perjantai, 30. elokuu 2019

Scientists Crack A Key Enzyme for DNA Methylation Research

Summary: A team at the University of California, Riverside, analyzed the crystal structure of an enzyme that plays a key role in DNA methylation, which found to be of great importance for understanding "DNA methylation from scratch."

A team at the University of California, Riverside, analyzed the crystal structure of an enzyme that plays a key role in DNA methylation, which is of great importance for understanding "DNA methylation from scratch."

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The research was published in Nature. It was completed by Jikui Song of University and Gang Greg Wang of California Riverside.

DNA methylation alters gene expression. This basic cellular mechanism of action is important for the evolution of plants, animals and humans. Previous studies have found that DNA methylation can regulate genomic stability and cell differentiation.

In humans, errors in methylation are associated with various diseases, including cancer. In mammals, DNA methylation is initially established by two related enzymes: DNMT3A and DNMT3B during germ cell development and early embryonic development. Understanding how DNA methylation "starts from scratch" is difficult, and one of the challenges is to resolve the structure of these enzymes.

In this article, researchers at the University of California, Riverside, obtained the crystal structure of DNMT3A combined with a substrate. This breakthrough reveals important information about how this enzyme recognizes and methylates its substrate.

"This structure reveals how DNMT3A molecules target two adjacent substrate sites on the same DNA molecule. Our work presents the first structural point of view for de novo DNA methylation and to understand how DNMT3A mutations influence cancers, such as acute myelogenous leukemia. It provides models that provide important insights into the function of DNMT3B”, Dr. Song said.

Understanding the structure of DNMT3A can help scientists regulate DNA methylation levels, gene expression, and cell differentiation, all of which are associated with diseases.

"This is particularly important for the long-term treatment of cancer."

At the same time, this study also explained why DNA methylation in mammals mainly occurs in "CpG dinucleotides."

"Scientists have no idea why the DNA methylation of mammals mainly occurs at the CpG site. Our understanding of the nascent DNA methylation was purely based on computer simulations and cannot reliably explain how DNMT3A works. How DNMT3A successfully binds to its substrate is also unknown. Our study has solved the DNMT3A-DNA complex structure, solved all these problems and provided new insights into how to generate specific DNA methylation patterns."

Due to the difficulty in producing stable enzyme-substrate complexes, studies of DNMT3A structures and substrates have been difficult.

In order to overcome this challenge, these researchers successfully developed a method to capture the reaction intermediates of DNMT3A-substrate complexes and resolved the structure by X-ray crystallography.

Source:

http://www.ucr.edu/

https://www.cd-genomics.com/

About CD Genomics

CD Genomics is offering five platforms for genome-wide epigenomics analysis, each designed to accommodate a wide range of sample types and suit your specific research needs, allowing researchers to look at epigenetic alterations easily. This information will help us not only understand the role of DNA methylation but also identify targets for therapeutic treatment.

torstai, 29. elokuu 2019

Preparation procedures of antibody labeling - enzyme, fluorescein, isotope and biotin labeling (part two)

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  1. fluorescein labeling
  2. Fluorescein isothiocyanate (FITC) labeling

The principle of FITC-labeled antibody is that under alkaline conditions, the isothiocyanato group of FITC can bind to the free amino group of IgG to form the binding of IgG to fluorescein.

FITC is the most commonly used fluorescein, followed by TRITC (Rhodamine tetramethyl isothiocyanate). FITC and TRITC are commonly used double-labeling combinations. The labeling steps are as follows:

(1). The antibody concentration was adjusted to 10 mg/ml with a carbonate buffer (pH 9.3, Na2CO3 8.6 g, NaHCO3 17.3 g, distilled water 1000 ml).

(2). Take 5 ml of the antibody solution in a 10 ml small beaker.

(3). Weigh 1 mg of FITC dissolved in 0.2 ml of DMSO, and slowly add it to the antibody solution as soon as it is dissolved, while gently stirring it; then leave it at room temperature for 2 hours.

(4). The cross-linked product was subjected to Sephadex G25 or G50 column chromatography to remove free fluorescein; the first peak was collected as a labeled antibody.

(5). FITC conjugate quality identification

IgG amount (mg/ml) = (OD280-OD495 × 0.35) / 1.4

F/P = 2.87 × OD495 / (OD280 - OD495 × 0.35), in general, the FITC-to-antibody molar ratio of 3:1 is suitable for tissue section staining, and is suitable for cell suspension staining at 5-6:1. Various antigens were stained with a labeled antibody, and the degree of specific staining and non-specific staining were determined.

(6). Preservation of labeled antibody: should be stored at 4 ° C, add NaN3 antiseptic.

  1. Rhodamine isothiocyanate (TRITC) label: Basically, the same as the FITC label.
  2. Isotope labeling

It should be emphasized that knowledge of isotope manipulation and protection should be mastered before using isotope-labeled monoclonal antibodies or other proteins. Normally, physical contact should be avoided and effective protection against gamma-ray exposure. When operating and using isotope-labeled antibodies, guards should be used and radioactive waste should be disposed of properly.

  1. Iodine labeling

Labeling antibody with iodine is an effective method of labeling. The 125I decay produces low-energy gamma and xenon rays that are easily detected, and its 60-day half-life guarantees a sufficient useful life and is convenient for handling radioactive waste. The most commonly used iodine labeling method is the chloramine T method, in which the oxidant chloramine T is added to the solution of the antibody and the iodide, the Na125I is converted to I2 by the action of chloramine T, and the free I2 can be combined with the tyrosine in the antibody molecule. Some histidines undergo a halogenation reaction, the reaction is terminated with a reducing agent and free tyrosine, and the labeled antibody is separated from the iodinated tyrosine and the reducing agent by gel filtration. The main steps are as follows:

(1). Before labeling, a gel with a molecular weight cut off of 20,000-50,000 was used to prepare a 1 ml gel column, and then the column was washed with 10 volumes of 1% BSA/PBS/0.02% sodium azide and PBS, respectively. Seal the bottom of the column and set aside.

(2). 10 ug of purified mAb was added to a 1.5 ml finger tube containing 25 ul of 2.5 mol/L sodium phosphate (pH 7.5). Subsequently, 500uCi Na125I was added and mixed.

(3). Add 25 ul of freshly prepared 2 mg/ml chloramine T. Leave at room temperature for 60 seconds. An additional 50 ul of chloramine T stop solution (containing 2.4 mg/ml sodium metabisulfite, 10 mg/ml tyrosine, 10% glycerol and 0.1% cyclohexanexyl PBS).

(4). The above iodine label was applied to the surface of the gel column, and the lower opening of the column was carefully opened, and collected with a 1.5 ml finger tube. When the iodine label was all entered into the column, 0.3 ml of PBS containing 0.03% sodium azide was added, 0.3 ml was collected with another finger tube, then 0.3 ml of 0.03% NaN3 PBS was added, 0.3 ml of the lower mouth was collected, and the reaction was repeated. Labeled and unlabeled monoclonal antibodies were simultaneously determined using an isotope tester. The labeled antibody appears approximately in the second to fourth tubes. The column and non-mAb-labeled parts are treated harmlessly.

(5). The labeled mAb was stored at 4 ° C for 6 weeks.

  1. Biosynthesis labeling

The hybridoma cells are cultured in a medium containing a radioactive precursor. As the antibody molecule is synthesized and assembled, the isotope is labeled on the primary amino acid chain of the antibody molecule, and the method does not cause loss of antibody activity. The main steps are:

(1). The hybridoma cells in the logarithmic growth phase were collected by centrifugation, and about 2 x 106 cells were required. The above cells were washed with a medium preheated at 37 ° C without methionine.

(2). Hybridoma cells were suspended in a medium containing 2% PBS without methionine to 106 cells/ml, and 35S methionine was added (105-106 cpm antibody was produced by adding 100 uCi each time).

(3). After culturing overnight in a CO 2 incubator, after centrifugation, the culture supernatant was aspirated, and 1/20 volume of 1 mol/L Tris (pH 8.0) and sodium azide were added to a concentration of 0.02%. The labeled antibody can be carried out by a purification monoclonal antibody method.

  1. Biotin labeling

The biotin labeling reaction is usually carried out using biotin succinimide ester. Biotin is coupled to a free lysine of an antibody molecule to complete the labeling. The main steps are:

(1). Prepare 10 mg/ml N-hydroxysuccinimide biotin with dimethyl sulfoxide (biotinylated succinyl esters of different sizes and arm lengths should be used as needed).

(2). Dilute the monoclonal antibody solution to 1-3 mg/ml with sodium borate buffer (0.1 mol/L, pH 8.0).

(3). Add 25-250 ug of biotin ester per mg of antibody, mix and allow to react at room temperature for 4 hours.

(4). Stop the reaction by adding 20 ul of 1 mol/L NH4Cl per 250 ug of biotin ester and let stand for 10 minutes at room temperature.

(5). The free biotin was removed by thorough dialysis with PBS, and the labeled antibody was frozen.

  1. Other labeling technologies

Other labeling methods for proteins can also be used for monoclonal antibodies, such as gold labels, chemiluminescent labels, SPA labels, ferritin labels, etc.

Immunolabeling is a technique with simple operation and high sensitivity. The main principle is the cascade method effect. Monoclonal antibodies have been widely used in disease diagnosis with monoclonal antibody-labeled diagnostic kit.

Creative Diagnostics is a leading manufacturer and supplier of antibodies, viral antigens, innovative diagnostic components and critical assay reagents. The company provides contract biologic R&D and manufacturing services to the diagnostic manufacturers along with GMP biologics manufacturing for the biopharmaceutical market. Its goal is to provide a trusted source for all assay development and manufacturing needs. Creative Diagnostics offers labeled antibodies using its catalogue antibody products and a broad range of intensely fluorescent dyes and labels including HRP, biotin, ALP, Alexa Fluor® dyes, DyLight® Fluor dyes, R-phycoerythrin (R-PE), at scales from less than 100 μg up to 1 g of IgG antibody.

maanantai, 26. elokuu 2019

How much do you know about Surface Plasmon Resonance?

Do you know the history of SPR?

Surface Plasmon Resonance (SPR) is shorted for SPR which is a new technology developed from the 1990s. The interaction between ligand and analyte on biosensor chip has been detected by SPR principle which has been widely applied in various fields. In 1902, Wood discovered the SPR phenomenon for the first time in an optical experiment and made a simple record of it. But it wasn't until 1941, 39 years later, that a scientist named Fano actually explained the SPR phenomenon. Over the next 30 years, SPR technology has not developed substantially, nor has it been applied to practical applications. In 1971, Kretschmann laid the foundation for the structure of SPR sensor and began the experiment with SPR technology. In 1983, Liedberg first used SPRs for the reaction of IgG with its antigen and achieved success. In 1987, Knoll et al began to study SPR imaging. In 1990, Biacore AB developed the first commercial SPR instrument, which opened up a new movement for the wider application of SPR technology.

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In short, SPR is used for real-time analysis and monitoring between DNA and protein, between protein and protein, between drug and protein, between nucleic acid and nucleic acid, between antigen and antibody. Interactions between receptors and ligands and other biomolecules simple and quick. SPR has a wide range of applications in life sciences, medical testing, drug screening, food testing, environmental monitoring, drug testing and forensic identification.

Do you know the principles of SPR?

(I) Evanescent wave

According to the optical theorem proposed by French physicist Fresnel:

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It can be seen that when light is injected from the dense medium into the light thinning medium, the incident angle increases to a certain angle. So that the refraction angle reaches 90 °, the refraction light will disappear completely and only the reflected light will be left. This phenomenon is called total reflection. When the total reflection is studied from the angle of wave optics, it is found that when the incident light reaches the interface. And it does not directly produce the reflected light, but first passes through the light thinning medium at a depth of about one wavelength. Then it flows along the interface about half the wavelength and then returns to the dense medium. Waves passing through light-sparsely media are called evanescent waves.

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(II)  SPR optical principle

We mentioned earlier that when the light is reflected on the surface of the prism and the metal film, it forms the evanescent wave and enters the light thinning medium. If it is a metal medium, there is a certain plasma wave in the medium. Resonance may occur when two waves meet.

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When the evanescent wave resonates with the surface plasmon wave, the detected reverberation intensity will be greatly weakened. The energy is transferred from photon to surface plasma meanwhile most of the energy of incident light is absorbed by surface plasma wave, which reduces the energy of reflected light.

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We can see a minimal spike from the reflected intensity response curve on the left where shows the wavelength of incident light is resonant wavelength and its corresponding incident angle θ is SPR angle. The electron absorbs light energy so that the reflected light intensity is greatly weakened at a certain angle. The angle in which the reflected light disappears completely is the SPR angle. The SPR angle varies with the refractive index of the metal surface meanwhile the change of the refractive index is proportional to the molecular mass of the metal surface. Therefore, the specific signal of biomolecules interaction can be obtained by the dynamic change of SPR angle during biological reaction.

(III) Analysis of bio-molecular interaction based on SPR principle

Bio-molecular interaction analysis is a novel bio-sensor analysis technology based on SPR principle which doesn’t need for labeling or purification of various biological components. It is a process of interaction between a variety of biological molecules, such as peptides, proteins, oligonucleotides, oligosaccharides, and viruses, bacteria, cells, and small molecules, in real time and in real time, through a sensor chip. Surface plasma resonance is one of the important enhancement mechanisms of surface enhanced Raman spectroscopy. Because the size effect and quantum effect of noble metal nanoparticles can cause surface plasmon resonance by exciting light irradiation, Raman scattering signal is greatly enhanced to achieve the purpose of trace detection.

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Do you know the character of SPR?

After 20 years of development, SPR optical bio-sensor has become an important research tool in the field of life sciences and pharmaceuticals. Compared with traditional interaction techniques such as ultracentrifugation, fluorescence and calorimetry, SPR biosensors have the following remarkable characteristics:

1. Real - time detection. Dynamically monitor the whole process of biological molecule interaction.

2. The molecular activity was maintained without labeling the sample.

3. Very few samples are required and only 1 mg of protein is required for one surface.

4. The detection process is convenient and quick and the sensitivity is high.

5. It has a very wide range of applications.

6. High quality analytical data.

7. Track and monitor the stability of fixed ligands.

8. Quantitative determination of the complex does not interfere with the equilibrium of the reaction.

9. In most cases, there is no need to treat the sample.

10. Because the SPR is based on the measurement of the reflected light of the unpenetrated sample, it can be carried out in the opacity or even opacity.

Do you know the future of SPR?

As SPR technology becomes analytical biochemistry, the application of SPR bio-sensor will be more diversified in the field of drug development and food monitoring. Especially, its new application in the field of small molecule detection and lipid membrane will make the SPR bio-sensor more widely used in the future. It will play an increasingly important role on drug discovering and membrane biology. In recent years, its development has been particularly rapid. With the continuous improvement of SPR instruments and the continuous enhancement of bio-molecular membrane construction ability, SPR bio-sensors have a very broad application prospect. Because the SPR is based on the measurement of the reflected light of the unpenetrated sample, it can be carried out in the opacity or even opacity. However, compared with the traditional analytical methods, especially the immunoassay, the existing SPR sensing technology still has some shortcomings in the detection cost, ease of use, stability, detection efficiency and so on which also determines the main development trend of this technology in the next few years.

lauantai, 24. elokuu 2019

Why Enzymes Are Important in Clinical Diagnosis and Treatment?

Enzymes, early referred to the meaning of “in yeast”, is actually a kind of biological catalysts produced by living cells in organisms, most of which are generally globular proteins. The enzymes can efficiently catalyze various biochemical reactions and promote the metabolism of organisms under conditions in the normal body. Life activities such as digestion, absorption, respiration, exercise and reproduction are all enzymatic reactions. Therefore, Enzymes are the basis for cells to survive.

 

There are numerous enzymes in the human body, which are complex in structure and various in variety. As far as we know, more than 3,000 kinds of enzymes have been discovered, such as pepsin, trypsin and other hydrolases. The protein that the human body takes from food must be hydrolyzed into amino acids under the activities of pepsin. More than 20 kinds of the amino acids required are selected under the function of other enzymes, which then recombined into other various proteins needed by the human body. Many complicated reactions occurred in the whole process. It can be said that without enzymes biological metabolism will not continue and thus the whole colorful biological communities in the natural world will disappear. Now we have known how crucial the enzymes are in living organisms, and that is why enzymes can be applied to clinical diagnosis and treatment.

 

Enzymes in the Diagnosis of Disease

 

Enzymatic diagnosis is a method to diagnose diseases by measuring the content and changes of certain substances in the body, or through the changes of the original enzyme activity in the body. It is obvious that enzymatic diagnostic methods based on two aspects: changes in the original enzyme activity in the body and changes of certain substances in body fluids, which is detected by enzymes. Phosphatase is a hydrolase that catalyzes the hydrolysis of phosphate monoester into inorganic phosphate under acidic conditions. Serum acid phosphatase activity will be elevated in the body of patients with prostate cancer or hyperparathyroidism. Another examples: glucose oxidase can be used to detect glucose content in diabetes diagnosis; urease can be used to measure the urea content to diagnose liver and kidney lesions; cholesterol oxidase can be used to measure the content of cholesterol in the blood to diagnose hyperlipidemia; glutaminase can be used to measure glutamine content in cerebrospinal fluid to diagnose cirrhosis and hepatic coma; DNA polymerase can be used to test whether the gene is normal or whether there is any oncogene exists in the body. Due to the unique enzyme characteristics of specificity, high-catalytic efficiency and mild action conditions, the enzymatic diagnosis has become a reliable, simple and rapid diagnostic method.

 

Enzymes in Diseases Prevention and Treatment

You may not know that enzymes can be used as medicines to treat a range of diseases which is called medicinal enzyme. For example, trypsin can be used not only to accelerate wound healing and dissolve blood clots, but also to remove necrotic tissue and inhibit the proliferation of contaminating microorganisms; L-asparaginase can be used to treat cancer by depriving nutrients needed by the growth of cancer cells and protease (like multi-enzyme tablets) can be used to treat dyspepsia and have an anti-inflammatory effect. In addition, lipase, superoxide dismutase, L-asparaginase, nattokinase, soybean meal plasmin, thrombin, etc. can be used for the treatment of diseases. Furthermore, many enzymes are used as diagnostic reagents in medical treatment. Because of its characteristics of remarkable curative effect and insignificant side effects, the medicinal enzymes have more and more applications in the health area.

 

Enzymes in Medicine Production

 

The process of producing various drugs by enzymes is to convert precursor substances into drugs by enzyme catalytic activities, which is called enzymatic production of drugs. The use of enzymes in the manufacture of pharmaceuticals is augmentative. A number of drugs, including some rare and expensive drugs, are now produced by enzymatic methods. For example, penicillin acylase can be used to produce semi-synthetic antibiotics; β-tyrosinase can be used to produce dopa; arabinoadenosine is produced by nucleoside phosphorylase; Achromobacter protease produces human insulin; polynucleotide phosphorylase produces polymyocytes; and β-D-glucosadenylase produces anti-tumor ginsenosides.

 

It is obvious from the above that enzymes play an irreplaceable and significant role in the field of clinical diagnosis and treatment. As a part of bioengineering, the important function and research results of enzyme have been recognized by the world. It has become our main goal to fully utilize the catalytic function, expand the application range, and improve the application efficiency of enzymes, and Creative Enzymes, as an enzyme manufacturer, are endeavoring to do so.

lauantai, 24. elokuu 2019

Hi, I’m Ghrelin

Hi, I’m Ghrelin peptide!

My name is Ghrelin peptide. My MW is 3314.8 Da. My formula is C147H245N45O42 and I was reacting with rat. My tested applications are ESI-MS and HPLC.  So, you may feel free to use me. My purity is greater than 98% purified which is mean I’m very pure. However, I need to be stored aliquot and store at-20°C. Avoid repeated freeze/thaw cycles. To tell you a secret, my sequence is

Gly-Ser-[Ser(n-octanoyl)]-Phe-Leu-Ser-Pro-Glu-His-Gln-Lys-Ala-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg. My target is Ghrelin who is my best partner. That’s me. I’m Ghrelin peptide!

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Do you know what is Ghrelin?

Ghrelin is a hormone produced mainly by P/D1 cells lining the fundus of the human stomach and epsilon cells of the pancreas that stimulates hunger. Ghrelin levels increase before meals and decrease after meals. It is considered the counterpart of the hormone leptin, produced by adipose tissue, which induces satiation when present at higher levels. In some bariatric procedures, the level of ghrelin is reduced in patients, thus causing satiation before it would normally occur.

Do you know how Ghrelin generated?

Ghrelin is also produced in the hypothalamic arcuate nucleus, where it stimulates the secretion of growth hormone from the anterior pituitary gland. Receptors for ghrelin are expressed by neurons in the arcuate nucleus and the lateral hypothalamus. The ghrelin receptor is a G protein-coupled receptor, formerly known as the GHS receptor (growth hormone secretagogue receptor).

Do you know what’s the function of Ghrelin?

Ghrelin plays a significant role in neurotrophy, particularly in the hippocampus, and is essential for cognitive adaptation to changing environments and the process of learning. Recently, ghrelin has been shown to activate the endothelial isoform of nitric oxide synthase in a pathway that depends on various kinases including Akt.

Do you know how the Ghrelin come into effect?

Small synthetic molecules called growth-hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary. They act through GHS-R, a G-protein-coupled receptor for which the ligand is unknown. Recent cloning of GHS-R strongly suggests that an endogenous ligand for the receptor does exist and that there is a mechanism for regulating GH release that is distinct from its regulation by hypothalamic growth-hormone-releasing hormone (GHRH). We now report the purification and identification in rat stomach of an endogenous ligand specific for GHS-R. The purified ligand is a peptide of 28 amino acids, in which the serine 3 residue is n-octanoylated. The acylated peptide specifically releases GH both in vivoand in vitro, and O-n-octanoylation at serine 3 is essential for the activity. We designate the GH-releasing peptide ‘ghrelin’ (ghre is the Proto-Indo-European root of the word ‘grow’). Human ghrelin is homologous to rat ghrelin apart from two amino acids. The occurrence of ghrelin in both rat and human indicates that GH release from the pituitary may be regulated not only by hypothalamic GHRH, but also by ghrelin.

Ghrelin receptors and diseases

Diabetic encephalopathy (DE) is a major central nervous system complication of diabetes mellitus, manifested as acquired cognitive dysfunction and physiological changes of brain disease, resulting in the complete loss of cognitive ability and self-care ability. DE and prognosis are not only affected by the overall blood sugar level, but also closely related to the fluctuation of blood glucose. In vitro, experiments have also shown that glucose levels with fluctuations in extracellular are more harmful to cells than sustained high glucose levels. Besides, the abnormal changes of hippocampal structure and function are the direct cause of DE, and the fluctuation of blood glucose is more obvious as a kind of stimulation signal, which is an independent damage of hippocampal neurons. Studies show that persistent hyperglycemia and diabetic blood glucose fluctuations all cause cognitive dysfunction in rats, and the disorder caused by blood glucose fluctuation is more serious, which is closely related to the expression of ghrelin and GHSR1a in rat's hippocampus. Therefore, the changes of ghrelin and GHSR1a play an important role in the process of speeding up the incidence of blood glucose fluctuation.