THE ACTION OF SERUM GONADOTROPHIN ON GLUTAMIC-OXALACETIC TRANSAMINASE IN THE RAT OVARY

1965 ◽  
Vol 49 (3) ◽  
pp. 379-387 ◽  
Author(s):  
B. Eckstein ◽  
Z. Paster
Keyword(s):  
Electrophoretic Mobility ◽  
Soluble Fraction ◽  
Mitochondrial Fraction ◽  
Supernatant Fraction ◽  
List Type ◽  
Differential Centrifugation ◽  
Glutamic Oxalacetic Transaminase ◽  
Enzyme Form ◽  
Rat Ovary

ABSTRACT The mechanism by which glutamic-oxalacetic transaminase (GOT) activity is increased in the rat ovary, following gonadotrophin treatment, was studied. It was found: Differential centrifugation of ovarian homogenate showed that there was an increased enzyme activity in the supernatant fraction. The GOT located in the mitochondrial fraction could be activated in vitro by incubating this fraction for several hours. Such treatment did not alter the activity of the enzyme in the supernatant fraction. Electrophoretic separation of the subcellular fractions showed the presence of the same two isoenzymes both in the mitochondrial and in the supernatant fractions. Only small amounts of the fast moving isoenzyme (GOT II) were present in the supernatant fraction, while the difference in quantity between the two isoenzymes in the mitochondria was less marked. Gonadotrophin stimulation intensified only the slow moving enzyme (GOT I) in the supernatant. Examination of the electrophoretic mobility of the enzyme released by the mitochondria into the incubation medium after in vitro activation showed that only GOT I was released by this treatment. The apparent Km for both L-aspartate as well as for α-ketoglutarate in the supernatant fraction remained unaltered by gonadotrophin stimulation, while the apparent Km were changed in the mitochondrial fraction of the hormone treated rats. These observations led to the hypothesis that gonadotrophin stimulation activates GOT in the rat ovary by transfering a latent mitochondrial GOT to the cell sap. In previous publications from this laboratory (Eckstein & Shain 1963; Eckstein 1963; and others), it was reported that gonadotrophins with follicle stimulating activity considerably enhance the glutamic-oxalacetic transaminase (GOT) activity in the rat ovary, while purified LH (NIH-LH-Sl) is inactive in this respect. Isoenzymes* of GOT have been reported for a great variety of organs (Boyd 1961, 1962; Katunuma 1962; Decker & Rau 1963; Fleisker et al. 1960; Augustinsson & Erne 1961). It has been shown (Boyd 1961), that two forms of GOT in the liver can be separated by differential centrifugation. One form adheres to the mitochondria, while the second enzyme is found in the soluble fraction. These two isoenzymes differ in their substrate affinities, pH dependance and electrophoretic mobility. The GOT in the mitochondria is in a latent form, and can be activated by release into the soluble fraction. The following experiments show that the rat ovary also contains at least two GOT isoenzymes; and that serum gonadotrophin (PMS) probably activates GOT by the release of one enzyme form from the mitochondria into the soluble fraction.

Ovarian production of progesterone and 20α-dihydroprogesterone in vitro following prostaglandin F2α induced luteolysis in the superluteinized rat

1984 ◽  
Vol 105 (2) ◽  
pp. 258-265 ◽  
Author(s):  
P. A. Torjesen ◽  
A. Aakvaag
Keyword(s):  
Adenylyl Cyclase ◽  
Prostaglandin F2α ◽  
Mitochondrial Fraction ◽  
Supernatant Fraction ◽  
Production Rates ◽  
Cyclic Monophosphate ◽  
Progesterone Production ◽  
Progesterone Secretion ◽  
Prostaglandin F

Abstract. Superluteinized rats were injected with the prostaglandin F2α (PGF2α) analogue cloprostenol to induce luteolysis. The treatment decreased progesterone production of ovarian homogenates from 8.9 ± 0.5 to 4.0 ± 0.7 nmol/ovary/10 min (mean ± sem) within 40 min. tochondrial fractions isolated from control and cloprostenol treated animals produced 4.7 ± 0.4 and 2.8 ± 0.3 nmol progesterone/ovary/10 min, respectively. Thus, the PGF2α analogue treatment significantly reduced mitochondrial progesterone production. Addition of the 15 000 × g supernatant fraction did not influence the progesterone production rates of the mitochondrial fraction. The basal progesterone secretion from quartered ovaries decreased from 1.50 ± 0.15 to 0.38 ± 0.05 nmol/ovary during the initial 15 min of incubation following cloprostenol administration. hCG and N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (DBC) stimulated the progesterone secretion from quartered ovaries, but the response was delayed in ovaries obtained from cloprostenol treated animals. Although the response was delayed, the progesterone secretion following cloprostenol treatment was re-activated with cAMP either directly or via hCG. The increment in progesterone secretion above unstimulated controls in response to DBC was not influenced by the cloprostenol treatment while the increment caused by hCG was decreased. Our data suggest that: 1) PGF2α deactivates mitochondrial progesterone production, 2) this deactivation may be overcome by cAMP, and 3) PGF2α decreases gonadotrophin responsive adenylyl cyclase.

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.

scholarly journals A BIOCHEMICAL AND HISTOCHEMICAL STUDY OF GLUTAMIC OXALACETIC TRANSAMINASE ACTIVITY OF RAT HEPATIC MITOCHONDRIA FIXED IN SITU AND IN VITRO

1968 ◽  
Vol 39 (3) ◽  
pp. 725-732 ◽  
Author(s):  
Sin Hang Lee ◽  
Richard M. Torack
Keyword(s):  
Histochemical Study ◽  
Intact Cell ◽  
Mitochondrial Fraction ◽  
Tissue Homogenate ◽  
Transaminase Activity ◽  
Glutamic Oxalacetic Transaminase ◽  
Histochemical Methods ◽  
Liver Tissue Homogenate

Rat liver perfused in situ briefly with a glutaraldehyde-formaldehyde mixture was homogenized in isotonic sucrose. The mitochondria, isolated from a homogenate of the perfused liver by differential centrifugation, assumed a slender and compact appearance similar to those often seen in an intact cell. The glutamic oxalacetic transaminase (GOT) activity of this mitochondrial fraction survived an additional formaldehyde fixation and was studied by biochemical and histochemical methods. The biochemical assay of the enzyme activity revealed that the activity was only slightly less than that of an unfixed mitochondrial fraction. The reaction product due to mitochondrial GOT activity was found to be localized to the cristae, as had been demonstrated in an intact liver cell. GOT activity of the mitochondrial fraction isolated from fresh liver tissue homogenate in 0.25 M sucrose was inactivated readily by either glutaraldehyde or formaldehyde and was no longer demonstrable by biochemical and histochemical methods after fixation.

scholarly journals Exchange of phospholipids between brain membranes in vitro

1972 ◽  
Vol 126 (4) ◽  
pp. 823-835 ◽  
Author(s):  
E. K. Miller ◽  
R. M. C. Dawson
Keyword(s):  
Mitochondrial Fraction ◽  
Supernatant Fraction ◽  
Exchange Processes ◽  
Brain Microsomes ◽  
Phospholipid Transfer ◽  
Myelin Fraction ◽  
Macromolecular Component ◽  
The Brain ◽  
Guinea Pig Brain

1. When unlabelled mitochondria from guinea-pig brain were incubated with a 32P-labelled microsomal fraction from brain there was a transfer of phospholipid to the mitochondria, which could not be accounted for by an aggregation of microsomes and mitochondria or an exchange with microsomes contaminating the mitochondria. Under similar circumstances there was a transfer of phospholipid from 32P-labelled mitochondria to microsomes, indicating that the process was one of exchange. 2. The transfer from microsomes was greatly stimulated by a non-dialysable heat-labile macromolecular component in the brain supernatant fraction but not by the concentration of the particulate fractions. 3. Phospholipid-exchange processes occurred most readily between pH7 and 7.5 and were inhibited by the presence of myelin and on the addition of lysophosphatidylcholine. 4. The rates of transfer of individual phospholipids from brain microsomes to mitochondria were similar. 5. 32P-labelled microsomes could slowly donate phospholipid to the isolated synaptosomal (nerve-ending) fraction but the phospholipids of the myelin fraction did not exchange. 6. Subfractionation of the synaptosomal fraction after [32P]phospholipid transfer showed that the mitochondria were most actively labelled during the incubation. All of the isolated individual synaptosomal membranes were capable of acquiring phospholipid on incubation with a 32P-labelled brain supernatant fraction although a greater percentage was again exchanged by the mitochondrial fraction.

DER EINFLUSS VON RESERPIN AUF DIE ANTITHYREOIDALE WIRKUNG VON KALIUMPERCHLORAT

1962 ◽  
Vol 39 (3) ◽  
pp. 423-430
Author(s):  
H. L. Krüskemper ◽  
F. J. Kessler ◽  
E. Steinkrüger
Keyword(s):  
Thyroid Gland ◽  
Male Rats ◽  
Normal Male ◽  
Tissue Respiration ◽  
List Type ◽  
Morphological Alterations ◽  
Hormone Synthesis ◽  
Stimulation Of

ABSTRACT 1. Reserpine does not inhibit the tissue respiration of liver in normal male rats (in vitro). 2. The decrease of tissue respiration of the liver with simultaneous morphological stimulation of the thyroid gland after long administration of reserpine is due to a minute inhibition of the hormone synthesis in the thyroid gland. 3. The morphological alterations of the thyroid in experimental hypothyroidism due to perchlorate can not be prevented with reserpine.

LACK OF EFFECT OF CORTICOSTEROIDS ON THE IN VIVO DISAPPEARANCE OF SULFHYDRYL-INHIBITED AND ANTIBODY-COATED ERYTHROCYTES

1964 ◽  
Vol 47 (3_Suppl) ◽  
pp. S28-S36
Author(s):  
Kailash N. Agarwal
Keyword(s):  
Red Cells ◽  
List Type ◽  
Red Cell

ABSTRACT Red cells were incubated in vitro with sulfhydryl inhibitors and Rhantibody with and without prior incubation with prednisolone-hemisuccinate. These erythrocytes were labelled with Cr51 and P32 and their disappearance in vivo after autotransfusion was measured. Prior incubation with prednisolone-hemisuccinate had no effect on the rate of red cell disappearance. The disappearance of the cells was shown to take place without appreciable intravascular destruction.

ÉTUDE IN VITRO DU MÉTABOLISME DES HYDRATES DE CARBONE DANS LE LEUCOCYTE CIRCULANT DU COBAYE TRAITÉ À LA CORTISONE

1966 ◽  
Vol 51 (2) ◽  
pp. 193-202
Author(s):  
J. A. Antonioli ◽  
A. Vannotti
Keyword(s):  
Glucose Concentration ◽  
Intramuscular Injection ◽  
Acute Inflammation ◽  
Glycogen Content ◽  
Glycogen Synthesis ◽  
Lactate Production ◽  
Glucose Consumption ◽  
List Type ◽  
Hydrates De Carbone

ABSTRACT 1. The metabolism of suspensions of circulating leucocytes has been studied after intramuscular injection of a dose of 50 mg/kg of a corticosteroid (cortisone acetate). The suspensions were incubated under aerobic conditions in the presence of a glucose concentration of 5.6 mm. Glucose consumption, lactate production, and variations in intracellular glycogen concentration were measured. After the administration of the corticosteroid, the anabolic processes of granulocyte metabolism were reversibly stimulated. Glucose consumption and lactate production increased 12 hours after the injection, but tended to normalize after 24 hours. The glycogen content of the granulocytes was enhanced, and glycogen synthesis during the course of the incubation was greatly stimulated. The action of the administered corticosteroid is more prolonged in females than in males. The injection of the corticosteroid caused metabolic modifications which resemble in their modulations and in their chronological development those found in circulating granulocytes of guinea-pigs suffering from sterile peritonitis. These results suggest, therefore, that, in the case of acute inflammation, the glucocorticosteroids may play an important role in the regulation of the metabolism of the blood leucocytes.

ÉTUDE DE L'ACTIVITÉ BIOLOGIQUE IN VITRO DES HORMONES GONADOTROPES

1960 ◽  
Vol XXXIII (III) ◽  
pp. 444-450 ◽  
Author(s):  
Maria de la Luz Suarez Soto ◽  
Jean Legault Démare
Keyword(s):  
Paper Chromatography ◽  
Incubation Medium ◽  
Ultraviolet Absorption ◽  
Rat Ovary

ABSTRACT Serum gonadotrophin (PMS) when added to the incubation medium of rat ovary slices increases the amount of Δ4-3-ketosteroids produced. This enhancement is proportional to the logarithm of dose. The ketosteroids were determined by their ultraviolet absorption; paper chromatography has shown that only androst-4-en-3,17-dione is present.

184 Two types of anti-TIGIT antibodies with distinct binding epitope and functional activities

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A198-A198
Author(s):  
Tingting Zhong ◽  
Xinghua Pang ◽  
Zhaoliang Huang ◽  
Na Chen ◽  
Xiaoping Jin ◽  
...  
Keyword(s):  
T Cells ◽  
Mixed Culture ◽  
Cell Activity ◽  
Cross Reactivity ◽  
Binding Activity ◽  
Jurkat Cells ◽  
List Type ◽  
Dependent Manner ◽  
Vitro Assay

BackgroundTIGIT is an inhibitory receptor mainly expressed on natural killer (NK) cells, CD8+ T cells, CD4+ T cells and Treg cells. TIGIT competes with CD226 for binding with CD155. In cancers, CD155 has been reported to up-regulate on tumor cells, and TIGIT was found to increase on TILs.1 Activation of TIGIT/CD155 pathway would mediate immunosuppression in tumor; while blockade of TIGIT promotes anti-tumor immune response.MethodsAK126 and AK113 are two humanized anti-human TIGIT monoclonal antibodies developed by Akesobio. Binding activity of AK126 and AK113 to human TIGIT, and competitive binding activity with CD155 and CD112, were performed by using ELISA, Fortebio, and FACS assays. Cross-reactivity with cynomolgus monkey TIGIT and epitope binning were also tested by ELISA assay. In-vitro assay to investigate the activity to promote IL-2 secretion was performed in mixed-culture of Jurkat-TIGIT cells and THP-1 cells.ResultsAK126 and AK113 could specifically bind to human TIGIT with comparative affinity and effectively blocked the binding of human CD155 and CD112 to human TIGIT. X-ray crystal structure of TIGIT and PVR revealed the C’-C’’ loop and FG loop regions of TIGIT are the main PVR interaction regions.2 The only amino acid residue differences in these regions between human and monkey TIGIT are 70C and 73D. AK126 binds to both human and monkey TIGIT, AK113 binds only to monkey TIGIT. This suggests that these residues are required for AK113 binding to human TIGIT, but not required for AK126. Interestingly, results from cell-based assays indicated that AK126 and AK113 showed significantly different activity to induce IL-2 secretion in mixed-culture of Jurkat-TIGIT cells and THP-1 cells (figure 1A and B), in which AK126 had a comparable capacity of activity to 22G2, a leading TIGIT mAb developed by another company, to induce IL-2 secretion, while, AK113 showed a significantly higher capacity than 22G2 and AK126.Abstract 184 Figure 1Anti-TIGIT Antibodies Rescues IL-2 Production in Vitro T-Cell Activity Assay in a dose dependent manner. Jurkat-TIGIT cells (Jurkat cells engineered to over-express human TIGIT) were co-cultured with THP-1 cells, and stimulated with plate-bound anti-CD3 mAb in the presence of TIGIT ligand CD155 (A) or CD112 (B) with anti-TIGIT antibodies. After incubated for 48h at 37° C and 5.0% CO2, IL-2 levels were assessed in culture supernatants by ELISA. Data shown as mean with SEM for n = 2.ConclusionsWe discovered two distinct types of TIGIT antibodies with differences in both epitope binding and functional activity. The mechanism of action and clinical significance of these antibodies require further investigation.ReferencesSolomon BL, Garrido-Laguna I. TIGIT: a novel immunotherapy target moving from bench to bedside. Cancer Immunol Immunother 2018;67:1659–1667.Stengel KF, Harden-Bowles K, Yu X, et al. Structure of TIGIT immunoreceptor bound to poliovirus receptor reveals a cell-cell adhesion and signaling mechanism that requires cis-trans receptor clustering. Proc Natl Acad Sci USA 2012;109:5399–5404.

scholarly journals In Vitro Assessment of Antioxidant, Thrombolytic, Antimicrobial Activities of Medicinal Plant Pandanus odoratissimus L. Leaves Extract

2020 ◽  
Vol 12 (3) ◽  
pp. 379-390
Author(s):  
F. I. Penu ◽  
S. M. Ivy ◽  
F. Ahmed ◽  
J. Uddin ◽  
M. S. Hossain ◽  
...  
Keyword(s):  
Soluble Fraction ◽  
Blood Clot ◽  
Folk Medicine ◽  
Maximum Level ◽  
Antimicrobial Activities ◽  
Total Phenolic ◽  
Thrombolytic Activity ◽  
Standard Drug ◽  
Pandanus Odoratissimus

The present study was carried out to investigate phytochemical, antioxidant; antimicrobial, thrombolytic activity and estimate total phenolic, total flavonoid content of Pandanus odoratissimus (p.odoratissimus) leaves of methanol extract. In thrombolytic activity, aqueous soluble fraction (AQSF) exhibited highest percentage (46.58 %) of potential to lyse blood clot compared to standard drug streptokinase (69.52 %). In antimicrobial assay, dichloromethane soluble fraction (DCMSF) explored the highest diameter of clear zone of inhibition against both gram positive (19.60 ± 0.12 mm) and gram negative (20.00 ± 0.20 mm) bacteria compared to standard antibiotic, Kanamycin (50.00 ± 0.19). Levels of antioxidant were determined by DPPH assay followed by calculated IC50 values of different Kupchan extracts. The methyl soluble fraction (MSF) showed the lowest level of IC50 value (36.70 ± 0.32 µg/mL) in comparison to ascorbic acid (12.48 ± 0.09 µg/mL) while MSF disclosed the maximum level (62.19 ±  0.26 mg of GAE/g of extract) of total phenolic content in the extracts of P. odoratissimus. This study was conducted to validate the P. odoratissimus leaves used as a folk medicine such as, antioxidant, thrombolytic, and antimicrobial potential.

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