scholarly journals Mode of action of α-chlorohydrin as a male anti-fertility agent. Inhibition of the metabolism of ram spermatozoa by α-chlorohydrin and location of block in glycolysis

1978 ◽  
Vol 170 (1) ◽  
pp. 23-37 ◽  
Author(s):  
Patricia D. C. Brown-Woodman ◽  
Hideo Mohri ◽  
Toshiko Mohri ◽  
Dai Suter ◽  
Ian G. White
Keyword(s):  
Dehydrogenase Activity ◽  
Recovery Period ◽  
Triose Phosphate Isomerase ◽  
Glycolytic Enzymes ◽  
Unexpected Finding ◽  
Glycolytic Intermediate ◽  
Enzyme Preparations ◽  
Triose Phosphate

1. The effect of α-chlorohydrin on the metabolism of glycolytic and tricarboxylate-cycle substrates by ram spermatozoa was investigated. The utilization and oxidation of fructose and triose phosphate were much more sensitive to inhibition by α-chlorohydrin (0.1–1.0mm) than lactate or pyruvate. Inhibition of glycolysis by α-chlorohydrin is concluded to be between triose phosphate and pyruvate formation. Oxidation of glycerol was not as severely inhibited as that of the triose phosphate. This unexpected finding can be explained in terms of competition between glycerol and α-chlorohydrin. A second, much less sensitive site, of α-chlorohydrin inhibition appears to be associated with production of acetyl-CoA from exogenous and endogenous fatty acids. 2. Measurement of the glycolytic intermediates after incubation of spermatozoal suspensions with 15mm-fructose in the presence of 3mm-α-chlorohydrin showed a ‘block’ in the conversion of glyceraldehyde 3-phosphate into 3-phosphoglycerate. α-Chlorohydrin also caused conversion of most of the ATP in spermatozoa into AMP. After incubation with 3mm-α-chlorohydrin, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase activities were decreased by approx. 90% and 80% respectively, and in some experiments aldolase was also inhibited. Other glycolytic enzymes were not affected by a low concentration (0.3mm) of α-chlorohydrin. Loss of motility of spermatozoa paralleled the decrease in glyceraldehyde 3-phosphate dehydrogenase activity. α-Chlorohydrin, however, did not inhibit glyceraldehyde 3-phosphate dehydrogenase or triose phosphate isomerase in sonicated enzyme preparations when added to the assay cuvette. 3. Measurement of intermediates and glycolytic enzymes in ejaculated spermatozoa before, during and after injection of rams with α-chlorohydrin (25mg/kg body wt.) confirmed a severe block in glycolysis in vivo at the site of triose phosphate conversion into 3-phosphoglycerate within 24h of the first injection. Glyceraldehyde 3-phosphate dehydrogenase activity was no longer detectable and both aldolase and triose phosphate isomerase were severely inhibited. Spermatozoal ATP decreased by 92% at this time, being quantitatively converted into AMP. At 1 month after injection of α-chlorohydrin glycolytic intermediate concentrations returned to normal in the spermatozoa but ATP was still only 38% of the pre-injection concentration. Motility of spermatozoa was, however, as good as during the pre-injection period. The activity of the inhibited enzymes also returned to normal during the recovery period and 26 days after injection were close to pre-injection values. 4. An unknown metabolic product of α-chlorohydrin is suggested to inhibit glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase of spermatozoa. This results in a lower ATP content, motility and fertility of the spermatozoa. Glycidol was shown not to be an active intermediate of α-chlorohydrin in vitro.

scholarly journals A model study of the fructose diphosphatase–phosphofructokinase substrate cycle

1973 ◽  
Vol 134 (2) ◽  
pp. 581-586 ◽  
Author(s):  
David P. Bloxham ◽  
Michael G. Clark ◽  
Paul C. Holland ◽  
Henry A. Lardy
Keyword(s):  
Triose Phosphate Isomerase ◽  
Theoretical Treatment ◽  
Glucose Phosphate Isomerase ◽  
Triose Phosphate ◽  
Glucose Phosphate ◽  
Model Study

A fructose diphosphatase–phosphofructokinase substrate cycle has been reconstructed in vitro to provide a system that recycles fructose 6-phosphate and hydrolyses ATP to ADP and Pi. The concerted actions of glucose phosphate isomerase, phosphofructokinase, aldolase and triose phosphate isomerase catalysed the loss of 3H from [5-3H,U-14C]glucose 6-phosphate. This was used as the basis of a method for the estimation of the fructose diphosphatase–phosphofructokinase substrate cycle. For the reconstructed cycle, the rate of decrease of the 3H/14C ratio in [5-3H,U-14C]hexose 6-phosphate was proportional to the rate of fructose 6-phosphate substrate cycling. A detailed theoretical treatment of this relationship is developed, which enables the rate of substrate cycling to be determined in vivo.

Characterization of the Metamitron Deaminating Enzyme Activity from Sugar Beet ( Beta vulgaris L.) Leaves

1979 ◽  
Vol 34 (11) ◽  
pp. 948-950 ◽  
Author(s):  
Carl Fedtke ◽  
Robert R. Schmidt
Keyword(s):  
Cytochrome C ◽  
Sugar Beet ◽  
Electron Donor ◽  
Nitrogen Atmosphere ◽  
Enzyme Preparations ◽  
Reducing Conditions ◽  
Trade Name ◽  
Membrane Bound

Abstract The enzymatic activity from sugar beet leaves which is responsible for the detoxification of the herbicide metamitron (4-amino-4,5-dihydro-3-methyl-6-phenyl-1, 2, 4-triazin-5-one, trade name Goltix®) has been characterized in vitro. The detoxification occurs by rapid deamination in vivo as well as in vitro. However, the deamination in vitro is only maximal under reducing conditions, i. e. with an electron donor and in a nitrogen atmosphere. The electron donor may be cystein, glutathione, dithionite or ascorbate. The enzymatic deamination further requires the addition of cytochrome c and a “supernatant factor”, which may be replaced by FMN, FAD or DCPIP. However, in the presence of FMN or DCPIP cytochrome c is not essential but only stimulatory. The partic­ulate as well as the soluble metamitron deaminating enzyme preparations obtained take up oxygen when supplied with cysteine and FMN. The particulate enzyme appears in the peroxysome-fraction. It is therefore suggested, that the enzymatic deamination of metamitron in sugar beet leaves is mediated by a proxisomal membrane bound electron transport system which alternatively may reduce oxygen or metamitron (deaminating).

scholarly journals Proton export drives the Warburg Effect

2021 ◽  
Author(s):  
Shonagh Russell ◽  
Liping Xu ◽  
Yoonseok Kam ◽  
Dominique Abrahams ◽  
Bryce Ordway ◽  
...  
Keyword(s):  
Warburg Effect ◽  
Cancer Metabolism ◽  
Aerobic Glycolysis ◽  
Lactate Production ◽  
Glycolytic Enzymes ◽  
Hek293 Cells ◽  
Driver Mutations ◽  
The Warburg Effect

Aggressive cancers commonly ferment glucose to lactic acid at high rates, even in the presence of oxygen. This is known as aerobic glycolysis, or the “Warburg Effect”. It is widely assumed that this is a consequence of the upregulation of glycolytic enzymes. Oncogenic drivers can increase the expression of most proteins in the glycolytic pathway, including the terminal step of exporting H+ equivalents from the cytoplasm. Proton exporters maintain an alkaline cytoplasmic pH, which can enhance all glycolytic enzyme activities, even in the absence of oncogene-related expression changes. Based on this observation, we hypothesized that increased uptake and fermentative metabolism of glucose could be driven by the expulsion of H+ equivalents from the cell. To test this hypothesis, we stably transfected lowly-glycolytic MCF-7, U2-OS, and glycolytic HEK293 cells to express proton exporting systems: either PMA1 (yeast H+-ATPase) or CAIX (carbonic anhydrase 9). The expression of either exporter in vitro enhanced aerobic glycolysis as measured by glucose consumption, lactate production, and extracellular acidification rate. This resulted in an increased intracellular pH, and metabolomic analyses indicated that this was associated with an increased flux of all glycolytic enzymes upstream of pyruvate kinase. These cells also demonstrated increased migratory and invasive phenotypes in vitro, and these were recapitulated in vivo by more aggressive behavior, whereby the acid-producing cells formed higher grade tumors with higher rates of metastases. Neutralizing tumor acidity with oral buffers reduced the metastatic burden. Therefore, cancer cells with increased H+ export increase intracellular alkalization, even without oncogenic driver mutations, and this is sufficient to alter cancer metabolism towards a Warburg phenotype.

scholarly journals Expression and Role of Parathyroid Hormone-Related Protein in Human Renal Proximal Tubule Cells during Recovery from ATP Depletion

1999 ◽  
Vol 10 (2) ◽  
pp. 238-244
Author(s):  
ADOLFO GARCÍA-OCAÑA ◽  
SUSAN C. GALBRAITH ◽  
SCOTT K. VAN WHY ◽  
KAI YANG ◽  
LINA GOLOVYAN ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Recovery Period ◽  
Ischemic Injury ◽  
Atp Depletion ◽  
Proximal Tubule Cell ◽  
Pthrp Expression ◽  
Human Proximal Tubule Cell

Abstract. Parathyroid hormone (PTH)-related protein (PTHrP) is widely expressed in normal fetal and adult tissues and regulates growth and differentiation in a number of organ systems. Although various renal cell types produce PTHrP, and PTHrP expression in rat proximal renal tubules is upregulated in response to ischemic injury in vivo, the role of PTHrP in the kidney is unknown. To study the effects of injury on PTHrP expression and its consequences in more detail, the immortalized human proximal tubule cell line HK-2 was used in an in vitro model of ATP depletion to mimic in vivo renal ischemic injury. These cells secrete PTHrP into conditioned medium and express the type I PTH/PTHrP receptor. Treatment of confluent HK-2 cells for 2 h with substrate-free, glucose-free medium containing the mitochondrial inhibitor antimycin A (1 μM) resulted in 75% depletion of cellular ATP. After an additional 2 h in glucose-containing medium, cellular ATP levels recovered to approximately 75% of baseline levels. PTHrP mRNA levels, as measured in RNase protection assays, peaked at 2 h into the recovery period (at four times baseline expression). The increase in PTHrP mRNA expression was correlated with an increase in PTHrP protein content in HK-2 cells at 2 to 6 h into the recovery period. Heat shock protein-70 mRNA expression was not detectable under baseline conditions but likewise peaked at 2 h into the recovery period. Treatment of HK-2 cells during the recovery period after injury with an anti-PTHrP(1-36) antibody (at a dilution of 1:250) resulted in significant reductions in cell number and uptake of [3H]thymidine, compared with nonimmune serum at the same titer. Similar results were observed in uninjured HK-2 cells. It is concluded that this in vitro model of ATP depletion in a human proximal tubule cell line reproduces the pattern of gene expression previously observed in vivo in rat kidney after ischemic injury and that PTHrP plays a mitogenic role in the proliferative response after energy depletion.

scholarly journals In Vitro and In Vivo Evidence That Ex Vivo Cytokine Priming of Donor Marrow Cells May Ameliorate Posttransplant Thrombocytopenia

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 353-359 ◽  
Author(s):  
Mariusz Z. Ratajczak ◽  
Janina Ratajczak ◽  
Boguslaw Machalinski ◽  
Rosemarie Mick ◽  
Alan M. Gewirtz
Keyword(s):  
Mononuclear Cells ◽  
Recovery Period ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Serum Free ◽  
Cd34 Cells ◽  
In Vivo Animal Model ◽  
Marrow Cells

AbstractThrombocytopenia is typically observed in patients undergoing hematopoietic stem cell transplantation. We hypothesized that delayed platelet count recovery might be ameliorated by increasing the number of megakaryocyte colony- forming units (CFU-Meg) in the hematopoietic cell graft. To test this hypothesis, we evaluated cytokine combinations and culture medium potentially useful for expanding CFU-Meg in vitro. We then examined the ability of expanded cells to accelerate platelet recovery in an animal transplant model. Depending on the cytokine combination used, we found that culturing marrow CD34+cells for 7 to 10 days in serum-free cultures was able to expand CFU-Meg ∼40 to 80 times over input number. Shorter incubation periods were also found to be effective and when CD34+ cells were exposed to thrombopoietin (TPO), kit ligand (KL), interleukin-1α (IL-1α), and IL-3 in serum-free cultures for as few as 48 hours, the number of assayable CFU-Meg was still increased ∼threefold over input number. Of interest, cytokine primed marrow cells were also found to form colonies in vitro more quickly than unprimed cells. The potential clinical utility of this short-term expansion strategy was subsequently tested in an in vivo animal model. Lethally irradiated Balb-C mice were transplanted with previously frozen syngeneic marrow mononuclear cells (106/mouse), one tenth of which (105) had been primed with [TPO, KL, IL-1a, and IL-3] under serum-free conditions for 36 hours before cryopreservation. Mice receiving the primed frozen marrow cells recovered their platelet and neutrophil counts 3 to 5 days earlier than mice transplanted with unprimed cells. Mice which received marrow cells that had been primed after thawing but before transplantation had similar recovery kinetics. We conclude that pretransplant priming of hematopoietic cells leads to faster recovery of all hematopoietic lineages. Equally important, donor cell priming before transplant may represent a highly cost-effective alternative to constant administration of cytokines during the posttransplant recovery period.

Critical Assessment of In Vitro Screening of α-Glucosidase Inhibitors from Plants with Acarbose as a Reference Standard

Planta Medica ◽  
2021 ◽  
Author(s):  
Neil Miller ◽  
Elizabeth Joubert
Keyword(s):  
Reference Standard ◽  
Screening Assay ◽  
Enzyme Preparations ◽  
Glucosidase Inhibitor ◽  
Biological Source ◽  
Oral Antidiabetic ◽  
Glucosidase Inhibitors ◽  
Plant Sources

AbstractPostprandial hyperglycemia is treated with the oral antidiabetic drug acarbose, an intestinal α-glucosidase inhibitor. Side effects of acarbose motivated a growing number of screening studies to identify novel α-glucosidase inhibitors derived from plant extracts and other natural sources. As “gold standard”, acarbose is frequently included as the reference standard to assess the potency of these candidate α-glucosidase inhibitors, with many outperforming acarbose by several orders of magnitude. The results are subsequently used to identify suitable compounds/products with strong potential for in vivo efficacy. However, most α-glucosidase inhibitor screening studies use enzyme preparations obtained from nonmammalian sources (typically Saccharomyces cerevisiae), despite strong evidence that inhibition data obtained using nonmammalian α-glucosidase may hold limited value in terms of identifying α-glucosidase inhibitors with actual in vivo hypoglycemic potential. The aim was to critically discuss the screening of novel α-glucosidase inhibitors from plant sources, emphasizing inconsistencies and pitfalls, specifically where acarbose was included as the reference standard. An assessment of the available literature emphasized the cruciality of stating the biological source of α-glucosidase in such screening studies to allow for unambiguous and rational interpretation of the data. The review also highlights the lack of a universally adopted screening assay for novel α-glucosidase inhibitors and the commercial availability of a standardized preparation of mammalian α-glucosidase.

Recent Advances in the Development of Triose Phosphate Isomerase Inhibitors as Antiprotozoal Agents

2021 ◽  
Vol 28 ◽  
Author(s):  
Lenci K. Vázquez-Jiménez ◽  
Antonio Moreno-Herrera ◽  
Alfredo Juárez-Saldivar ◽  
Alonzo González-González ◽  
Eyra Ortiz-Pérez ◽  
...  
Keyword(s):  
Trichomonas Vaginalis ◽  
Tertiary Structure ◽  
Triose Phosphate Isomerase ◽  
Interfacial Region ◽  
Parasitic Diseases ◽  
Binding Modes ◽  
Design Strategies ◽  
Triose Phosphate ◽  
Glycolysis Pathway

Background: Parasitic diseases caused by protozoa such as Chagas disease, leishmaniasis, malaria, African trypanosomiasis, amebiasis, trichomoniasis, and giardiasis are considered serious public health problems in developing countries. Drug-resistance among parasites justifies the search for new therapeutic drugs and the identification of new targets becomes a valuable approach. In this scenario, glycolysis pathway which consists of the conversion of glucose into pyruvate plays an important role in the protozoa energy supply and it is therefore considered as a promising target. In this pathway, triose phosphate isomerase (TIM) plays an essential role in efficient energy production. Furthermore, protozoa TIM show structural differences with human enzyme counterparts suggesting the possibility of obtaining selective inhibitors. Therefore, TIM is considered a valid approach to develop new antiprotozoal agents, inhibiting the glycolysis in the parasite. Objective: In this review, we discuss the drug design strategies, structure-activity relationship, and binding modes of outstanding TIM inhibitors against Trypanosoma cruzi, Trypanosoma brucei, Plasmodium falciparum, Giardia lamblia, Leishmania mexicana, Trichomonas vaginalis, and Entamoeba histolytica. Results: TIM inhibitors showed mainly aromatic systems and symmetrical structure, where the size and type of heteroatom are important for enzyme inhibition. This inhibition is mainly based on the interaction with i) the interfacial region of TIM inducing changes on the quaternary and tertiary structure or ii) with the TIM catalytic region were the main pathways that disabled the catalytic activity of the enzyme. Conclusion: Benzothiazole, benzoxazole, benzimidazole, and sulfhydryl derivatives stand out as TIM inhibitors. In silico and in vitro studies demonstrate that the inhibitors bind mainly at the TIM dimer interface. In this review, the development of new TIM inhibitors as antiprotozoal drugs is demonstrated as an important pharmaceutical strategy that may lead to new therapies for these ancient parasitic diseases.

Über den Abbau von Flavonolen in pflanzlichen Zellsuspensionskulturen / Degradation of Flavonols in Plant Suspension Cultures

1972 ◽  
Vol 27 (8) ◽  
pp. 946-954 ◽  
Author(s):  
Wolfgang Hösel ◽  
Paul D. Shaw ◽  
Wolfgang Barz
Keyword(s):  
Salicylic Acid ◽  
Glycine Max ◽  
Cell Suspension ◽  
Cicer Arietinum ◽  
Cell Suspension Cultures ◽  
Suspension Cultures ◽  
Enzyme Preparations ◽  
Cicer Arietinum L

The flavonols kaempferol, quercetin and isorhamnetin were labelled with 14C by keeping seven day old Cicer arietinum L. plants in an atmosphere of 14CO2 for five days. The purified (U-14C) flavonols were applied to cell suspension cultures of Cicer arietinum L., Phaseolus aureus Roxb., Glycine max and Petroselinum hortense. Based on the rates of 14CO2 formation and distribution of radioactivity after fractionation of the cells, the flavonols were shown to be catabolized to a very high extent.All four cell suspension cultures possess the enzymatic activity transforming flavonols to the recently discovered 2,3-dihydroxyflavanones. Upon incubation of the flavonols datiscetin and kaempferol with enzyme preparations from Cicer arietinum L. cell suspension cultures, it was demonstrated that the enzymatically formed 2,3-dihydroxyflavanones are further transformed in an enzyme catalyzed reaction. Salicylic acid was found as a degradation fragment of ring B of the 2,3,5,7,2′-pentahydroxyflavanone derived from datiscetin. Neither phloroglucinol nor phloroglucinol carboxylic acid were observed as metabolites of ring A. These in vitro findings were further substantiated by in vivo data because the flavonols kaempferol, quercetin and datiscetin when applied to cell suspension cultures of Cicer arietinum L. and Glycine max gave rise to para-hydroxybenzoic acid, protocatechuic acid and salicylic acid, respectively. It was thus concluded that flavonols are catabolized via 2,3-dihydroxyflavanones with the B-ring liberated as the respective benzoic acid. The data are discussed in connection with earlier findings on the catabolism of chalcones, cinnamic and benzoic acids.

scholarly journals Inhibition of glycolytic enzymes by 2-phosphotartronate

1976 ◽  
Vol 153 (3) ◽  
pp. 741-744 ◽  
Author(s):  
M K Thomas ◽  
T G Spring
Keyword(s):  
Pyruvate Kinase ◽  
Competitive Inhibition ◽  
Phosphoglycerate Kinase ◽  
Triose Phosphate Isomerase ◽  
Glycolytic Enzymes ◽  
Phosphoglycerate Mutase ◽  
Rabbit Muscle ◽  
Triose Phosphate ◽  
Permanganate Oxidation

2-Phosphotartronate has been synthesized by permanganate oxidation of glycerol 2-phosphate and has been tested as an inhibitor of five glycolytic enzymes that bind phosphoglycerate or phosphoglycollate. Competitive inhibition of rabbit muscle phosphoglycerate mutase, enolase and pyruvate kinase was observed. Triose phosphate isomerase and 3-phosphoglycerate kinase were not inhibited.

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