❶: Functional testing of mutant genes, especially to help confirm the genetic diagnosis of hyperinsulinemic hypoglycemia (HH) after an unknown mutation is found in genetic diagnosis, functional verification of the mutant gene is required to help confirm the pathogenicity of the mutant gene;
❷: Preparation of mutant proteins: Through point mutation and protein expression basis, the preparation of mutant proteins of some genes can help with functional verification and drug screening.
Glucokinase (GCK) plays a vital role both in both diabetes and hypoglycemia, and it is the glucose receptor for the body which regulated the metabolism of glucose by feeling the changes in the blood sugar and plays a key role in the maintenance of glucose homeostasis. We have already expressed and prepared the purified protein of GST-GCK, and assessment of the function has proved the activity.
Glutamate dehydrogenase(GDH) is a key enzyme for the decomposition and metabolism of amino acid, GDH gain-of-function mutation can cause child patents to have 🅐: Hyperinsulinemia hypoglycemia; 🅑：Hyperprotein-induced hypoglycemia; 🅒：Hyperaminemima; 🅓：Epilepsy, cerebral dysgenesis and decreased learning ability and etc, causing systemic damage throughout the body, and we can do systemic enzyme kinetics measurement based on the cellular system.
Alpha-glucoside enzyme（α-GC）can catalyze the hydrolysis of alpha-1.4 glycosidic bonds and make the maltose, sucrose, and other oligosaccharides inside the small intestine to hydrolyze. It is a vital link in the process of digesting carbohydrates. It can inhibit the activity of ∂-glucose enzymes, slow the production and absorption of glucose, reduce the peak value of blood glucose after meals, regulate blood sugar level, especially regulating blood sugar after eating carbohydrates.
Recently, α-glucosidases inhibitors have become a hot spot of research topic in pharmaceutical chemistry and diabetes medication. The enzyme kinetics detection of α-GC is an enzyme catalytic reaction with PNPG as the substrate, adding Na2CO3 while completing the reaction, and it will have a chromogenic reaction at the same time as the reaction is terminated, and then use enzyme marker to measure the light absorbent value at 405 nm.
We can provide collaboration of New Pharmaceutical research and development for α-GC inhibitors, and screen the new drugs through enzyme kinetics measurement of α-GC. As shown in the figures below, the Dose Dependence Curve of PNPG Substrate and with positive control of the Inhibition Curve of α-GC of the apo sugar.
Our Organization provides services for determination of ammonia content in animal serum or other biological samples.
Ammonia is an important amino acid metabolites. Normal human blood contains trace amounts of free ammonia. Endogenous ammonia is produced in the process of protein metabolism in the body and is decomposed by amino acids through deamination. It is the main source of ammonia in the blood. In general, ammonia from muscles and organs such as kidneys and etc interacts with glutamate to produce glutamine and then be transported to the liver. After it is converted into urea or other nitrogen-containing chemicals compounds in the liver, it will be excreted by the kidneys or forms ammonium salts and excretes in the urine.
An increase in the source of blood ammonia and a decrease in the way out will cause an increase in blood ammonia. Measurement of ammonia plays a critical role in studying amino acid metabolism and the diagnosis of hepatic encephalopathy.
NH4+ + NADH + α-ketoglutaric acid Glutamic Acid+ NAD+
*The detection principle is based on the biochemical reaction above. The change in NADH absorbance (ΔOD) is measured under the wavelength of 340 nm during the detection time, and the ammonia content of the test sample is calculated according to the formula of the standard curve.
Lipase is triacylglycerol which catalyzes the hydrolysis of natural substrate oils to generate fatty acids, glycerol, and mono- or di-glycerides. At present, the incidence of obesity-related diseases such as hypertension, hyperlipidemia, and diabetes is on the rise.
Lipase inhibitors can achieve weight loss by inhibiting lipase activity and reducing the digestion and absorption of fats. 4-Nitrophenyl ester (4-Nitrophenyl ester) is one of the most widely used substrates in the determination of lipase hydrolysis activity. Lipase will hydrolyze into PNP (p-nitrophenol). PNP will appear yellow under alkaline conditions. Based on this principle, we established a lipase enzyme kinetics assay method, with the weight-loss drug orlistat as a positive control. Our organization can provide new drug research and development cooperation for lipase inhibitors, and screen new compounds through lipase enzyme kinetic assay.
Sialic acid (SA), also known as “N-acetylneuraminic acid”, is a type of carbohydrate compound that is widely existing in biological systems. It widely exists inside the cell membrane glycoproteins and lipoproteins of organisms and plays a role in many important physiological and biochemical processes of the living body, such as participating in cell recognition, survival, reproduction, biofilm flow, cell endocytosis and etc. Infections, tumors, coronary heart disease, and other diseases that endanger human health are mostly associated with abnormalities in SA. For healthy people, sialic acid is a crucial nutrient for neurodevelopment. The level of sialic acid is of great significance in the diagnosis of early childhood autism, cognitive impairment in the elderly, gestational diabetes, and monitoring of sialic acid in pregnant women or nursing mothers. Our organization can provide a test paper by taking oral saliva as a sample and using the neuraminidase method to develop the relationship between the concentration of sialic acid and the absorbance value, which can be used to measure the content of sialic acid in the saliva.
We have a well-developed platform for cell culture that can achieve:
❶. Cell Transfection:
Cell transfection technology refers to the technology of introducing external molecules such as DNA, RNA, and other to eukaryotic cells. The commonly adopted methodologies of transfection are liposome transfection, virus transfection, and so on. As it is shown in the Figures below, plasmid transfection of 293T cells (left) and lentivirus transfection (right);
❷. Cytotoxicity Measurement:
Cytotoxicity is a pure cell killing incident caused by cells or chemicals and does not depend on the mechanism of death of apoptosis or necrotic cells. Sometimes it needs to conduct measurement of cytotoxicity for specific substances, such as drug screening. Measurement for cytotoxicity is mainly based on measuring the changes of the Cell Membrane Permeability, commonly apply MTT and XTT method. By utilizing the activity of the internal enzyme of the mitochondria, a specific tetraphosphate salt can be converted to measure by an enzyme marker.
❸. Measurement for Cell Proliferation
Cell proliferation is a vital living characteristic of organisms, and proliferate by cell division. Through cell division, the replicated genetic materials can be evenly distributed to two subcells. Cell proliferation measurements include MTT and CCK8 method.
❹. Cell Migration /Invasion
Cell migration/invasion refers to the movement produced by the cells after they receiving signals or feeling the gradient of certain substances, commonly apply the Transwell method. The main material of the Transwell experiment is the small chamber of transwell, with a membrane with micropores on the bottom of the nest, with a diameter size of 8-12um. Matrigel is needed to cover the micropores on the membrane when conductin the Cell invasion experiments. However, Cell migration does not require Matrigel, we can analyze the mobility of the cell by using crystalline purple staining to calculate the number of cells which pass through the filter membranes.
At present, we can develop cooperation on two mouse models, including ❶ The gain-of-mutant gene of GDH knocking into the whole body of mouse(GDH-H454Y-KI), the mouse presents hyperammonemia, fasting hypoglycemia and brain dysfunction characterized by abnormal behavior；❷ Whole-body ATP relies on potassium ion channels knockout mouse (SUR1-KO): is a mouse model of hyperinsulinemia hypoglycemia, manifests fasting hypoglycemia, amino acid-induced hypoglycemia, and islet shows GSIS damage, gain sensitivity to insulin secretion stimulated by amino acid and etc.
Utilizing the interaction of the simulated target spot and the candidate compounds simulated by the molecular docking software on a computer to calculate the affinity between them, and selecting seed compounds from a large number of chemical compounds. Computer High-throughput virtual screening does not consume chemical samples, but reduce the cost of screening, and shortens the cycle of research and development for the new drugs.
Computer-aided Drug Design is a method of designing and optimizing lead compounds by computer simulation, calculation, and budgeting of the relationship between the drugs and the receptors. Essentially, Computer-aided Drug Design is the optimization and design of lead compounds that are conducted through simulation and calculation of the interaction between receptors and ligands. In recent years, Computer-aided Drug Design is a brand-new technology which is developed to research and develop new drugs, it dramatically accelerates the speed of new drug design and saves the manpower and material resources for creating the new drug, and enables the pharmacologists to purposefully develop the new drugs under the guidance of theory.
❶. Customer Demand: The customers request the content of service according to their own needs and then discuss the specific experimental process and details with our specific project manager, later one we put forward a specific plane and cycle of the experiment, and at the end enter the implementation phase after confirmation of both sides.
❷. Experimental Cycle: The experimental cycle varies from case to case. We communicate with customers timely in different stages of the experiment, discuss the results and process of the experiment, make adjustments accordingly based on the condition of the specific experiment, and finally we provide complete results of data analysis of the final product, also detailed experiment report.
Glucokinase(GCK) Purified Protein
Glucose kinase is an enzyme that catalyzes glucose phosphorylation to produce an enzyme of glucose-6-phosphate. Glucose kinase is present in the liver and pancreas cells of humans and most other vertebrates. It serves as a glucose sensor and plays a vital role in regulating the glucose metabolism in these organs. The mutation in the gene of the enzyme may cause diabetes or hypoglycemia (View Figure ❶).
We have completed the preparation of the purified protein of GST_GCK, activity of enzyme demonstrated by the research of enzyme kinetics, and we are able provide different packages of the GCK protein for customers according to their needs, research services and collaborations of enzyme kinetics.
Glutamate Dehydrogenase (GLDH/GDH) is an enzyme found in the mitochondria of most microorganisms and eukaryotes and is also the enzyme needed for urea synthesis. It converts glutamate into α-ketoglutaric acid, which is involved in the tricarboxylic acid cycle. The mutation in the enzyme gene may induce diabetes or hypoglycemia.
The mutation of Human GDH H454Y is the mutation of the GTP binding site and shows a reduced sensitivity to GTP inhibition. We have successfully constructed the a full-body knock out mouse model of the H454Y human mutation GDH, which means to replace mouse’s own GDH with the human mutation GDH(H454Y). The study of the enzyme kinetics of liver tissue homogenate has been completed, and it shows severe damage to GTP sensitivity which is as expected.