scholarly journals Preparation and Purification of Tritiated Phytohemagglutinin and Studies of Cellular Localization in Human Leukocyte Cultures

Blood ◽  
1970 ◽  
Vol 35 (1) ◽  
pp. 44-55 ◽  
Author(s):  
ROBERT A. CONARD ◽  
CHARLES F. DEMOISE
Keyword(s):  
Cellular Localization ◽  
Mononuclear Cells ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Human Leukocyte ◽  
Chemical Extraction ◽  
Mitochondrial Activity ◽  
Electron Microscopic ◽  
Grain Distribution ◽  
Rna And Dna

Abstract A tritiated bean extract was obtained from bean plants (Phaseolus vulgaris) grown hydroponically in nutrient media containing various concentrations of tritium water. Purification was accomplished by ammonium sulfate precipitation and column chromatography using DEAE, CM-52 cellulose columns, and finally by Sephadex G-100 gel filtration. The purified product showed greatly increased mitogenic activity and electrophoretically showed a single labeled protein band on electrophoresis. Radioactivity of the purified PHA varied between 0.1-0.5 µCi/mg. which, though not as high as desirable, was sufficient for autoradiographic and subcellular fractionation studies in human leukocyte cultures. Within a few hours cytoplasmic localization of the label was noted in most of the cells in culture, including neutrophils and larger mononuclear cells. Breakdown of many of these cells during the first 24 hr. resulted in labeled amorphous basophilic staining masses of cell particles. Though agglutination of cells was commonly seen, the lack of label in red cells or surface label of leukocytes was notable. Blast forms which appeared by the second day showed prominent labeling which appeared to be largely cytoplasmic as evidenced by grain distribution. Several reasons were discussed which made it seem likely that the mitogenic molecule was represented in the cell label. Assay of subcellular fractions showed the major portion of radioactivity in the mitochondrial fraction. Specific activity, however, was too low to permit electron microscopic localization of label in individual organelles. Lack of label of RNA and DNA proteins was demonstrated by cellular distribution of the label and chemical extraction of RNA and DNA. Interesting speculation arises as to whether the PHA may stimulate mitochondrial activity and induce RNA synthesis and blastogenesis. Further experiments are in progress to obtain a purer PHA with higher specific radioactivity in order to more precisely define the site and mechanism of action of the mitogen.

scholarly journals Parathyroid hormone receptor in intact embryonic chicken bone: characterization and cellular localization.

1982 ◽  
Vol 94 (2) ◽  
pp. 379-386 ◽  
Author(s):  
C M Silve ◽  
G T Hradek ◽  
A L Jones ◽  
C D Arnaud
Keyword(s):  
Parathyroid Hormone ◽  
Cellular Localization ◽  
Adenosine Monophosphate ◽  
Biologically Active ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Parathyroid Hormone Receptor ◽  
Specific Localization ◽  
Grain Distribution

The specific localization and the characterization of the parathyroid hormone (PTH) receptor in bone have been studied using 18-d embryonic chick calvariae and biologically active, electrolytically labeled [125I] bovine PTH(1-34). Binding was initiated by adding [125I]-bPTH(1-34) to bisected calvariae at 30 degrees C. Steady state binding was achieved at 90 min at which time 10 mg drg wt of calvaria specifically bound 17% of the added [125I]bPTH(1-34). Nonspecific binding in the presence of 244 nM unlabeled bPTH(1-34) was less than 2%. Insulin, glucagon, and calcitonin (1 microgram/ml) did not compete for PTH binding sites. Half-maximal inhibition of binding was achieved at concentrations of unlabeled bPTH(1-34) or bPTH(1-84) of about 10 nM. The range of concentration (2-100 nM) over which bPTH(1-34) and bPTH(1-84) stimulated cyclic 3'5'adenosine monophosphate (cAMP) production was similar to that which inhibited the binding of [125I]bPTH(1-34). Light microscope autoradiograms showed that grains were concentrated over cells (osteoblasts and progenitor cells) at the external surface of the calvariae and in trabeculae. In the presence of excess unlabeled PTH, labeling of control autoradiograms was reduced to near background levels. No labeling of osteocytes or osteoclasts was observed. At the electron microscopic level, grains were localized primarily over cell membranes. A quantitative analysis of grain distribution suggested that cellular internalization of PTH occurred.

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.

The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

1973 ◽  
Vol 31 ◽  
pp. 574-575
Author(s):  
J. T. Stasny ◽  
R. C. Burns ◽  
R. W. F. Hardy
Keyword(s):  
Cellular Localization ◽  
Direct Evidence ◽  
Azotobacter Vinelandii ◽  
Intracellular Localization ◽  
Protein Component ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Fe Protein ◽  
Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

Newborn Platelet Dysfunction: a Storage Pool and Release Defect

1976 ◽  
Vol 36 (01) ◽  
pp. 200-207 ◽  
Author(s):  
Donald G. Corby ◽  
Thomas F. Zuck
Keyword(s):  
Specific Activity ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Newborn Infants ◽  
Specific Radioactivity ◽  
High Dose ◽  
Platelet Dysfunction ◽  
Paired Samples ◽  
Normal Adults

SummaryPer cent aggregation, release and content of adenine nucleotides, and specific radioactivity were evaluated in citrated platelet-rich plasma (PRP) prepared from paired samples of maternal and cord blood. Platelets of newborn infants aggregated normally in response to high dose ADP (20 μM), strong collagen suspensions, and thrombin; however, when compared with PRP from the mothers or from normal adults, per cent aggregation in response to lower concentrations of ADP (2 μM), weak collagen, and part particularly epinephrine was markedly reduced. Nucleotide release after stimulation of the newborns’ PRP with the latter two inducers was also impaired. ATP and ADP content of the newborns’ platelets was also significantly less than that of their mothers or of normal adults, but specific activity was normal. The data suggest that the impairment of ADP release in the platelets of newborn infants is due to decreased sensitivity to external stimuli. Since metabolic ATP is necessary for the platelet release reaction, it is postulated that the platelet dysfunction results from a lack of metabolic ATP.

THE INCORPORATION OF PALMITATE-1-14C BY RAT LUNG IN VITRO

1967 ◽  
Vol 45 (4) ◽  
pp. 597-607 ◽  
Author(s):  
A. Naimark ◽  
D. Klass
Keyword(s):  
Specific Activity ◽  
Specific Radioactivity ◽  
Phosphatidyl Inositol ◽  
Phosphatidyl Serine ◽  
Rat Lung ◽  
Exchange Reactions ◽  
Ethanolamine Phosphatidyl ◽  
Exogenous Glucose ◽  
Lipid Fractions

The incorporation of palmitate-1-14C into various lipid fractions was studied in rat lung in vitro. Most of the radioactivity was found in phospholipid, although in terms of decreasing specific activity the lipids were ranked: free fatty acid (FFA), glycerides, phospholipid. In addition to the usual glycerophosphatides, rat lung contained a substance with some of the chromatographic characteristics of phosphatidyl dimethylethanolamine. In terms of decreasing specific activities the phospholipids were ranked: phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl dimethylethanolamine, phosphatidyl serine plus phosphatidyl inositol, sphingomyelin plus lysophosphatidyl choline. Inhibition of oxidative energy production by hypoxia, cyanide, or low temperature markedly depressed the esterification of palmitate-1-14C. Less marked depression was observed in the absence of exogenous glucose. In all cases the decreased incorporation was associated with an increase in the total and specific radioactivity in tissue FFA. It is concluded that energy-independent exchange reactions are probably of little importance in the incorporation of FFA into esterified lipids of lung tissue. Under conditions of metabolic inhibition the penetration of labelled FFA into the tissue FFA pool does not appear to limit esterification.

scholarly journals Xylanase Attachment to the Cell Wall of the Hyperthermophilic Bacterium Thermotoga maritima

2007 ◽  
Vol 190 (4) ◽  
pp. 1350-1358 ◽  
Author(s):  
Wolfgang Liebl ◽  
Christoph Winterhalter ◽  
Wolfgang Baumeister ◽  
Martin Armbrecht ◽  
Michael Valdez
Keyword(s):  
Outer Membrane ◽  
Signal Peptide ◽  
Cellular Localization ◽  
Culture Supernatant ◽  
Thermotoga Maritima ◽  
Surrounding Medium ◽  
Hydrophobic Core ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Amino Terminal

ABSTRACT The cellular localization and processing of the endo-xylanases (1,4-β-d-xylan-xylanohydrolase; EC 3.2.1.8) of the hyperthermophile Thermotoga maritima were investigated, in particular with respect to the unusual outer membrane (“toga”) of this gram-negative bacterium. XynB (40 kDa) was detected in the periplasmic fraction of T. maritima cells and in the culture supernatant. XynA (120 kDa) was partially released to the surrounding medium, but most XynA remained cell associated. Immunogold labeling of thin sections revealed that cell-bound XynA was localized mainly in the outer membranes of T. maritima cells. Amino-terminal sequencing of purified membrane-bound XynA revealed processing of the signal peptide after the eighth residue, thereby leaving the hydrophobic core of the signal peptide attached to the enzyme. This mode of processing is reminiscent of type IV prepilin signal peptide cleavage. Removal of the entire XynA signal peptide was necessary for release from the cell because enzyme purified from the culture supernatant lacked 44 residues at the N terminus, including the hydrophobic part of the signal peptide. We conclude that toga association of XynA is mediated by residues 9 to 44 of the signal peptide. The biochemical and electron microscopic localization studies together with the amino-terminal processing data indicate that XynA is held at the cell surface of T. maritima via a hydrophobic peptide anchor, which is highly unusual for an outer membrane protein.

Rates of de novo Synthesis of Malate Synthase and Albumins during the Very Early Phase of Germination

1979 ◽  
Vol 34 (12) ◽  
pp. 1237-1242 ◽  
Author(s):  
Wolfram Köller ◽  
Helmut Kindl
Keyword(s):  
Early Phase ◽  
De Novo ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Malate Synthase ◽  
Polyacrylamide Gel ◽  
De Novo Synthesis ◽  
Albumin Fraction ◽  
Average Value ◽  
Large Pool

Abstract Malate synthase is synthesized de novo in the very early phase of germination. Its molecular and immunological properties do not differ from those of malate synthase from fully developed cotyledons. Radioactive leucine was administered to dry seeds of cucumber, and its incorporation into proteins of cotyledons was examined after 2 days of germination. The specific radioactivity of malate synthase, purified by immunoprecipitation and electrophoresis on polyacrylamide gel, was only 1/20 the average value of the total albumin fraction. The minimal incorporation documented by the comparatively low specific activity of isolated malate synthase is discussed in relation to the large pool of malate synthase already present in dry seeds.

scholarly journals Selective cytotoxicity of 125I-labeled monoclonal antibody T101 in human malignant T cell lines

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 429-435
Author(s):  
E Boven ◽  
T Lindmo ◽  
JB Mitchell ◽  
PA Jr Bunn
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
High Specific Activity ◽  
Tumor Localization ◽  
Cytotoxic Effects ◽  
High Specific Radioactivity ◽  
T Cell Lines

The radiolabeled anti-T cell antibody T101 can be used for specific tumor localization, but unlabeled T101 produces limited cytotoxicity in patients. We thus studied the in vitro cytotoxic effects of T101 labeled with 125I, a radionuclide known for its short-range, high- linear-energy electrons. We showed that 125I-T101 could be readily prepared at high specific activity with high immunoreactivity. Human malignant T cell lines HUT 102, MOLT-4, and HUT 78 were found to differ in the number of T65 determinants (the antigen recognized by T101) and the sensitivity to external x-ray radiation, which were of significance for the cytotoxicity of 125I-T101 in vitro. The cytotoxic effects of 125I-T101 were also found to be dose dependent and increased with exposure time under frozen conditions. As controls, unlabeled T101 had no cytotoxic effect, while free Na 125I or the 125I-labeled irrelevant antibody 9.2.27 exerted minor cytotoxicity. In HUT 102 and MOLT-4, more than 3 logs' cell killing was achieved within four weeks. Because considerable cytotoxicity was demonstrated in vitro by 125I-T101 on T65- positive malignant cells, and because low-dose 111In-T101 can be used successfully for tumor localization, future trials using 125I-T101 at high specific radioactivity may improve therapeutic results in patients with T65-positive malignancies.

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