scholarly journals Adsorptive pinocytosis of 125I-labelled lactate dehydrogenase isoenzymes H4 and M4 by rat yolk sacs incubated in vitro

1981 ◽  
Vol 198 (3) ◽  
pp. 587-593 ◽  
Author(s):  
T Kooistra ◽  
K E Williams
Keyword(s):  
Lactate Dehydrogenase ◽  
Hydrophobic Interactions ◽  
Dissociation Constants ◽  
Yolk Sac ◽  
Sinusoidal Cells ◽  
Membrane Complex ◽  
Liver Sinusoidal Cells ◽  
Lactate Dehydrogenase Isoenzymes

The pinocytic uptake of 125I-labelled porcine lactate dehydrogenase isoenzymes H4 and M4 by 17.5-day rat visceral yolk sac incubated in vitro was saturable and binding obeyed Michaelis-Menten kinetics. The uptake characteristics of the two isoenzymes were very similar. For the H4 and the M4 isoenzymes, the dissociation constants of the protein-plasma-membrane complex were 0.62 microM and 0.84 microM respectively, and the maximum rates of uptake 0.13 and 0.26 nmol/mg of yolk-sac protein per h respectively. These findings contrast with those from studies in vivo, which show the M4 form is taken up by rat liver sinusoidal cells at a much higher rate than the H4 form, and point to different recognition systems for the adsorptive pinocytosis of simple non-conjugate proteins in yolk-sac epithelial cells and liver sinusoidal cells. Competition experiments indicate that binding of the H4 isoenzyme to the yolk-sac cells is restricted to hydrophobic interactions, whereas the binding of the M4 isoenzyme involves hydrophobic as well as positively charged sites on the protein molecules.

scholarly journals Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas.

1987 ◽  
Vol 105 (3) ◽  
pp. 1463-1469 ◽  
Author(s):  
C Fornieri ◽  
M Baccarani-Contri ◽  
D Quaglino ◽  
I Pasquali-Ronchetti
Keyword(s):  
Hydrophobic Interactions ◽  
Isonicotinic Acid ◽  
Lysyl Oxidase ◽  
Acid Hydrazide ◽  
Elastic Fibers ◽  
Amino Groups ◽  
Lysine Residues ◽  
Oxidase Inhibition

Hydrophobic tropoelastin molecules aggregate in vitro in physiological conditions and form fibers very similar to natural ones (Bressan, G. M., I. Pasquali Ronchetti, C. Fornieri, F. Mattioli, I. Castellani, and D. Volpin, 1986, J. Ultrastruct. Molec. Struct. Res., 94:209-216). Similar hydrophobic interactions might be operative in in vivo fibrogenesis. Data are presented suggesting that matrix glycosaminoglycans (GAGs) prevent spontaneous tropoelastin aggregation in vivo, at least up to the deamination of lysine residues on tropoelastin by matrix lysyl oxidase. Lysyl oxidase inhibitors beta-aminopropionitrile, aminoacetonitrile, semicarbazide, and isonicotinic acid hydrazide were given to newborn chicks, to chick embryos, and to newborn rats, and the ultrastructural alterations of the aortic elastic fibers were analyzed and compared with the extent of the enzyme inhibition. When inhibition was greater than 65% all chemicals induced alterations of elastic fibers in the form of lateral aggregates of elastin, which were always permeated by cytochemically and immunologically recognizable GAGs. The number and size of the abnormal elastin/GAGs aggregates were proportional to the extent of lysyl oxidase inhibition. The phenomenon was independent of the animal species. All data suggest that, upon inhibition of lysyl oxidase, matrix GAGs remain among elastin molecules during fibrogenesis by binding to positively charged amino groups on elastin. Newly synthesized and secreted tropoelastin has the highest number of free epsilon amino groups, and, therefore, the highest capability of binding to GAGs. These polyanions, by virtue of their great hydration and dispersing power, could prevent random spontaneous aggregation of hydrophobic tropoelastin in the extracellular space.

scholarly journals Screening marine algae metabolites as high-affinity inhibitors of SARS-CoV-2 main protease (3CLpro): an in silico analysis to identify novel drug candidates to combat COVID-19 pandemic

2020 ◽  
Vol 63 (1) ◽  
Author(s):  
Ghazala Muteeb ◽  
Adil Alshoaibi ◽  
Mohammad Aatif ◽  
Md. Tabish Rehman ◽  
M. Zuhaib Qayyum
Keyword(s):  
Hydrophobic Interactions ◽  
In Silico Analysis ◽  
Salt Bridge ◽  
Competitive Inhibitor ◽  
Binding Energies ◽  
Dynamics Simulation ◽  
In Vivo Studies ◽  
Energy Calculation

AbstractThe recent dissemination of SARS-CoV-2 from Wuhan city to all over the world has created a pandemic. COVID-19 has cost many human lives and created an enormous economic burden. Although many drugs/vaccines are in different stages of clinical trials, still none is clinically available. We have screened a marine seaweed database (1110 compounds) against 3CLpro of SARS-CoV-2 using computational approaches. High throughput virtual screening was performed on compounds, and 86 of them with docking score <  − 5.000 kcal mol−1 were subjected to standard-precision docking. Based on binding energies (< − 6.000 kcal mol−1), 9 compounds were further shortlisted and subjected to extra-precision docking. Free energy calculation by Prime-MM/GBSA suggested RC002, GA004, and GA006 as the most potent inhibitors of 3CLpro. An analysis of ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties of RC002, GA004, and GA006 indicated that only RC002 (callophysin A, from red alga Callophycus oppositifolius) passed Lipinski’s, Veber’s, PAINS and Brenk’s filters and displayed drug-like and lead-like properties. Analysis of 3CLpro-callophysin A complex revealed the involvement of salt bridge, hydrogen bonds, and hydrophobic interactions. callophysin A interacted with the catalytic residues (His41 and Cys145) of 3CLpro; hence it may act as a mechanism-based competitive inhibitor. Docking energy and docking affinity of callophysin A towards 3CLpro was − 8.776 kcal mol−1 and 2.73 × 106 M−1, respectively. Molecular dynamics simulation confirmed the stability of the 3CLpro-callophysin A complex. The findings of this study may serve as the basis for further validation by in vitro and in vivo studies.

Study of yolk-sac endoderm organogenesis in the chick using a specific enzyme (cysteine lyase) as a marker of cell differentiation

Development ◽  
1973 ◽  
Vol 29 (1) ◽  
pp. 159-174
Author(s):  
Nelly Bennett
Keyword(s):  
Cell Differentiation ◽  
Blood Cells ◽  
Primitive Streak ◽  
Yolk Sac ◽  
Specific Enzyme ◽  
Cell Proliferation And Differentiation ◽  
Lyase Activity ◽  
Morphological Specialization

The detection of a specific enzyme (cysteine lyase) of the yolk-sac endoderm by a very sensitive method is employed to characterize cell differentiation during the early stages of endoderm organogenesis in the chick. The first cells to contain active cysteine lyase are found in the germ wall at the primitive streak stage. In vivo observations establish a relation between the morphological specialization and organization of endodermal cells, their loss of mitotic activity and the increase in cysteine lyase activity. They suggest an influence of the mesoderm on endoderm differentiation. In vitro experiments confirm the existence in the yolk-sac endoderm of an incompatibility between cell proliferation and differentiation, as well as the action of the mesoderm on both the structural organization of the endoblast and the appearance of cysteine lyase; this last action seems to be due mainly to blood cells; chicken and rabbit blood cells are equally active. The problems of the origin of the endoderm and of the interactions occurring during the organogenesis of the yolk-sac endoderm are discussed.

Diagnosis of hemolytic disease by electrophoresis of erythrocyte lactate dehydrogenase isoenzymes on cellulose acetate or Agarose.

1981 ◽  
Vol 27 (8) ◽  
pp. 1453-1455 ◽  
Author(s):  
F Van Lente ◽  
A Marchand ◽  
R S Galen
Keyword(s):  
Lactate Dehydrogenase ◽  
Cellulose Acetate ◽  
Predictive Value ◽  
In Vitro Models ◽  
Hemolytic Disease ◽  
Cellulose Acetate Electrophoresis ◽  
Lactate Dehydrogenase Isoenzymes

Abstract We determined the LD-1/LD-2 isoenzyme ratio in hemolysates of erythrocytes by electrophoresis on cellulose acetate and on agarose. A ratio exceeding 1.0 was found with the former but not the latter. Results were similar for in vitro models of hemolytic disorders. Using cellulose acetate electrophoresis, we determined the predictive value of data on total LD activity and of the LD-1/LD-2 ratio in diagnosis of hemolytic disease in 100 patients. The sensitivity of the "flipped" LD-1/LD-2 ratio was only 58%, the specificity was 93%, and the predictive value was 74% for diagnosis of hemolytic disease. A normal total LD activity is highly predictive (92%) for ruling at the presence of hemolytic disease.

Bulk isolation of renal PCT and PST. II. Differential responses to anoxia or hypoxia

1990 ◽  
Vol 259 (1) ◽  
pp. F176-F185 ◽  
Author(s):  
C. E. Ruegg ◽  
L. J. Mandel
Keyword(s):  
Lactate Dehydrogenase ◽  
O2 Consumption ◽  
Atp Content ◽  
Hypoxic Injury ◽  
Biochemical Responses ◽  
In Vitro Exposure ◽  
Differential Responses

Innate biochemical responses of rabbit renal proximal convoluted (PCT) and straight (PST) segments following in vitro exposure to anoxia or hypoxia were investigated to delineate the mechanisms responsible for segment-selective injury in vivo. After bulk isolation, suspensions (1 mg/ml) enriched in either PCT or PST were preincubated in Dulbecco's modified Eagle's-Ham's F-12 medium for 1 h before being exposed to either 40 min of anoxia (N2) or 120 min of hypoxia (1% O2) and 1 h of recovery under air-CO2 conditions. After recovery from anoxia, the percent of control values for each viability indicator in PCT and PST, respectively, were as follows: O2 consumption (QO2), 30/50; ATP content, 22/49; K+ content, 60/70; and percent lactate dehydrogenase (LDH) release, 66/45. Likewise, following recovery from hypoxia, the percent of control values for PCT and PST, respectively, were as follows: QO2, 50/90; ATP, 16/57; K+, 52/79; LDH, 45/17. These differential responses indicate that PCT segments were innately more susceptible to anoxic and hypoxic injury than PST segments. Because ATP content was significantly higher in PST segments immediately after anoxia and hypoxia, we investigated glucose-dependent responses during anoxia by exposing these segments to 30 min of anoxia in nutrient buffer with or without glucose. Results from these experiments demonstrate that the PST protection from anoxia was glucose dependent because removal of glucose from the nutrient buffers during anoxia abolishes the differential responses between PCT and PST. The in vitro PCT sensitivity observed here contrasts with the PST sensitivity observed following in vivo ischemia, suggesting that hemodynamic factors present in vivo may ultimately determine the overall susceptibility of PST segments in situ.

scholarly journals In Vitro and In Vivo Effects of Fermented Oyster-Derived Lactate on Exercise Endurance Indicators in Mice

2020 ◽  
Vol 17 (23) ◽  
pp. 8811
Author(s):  
Storm N. S. Reid ◽  
Joung-Hyun Park ◽  
Yunsook Kim ◽  
Yi Sub Kwak ◽  
Byeong Hwan Jeon
Keyword(s):  
Lactate Dehydrogenase ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Biochemical Analysis ◽  
Positive Control ◽  
Exercise Endurance

Exogenous lactate administration has more recently been investigated for its various prophylactic effects. Lactate derived from potential functional foods, such as fermented oyster extract (FO), may emerge as a practical and effective method of consuming exogenous lactate. The current study endeavored to ascertain whether the lactate derived from FO may act on muscle cell biology, and to what extent this may translate into physical fitness improvements. We examined the effects of FO in vitro and in vivo, on mouse C2C12 cells and exercise performance indicators in mice, respectively. In vitro, biochemical analysis was carried out to determine the effects of FO on lactate content and muscle cell energy metabolism, including adenosine triphosphate (ATP) activity. Western blot analysis was also utilized to measure the protein expression of total adenosine monophosphate-activated protein kinase (AMPK), p-AMPK (Thr172), lactate dehydrogenase (LDH), succinate dehydrogenase (SDHA) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in response to FO administration. Three experimental groups were formed: a positive control (PC) treated with 1% horse serum, FO10 treated with 10 μg/mL and FO50 treated with 50 μg/mL. In vivo, the effects of FO supplementation on exercise endurance were measured using the Rota-rod test, and Western blot analysis measured myosin heavy-chain 2 (MYH2) to assess skeletal muscle growth, alongside p-AMPK, total-AMPK, PGC-1α, cytochrome C and UCP3 protein expression. Biochemical analysis was also performed on muscle tissue to measure the changes in concentration of liver lactate, lactate dehydrogenase (LDH), glycogen and citrate. Five groups (n = 10/per group) consisted of a control group (CON), exercise group (Ex), positive control treated with Ex and 500 mg/kg Taurine (Ex-Tau), Ex and 100 mg/kg FO supplementation (Ex-FO100) and Ex and 200 mg/kg FO supplementation (Ex-FO200) orally administered over the 4-week experimental period.FO50 significantly increased PGC-1α expression (p < 0.001), whereas both FO10 and FO50 increased the expression of p-AMPK (p < 0.001), in C2C12 muscle cells, showing increased signaling important for mitochondrial metabolism and biogenesis. Muscle lactate levels were also significantly increased following FO10 (p < 0.05) and FO50 (p < 0.001). In vivo, muscle protein expression of p-AMPK (p < 0.05) and PGC-1α were increased, corroborating our in vitro results. Cytochrome C also significantly increased following FO200 intake. These results suggest that the effects of FO supplementation may manifest in a dose-response manner. FO administration, in vitro, and supplementation, in vivo, both demonstrate a potential for improvements in mitochondrial metabolism and biogenesis, and even for potentiating the adaptive effects of endurance exercise. Mechanistically, lactate may be an important molecule in explaining the aforementioned positive effects of FO.

A Mathematical Treatment of the Blood Dissociation Curve for Oxygen

1963 ◽  
Vol 9 (6) ◽  
pp. 745-762 ◽  
Author(s):  
Rodolfo Margaria
Keyword(s):  
Dissociation Constants ◽  
Quantitative Description ◽  
Mathematical Treatment ◽  
Physiological Effects ◽  
Functional Importance ◽  
Acidic Group ◽  
Oxygen Dissociation ◽  
Dissociation Curves

Abstract A quantitative description of Bohr's effect can be made from the oxygen dissociation curves of hemoglobin at different pHs, and the dissociation constants of the 02-linked acidic group of Hb (KR) and Hb02 (Ko) have been calculated as, respectively, 1.29 1O-8 and 3.42 10-7 at37°. On the assumption that the oxygenation of hemoglobin takes place in four successive steps, the constant for each equilibrium can easily be calculated, and the values given. It appears that the oxygenation takes place with the same affinity for the first three steps, while the affinity for the fourth oxygenation is 125 times greater. On the basis of these results a simplification of Adair's formula is given, containing only two constants, one (K) being representative of the affinity of the oxygen for the first three hemes, and the other (in)being the increase of affinity for the fourth oxygenation. This formula seems to fit most data in the literature of hemoglobin solutions and of blood in vivo and in vitro. The physiological effects and the functional importance of the increased affinity for the fourth oxygenation are described, and the possibility that disturbances of the Hb oxygenation process may be due to the lack of this process is considered.

Effect of reticulocytosis on lactate dehydrogenase isoenzyme distribution in serum: in vivo and in vitro studies

1990 ◽  
Vol 36 (9) ◽  
pp. 1638-1641 ◽  
Author(s):  
S C Kazmierczak ◽  
W J Castellani ◽  
F Van Lente ◽  
E D Hodges ◽  
B Udis
Keyword(s):  
Flow Cytometry ◽  
Lactate Dehydrogenase ◽  
Cell Types ◽  
In Vitro Studies ◽  
Hemolytic Disease ◽  
Dehydrogenase Isoenzyme ◽  
Disease Analysis

Abstract We investigated the effect of reticulocytosis on the lactate dehydrogenase (LD; EC 1.1.1.27) isoenzyme LD1/LD2 ratio in patients with and without evidence of hemolytic disease. Analysis of sera from patients with reticulocytosis and in vivo hemolysis showed a mean LD1/LD2 ratio of 0.92 compared with a ratio of 0.69 in patients with in vivo hemolysis and normal reticulocyte counts. Determination of LD isoenzymes in erythrocyte lysate revealed significantly increased LD1/LD2 ratios for patients with marked reticulocytosis compared with those for patients with normal-to-minimal increases in reticulocytes. Finally, separation of mature erythrocytes and reticulocytes by flow cytometry revealed marked differences in the LD1/LD2 isoenzyme distribution between these two cell types. The ability of hemolysis to cause a "flipped" LD1/LD2 ratio is dependent on the proportion of the hemolyzed cells that are reticulocytes.

Deferiprone Stimulates Aged Dermal Fibroblasts via HIF-1α Modulation

2020 ◽  
Author(s):  
Andrea Pagani ◽  
B Manuela Kirsch ◽  
Ursula Hopfner ◽  
Matthias M Aitzetmueller ◽  
Elizabeth A Brett ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Cell Metabolism ◽  
Hypoxia Inducible Factor ◽  
Survival Rates ◽  
Iron Chelation ◽  
Iron Chelator ◽  
Dermal Fibroblasts ◽  
Topical Formulation

Abstract Background Hypoxia-inducible factor 1α (HIF-1α), a transcription factor responsible for tissue homeostasis and regeneration, presents reduced functionality in advanced age. In addition to absence of oxygen, sequestration of iron also stimulates HIF-1α. Therefore, we analyzed the efficacy of the iron-chelator deferiprone (DFP) at stimulating dermal fibroblasts. Objectives The main objective of this study was to quantify the DFP concentrations capable of stimulating dermal fibroblasts in vitro and to correlate the effective DFP concentrations with the ability of DFP to penetrate the epidermis, reach the dermis, and activate HIF-1α in vivo. Methods We measured cell proliferation, metabolic activity, HIF-1α expression, and lactate dehydrogenase levels of both young and aged fibroblasts after a 24-hour in vitro preconditioning with DFP. In addition, we evaluated cell survival rates and morphology with different cellular stainings. Finally, we performed a transdermal permeation study with a 1% DFP topical formulation to quantify the concentration required to reach the dermis. Results In vitro administration of iron-chelation therapy (156-312.5 µg/mL DFP ) on aged fibroblasts resulted in activation of various antiaging processes. The concentration required to reach the dermis within 24 hours was 1.5% (0.15 mg/mL), which corresponds well with the effective doses of our laboratory analyses. Conclusions The activation of HIF-1α by DFP enhances cell metabolism, proliferation, and survival of fibroblasts while reducing lactate dehydrogenase levels. Modulation of HIF-1α is linked to activation of key regeneration enzymes and proteins, and by proxy, antiaging. Therefore, the antiaging properties of DFP and its satisfactory dermal penetration make it a promising regenerative agent.

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