scholarly journals A Cytochemical Study on the Pancreas of the Guinea Pig

1958 ◽  
Vol 4 (5) ◽  
pp. 557-566 ◽  
Author(s):  
Philip Siekevitz ◽  
George E. Palade
Keyword(s):  
Guinea Pigs ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Pancreatic Cell ◽  
Cytochemical Study ◽  
Mitochondrial Fractions ◽  
Liver And Pancreas ◽  
Intracardiac Injection ◽  
Cell Fractions

DL-leucine-1-C14 was administered by intracardiac injection to guinea pigs and its in vivo incorporation into the proteins of various pancreatic cell fractions followed over a period of 2 hours. The pancreas was homogenized in 0.88 M sucrose and fractionated by differential centrifugation to give nuclear, zymogen, mitochondrial, microsomal, postmicrosomal, and final supernatant fractions. The proteins of these fractions, obtained by precipitation with trichloroacetic acid followed by washing, were counted. The proteins of the microsomal fraction showed the highest early specific activity and were followed by those of the zymogen and mitochondrial fractions. The microsomal fraction was broken up into two subfractions: one consisting of detached RNP particles, the other representing mainly the microsomal content and membranes. The incorporation of labelled leucine into the proteins of microsomal subtractions and in those of postmicrosomal fractions was studied comparatively in the pancreas of fasted and fed guinea pigs as well as in the liver and pancreas of fasted animals. A tentative cytological picture of protein synthesis and transport based on these findings is presented.

scholarly journals Studies on bone enzymes. Distribution of acid hydrolases, alkaline phenylphosphatase, cytochrome oxidase and catalase in subcellular fraction of bone tissue homogenates

1965 ◽  
Vol 97 (2) ◽  
pp. 389-392 ◽  
Author(s):  
G Vaes ◽  
P Jacques
Keyword(s):  
Cytochrome Oxidase ◽  
Subcellular Fraction ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Distribution Patterns ◽  
Mitochondrial Fraction ◽  
Acid Hydrolases ◽  
Mitochondrial Fractions ◽  
Tissue Homogenates ◽  
Isopycnic Centrifugation

1. When bone homogenates were fractionated according to the scheme developed for liver by de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955), all the enzymes assayed except cytochrome oxidase were found to occur partly in soluble and partly in particulate fractions. Among the particle-bound enzymes, the highest specific activity was found in the heavy-mitochondrial fraction for cytochrome oxidase, in the microsomal fraction for alkaline phenylphosphatase and in the light-mitochondrial fraction for eight acid hydrolases and for catalase. 2. Combined heavy-mitochondrial and light-mitochondrial fractions were subfractionated by isopycnic centrifugation in density gradients of sucrose or glycogen. In the various systems tried, cytochrome oxidase showed a relatively narrow distribution range with a sharp peak; the acid hydrolases and catalase showed flat and irregular distribution patterns, differing slightly in shape from one enzyme to the other. However, it was not possible to achieve a marked separation between the various enzymes under study. 3. It is concluded from these results that the acid hydrolases belong to special cytoplasmic particles, probably lysosomes, and that these particles are physically and enzymically heterogeneous. Catalase appears to be non-mitochondrial and could also belong to the lysosomes; but the possibility of an association with another type of particle must be kept in mind in view of what is known of liver catalase. Alkaline phenylphosphatase is largely attached to microsomal elements.

scholarly journals Effect of b-Propiolactone treatment on the complement activation mediated by equine antisera

1997 ◽  
Vol 39 (2) ◽  
pp. 119-122 ◽  
Author(s):  
Rosalvo GUIDOLIN ◽  
Josefina Farina MORAIS ◽  
Marco Antonio STEPHANO ◽  
José Roberto MARCELINO ◽  
Ivone Kazuko YAMAGUCHI ◽  
...  
Keyword(s):  
Biological Activity ◽  
Ammonium Sulfate ◽  
Complement Activation ◽  
Guinea Pigs ◽  
Specific Activity ◽  
Enzymatic Digestion ◽  
Significant Loss ◽  
Pepsin Digestion ◽  
Human Use

Reduction of complement activation through an alteration of the Fc fragment of immunoglobulins by <FONT FACE="Symbol">b</font>-propiolactone treatment was carried out in equine antisera raised against rabies virus, Bothrops venoms and diphtherial toxin. Results were evaluated by means of an anaphylactic test performed on guinea-pigs, and compared to the ones obtained with the same sera purified by saline precipitation (ammonium sulfate), followed or not by enzymatic digestion with pepsin. Protein purity levels for antibothropic serum were 184.5 mg/g and 488.5 mg/g in <FONT FACE="Symbol">b</font>-propiolactone treated and pepsin-digested sera, respectively. The recovery of specific activity was 100% and 62.5% when using antibothropic serum treated by<FONT FACE="Symbol"> b</font>-propiolactone and pepsin digestion, respectively. The antidiphtherial and anti-rabies sera treated with <FONT FACE="Symbol">b</font>-propiolactone and pepsin presented protein purity levels of 5,698 and 7,179 Lf/g, 16,233 and 6,784 IU/g, respectively. The recovery of specific activity for these antisera were 88.8%, 77.7%, 100% and 36,5%, respectively. <FONT FACE="Symbol">b</font>-propiolactone treatment induced a reduction in complement activation, tested "in vivo", without significant loss of biological activity. This treatment can be used in the preparation of heterologous immunoglobulins for human use.

scholarly journals A Cytochemical Study on the Pancreas of the Guinea Pig

1960 ◽  
Vol 7 (4) ◽  
pp. 619-630 ◽  
Author(s):  
Philip Siekevitz ◽  
George E. Palade
Keyword(s):  
Guinea Pig ◽  
Microsomal Fraction ◽  
Specific Radioactivity ◽  
Early Time ◽  
Acid Extraction ◽  
Time Intervals ◽  
Cytochemical Study ◽  
Cell Compartments ◽  
Exocrine Cell ◽  
Cell Fractions

Chymotrypsinogen synthesis in the exocrine cell of the guinea pig pancreas was studied under the following conditions: Animals fed after a fast of ∼48 hours received ∼1 hour after feeding an intravenous injection of DL-leucine-1-C14. At various time intervals (1 to 45 minutes) after the injection, the glands were removed and fractionated into a series of cell fractions of known cytological significance. Ten to twelve animals were used for each time point. From each cell fraction, the chymotrypsinogen was isolated by acid extraction and purified by (NH4)2SO4 fractionation, isoelectric precipitation, and chromatography. Because of the minuteness of the quantities involved, chymotrypsinogen amounts were calculated from enzymatic activity figures, and a carrier method was used to precipitate and count the enzyme. The chymotrypsinogen isolated from the attached ribonucleoprotein particles of the microsomal fraction had the highest specific radioactivity at the early time points (1 to 3 minutes). After long intervals (at 15 to 45 minutes), the specific radioactivity of the enzyme increased in the microsomal contents and finally in the zymogen granules. The results are compatible with the view that the chymotrypsinogen is synthesized in or on the attached RNP particles and subsequently transported to other cell compartments.

Studies on the toxicity of copper. I. The toxic action of copper in vivo and in vitro

1966 ◽  
Vol 166 (1004) ◽  
pp. 273-284 ◽  
Keyword(s):  
Microsomal Fraction ◽  
Specific Activity ◽  
Membrane Surface ◽  
High Specific Activity ◽  
Rapid Onset ◽  
Significant Inhibition ◽  
Low Concentrations ◽  
The Brain

With the object of throwing light upon the brain damage found in patients with Wilson’s disease (hepato-lenticular degeneration) due to the accumulation of copper, the effect of Cu 2+ has been investigated in pigeons. Subarachnoid injections of Cu 2+ (10 to 25 µ g) led to rapid onset of convulsions and death. These concentrations of Cu 2+ inhibited pigeon and rat b rain mitochondria; more organized tissue breis or slices showed no significant inhibition of oxygen up take at Cu +2 concentration inducing convulsions in vivo . Studies with radioactive copper ( 64 Cu) showed that the injected copper was widely distributed in the brain, though maximal near the site of injection. Centrifugation showed a high specific activity in the ATP -ase-rich microsomal fraction. Thorium in concentrations similar to Cu 2+ was not toxic. From this we suggest that the Cu 2+ does not alter the charge on some membrane surface. Since the effect of the copper is immediate, and since it does not affect respiration of slices in these low concentrations, we conclude that it is exerting its convulsive effect directly upon the cell surfaces.

Effects of hypoxia on in vivo glycine-C14 incorporation into pancreatic cell proteins

1965 ◽  
Vol 208 (6) ◽  
pp. 1177-1182 ◽  
Author(s):  
M. Don Turner ◽  
Anne C. Turner
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Liquid Scintillation ◽  
Experimental Series ◽  
Male Rats ◽  
Supernatant Fraction ◽  
Zymogen Granule ◽  
Pancreatic Cell ◽  
Cell Fractions

The effects of graded hypoxia on glycine-C14 incorporation into subcellular components were measured in the intact mammal. Groups of three fasted male rats were injected intraperitoneally with 5 µc of glycine-2-C14 and sacrificed at 5 (or 10), 15, 20, 30, and 45 min by decapitation. In one experimental series the environmental pO2 was maintained at 35 mm Hg for 2 hr before injection and throughout the experiment. In three other experimental series, the pO2 in the sealed chamber was maintained at 58, 48, and 38 mm Hg for 45 min before injection and for the duration of the experiment. The whole pancreases were rapidly removed, cooled, homogenized, and pooled before separation of cell fractions by ultracentrifugation. The specific activities (counts/min per µg of amino acid or protein N) were obtained for plasma and supernatant fraction ("cell sap") amino acids and for the purified proteins of zymogen granule, mitochondrial, microsomal, and cell sap fractions from micro-Kjeldahl analyses and liquid-scintillation counting. No detectable changes were found in the turnover of plasma amino acids during graded hypoxia. Amino acid incorporation into the proteins of all cell fractions was depressed stepwise with increasing degrees of hypoxia.

Ontogeny of gastric H(+)-K(+)-ATPase in suckling rabbits

1990 ◽  
Vol 259 (6) ◽  
pp. G913-G921 ◽  
Author(s):  
J. M. Crothers ◽  
W. W. Reenstra ◽  
J. G. Forte
Keyword(s):  
Microsomal Fraction ◽  
Apical Membrane ◽  
Specific Activity ◽  
Oxyntic Cell ◽  
Age Related ◽  
Nitrophenyl Phosphate ◽  
Weaning Age ◽  
Age Range ◽  
Hydrolysis Of

Gastric mucosal homogenates were prepared from resting and stimulated stomachs of rabbits, age 3-57 days postnatal, and fractionated by differential centrifugation. Total H(+)-K(+)-adenosinetriphosphatase (ATPase) (assayed as K(+)-dependent ouabain-insensitive hydrolysis of p-nitrophenyl phosphate) was low in the first 3 wk but rapidly accumulated between days 20 and 43. Specific activity rose eightfold from day 3 to a typically adult level of 2 mumol.mg-1.h-1 by day 43. The microsomal fraction (P3) was subfractionated on sucrose gradients (20, 27, and 33% steps or 10-40% continuous gradient). H(+)-K(+)-ATPase from P3 of resting stomachs was distributed bimodally on the continuous gradients, with activity mainly in the denser peak (or on the 33% sucrose step) before day 20, but accumulating mainly in the lighter peak (or in the lighter step-gradient fractions) after day 20. Throughout the age range tested, in vivo stimulation with histamine just before removal of the stomach caused a loss of most H(+)-K(+)-ATPase from P3 and an increase in H(+)-K(+)-ATPase in a lower-speed fraction P1. Thus, even in the 1st postnatal wk, when H(+)-K(+)-ATPase is low, most of the enzyme occurs in cells with histamine H2 receptors and all the intracellular mechanisms for fusion of oxyntic cell tubulovesicles (enriched in P3) with the apical membrane (enriched in P1). These studies delineate a 3-wk period of sharply accelerated synthesis of H(+)-K(+)-ATPase before weaning. Age-related changes in distribution of H(+)-K(+)-ATPase among microsomal density subfractions suggest maturational changes either in the intracellular partitioning of the enzyme or in properties of the membranes containing the enzyme.

Evidence for the existence of a C25-sesterterpene pathway of steroidogenesis not involving cholesterol as an obligatory intermediate

1983 ◽  
Vol 61 (7) ◽  
pp. 714-721 ◽  
Author(s):  
Bhagu R. Bhavnani ◽  
Tony Wong
Keyword(s):  
Isotope Dilution ◽  
Guinea Pigs ◽  
Radiochemical Purity ◽  
Specific Activity ◽  
Dilution Technique ◽  
Isotope Dilution Technique ◽  
Alternate Pathway

Previous in vitro studies had indicated the possibility of steroidogenesis through a C25-sesterterpene pathway in which squalene and cholesterol are not required as obligatory intermediates. To investigate whether such a pathway exists in vivo, the precursor role of [7-3H]23,24-dinor-5-cholene-3β,20-diol in the in vivo formation of cortisol by the guinea pigs was studied. The [3H]23,24-dinor-5-cholene-3β,20-diol was synthesized by reacting [3H]pregnenolone acetate with a Grignard reagent. The product was purified by chromatography and its radiochemical purity was established by the isotope dilution technique. In the first experiment a total of 134 × 106 dpm of [3H]23,24-dinor-5-cholene-3β,20-diol was injected subcutaneously into three guinea pigs. Urine was collected for 8 days and was pooled. Only 12% of the administered dose was excreted in the urine. The urine was extracted and a neutral extract (3 × 106 dpm) was obtained. From this extract 2.3 mg of cortisol containing 2.9 × 104 dpm was isolated. Radiochemical purity of the isolated cortisol was established by the isotope dilution technique. Radiochemical purity was further confirmed by conversion to cortisol 21-acetate and subsequently to 11β-hydroxyandrost-4-ene-3,17-dione and recrystallization to constant specific activity. The results of this experiment were confirmed by repeating the experiment with a higher specific activity [3H]23,24-dinor-5-cholene-3β,20-diol. These results indicate that the C25-sesterterpene pathway is a possible in vivo alternate pathway of steroidogenesis, not involving either squalene or cholesterol as obligatory intermediates.

scholarly journals Assimilation of glucose carbon in subcellular rat brain particles in vivo and the problems of axoplasmic flow

1967 ◽  
Vol 105 (3) ◽  
pp. 927-936 ◽  
Author(s):  
R. Vrba
Keyword(s):  
Particulate Matter ◽  
High Speed ◽  
Specific Activity ◽  
Sucrose Density Gradient ◽  
Mitochondrial Fraction ◽  
Axoplasmic Flow ◽  
Brain Lipids ◽  
Mitochondrial Fractions ◽  
High Speed Supernatant

1. Rats were injected with [U−14C]glucose and the content of 14C in proteins and lipids of the cerebral P1 (‘nuclear’), P2 (‘mitochondrial’), P3 (‘microsomal’) and high-speed supernatant fractions was measured 7, 22 and 93hr. after injection of labelled glucose. 2. The crude brain mitochondrial fractions (P2) were subfractionated on continuous sucrose gradients (0·32–1·8m-sucrose) and the 14C content of the proteins and lipids of about 20 subfractions was measured. 3. About 40–50% of the 14C assimilated by brain proteins was found in the P2 (‘mitochondrial’) fraction. About 68–70% of the 14C assimilated by brain lipids was also recovered from the lipids of the P2 fraction. 4. Between 22 and 93hr. after injection of [U−14C]glucose both the amount of 14C in the protein of the P2 (‘mitochondrial’) fraction and the specific activity of this protein increased. The specific activity of the protein of all other particulate fractions (P1, P2 and P3) and subfractions (obtained from sucrose-density-gradient subfractionation of fraction P2) when related to the specific activity of the high-speed supernatant protein also increased during 93hr. after injection of [U−14C]glucose. The amount of 14C in the protein of the high-speed supernatant and the specific activity of this protein decreased during the same period. 5. The distribution of 14C in the lipids of all subcellular particulate fractions remained unchanged during the period 22–93hr. after injection of [U−14C]glucose. 6. It was concluded that a diffusion occurs of some supernatant proteins into subcellular particulate matter of the cerebrum and no significant preference for any subcellular particulate matter was observed. The lipids occur in the cerebrum mainly in a non-diffusible state, which is consistent with the view that they form almost entirely a part of the structure of the cerebrum. 7. The data obtained do not lend further support to the concept of axoplasmic flow within the cerebrum or the concept of a one-directional flow of mitochondria or other subcellular particles within the cerebrum.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Mitochondrial matrix calcium ions regulate energy production in myocardium: A cytochemical study

1990 ◽  
Vol 48 (2) ◽  
pp. 166-167
Author(s):  
W.A. Jacob ◽  
R. Hertsens ◽  
A. Van Bogaert ◽  
M. De Smet
Keyword(s):  
Energy Metabolism ◽  
Calcium Ions ◽  
Energy Production ◽  
Regulation Of Respiration ◽  
Free Calcium ◽  
Mitochondrial Matrix ◽  
Cytochemical Study ◽  
Nmr Techniques

In the past most studies of the control of energy metabolism focus on the role of the phosphorylation potential ATP/ADP.Pi on the regulation of respiration. Studies using NMR techniques have demonstrated that the concentrations of these compounds for oxidation phosphorylation do not change appreciably throughout the cardiac cycle and during increases in cardiac work. Hence regulation of energy production by calcium ions, present in the mitochondrial matrix, has been the object of a number of recent studies.Three exclusively intramitochondnal dehydrogenases are key enzymes for the regulation of oxidative metabolism. They are activated by calcium ions in the low micromolar range. Since, however, earlier estimates of the intramitochondnal calcium, based on equilibrium thermodynamic considerations, were in the millimolar range, a physiological correlation was not evident. The introduction of calcium-sensitive probes fura-2 and indo-1 made monitoring of free calcium during changing energy metabolism possible. These studies were performed on isolated mitochondria and extrapolation to the in vivo situation is more or less speculative.

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