphasizes the phagolysosomal nature of Leishmania parasitophorous vacuoles. In addition, the results suggest that appropriate mannosylated molecules may be used as carriers for targeting of leishmanicidal drugs to the parasitophorous vacuoles of infected macrophages.

scholarly journals CONTROLLED PROTEOLYSIS OF NASCENT POLYPEPTIDES IN RAT LIVER CELL FRACTIONS

1970 ◽  
Vol 45 (1) ◽  
pp. 146-157 ◽  
Author(s):  
D. D. Sabatini ◽  
G. Blobel
Keyword(s):  
Amino Acid ◽  
Messenger Rna ◽  
Close Association ◽  
Membrane Integrity ◽  
Sucrose Density Gradient ◽  
Electron Microscopic ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Cell Fractions

Rough microsomes were incubated in an in vitro amino acid-incorporating system for labeling the nascent polypeptide chains on the membrane-bound ribosomes. Sucrose density gradient analysis showed that ribosomes did not detach from the membranes during incorporation in vitro. Trypsin and chymotrypsin treatment of microsomes at 0° led to the detachment of ribosomes from the membranes; furthermore, trypsin produced the dissociation of released, messenger RNA-free ribosomes into subunits. Electron microscopic observations indicated that the membranes remained as closed vesicles. In contrast to the situation with free polysomes, nascent chains contained in rough microsomes were extensively protected from proteolytic attach. By separating the microsomal membranes from the released subunits after proteolysis, it was found that nascent chains are split into two size classes of fragments when the ribosomes are detached. These were shown by column chromatography on Sephadex G-50 to be: (a) small (39 amino acid residues) ribosome-associated fragments and (b) a mixture of larger membrane-associated fragments excluded from the column. The small fragments correspond to the carboxy-terminal segments which are protected by the large subunits of free polysomes. The larger fragments associated with the microsomal membranes depend for their protection on membrane integrity. These fragments are completely digested if the microsomes are subjected to proteolysis in the presence of detergents. These results indicate that when the nascent polypeptides growing in the large subunits of membrane-bound ribosomes emerge from the ribosomes they enter directly into a close association with the microsomal membrane.

The rhodopsin cycle is preserved in IRBP “knockout” mice despite abnormalities in retinal structure and function

2000 ◽  
Vol 17 (1) ◽  
pp. 97-105 ◽  
Author(s):  
HARRIS RIPPS ◽  
NEAL S. PEACHEY ◽  
XIAOPING XU ◽  
SUSAN E. NOZELL ◽  
SYLVIA B. SMITH ◽  
...  
Keyword(s):  
Binding Protein ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Indirect Evidence ◽  
Significant Loss ◽  
Subretinal Space ◽  
Electron Microscopic ◽  
Outer Segments ◽  
Structural And Functional Properties

In the vertebrate retina, vision is initiated and maintained by the photolysis and regeneration, respectively, of light-sensitive pigments in the disk membranes of the photoreceptor outer segments. This cyclical process depends on an exchange of retinoids between the photoreceptors and the retinal pigment epithelium (RPE). There is a great deal of indirect evidence that the transport of retinoids between these cellular compartments is mediated by the interphotoreceptor retinoid-binding protein (IRBP), a large glycoprotein synthesized in the photoreceptors and extruded into the interphotoreceptor matrix (IPM) that fills the subretinal space. Nevertheless, a number of in vitro experiments have demonstrated that an intermembranous transfer of retinoids can occur through an aqueous medium independent of any retinoid-binding protein. This led to the suggestion that IRBP may play the more passive role of an extracellular buffer, serving to prevent the degradation and potentially cytotoxic effects of free retinoids when large amounts are released into the IPM. We have studied the structural and functional properties of transgenic mice in which homologous recombination was used to delete the IRBP gene. Light- and electron-microscopic examination of the retinas of “knockout” (IRBP−/−) mice revealed a significant loss of photoreceptor nuclei, and profound changes in the structure and organization of the receptor outer segments. Consistent with these observations, electroretinographic recordings showed a marked reduction in response amplitude for both rod- and cone-mediated potentials. However, despite the histological and electrophysiological changes, there was no evidence of gross abnormalities in the visual cycle. After bleaching a significant fraction of the available rhodopsin, electroretinogram amplitude and rhodopsin density gradually increased toward their pre-bleach levels, and the rates of recovery were even more rapid than those seen in wild-type (IRBP+/+) mice.

A simplified method of emulsion coating and development for quantitative electron microscopic radioautography

1978 ◽  
Vol 36 (2) ◽  
pp. 64-65
Author(s):  
K. Yoshida ◽  
F. Murata ◽  
S. Ohno ◽  
T. Nagata
Keyword(s):  
Mass Production ◽  
Identical Condition ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Simplified Method ◽  
Complicated Process ◽  
Critical Problems ◽  
Electron Microscopic Radioautography ◽  
Quantitative Radioautography

IntroductionSeveral methods of mounting emulsion for radioautography at the electron microscopic level have been reported. From the viewpoint of quantitative radioautography, however, there are many critical problems in the procedure to produce radioautographs. For example, it is necessary to apply and develop emulsions in several experimental groups under an identical condition. Moreover, it is necessary to treat a lot of grids at the same time in the dark room for statistical analysis. Since the complicated process and technical difficulties in these procedures are inadequate to conduct a quantitative analysis of many radioautographs at once, many factors may bring about unexpected results. In order to improve these complicated procedures, a simplified dropping method for mass production of radioautographs under an identical condition was previously reported. However, this procedure was not completely satisfactory from the viewpoint of emulsion homogeneity. This paper reports another improved procedure employing wire loops.

Cytoplasmic Granules in Lymphocytes from Rats Dosed with SK&F 14336-D

1971 ◽  
Vol 29 ◽  
pp. 556-557
Author(s):  
T. G. Merrill ◽  
B. J. Payne ◽  
A. J. Tousimis
Keyword(s):  
Lipid Storage ◽  
Pokeweed Mitogen ◽  
Electron Microscopic ◽  
Cytoplasmic Granules ◽  
Storage Diseases ◽  
Tay Sachs Disease ◽  
Large Numbers ◽  
Microscopic Studies ◽  
Neurovisceral Lipidosis

Rats given SK&F 14336-D (9-[3-Dimethylamino propyl]-2-chloroacridane), a tranquilizing drug, developed an increased number of vacuolated lymphocytes as observed by light microscopy. Vacuoles in peripheral blood of rats and humans apparently are rare and are not usually reported in differential counts. Transforming agents such as phytohemagglutinin and pokeweed mitogen induce similar vacuoles in in vitro cultures of lymphocytes. These vacuoles have also been reported in some of the lipid-storage diseases of humans such as amaurotic familial idiocy, familial neurovisceral lipidosis, lipomucopolysaccharidosis and sphingomyelinosis. Electron microscopic studies of Tay-Sachs' disease and of chloroquine treated swine have demonstrated large numbers of “membranous cytoplasmic granules” in the cytoplasm of neurons, in addition to lymphocytes. The present study was undertaken with the purpose of characterizing the membranous inclusions and developing an experimental animal model which may be used for the study of lipid storage diseases.

Electron microscopic study of the membrane glycoproteins in the rat kidney after cisplatin treatment

1989 ◽  
Vol 47 ◽  
pp. 912-913
Author(s):  
S.K. Aggarwal
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Kidney ◽  
Light And Electron Microscopy ◽  
Kidney Tissue ◽  
Membrane Glycoproteins ◽  
Electron Microscopic ◽  
Before And After ◽  
Interstrand Cross Links ◽  
Cross Links

The proposed primary mechanism of action of the anticancer drug cisplatin (Cis-DDP) is through its interaction with DNA, mostly through DNA intrastrand cross-links or DNA interstrand cross-links. DNA repair mechanisms can circumvent this arrest thus permitting replication and transcription to proceed. Various membrane transport enzymes have also been demonstrated to be effected by cisplatin. Glycoprotein alkaline phosphatase was looked at in the proximal tubule cells before and after cisplatin both in vivo and in vitro for its inactivation or its removal from the membrane using light and electron microscopy.Outbred male Swiss Webster (Crl: (WI) BR) rats weighing 150-250g were given ip injections of cisplatin (7mg/kg). Animals were killed on day 3 and day 5. Thick slices (20-50.um) of kidney tissue from treated and untreated animals were fixed in 1% buffered glutaraldehyde and 1% formaldehyde (0.05 M cacodylate buffer, pH 7.3) for 30 min at 4°C. Alkaline phosphatase activity and carbohydrates were demonstrated according to methods described earlier.

Cytological changes induced by platinum-thymine in sarcoma-180 cells: Electron microscopy study

1990 ◽  
Vol 48 (3) ◽  
pp. 290-291
Author(s):  
Gustav Ofosu
Keyword(s):  
Mammalian Cells ◽  
Antitumor Agent ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Limiting Factor ◽  
Sarcoma 180 ◽  
Electron Microscopic ◽  
Cytological Changes

Platinum-thymine has been found to be a potent antitumor agent, which is quite soluble in water, and lack nephrotoxicity as the dose-limiting factor. The drug has been shown to interact with DNA and inhibits DNA, RNA and protein synthesis in mammalian cells in vitro. This investigation was undertaken to elucidate the cytotoxic effects of piatinum-thymine on sarcoma-180 cells in vitro ultrastructurally, Sarcoma-180 tumor bearing mice were treated with intraperitoneal injection of platinum-thymine 40mg/kg. A concentration of 60μg/ml dose of platinum-thymine was used in in vitro experiments. Treatments were at varying time intervals of 3, 7 and 21 days for in vivo experiments, and 30, 60 and 120 min., 6, 12, and 24th in vitro. Controls were not treated with platinum-thymine.Electron microscopic analyses of the treated cells in vivo and in vitro showed drastic cytotoxic effect.

Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

1991 ◽  
Vol 66 (05) ◽  
pp. 609-613 ◽  
Author(s):  
I R MacGregor ◽  
J M Ferguson ◽  
L F McLaughlin ◽  
T Burnouf ◽  
C V Prowse
Keyword(s):  
High Purity ◽  
Time Course ◽  
Prothrombin Complex Concentrate ◽  
Degradation Products ◽  
Canine Model ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

Platelet Incorporation of Labelled Adenosine and Adenosine Diphosphate

1969 ◽  
Vol 22 (02) ◽  
pp. 304-315 ◽  
Author(s):  
E. W Salzman ◽  
T. P Ashford ◽  
D. A Chambers ◽  
Lena L. Neri
Keyword(s):  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Platelet Inhibition ◽  
Electron Microscopic ◽  
Platelet Binding ◽  
Minor Degree ◽  
A Minor ◽  
Plasma Marker ◽  
Electron Microscopic Radioautography ◽  
Simultaneous Assessment

SummaryAfter incubation of platelet-rich plasma with labelled adenosine or ADP, platelet incorporation of radioactivity was assessed. Platelets were rapidly separated for counting by filtration through cellulose acetate Millipore. Inulin-H3 served as a plasma marker, and triple isotope techniques permitted simultaneous assessment of the behavior of the adenine and phosphate moieties of ADP without washing of platelets. In other experiments, electron microscopic radioautography was employed to trace the label after platelet incorporation.The results were consistent with previous reports that ADP is dephosphorylated in plasma and is incorporated by platelets only as a dephosphorylated residue, probably adenosine. The label crossed the platelet membrane and entered the platelet, where it was distributed in platelet granules and the agranular cell sap. Concentration within granules occurred to a minor degree.The results support the hypothesis that platelet aggregation by ADP occurs without a persistent bond of ADP to the platelet. Inhibition of aggregation by adenosine probably depends on a metabolic or transport process rather than on competition between adenosine and ADP for platelet binding sites.

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