Investigating interactions between chloroquine/hydroxychloroquine and their single enantiomers and angiotensin-converting enzyme 2 by a cell membrane chromatography method

Chloroquine and hydroxychloroquine have been studied since the early clinical treatment of SARS-CoV-2 outbreak. Considering these two chiral drugs are currently in use as the racemate, high-expression angiotensin-converting enzyme 2 cell membrane chromatography was established for investigating the differences of two paired enantiomers binding to angiotensin-converting enzyme 2 receptor. Molecular docking assay and detection of SARS-CoV-2 spike pseudotyped virus entry into angiotensin-converting enzyme 2-HEK293T cells were also conducted for further investigation. Results showed that each single enantiomer could bind well to angiotensin-converting enzyme 2, but there were differences between the paired enantiomers and corresponding racemate in frontal analysis. R-Chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine. Moreover, each single enantiomer was proved effective compared with the control group; compared with S-chloroquine or the racemate, R-chloroquine showed better inhibitory effects at the same concentration. As for hydroxychloroquine, R-hydroxychloroquine showed better inhibitory effects than S-hydroxychloroquine, but it slightly worse than the racemate. In conclusion, R-chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability and inhibitory effects compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine (racemate), while the effect of preventing SARS-CoV-2 pseudovirus from entering cells was weaker than R-hydroxychloroquine/hydroxychloroquine (racemate).     

Determination in plasma of angiotensin-converting enzyme inhibitor by inhibitor-binding assay.

A method of determining a new angiotensin-converting enzyme inhibitor (CS-622) and its active metabolite (RS-5139) in plasma by inhibitor-binding assay has been developed using high-performance liquid chromatography. The assay is based on the principle that the amount of inhibitor bound to the enzyme is inversely related to the amount of hippuric acid liberated on hydrolysis from the artificial substrate (hippuryl-L-histidyl-L-leucine). Plasma was heated at 60 degrees C for 15 min, to inactivate endogenous enzyme, and preincubated with rabbit-lung angiotensin-converting enzyme at 37 degrees C for 3 min. The artificial substrate (5.75 mg/ml in pH 8.3 phosphate buffer containing sodium chloride) was added to the resulting solution, and the mixture was incubated for 30 min. The reaction was terminated by the addition of 2 M hydrochloric acid. The hippuric acid liberated on hydrolysis was extracted with ethyl acetate and determined by reversed-phase chromatography using methylparaben as an internal standard. The total concentration of the inhibitor and its metabolite were determined by this method after de-esterification by rat-plasma esterase. The standard curve was obtained by the regression analysis of log concentration against logit response. The within-day and day-to-day precision were satisfactory. The proposed method is simple, rapid and sensitive enough to determine angiotensin-converting enzyme inhibitor in plasma.     

Some aspects of the angiotensin-converting enzyme for leukemias

Russian Chemical Bulletin - The present article shows the diagnosis of different type leukemias in children by the activity of angiotensin-converting enzyme in blood serum to be not promising....     

Statistical Optimization of Alpha-Amylase Production with Immobilized Cells of <Emphasis Type="Italic">Streptomyces erumpens</Emphasis> MTCC 7317 in <Emphasis Type="Italic">Luffa cylindrica</Emphasis> L. Sponge Discs

The purpose of this investigation was to study the effect of Streptomyces erumpens cells immobilized in various matrices, i.e., agar–agar, polyacrylamide, and luffa (Luffa cylindrica L.) sponge for production of α-amylase. Luffa sponge was found to be 21% and 51% more effective in enzyme yield than agar–agar and polyacrylamide, respectively. Response surface methodology was used to evaluate the effect of three main variables, i.e., incubation period, pH, and temperature on enzyme production with immobilized luffa cells. The experimental results showed that the optimum incubation period, pH, and temperature were 36h, 6.0, and 50 °C, respectively. The repeated batch fermentation of immobilized cells in shake flasks showed that S. erumpens cells were more or less equally physiologically active on the support even after three cycles of fermentation (3,830–3,575 units). The application of S. erumpens crude enzyme in liquefying cassava starch was studied. The maximum hydrolysis of cassava starch (85%) was obtained with the application of 4ml (15,200 units) of crude enzyme after 5 h of incubation.     

Description of the activation-inactivation processes in enzymes

The process of activation of an angiotensin-converting enzyme by ionizing radiation was studied in terms of a kinetic model suggesting the existence of at least one activated form of the enzyme. The kinetic decaying oscillations induced by an exciting force were obtained and analyzed. It was shown that the probability of a periodic process is lower than that of the appearance and reactions of one activated form of the enzyme.     

Behavior of angiotensin-converting enzyme containing 67Zn

Magnetic nuclei of isotope ⁶⁷Zn (S ?5/2) were introduced into the active center of an angiotensin-converting enzyme instead of the zinc of usual isotope composition in order to consider their possible influence on the enzyme activity toward peptide substrates. No changes were observed in vitro, that gives us a possibility to study the influence of the zinc magnetic isotope on lymphocytes in various blood cell lines in vitro and ex vivo, neglecting contribution of this enzyme.     

Analysis of Several Angiotensin-Converting Enzyme Inhibitors using Potentiometric,Enantioselective Membrane Electrodes

ABSTRACT

The construction of potentiometric, enantioselective membrane electrodes for several angiotensin-converting enzyme inhibitors as well as the mechanism of potential development and enantioselectivity are described.

The response characteristics for angiotensin-converting enzyme inhibitors and the reliability of their enantiopurity tests are highlighted.     

稀土离子与牛血Cu(Zn)-SOD的作用

采用ESR、CD谱和荧光光谱研究了pH=6.3时六次甲基四胺-HCl缓冲溶液中LaCl_3和TbCl_3与Cu(Zn)-SOD的配位作用和结构。Cu(Zn)-SOD可增强Tb~(3+)的荧光发射,Tb~(3+)与Cu(Zn)-SOD有多个配位位置,其中有2个强结合位点,La~(3+)与Tb~(3+)可竞争Cu(Zn)…SOD上相同结合位点,77K下La~(3+)与Tb~(3+)使Cu(Zn)-SOD的Cu~(2+)活性中心的配位环境由菱形对称结构向轴对称结构转变,使Cu(Zn)-SOD的局部结构变松散,但对SOD酶活性基本无影响,表明稀土离子主要与酶蛋白分子中的酸性氨基酸羧基配位,对酶蛋白二级结构仅产生微弱扰动,对活性中心空间结构影响较小,基本不影响Cu(Zn)-SOD酶活性。     

Determination of Glucose in Diluted Blood with a Thermal Flow Injection Analysis Biosensor

Abstract

The glucose concentration in diluted whole blood has been measured, using a miniaturized thermal biosensor based on the enzyme thermistor principle. The biosensor is a small flow injection system. A sample volume of 20μl is injected into a flow of 50μl/min. The heat produced when the sample passes the enzyme column is measured with a thermistor connected to a Wheatstone bridge. The enzyme column contains glucose oxidase and catalase co-immobilized on a solid support material. Samples of whole blood usually cause problems in flow-systems. The blood cells tend to block the enzyme column and the back pressure increases. We have tested a superporous agarose material as enzyme support material using tenfold diluted samples of whole human blood. The blood was collected from the finger-tip and diluted with buffer containing an anticoagulant and sodium fluoride. The number of samples possible to inject and the accuracy compared to the Boehringer Mannheim Reflolux have been determined. At least 100 ten-fold diluted blood samples could be injected on a micro-column of superporous agarose. The obtained glucose concentration correlated well with the one obtained with the reference instrument.     

Effects of a chemical chaperone on genetic mutations in ��-galactosidase A in Korean patients with Fabry disease

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the gene encoding the α-galactosidase A (GLA) enzyme. We have identified 15 distinct mutations in the GLA gene in 13 unrelated patients with classic Fabry disease and 2 unrelated patients with atypical Fabry disease. Two of the identified mutations were novel (i.e., the D231G missense mutation and the L268delfsX1 deletion mutation). This study evaluated the effects of the chemical chaperones 1-deoxygalactonojirimycin (DGJ) on the function of GLA in vitro, in cells containing missense mutations in the GLA gene. Nine missense and a nonsense mutations, including one novel mutation were cloned into mammalian expression vectors. After transient expression in COS-7 cells, GLA enzyme activity and protein expression were analyzed using fluorescence spectrophotometry and Western blot analysis, respectively. DGJ enhanced GLA enzyme activity in the M42V, I91T, R112C and F113L mutants. Interestingly, the I91T and F113L mutations are associated with the atypical form of Fabry disease. However, DGJ treatment did not have any significant effect on the GLA enzyme activity and protein expression of other mutants, including C142W, D231G, D266N, and S297F. Of note, GLA enzyme activity was not detected in the novel mutant (i.e., D231G), although protein expression was similar to the wild type. In the absence of DGJ, the E66Q mutant had wild-type levels of GLA protein expression and approximately 40% GLA activity, indicating that E66Q is either a mild mutation or a functional single nucleotide polymorphism (SNP). Thus, the results of this study suggest that the chemical chaperone DGJ enhances GLA enzyme activity and protein expression in milder mutations associated with the atypical form of Fabry disease.     

Mode change in the self-motion of a benzoquinone disk coupled with a NADPH system

The self-motion of a benzoquinone (BQ) disk on NADPH was investigated as the coupling of an autonomous motor and an enzyme reaction. In the absence of the enzyme reaction, features of motion changed depending on the concentration of NADPH, that is, continuous motion→ intermittent oscillatory motion→ no motion. When the reverse reaction from NADP(+) to NADPH was introduced into the system with the addition of an enzyme reaction, continuous motion changed to intermittent oscillatory motion with small amplitude. The mechanism of this mode change is discussed in relation to the surface tension as a driving force and the time course of UV spectra as a window to the progress of the reaction. Characteristic features of the mode change were qualitatively reproduced by a numerical calculation.     

Optimization of capillary electrophoretic separation of several inhibitors of the angiotensin-converting enzyme

Capillary electrophoretic separation of eight inhibitors of the angiotensin-converting enzyme, viz., enalapril, lisinopril, quinapril, fosinopril, perindopril, ramipril, benazepril and cilazapril, was investigated with respect to the following parameters: pH of the running buffer, organic modifiers and surfactants. The most critical parameter is the pH of the running buffer. The addition of sodium dodecyl sulfate had a negative influence on the peak symmetry, and selectivity was not improved. The separation of the eight compounds can be performed by means of two phosphate buffers (each 100 mM) at pH 7.0 and pH 6.25, respectively. This combination is necessary for the selective identification of structurally related substances because of their similar pKa values.     

Paper-based device for rapid typing of secondary human blood groups

We report the use of bioactive paper for typing of secondary human blood groups. Our recent work on using bioactive paper for human blood typing has led to the discovery of a new method for identifying haemagglutination of red blood cells. The primary human blood groups, i.e., ABO and RhD groups, have been successfully typed with this method. Clinically, however, many secondary blood groups can also cause fatal blood transfusion accidents, despite the fact that the haemagglutination reactions of secondary blood groups are generally weaker than those of the primary blood groups. We describe the design of a user-friendly sensor for rapid typing of secondary blood groups using bioactive paper. We also present mechanistic insights into interactions between secondary blood group antibodies and red blood cells obtained using confocal microscopy. Haemagglutination patterns under different conditions are revealed for optimization of the assay conditions.     

Progress in Articular Cartilage Tissue Engineering: A Review on Therapeutic Cells and Macromolecular Scaffolds

Repair and regeneration of articular cartilage lesions have always been a major challenge in the medical field due to its peculiar structure (e.g., sparsely distributed chondrocytes, no blood supply, no nerves). Articular cartilage tissue engineering is considered as one promising strategy to achieve reconstruction of cartilage. With this perspective, the articular cartilage tissue engineering has been widely studied. Here, the recent progress of articular cartilage tissue engineering is reviewed. The ad hoc therapeutic cells and growth factors for cartilage regeneration are summarized and discussed. Various types of bio/macromolecular scaffolds together with their pros and cons are also reviewed and elaborated.     

Determination of Imatinib in the Blood Cells of Chronic Myelogenous Leukemia Patients by Ion-Trap Mass Spectrometry

Imatinib mesylate is a standard first-line therapy for patients with chronic myelogenous leukemia. However, there is still a significant proportion of these patients who reflect sub-optimal responses or fail imatinib therapy. Knowledge of the distribution within the studied system (e.g., peripheral blood) may be of high importance for understanding the principles of drug action and possible patient resistance to treatment. Intracellular or more precisely cell-associated, imatinib concentrations in patients, were shown to be higher compared to those in plasma, but still only limited data related to the methodology aspects of cell-associated concentrations are available. Herein is presented an assessment of the cell-associated imatinib determination assay by mass spectrometry. Three approaches were evaluated to isolate cells from the peripheral blood of chronic myelogenous leukemia patients. Erythrocyte lysis was found to cause substantial leakage of cell-associated imatinib in the first step. Selected alternative procedures utilizing density gradients did not affect the cell-associated imatinib concentration significantly. Cell isolates were subjected to flow cytometry which revealed differences in the population composition of peripheral blood cell isolates among individual patients indicating that the cell isolate composition should be addressed with the cell-associated imatinib concentration. The proposed approach may be utilized for the determination of intracellular concentration of imatinib and for other drugs in which the intracellular concentration plays a key role in the therapy.     

Affinophoresis of red blood cells: surface antigen-specific acceleration of electrophoresis

A method employing the technique of affinophoresis to increase the electrophoretic mobility of specific cells according to their surface antigens was developed. Red blood cells were treated consecutively with the maximum subagglutinating dose of an anti-red blood cell serum, a biotinylated second antibody, avidin and finally with a negatively charged biotin-affinophore which was prepared by coupling biotin to polylysine (average degree of polymerization, 270 or 1150), followed by complete succinylation. The electrophoretic mobility of cells was analyzed with an automatic cell electrophoresis analyzer. The use of a homologous anti-serum increased the electrophoretic mobility of rabbit, human and rat blood cells by 2.9, 1.7 and 1.6 times, respectively. A larger affinophore containing fewer biotin moieties was more effective. In the case of a mixture of red blood cells from two species, cells from only one species could be accelerated by using homologous antiserum, e.g., affinophoresis of a mixture of human and rat red blood cells by using either homologous antiserum gave two separate peaks on the histogram, whereas a single peak would be obtained in usual electrophoresis because there is little difference in the original migration velocities of the two cell types.     

Visualization of microscale particle focusing in diluted and whole blood using particle trajectory analysis

Inertial microfluidics has demonstrated the potential to provide a rich range of capabilities to manipulate biological fluids and particles to address various challenges in biomedical science and clinical medicine. Various microchannel geometries have been used to study the inertial focusing behavior of particles suspended in simple buffer solutions or in highly diluted blood. One aspect of inertial focusing that has not been studied is how particles suspended in whole or minimally diluted blood respond to inertial forces in microchannels. The utility of imaging techniques (i.e., high-speed bright-field imaging and long exposure fluorescence (streak) imaging) primarily used to observe particle focusing in microchannels is limited in complex fluids such as whole blood due to interference from the large numbers of red blood cells (RBCs). In this study, we used particle trajectory analysis (PTA) to observe the inertial focusing behavior of polystyrene beads, white blood cells, and PC-3 prostate cancer cells in physiological saline and blood. Identification of in-focus (fluorescently labeled) particles was achieved at mean particle velocities of up to 1.85 m s(-1). Quantitative measurements of in-focus particles were used to construct intensity maps of particle frequency in the channel cross-section and scatter plots of particle centroid coordinates vs. particle diameter. PC-3 cells spiked into whole blood (HCT = 45%) demonstrated a novel focusing mode not observed in physiological saline or diluted blood. PTA can be used as an experimental frame of reference for understanding the physical basis of inertial lift forces in whole blood and discover inertial focusing modes that can be used to enable particle separation in whole blood.     

A Micro-Scale Model to Monitor the Major Proteins in Human Urine Before and After Medication with Angiotensin-Converting Enzyme Inhibitor: A Preliminary Study

A simple and micro-scale liquid chromatographic (LC) method coupled with mass spectrometry was developed for analyzing major proteins in human urine. After one-step sample preparation, proteins were precipitated, redissolved, and digested. Only micro-liter level (10 μL) of urine was sufficient for major protein identification. This method was applied in clinical study, and urine proteins were monitored after medication with angiotensin-converting enzyme inhibitor within 24 hr. This method can identify many important proteins in human urine. Bioactive peptides associated with blood pressure control can also be identified simultaneously. We hope this simple method may prove useful in clinical research and disease diagnosis.