scholarly journals Effects of Cobalt on the Renal Erythropoietic Factor and Kidney Hydrolase Activity in the Rat

Blood ◽  
1973 ◽  
Vol 42 (6) ◽  
pp. 893-905 ◽  
Author(s):  
Robert J. Smith ◽  
James W. Fisher
Keyword(s):  
Enzyme Inhibitors ◽  
Mitochondrial Fraction ◽  
Hydrolase Activity ◽  
Marker Enzyme ◽  
Factor Activity ◽  
Rat Serum ◽  
Mitochondrial Fractions ◽  
Normal Rat ◽  
Mitochondrial Extract

Abstract In an experiment to determine the effects of cobalt on the renal erythropoietic factor and kidney hydrolase activity in the rat we obtained the following results: Cobalt produced significant increases in renal erythropoietic factor activity and plasma levels of erythropoietin which reached peak activity 12 hr after treatment. It also produced an increase in the activity of renal hydrolases, cathepsins A and B, which paralleled the increase in renal erythropoietic factor activity. Enzyme inhibitors which are specific for proteases, esterases, and metalloenzymes inhibited the activity of the renal erythropoietic factor in vitro. Polycythemic mice exposed to 7- and 8-day posthypoxic intervals still retained their ability to respond to in vitro generated erythropoietin when compared to mice treated on the fourth posthypoxic day. The erythropoietic activity generated by the light mitochondrial extract—normal rat serum (LME-NRS) reaction mixture was blocked by the antibody to erythropoietin. The relative concentrations of smooth and rough endoplasmic reticulum (microsomes) and vesicles (lysosomes) were approximately the same in the light mitochondrial fractions of kidneys from normal and cobalt-treated rats. Marker enzyme studies revealed primarily alkaline phosphatase activity in the light mitochondrial fraction. These studies correlate with electron micrographs of the LME which indicate a fraction composed mainly of microsomes. In addition, these data suggest a possible relationship between renal lysosomal hydrolase activity and the renal erythropoietic factor (Erythrogenin).

HORMONES AND PROTEIN BIOSYNTHESIS IN ISOLATED RAT DIAPHRAGM

1959 ◽  
Vol 18 (4) ◽  
pp. 381-394 ◽  
Author(s):  
K. L. MANCHESTER ◽  
F. G. YOUNG
Keyword(s):  
Amino Acids ◽  
Glucose Uptake ◽  
Protein Biosynthesis ◽  
Metabolic Inhibitors ◽  
Minimal Amount ◽  
Rat Diaphragm ◽  
Rat Serum ◽  
Significant Stimulation ◽  
Normal Rat

SUMMARY 1. With rat diaphragm in vitro, addition of insulin to the medium so as to give a concentration as low as 0·05 mu./ml. of the hormone, stimulated the incorporation of [14C]glycine into protein of tissue. Simultaneous addition of glucose to the medium did not affect either the minimal amount of insulin required to produce a significant stimulation of incorporation of glycine, or the magnitude of the effect of the small concentration of insulin used. 2. Addition of a mixture of oxidized A and B chains of the insulin molecule did not affect incorporation of a mixture of labelled amino acids into the protein of isolated diaphragm, but a degraded insulin (DHA-insulin), which has about 15% of the activity of insulin in stimulating glucose uptake by diaphragm, was found to stimulate incorporation of [14C]glycine to an extent comparable with its effect in stimulating glucose-uptake. 3. Addition of rat serum, or the dipping of diaphragm in a medium containing insulin, stimulated incorporation of [14C]glycine into protein of diaphragm. Both these effects and the stimulation produced by insulin in vitro were abolished when the medium contained an antiserum to insulin. 4. Addition in vitro of growth hormone (GH) stimulated incorporation of [14C]glycine into protein of diaphragm from the hypophysectomized rat but had no effect on diaphragm from the normal rat, whether or not a small dose of insulin was also added in vitro. The action of GH in promoting incorporation of [14C]glycine into protein of diaphragm from the hypophysectomized rat was not neutralized by insulin antiserum. 5. Corticotrophin, cortisol, thyroxine, vitamin B12, vitamin D2 and linoleic acid all had no observable effect on incorporation of labelled amino acids into diaphragm. Glucagon stimulated incorporation, but the stimulation was abolished by the in vitro addition of antiserum to insulin and was probably attributable to the presence of a trace of insulin in the glucagon. 6. Anaerobiosis, and the addition of various metabolic inhibitors, were found to suppress incorporation of [14C]glycine into diaphragm protein almost entirely.

scholarly journals Extraction of an Erythropoietin-Producing Factor from a Particulate Fraction of Rat Kidney

Blood ◽  
1966 ◽  
Vol 28 (3) ◽  
pp. 330-343 ◽  
Author(s):  
JOSEPH F. CONTRERA ◽  
ALBERT S. GORDON ◽  
ARTHUR H. WEINTRAUB
Keyword(s):  
Rat Kidney ◽  
Deae Cellulose ◽  
Mitochondrial Fraction ◽  
Normal Serum ◽  
The Other ◽  
Step Method ◽  
Renal Vasculature ◽  
Rat Serum ◽  
Normal Rat ◽  
Strong Resemblance

Abstract 1. A two-step method for the extraction of erythropoietin from hypoxic kidneys has been developed which allows residual plasma erythropoietin in renal vasculature to be separated from that of intracellular origin. 2. Renal extracts have been purified by DEAE cellulose chromatography and found to contain 2 major erythropoietically active fractions. One bears strong resemblance to plasma erythropoietin. The other component is unique in that it has practically no erythropoietic-stimulating activity unless previously incubated with normal rat serum. This activation phenomenon is used to identify this kidney component as the renal erythropoietic factor (REF). The REF has the capacity to produce erythropoietin or become erythropoietically active when incubated with normal rat serum. 3. Differential centrifugation technics revealed that the REF is confined to particles present in the light mitochondrial fraction of kidney. 4. Extracts of the light mitochondrial fraction of kidneys from normal rats produced significant amounts of erythropoietin when incubated with normal serum. The quantity found, however, was less than that evoked by similar extracts of kidneys from hypoxic rats. 5. The product of the incubation extracts of the renal light mitochondrial fraction with normal rat serum showed the same log dose/response regression as sheep plasma erythropoietin standard. 6. It is hypothesized that either (a) the REF is a precursor of erythropoietin which must be complexed with a carrier present in normal serum in order to become physiologically active, or (b) the renal factor is an enzyme which produces erythropoietin by its action on a particular serum protein.

Effect of Liver on Lipolysis by Normal and Postheparin Sera in the Rat

1956 ◽  
Vol 185 (1) ◽  
pp. 18-22 ◽  
Author(s):  
Judy A. Spitzer ◽  
John J. Spitzer
Keyword(s):  
Rat Liver ◽  
Lipolytic Activity ◽  
Factor Activity ◽  
Rat Serum ◽  
Oil Emulsion ◽  
Isolated Rat Liver ◽  
Normal Rat ◽  
Isolated Liver ◽  
Isolated Rat

Lipolysis by sera heparinized in vivo and by normal sera was studied after perfusion through isolated rat liver. The rate of clearing of an oil emulsion by serum containing clearing factor was decreased after perfusion through the liver. The extent of clearing was not always affected. Clearing factor activity was lessened by incubation with heparinase (prepared from rabbit livers). Normal rat serum exhibited consistent lipolytic activity following perfusion through the isolated liver. This activity was not due to clearing factor.

Properties of skeletal muscle mitochondria isolated from subsarcolemmal and intermyofibrillar regions

1993 ◽  
Vol 264 (2) ◽  
pp. C383-C389 ◽  
Author(s):  
A. M. Cogswell ◽  
R. J. Stevens ◽  
D. A. Hood
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Mitochondrial Protein ◽  
Mitochondrial Fraction ◽  
Biochemical Properties ◽  
Leucine Incorporation ◽  
Mitochondrial Protein Synthesis ◽  
Skeletal Muscle Mitochondria ◽  
Mitochondrial Fractions

Two mitochondrial fractions, termed intermyofibrillar (IMF) and subsarcolemmal (SS), were isolated from skeletal muscle, and their biochemical properties were related to differences in respiration and mitochondrial protein synthesis. State III respiration was 2.3- to 2.8-fold greater in IMF than in SS mitochondria. Site 1 inhibition of respiration with rotenone reduced this difference to 1.4-fold. When sites 1 and 2 were inhibited with antimycin, the 1.4-fold differences remained. The activities of cytochrome-c oxidase (CYTOX) and succinate dehydrogenase (SDH) could account for some of these differences, since CYTOX was 20% greater (P < 0.05) in IMF mitochondria, and SDH was 40% greater (P < 0.05) in SS mitochondria. Cytochromes a, b, c, and c1 contents were similar in the two fractions. Cardiolipin (CL) content was higher (P < 0.05) in SS mitochondria, indicating a less dense mitochondrial fraction with respect to CL. In vitro [3H]leucine incorporation was 1.8-fold higher (P < 0.05) in IMF than in SS mitochondria. Thus compositional differences between IMF and SS fractions exist, perhaps representing mitochondria at different stages of biogenesis. The biochemical and functional differences could not solely be due to differences in mitochondrial protein synthesis but could also be due to nuclear-directed protein synthesis specific to each mitochondrial fraction.

scholarly journals Partial purification and immunoreactivity of an 80000-molecular-weight polypeptide associated with peroxisome proliferation in rat liver

1980 ◽  
Vol 188 (3) ◽  
pp. 731-740 ◽  
Author(s):  
M. Kumudavalli Reddy ◽  
Paul F. Hollenberg ◽  
Janardan K. Reddy
Keyword(s):  
Large Particle ◽  
Sodium Dodecyl ◽  
Mitochondrial Fraction ◽  
Single Step ◽  
Partial Purification ◽  
Peroxisome Proliferation ◽  
Peroxisome Proliferators ◽  
Marker Enzyme ◽  
Functional Protein ◽  
Mitochondrial Fractions

Hypolipidaemic drugs and industrial plasticizers such as di-(2-ethylhexyl) phthalate, which cause proliferation of hepatic peroxisomes, also cause an increase in an 80000-mol.wt. polypeptide in the liver of rats and mice. This polypeptide has been designated as PPA-80 (PPA, for peroxisome-proliferation-associated; 80 for 80000mol.wt.). The polypeptide PPA-80 was purified to over 90% purity from livers of rats treated with the peroxisome proliferators Wy-14,643, nafenopin, tibric acid and clofibrate by a single-step preparative sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic procedure. The antibodies raised against the PPA-80 polypeptide isolated from livers of rats treated with Wy-14,643 cross-reacted with polypeptide PPA-80 purified from the livers of rats treated with Wy-14,643, as well as from the livers of rats treated with nafenopin, tibric acid and clofibrate. The anti-(polypeptide PPA-80) antibodies did not cross-react with catalase, a marker enzyme for peroxisomes, or with NADPH–cytochrome P-450 reductase, which has the same approximate mol.wt., 80000. The intensity of immunoprecipitin bands formed with microsomal, large-particle and postnuclear fractions from livers of animals pretreated with peroxisome proliferators was significantly greater compared with equal amounts of protein from corresponding fractions obtained from control animals, suggesting that these agents all enhance the synthesis of the same 80000-mol.wt. polypeptide. Although the polypeptide PPA-80 was increased in the postnuclear, large-particle and microsomal fractions of livers of rats pretreated with peroxisome proliferators, the relative abundance of this peptide in the peroxisome-rich light-mitochondrial fraction and its lack in highly purified mitochondrial fractions suggest the localization of this polypeptide in peroxisomes and/or microsomal fraction. Additional studies are needed to establish unequivocally the subcellular localization of the polypeptide PPA-80 and to ascertain if this polypeptide is identical with the multi-functional protein displaying enoyl-CoA hydratase and β-hydroxyacyl-CoA dehydrogenase activities that was purified by Osumi & Hashimoto [(1979) Biochem. Biophys. Res. Commun.89, 580–584].

scholarly journals The significance of promitochondrial structures in rat liver for mitochondrial biogenesis

1973 ◽  
Vol 134 (3) ◽  
pp. 687-695 ◽  
Author(s):  
J. G. Satav ◽  
M. S. Rajwade ◽  
S. S. Katyare ◽  
M. S. Netrawali ◽  
P. Fatterpaker ◽  
...  
Keyword(s):  
Buoyant Density ◽  
Mitochondrial Fraction ◽  
The Other ◽  
Nucleic Acid Content ◽  
Differential Centrifugation ◽  
Thyroid Status ◽  
Double Membrane ◽  
Mitochondrial Fractions ◽  
Rna And Dna

1. The heavy, light and fluffy mitochondrial fractions obtained by differential centrifugation were further characterized with respect to their protein synthesizing ability in vitro, their nucleic acid content, buoyant density of their DNA and ultrastructure. 2. The light mitochondrial fraction synthesized proteins in vitro at a rate 4–5 times as high as heavy and fluffy mitochondria. The incorporation ability of this fraction was also maximally affected by the thyroid status of the animal. The radioactivity in leucyl-tRNA of the light mitochondrial fraction was about 3–4 times as high as that of the other two fractions. 3. The heavy, light and fluffy mitochondrial fractions contained small but consistent amounts of RNA and DNA. Although the DNA content was the same in all mitochondria fractions, the light mitochondria contained relatively more RNA. The buoyant density of DNA from all the fractions was 1.701g/cm3. 4. Electron microscopy revealed that the heavy mitochondria have a typical mitochondrial architecture, with densely packed cristae and a well developed double membrane. Light mitochondria were also surrounded by double membranes, but were smaller in size and contained less cristae. The fluffy fraction consisted of a mixture of well formed mitochondria and those in the process of degradation. 5. The significance of these findings in relation to mammalian mitochondrial genesis is discussed.

Ultrastructural evidence for complement and antibody-dependent damage to schistosomula of Schistosoma mansoni by rat eosinophils in vitro

Parasitology ◽  
1978 ◽  
Vol 77 (3) ◽  
pp. 313-324 ◽  
Author(s):  
Diane J. McLaren ◽  
F. J. Ramalho-Pinto ◽  
S. R. Smithers
Keyword(s):  
Schistosoma Mansoni ◽  
Surface Damage ◽  
The Body ◽  
Receptor Interaction ◽  
Focal Lesions ◽  
Rat Serum ◽  
Ultrastructural Evidence ◽  
Basal Plasma ◽  
Normal Rat

SummaryRat peritoneal eosinophils adhere to live Schistosoma mansoni schistosomula in vitro in the presence of fresh normal rat serum, or in heat-inactivated serum from rats immune to the parasite. When the eosinophils are present in sufficient numbers the worms show ultrastructural evidence of surface damage and are ultimately killed. It is believed that the appearance of focal lesions in the tegument of the schistosomulum follows the secretion of enzymes by the eosinophils onto the parasite surface. The cells have been observed within these lesions and later between the basal plasma membrane of the tegument and the underlying interstitial material. It is suggested that the cells are responsible for prising the tegument away from the body of the worm. The detached tegument shows evidence of further degradation. Adherent eosinophils which have released their secretions appear to degenerate and are eventually replaced by macrophages. Remnants of both the expended eosinophils and the disrupted tegument have been identified within the macrophages. Adherence of eosinophils through C3–C3 receptor interaction results in earlier and more severe damage to the schistosomula than when adherence occurs through Fe receptors. Rat eosinophils also adhere to C3-coated, glutaraldehyde-flxed schistosomula and C3-coated Sepharose beads. However, evidence of enzyme secretion is only obtained when the target is a schistosomulum.

scholarly journals The biosynthesis of brain gangliosides. Separation of membranes with different ratios of ganglioside sialylating activity to gangliosides

1977 ◽  
Vol 168 (3) ◽  
pp. 325-332 ◽  
Author(s):  
C A Landa ◽  
H J F Maccioni ◽  
A Arce ◽  
R Caputto
Keyword(s):  
Membrane Fraction ◽  
Time Course ◽  
Mitochondrial Fraction ◽  
Subcellular Fractions ◽  
Synthetase Activity ◽  
Acetylneuraminic Acid ◽  
Mitochondrial Fractions ◽  
Brain Gangliosides

Brain subcellular fractions were analysed for ganglioside-sialylating activity by measuring the incorporation of N-[3H]acetylneuraminic acid from CMP-N-[3H]acetylneuraminic acid into endogenous ganglioside acceptors (endogenous incorporation) and into exogenous lactosyceramide (haematoside synthetase activity). The ratios of endogenous incorporation to gangliosides and of haematoside synthetase to gangliosides for the synaptosomal and mitochondrial fractions from a washed crude mitochondrial fraction were lower than those obtained for other membrane fractions. The differences appear to reflect intrinsic characteristics of each membrane fraction. The results of labelling in vitro and the time course of labelling of gangliosides of the different subcellular fractions in vivo after injection of N-[3H]acetylmannosamine are consistent with the possibility of a subcellular site for synthesis of gangliosides different from that of ganglioside deposition.

THE ACTION OF THE GONADOTROPHINS ON THE RAT PROSTATE IN TISSUE CULTURE

1960 ◽  
Vol XXXIII (IV) ◽  
pp. 545-551 ◽  
Author(s):  
Stig Bengmark ◽  
Barbro Ingemanson ◽  
Bengt Källén
Keyword(s):  
Growth Rate ◽  
Culture Medium ◽  
Female Rats ◽  
Rat Serum ◽  
Male And Female Rats ◽  
Different Seasons ◽  
Normal Rat ◽  
Rat Prostate ◽  
Rates Of Growth

ABSTRACT The growth rate of rat prostatic tissue in vitro has been studied. It is significantly lower in cultures made with serum taken from castrated male and female rats during the first two weeks after castration as compared with the rate in cultures made from normal rat serum. This effect can be compensated for by substitution with testosterone propionate to the male serum donors. Addition of follicle stimulating hormone (FSH) to the culture medium causes a significant reduction of the growth rate. Effective concentrations are 0.01 to 0.1 μg/ml. This effect disappears after inactivation of the FSH solution at 70° for one hour. Higher concentrations of FSH do not produce such an arrest. It is suggested that the growth retarding effect of serum or plasma from castrated animals is due to the increased content of FSH. It is further suggested that this method might be of interest for the quantitative study of circulating gonadotrophins. An example is given in which the rates of growth in plasma from cockerels bled at different seasons of the year are compared.

Activated macrophages in highly irradiated cercariae-induced immunity toSchistosoma japonicumin rats

Parasitology ◽  
1991 ◽  
Vol 102 (1) ◽  
pp. 65-72
Author(s):  
C. Xu ◽  
S. Xu
Keyword(s):  
Alveolar Macrophages ◽  
Sheep Erythrocyte ◽  
Peritoneal Macrophages ◽  
Rat Serum ◽  
Sheep Erythrocytes ◽  
Proteose Peptone ◽  
Normal Rat ◽  
Induced Immunity

SUMMARYThe results of studies on the schistosomulicidal activity of activated peritoneal and alveolar macrophages (pMø and aMø) from rats immunized with highly irradiated (50 krad.)Schistosoma japonicumcercariae are reported. The authors have examined the activation of these macrophages in terms of spreading, adhesion and ingestion of sheep erythrocytes and pinocytosis of horse-radish peroxidase. Using three criteria, peritoneal macrophages and alveolar macrophages from immunized rats and from rats intraperitoneally injected with BCG were significantly more active than those from normal rats or rats stimulated with 10% proteose-peptone or 1% sodium thioglycolate. A significantly higher percentage of adhesion and ingestion was obtained with the sheep erythrocytes that were co-opsonized by heat-inactivated rat anti-sheep erythrocyte serum and fresh normal rat serum. Schistosomulicidal effects were observed with macrophages from irradiated cercariae-immunized rats in two activation systems:in vitroactivation in the presence of macrophage-activating factor (MAF), andin vivoactivation by the intraperitoneal challenge with sonicated cercarial antigens.

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