scholarly journals MITOCHONDRIAL LOCALIZATION OF OXIDATIVE ENZYMES: STAINING RESULTS WITH TWO TETRAZOLIUM SALTS

1961 ◽  
Vol 9 (1) ◽  
pp. 47-61 ◽  
Author(s):  
Alex B. Novikoff ◽  
Woo-Yung Shin ◽  
Joan Drucker
Keyword(s):  
Rat Kidney ◽  
Intracellular Localization ◽  
Oxidative Enzymes ◽  
Mitochondrial Morphology ◽  
Mitochondrial Localization ◽  
Enzyme Localization ◽  
Aqueous Interfaces ◽  
Physicochemical Factors ◽  
Lipofuscin Granules ◽  
In Cells

A comparison is made of the staining results obtained with Nitro-BT and MTT-Co++ as acceptors when DPNH is the substrate in frozen sections of cold formol-calcium-fixed rat kidney (normal and following ligation of the blood vessels) and human liver containing lipofuscin granules. The kidney results are evaluated in terms of mitochondrial morphology seen after classical mitochondrial stains and in electron micrographs. It is concluded that, except for formazan deposition on lipid-aqueous interfaces, Nitro-BT staining indicates the intracellular localization of oxidative enzymes, at least at the level of light microscopy. In contrast, the use of MTT-Co++ is not reliable for such intracellular localizations. The deposition of the formazan of MTT-Co++ is determined in large part by physicochemical factors other than enzyme localization. Despite marked abnormalities of the mitochondria in cells of the ligated kidney, MTT-Co++ formazan is generally deposited in the same dotlike fashion as in cells of normal kidney.

The Morphology of the Rat Kidney Following Folic Acid Administration

1970 ◽  
Vol 28 ◽  
pp. 200-201
Author(s):  
Aline Byrnes ◽  
Elsa E. Ramos ◽  
Minoru Suzuki ◽  
E.D. Mayfield
Keyword(s):  
Folic Acid ◽  
De Novo ◽  
Specific Activity ◽  
Rat Kidney ◽  
Present Report ◽  
Intracellular Localization ◽  
Male Rats ◽  
Renal Hypertrophy ◽  
Electron Microscopic ◽  
Biochemical Alterations

Renal hypertrophy was induced in 100 g male rats by the injection of 250 mg folic acid (FA) dissolved in 0.3 M NaHCO3/kg body weight (i.v.). Preliminary studies of the biochemical alterations in ribonucleic acid (RNA) metabolism of the renal tissue have been reported recently (1). They are: RNA content and concentration, orotic acid-c14 incorporation into RNA and acid soluble nucleotide pool, intracellular localization of the newly synthesized RNA, and the specific activity of enzymes of the de novo pyrimidine biosynthesis pathway. The present report describes the light and electron microscopic observations in these animals. For light microscopy, kidney slices were fixed in formalin, embedded, sectioned, and stained with H & E and PAS.

scholarly journals Synthesis of [1,2-3H2]cholecalciferol and metabolism of [4-14C,1,2-3H2]- and [4-14C,1-3H]-cholecalciferol in rachitic rats and chicks

1971 ◽  
Vol 121 (4) ◽  
pp. 673-682 ◽  
Author(s):  
D. E. M. Lawson ◽  
B. Pelc ◽  
P. A. Bell ◽  
P. W. Wilson ◽  
E. Kodicek
Keyword(s):  
Vitamin D ◽  
Substance P ◽  
Rat Kidney ◽  
Intracellular Localization ◽  
Specific Radioactivity ◽  
Experimental Period ◽  
Time Interval ◽  
High Concentration ◽  
Very High ◽  
25 Hydroxycholecalciferol

[1,2-3H2]Cholecalciferol has been synthesized with a specific radioactivity of 508mCi/mmol by using tristriphenylphosphinerhodium chloride, the homogeneous hydrogen catalyst. With doses of 125ng (5i.u.) of [4-14C,1-3H2]cholecalciferol the tissue distribution in rachitic rats of cholecalciferol and its metabolites (25-hydroxycholecalciferol and peak P material) was similar to that found in chicken with 500ng doses of the double-labelled vitamin. The only exceptions were rat kidney, with a very high concentration of vitamin D, and rat blood, with a higher proportion of peak P material, containing a substance formed from vitamin D with the loss of hydrogen from C-1. Substance P formed from [4-14C,1,2-3H2]cholecalciferol retained 36% of 3H, the amount expected from its distribution between C-1 and C-2, the 3H at C-1 being lost. 25-Hydroxycholecalciferol does not seem to have any specific intracellular localization within the intestine of rachitic chicks. The 3H-deficient substance P was present in the intestine and bone 1h after a dose of vitamin D and 30min after 25-hydroxycholecalciferol. There was very little 25-hydroxycholecalciferol in intestine at any time-interval, but bone and blood continued to take it up over the 8h experimental period. It is suggested that the intestinal 3H-deficient substance P originates from outside this tissue. The polar metabolite found in blood and which has retained its 3H at C-1 is not a precursor of the intestinal 3H-deficient substance P.

scholarly journals The C-terminus of connexin43 adopts different conformations in the Golgi and gap junction as detected with structure-specific antibodies

2007 ◽  
Vol 408 (3) ◽  
pp. 375-385 ◽  
Author(s):  
Gina E. Sosinsky ◽  
Joell L. Solan ◽  
Guido M. Gaietta ◽  
Lucy Ngan ◽  
Grace J. Lee ◽  
...  
Keyword(s):  
Gap Junction ◽  
Tertiary Structure ◽  
Mutational Analysis ◽  
Peptide Mapping ◽  
Rat Kidney ◽  
Intracellular Localization ◽  
Protein Protein Interaction ◽  
Binding Domains ◽  
C Terminus ◽  
Junction Protein

The C-terminus of the most abundant and best-studied gap-junction protein, connexin43, contains multiple phosphorylation sites and protein-binding domains that are involved in regulation of connexin trafficking and channel gating. It is well-documented that SDS/PAGE of NRK (normal rat kidney) cell lysates reveals at least three connexin43-specific bands (P0, P1 and P2). P1 and P2 are phosphorylated on multiple, unidentified serine residues and are found primarily in gap-junction plaques. In the present study we prepared monoclonal antibodies against a peptide representing the last 23 residues at the C-terminus of connexin43. Immunofluorescence studies showed that one antibody (designated CT1) bound primarily to connexin43 present in the Golgi apparatus, whereas the other antibody (designated IF1) labelled predominately connexin43 present in gap junctions. CT1 immunoprecipitates predominantly the P0 form whereas IF1 recognized all three bands. Peptide mapping, mutational analysis and protein–protein interaction experiments revealed that unphosphorylated Ser364 and/or Ser365 are critical for CT1 binding. The IF1 paratope binds to residues Pro375–Asp379 and requires Pro375 and Pro377. These proline residues are also necessary for ZO-1 interaction. These studies indicate that the conformation of Ser364/Ser365 is important for intracellular localization, whereas the tertiary structure of Pro375–Asp379 is essential in targeting and regulation of gap junctional connexin43.

scholarly journals Transformation-specific tyrosine phosphorylation of a novel cellular protein in chicken cells expressing oncogenic variants of the avian cellular src gene.

1989 ◽  
Vol 9 (2) ◽  
pp. 629-638 ◽  
Author(s):  
A B Reynolds ◽  
D J Roesel ◽  
S B Kanner ◽  
J T Parsons
Keyword(s):  
Tyrosine Phosphorylation ◽  
Intracellular Localization ◽  
Cellular Transformation ◽  
Cellular Protein ◽  
Morphological Transformation ◽  
Specific Antibodies ◽  
Phosphorylated Proteins ◽  
Src Gene ◽  
In Cells

We used myristylated and nonmyristylated c-src-based variants and phosphotyrosine-specific antibodies to reevaluate the role of tyrosine phosphorylation in cellular transformation by pp60src. Prior methods used to detect tyrosine-phosphorylated proteins failed to discriminate predicted differences in tyrosine phosphorylation which are clearly observed with phosphotyrosine-specific antibodies and Western blotting (immunoblotting). Here we report the observation of a 120,000-Mr protein whose phosphorylation on tyrosine correlates with the induction of morphological transformation. p120 was not observed in cells overexpressing the regulated, nononcogenic pp60c-src, whereas phosphorylation of p120 was greatly enhanced in cells expressing activated, oncogenic pp60527F. Furthermore, phosphorylation of p120 was not induced by expression of the activated but nonmyristylated src variant pp602A/527F, which is transformation defective. p120 partitioned preferentially with cellular membranes, consistent with the observation that transforming src proteins are membrane associated. Although a number of additional putative substrates were identified and partially characterized with respect to intracellular localization, tyrosine phosphorylation of these proteins was not tightly linked to transformation.

scholarly journals Human Cytomegalovirus UL36 Protein Is Dispensable for Viral Replication in Cultured Cells

1999 ◽  
Vol 73 (9) ◽  
pp. 7126-7131 ◽  
Author(s):  
Catherine E. Patterson ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Half Life ◽  
Cultured Cells ◽  
Intracellular Localization ◽  
State Level ◽  
Specific Variation ◽  
Infected Cells ◽  
Ad169 Strain ◽  
Expression Plasmids ◽  
In Cells

ABSTRACT Consistent with earlier analyses of human cytomegalovirus UL36 mRNA, we find that the UL36 protein is present throughout infection. In fact, it is delivered to the infected cell as a constituent of the virion. Curiously, much less UL36 protein accumulated in cells infected with the AD169 strain of human cytomegalovirus than in cells infected with the Towne or Toledo strain, and localization of the protein in cells infected with AD169 is strikingly different from that in cell infected with the Towne or Toledo strain. The variation in steady-state level of the proteins results from different stabilities of the proteins. The UL36 proteins from the three viral strains differ by several amino acid substitutions. However, this variability is not responsible for the different half-lives because the AD169 and Towne proteins, which exhibit very different half-lives within infected cells, exhibit the same half-life when introduced into uninfected cells by transfection with expression plasmids. We demonstrate that the UL36 protein is nonessential for growth in cultured cells, and we propose that the ability of the virus to replicate in the absence of UL36 function likely explains the striking strain-specific variation in the half-life and intracellular localization of the protein.

scholarly journals 11βHSD2 Efficacy in Preventing Transcriptional Activation of the Mineralocorticoid Receptor by Corticosterone

2021 ◽  
Vol 5 (11) ◽  
Author(s):  
Yusuf Ali ◽  
Maniselvan Kuppusamy ◽  
Carolina Velarde-Miranda ◽  
Clara M Gomez-Sanchez ◽  
Maria Plonczynski ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Transcriptional Activation ◽  
Mineralocorticoid Receptor ◽  
Nuclear Translocation ◽  
Rat Kidney ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Dose Dependent Fashion ◽  
The Right ◽  
In Cells

Abstract Affinity of the mineralocorticoid receptor (MR) is similar for aldosterone and the glucocorticoids (GC) cortisol and corticosterone, which circulate at concentrations far exceeding those of aldosterone. 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) inactivation of GC within the immediate vicinity of the MR is credited with prereceptor specificity for aldosterone in cells coexpressing MR and 11βHSD2. 11βHSD2 efficacy is also critical to other recently described 11βHSD2 substrates. The aim of this work was to address doubts that low levels of expression of 11βHSD2 in aldosterone target tissues suffice to prevent the initiation of gene transcription by the MR activated by physiological concentrations of corticosterone. Cell models stably expressing an MR/Gaussia luciferase reporter and various levels of constitutive or induced 11βHSD2 at concentrations lower than those in rat kidney homogenates and microsomes were produced. Aldosterone and corticosterone were equipotent transactivators of the MR reporter gene in cells without 11βHSD2. Rate of conversion of tritiated corticosterone to 11-dehydrocorticosterone increased and corticosterone-induced nuclear translocation of MR decreased, as 11βHSD2 expression increased. The 50% maximal MR activation for the reporter gene stimulation by corticosterone rose with increasing 11βHSD2 expression, shifting the steroid dose-response curve for corticosterone-induced MR transactivation to the right. Several stable cell lines expressing an easily and reproducibly measured MR reporter system and consistent incremental amounts of 11βHSD2 protein were produced and used to document that 11βHSD2 within low physiological levels inactivates relevant concentrations of GC and decreases MR transactivation by GC in a dose-dependent fashion, laying to rest doubts of the efficacy of this enzyme.

scholarly journals Intracellular localization of human Ins(1,3,4,5,6)P5 2-kinase

2007 ◽  
Vol 408 (3) ◽  
pp. 335-345 ◽  
Author(s):  
Maria A. Brehm ◽  
Tobias M. H. Schenk ◽  
Xuefei Zhou ◽  
Werner Fanick ◽  
Hongying Lin ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Rat Kidney ◽  
Intracellular Localization ◽  
Stress Granules ◽  
Site Directed Mutagenesis ◽  
Normal Rat ◽  
High Concentrations ◽  
Green Fluorescent ◽  
Sites Of Action

InsP6 is an intracellular signal with several proposed functions that is synthesized by IP5K [Ins(1,3,4,5,6)P5 2-kinase]. In the present study, we overexpressed EGFP (enhanced green fluorescent protein)–IP5K fusion proteins in NRK (normal rat kidney), COS7 and H1299 cells. The results indicate that there is spatial microheterogeneity in the intracellular localization of IP5K that could also be confirmed for the endogenous enzyme. This may facilitate changes in InsP6 levels at its sites of action. For example, overexpressed IP5K showed a structured organization within the nucleus. The kinase was preferentially localized in euchromatin and nucleoli, and co-localized with mRNA. In the cytoplasm, the overexpressed IP5K showed locally high concentrations in discrete foci. The latter were attributed to stress granules by using mRNA, PABP [poly(A)-binding protein] and TIAR (TIA-1-related protein) as markers. The incidence of stress granules, in which IP5K remained highly concentrated, was further increased by puromycin treatment. Using FRAP (fluorescence recovery after photobleaching) we established that IP5K was actively transported into the nucleus. By site-directed mutagenesis we identified a nuclear import signal and a peptide segment mediating the nuclear export of IP5K.

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