Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Uncatalysed, the hydrolysis of peptide bonds is extremely slow, taking hundreds of years. Proteolysis is typically catalysed by cellular enzymes called proteases, but may also occur by intra-molecular digestion.
Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting long chains of amino acids into smaller pieces. It is a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where it hydrolyzes proteins. Trypsin is formed in the small intestine when its proenzyme form, the trypsinogen produced by the pancreas, is activated. Trypsin cuts peptide chains mainly at the carboxyl side of the amino acids lysine or arginine. It is used for numerous biotechnological processes. The process is commonly referred to as trypsin proteolysis or trypsinization, and proteins that have been digested/treated with trypsin are said to have been trypsinized. Trypsin was discovered in 1876 by Wilhelm Kühne and was named from the Ancient Greek word for rubbing since it was first isolated by rubbing the pancreas with glycerin.
A protease is an enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products. They do this by cleaving the peptide bonds within proteins by hydrolysis, a reaction where water breaks bonds. Proteases are involved in many biological functions, including digestion of ingested proteins, protein catabolism, and cell signaling.
A thrombus, colloquially called a blood clot, is the final product of the blood coagulation step in hemostasis. There are two components to a thrombus: aggregated platelets and red blood cells that form a plug, and a mesh of cross-linked fibrin protein. The substance making up a thrombus is sometimes called cruor. A thrombus is a healthy response to injury intended to stop and prevent further bleeding, but can be harmful in thrombosis, when a clot obstructs blood flow through healthy blood vessels in the circulatory system.
Alpha-1 antitrypsin or α1-antitrypsin is a protein belonging to the serpin superfamily. It is encoded in humans by the SERPINA1 gene. A protease inhibitor, it is also known as alpha1–proteinase inhibitor (A1PI) or alpha1-antiproteinase (A1AP) because it inhibits various proteases. In older biomedical literature it was sometimes called serum trypsin inhibitor, because its capability as a trypsin inhibitor was a salient feature of its early study. As a type of enzyme inhibitor, it protects tissues from enzymes of inflammatory cells, especially neutrophil elastase, and has a reference range in blood of 0.9–2.3 g/L, but the concentration can rise manyfold upon acute inflammation.
Bradykinin (BK) (Greek brady-, slow; -kinin, kīn(eîn) to move) is a peptide that promotes inflammation. It causes arterioles to dilate (enlarge) via the release of prostacyclin, nitric oxide, and endothelium-derived hyperpolarizing factor and makes veins constrict, via prostaglandin F2, thereby leading to leakage into capillary beds, due to the increased pressure in the capillaries. Bradykinin consists of nine amino acids, and is a physiologically and pharmacologically active peptide of the kinin group of proteins.
Serine proteases are enzymes that cleave peptide bonds in proteins. Serine serves as the nucleophilic amino acid at the (enzyme's) active site. They are found ubiquitously in both eukaryotes and prokaryotes. Serine proteases fall into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.
Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This technique is also called micropropagation. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. They need to be kept at body temperature (37 °C) in an incubator. These conditions vary for each cell type, but generally consist of a suitable vessel with a substrate or rich medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH buffer, osmotic pressure, temperature). Most cells require a surface or an artificial substrate to form an adherent culture as a monolayer (one single-cell thick), whereas others can be grown free floating in a medium as a suspension culture. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar. Tissue culture commonly refers to the culture of animal cells and tissues, with the more specific term plant tissue culture being used for plants. The lifespan of most cells is genetically determined, but some cell-culturing cells have been “transformed” into immortal cells which will reproduce indefinitely if the optimal conditions are provided.
Tryptase is the most abundant secretory granule-derived serine proteinase contained in mast cells and has been used as a marker for mast cell activation. Club cells contain tryptase, which is believed to be responsible for cleaving the hemagglutinin surface protein of influenza A virus, thereby activating it and causing the symptoms of flu.
Zebrafish AB9 cells are a primary fibroblast cell line developed from fin tissue of the AB strain. These cells are commonly used for studies focusing on the biochemical and molecular properties of zebrafish. Cells are grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS) in a humid, 5% CO2-enriched atmosphere at 28 °C. Under these conditions, cells passaged 1 in 4 doubled every 72 hours when fed with fresh culture medium at 3-day intervals.
In biology, a subculture is either a new cell culture or a microbiological culture made by transferring some or all cells from a previous culture to fresh growth medium. This action is called subculturing or passaging the cells. Subculturing is used to prolong the lifespan and/or increase the number of cells or microorganisms in the culture.
A trypsin inhibitor (TI) is a protein and a type of serine protease inhibitor (serpin) that reduces the biological activity of trypsin by controlling the activation and catalytic reactions of proteins. Trypsin is an enzyme involved in the breakdown of many different proteins, primarily as part of digestion in humans and other animals such as monogastrics and young ruminants. Serpins – including trypsin inhibitors – are irreversible and suicide substrate-like inhibitors.
The drug aprotinin, is a small protein bovine pancreatic trypsin inhibitor (BPTI), or basic trypsin inhibitor of bovine pancreas, which is an antifibrinolytic molecule that inhibits trypsin and related proteolytic enzymes. Under the trade name Trasylol, aprotinin was used as a medication administered by injection to reduce bleeding during complex surgery, such as heart and liver surgery. Its main effect is the slowing down of fibrinolysis, the process that leads to the breakdown of blood clots. The aim in its use was to decrease the need for blood transfusions during surgery, as well as end-organ damage due to hypotension as a result of marked blood loss. The drug was temporarily withdrawn worldwide in 2007 after studies suggested that its use increased the risk of complications or death; this was confirmed by follow-up studies. Trasylol sales were suspended in May 2008, except for very restricted research use. In February 2012 the European Medicines Agency (EMA) scientific committee reverted its previous standpoint regarding aprotinin, and has recommended that the suspension be lifted. Nordic became distributor of aprotinin in 2012.
An enzyme inhibitor is a molecule that binds to an enzyme and blocks its activity. Enzymes are proteins that speed up chemical reactions necessary for life, in which substrate molecules are converted into products. An enzyme facilitates a specific chemical reaction by binding the substrate to its active site, a specialized area on the enzyme that accelerates the most difficult step of the reaction.
Protein metabolism denotes the various biochemical processes responsible for the synthesis of proteins and amino acids (anabolism), and the breakdown of proteins by catabolism.
Cell synchronization is a process by which cells in a culture at different stages of the cell cycle are brought to the same phase. Cell synchrony is a vital process in the study of cells progressing through the cell cycle as it allows population-wide data to be collected rather than relying solely on single-cell experiments. The types of synchronization are broadly categorized into two groups; physical fractionization and chemical blockade.
Amine oxidase, copper containing 3 (AOC3), also known as vascular adhesion protein (VAP-1) and HPAO is an enzyme that in humans is encoded by the AOC3 gene on chromosome 17. This protein is a member of the semicarbazide-sensitive amine oxidase family of enzymes and is associated with many vascular diseases.
Angiogenesis is the process of forming new blood vessels from existing blood vessels, formed in vasculogenesis. It is a highly complex process involving extensive interplay between cells, soluble factors, and the extracellular matrix (ECM). Angiogenesis is critical during normal physiological development, but it also occurs in adults during inflammation, wound healing, ischemia, and in pathological conditions such as rheumatoid arthritis, hemangioma, and tumor growth. Proteolysis has been indicated as one of the first and most sustained activities involved in the formation of new blood vessels. Numerous proteases including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase domain (ADAM), a disintegrin and metalloproteinase domain with throbospondin motifs (ADAMTS), and cysteine and serine proteases are involved in angiogenesis. This article focuses on the important and diverse roles that these proteases play in the regulation of angiogenesis.
A cell suspension or suspension culture is a type of cell culture in which single cells or small aggregates of cells are allowed to function and multiply in an agitated growth medium, thus forming a suspension. Suspension culture is one of the two classical types of cell culture, the other being adherent culture. The history of suspension cell culture closely aligns with the history of cell culture overall, but differs in maintenance methods and commercial applications. The cells themselves can either be derived from homogenized tissue or from heterogenous cell solutions. Suspension cell culture is commonly used to culture nonadhesive cell lines like hematopoietic cells, plant cells, and insect cells. While some cell lines are cultured in suspension, the majority of commercially available mammalian cell lines are adherent. Suspension cell cultures must be agitated to maintain cells in suspension, and may require specialized equipment and flasks. These cultures need to be maintained with nutrient containing media and cultured in a specific cell density range to avoid cell death.
Adherent cell cultures are a type of cell culture that requires cells to be attached to a surface in order for growth to occur. Most vertebrate derived cells can be cultured and require a 2 dimensional monolayer that to facilitate cell adhesion and spreading. Cell samples can be taken from tissue explants or cell suspension cultures. Adherent cell cultures with an excess of nutrient-containing growth medium will continue to grow until they cover the available surface area. Proteases like trypsin are most commonly used to break the adhesion from the cells to the flask. Alternatively, cell scrapers can be used to mechanically break the adhesion if introducing proteases could damage the cell cultures. Unlike suspension cultures, the other main type of cell culture, adherent cultures require regular passaging performed using mechanical or enzymatic dissociation. The culture can be visualized using an inverted microscope, however the growth of adherent cultures is dependent on the available surface area. For this reason, adherent cell cultures are not commonly used to obtain a high yield of cells, instead the use of suspension cultures is preferred.
