Synapto-pHluorin
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Synapto-pHluorin
Synapto-pHluorin is a genetically encoded optical indicator of vesicle release and recycling. It is used in neuroscience to study transmitter release. It consists of a pH-sensitive form of green fluorescent protein (GFP) fused to the luminal side of a vesicle-associated membrane protein (VAMP). At the acidic pH inside transmitter vesicles, synapto-pHluorin is non-fluorescent (quenched). When vesicles get released, synapto-pHluorin is exposed to the neutral extracellular space and the presynaptic terminal becomes brightly fluorescent. Following endocytosis, vesicles become re-acidified and the cycle can start again. Chemical alkalinization of all vesicles is often used for normalization of the synapto-pHluorin signals. Synapto-pHluorin sometimes consists of yellow fluorescent protein (YFP) to monitor the cytoplasm because its pKa is higher than GFP (7.1 versus 6.0). History Synapto-pHluorin was invented by Gero Miesenböck in 1998. In 2006, an improved version was published, using ...
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Neuroscience
Neuroscience is the scientific study of the nervous system (the brain, spinal cord, and peripheral nervous system), its functions and disorders. It is a multidisciplinary science that combines physiology, anatomy, molecular biology, developmental biology, cytology, psychology, physics, computer science, chemistry, medicine, statistics, and Mathematical Modeling, mathematical modeling to understand the fundamental and emergent properties of neurons, glia and neural circuits. The understanding of the biological basis of learning, memory, behavior, perception, and consciousness has been described by Eric Kandel as the "epic challenge" of the Biology, biological sciences. The scope of neuroscience has broadened over time to include different approaches used to study the nervous system at different scales. The techniques used by neuroscientists have expanded enormously, from molecular biology, molecular and cell biology, cellular studies of individual neurons to neuroimaging, imaging ...
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Synaptic Vesicle
In a neuron, synaptic vesicles (or neurotransmitter vesicles) store various neurotransmitters that are released at the synapse. The release is regulated by a voltage-dependent calcium channel. Vesicles are essential for propagating nerve impulses between neurons and are constantly recreated by the cell. The area in the axon that holds groups of vesicles is an axon terminal or "terminal bouton". Up to 130 vesicles can be released per bouton over a ten-minute period of stimulation at 0.2 Hz. In the visual cortex of the human brain, synaptic vesicles have an average diameter of 39.5 nanometers (nm) with a standard deviation of 5.1 nm. Structure Synaptic vesicles are relatively simple because only a limited number of proteins fit into a sphere of 40 nm diameter. Purified vesicles have a protein:phospholipid ratio of 1:3 with a lipid composition of 40% phosphatidylcholine, 32% phosphatidylethanolamine, 12% phosphatidylserine, 5% phosphatidylinositol, and 10% chole ...
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Recombinant Proteins
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome. Recombinant DNA is the general name for a piece of DNA that has been created by combining at least two fragments from two different sources. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure, and differ only in the nucleotide sequence within that identical overall structure. Recombinant DNA molecules are sometimes called chimeric DNA, because they can be made of material from two different species, like the mythical chimera. R-DNA technology uses palindromic sequences and leads to the production of sticky and blunt ends. The DNA sequences used in the construction of recombinant DNA molecules can originate from any species. For example, plant DNA may be joined to bacter ...
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Protein Methods
Protein methods are the techniques used to study proteins. There are experimental methods for studying proteins (e.g., for detecting proteins, for isolating and purifying proteins, and for characterizing the structure and function of proteins, often requiring that the protein first be purified). Computational methods typically use computer programs to analyze proteins. However, many experimental methods (e.g., mass spectrometry) require computational analysis of the raw data. Genetic methods Experimental analysis of proteins typically requires expression and purification of proteins. Expression is achieved by manipulating DNA that encodes the protein(s) of interest. Hence, protein analysis usually requires DNA methods, especially cloning. Some examples of genetic methods include conceptual translation, Site-directed mutagenesis, using a fusion protein, and matching allele with disease states. Some proteins have never been directly sequenced, however by translating codons from know ...
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Pancreas
The pancreas is an organ of the digestive system and endocrine system of vertebrates. In humans, it is located in the abdomen behind the stomach and functions as a gland. The pancreas is a mixed or heterocrine gland, i.e. it has both an endocrine and a digestive exocrine function. 99% of the pancreas is exocrine and 1% is endocrine. As an endocrine gland, it functions mostly to regulate blood sugar levels, secreting the hormones insulin, glucagon, somatostatin, and pancreatic polypeptide. As a part of the digestive system, it functions as an exocrine gland secreting pancreatic juice into the duodenum through the pancreatic duct. This juice contains bicarbonate, which neutralizes acid entering the duodenum from the stomach; and digestive enzymes, which break down carbohydrates, proteins, and fats in food entering the duodenum from the stomach. Inflammation of the pancreas is known as pancreatitis, with common causes including chronic alcohol use and gallstones. Becaus ...
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Beta Cells
Beta cells (β-cells) are a type of cell found in pancreatic islets that synthesize and secrete insulin and amylin. Beta cells make up 50–70% of the cells in human islets. In patients with Type 1 diabetes, beta-cell mass and function are diminished, leading to insufficient insulin secretion and hyperglycemia. Function The primary function of a beta cell is to produce and release insulin and amylin. Both are hormones which reduce blood glucose levels by different mechanisms. Beta cells can respond quickly to spikes in blood glucose concentrations by secreting some of their stored insulin and amylin while simultaneously producing more. Primary cilia on beta cells regulate their function and energy metabolism. Cilia deletion can lead to islet dysfunction and type 2 diabetes. Insulin synthesis Beta cells are the only site of insulin synthesis in mammals. As glucose stimulates insulin secretion, it simultaneously increases proinsulin biosynthesis, mainly through translational cont ...
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Insulin
Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolism of carbohydrates, fats and protein by promoting the absorption of glucose from the blood into liver, fat and skeletal muscle cells. In these tissues the absorbed glucose is converted into either glycogen via glycogenesis or fats (triglycerides) via lipogenesis, or, in the case of the liver, into both. Glucose production and secretion by the liver is strongly inhibited by high concentrations of insulin in the blood. Circulating insulin also affects the synthesis of proteins in a wide variety of tissues. It is therefore an anabolic hormone, promoting the conversion of small molecules in the blood into large molecules inside the cells. Low insulin levels in the blood have the opposite effect by promoting widespread catabolism, especially o ...
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Chemical Synapse
Chemical synapses are biological junctions through which neurons' signals can be sent to each other and to non-neuronal cells such as those in muscles or glands. Chemical synapses allow neurons to form circuits within the central nervous system. They are crucial to the biological computations that underlie perception and thought. They allow the nervous system to connect to and control other systems of the body. At a chemical synapse, one neuron releases neurotransmitter molecules into a small space (the synaptic cleft) that is adjacent to another neuron. The neurotransmitters are contained within small sacs called synaptic vesicles, and are released into the synaptic cleft by exocytosis. These molecules then bind to neurotransmitter receptors on the postsynaptic cell. Finally, the neurotransmitters are cleared from the synapse through one of several potential mechanisms including enzymatic degradation or re-uptake by specific transporters either on the presynaptic cell o ...
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Synaptophysin
Synaptophysin, also known as the major synaptic vesicle protein p38, is a protein that in humans is encoded by the ''SYP'' gene. Genomics The gene is located on the short arm of X chromosome (Xp11.23-p11.22). It is 12,406 bases in length and lies on the minus strand. The encoded protein has 313 amino acids with a predicted molecular weight of 33.845 kDa. Molecular biology The protein is a synaptic vesicle glycoprotein with four transmembrane domains weighing 38kDa. It is present in neuroendocrine cells and in virtually all neurons in the brain and spinal cord that participate in synaptic transmission. It acts as a marker for neuroendocrine tumors, and its ubiquity at the synapse has led to the use of synaptophysin immunostaining for quantification of synapses. The exact function of the protein is unknown: it interacts with the essential synaptic vesicle protein synaptobrevin, but when the synaptophysin gene is experimentally inactivated in animals, they still develop and ...
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Green Fluorescent Protein
The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea victoria'' and is sometimes called ''avGFP''. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods and lancelets. The GFP from ''A. victoria'' has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79. The GFP from the sea pansy (''Renilla reniformis'') has a single major excitation peak at 498 nm. GFP makes for an excellent tool in many forms of biology due to its ability to form an internal chromophore without requiring any accessory cofactors, gene products, or enzymes / substrates other than mo ...
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Gero Miesenböck
Gero Andreas Miesenböck (born 15 July 1965) is an Austrian scientist. He is currently Waynflete Professor of Physiology and Director of the Centre for Neural Circuits and Behaviour (CNCB) at the University of Oxford and a fellow of Magdalen College, Oxford. Education and early life A native of Austria, Miesenböck was educated at the University of Innsbruck and Umeå University in Sweden. He graduated sub auspiciis praesidentis rei publicae from the University of Innsbruck Medical School. Following his Doctor of Medicine (MD) in 1993, he undertook postdoctoral training with James Rothman. Research and career Miesenböck is known as the founder of optogenetics. He was the first scientist to modify nerve cells genetically so that their electrical activity could be controlled with light. This involved inserting DNA for light-responsive opsin proteins into the cells. Miesenböck used similar genetic modifications to breed animals whose brains contained light-responsive ne ...
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Acid Dissociation Constant
In chemistry, an acid dissociation constant (also known as acidity constant, or acid-ionization constant; denoted ) is a quantitative measure of the strength of an acid in solution. It is the equilibrium constant for a chemical reaction :HA A^- + H^+ known as dissociation in the context of acid–base reactions. The chemical species HA is an acid that dissociates into , the conjugate base of the acid and a hydrogen ion, . The system is said to be in equilibrium when the concentrations of its components will not change over time, because both forward and backward reactions are occurring at the same rate. The dissociation constant is defined by :K_\text = \mathrm, or :\mathrmK_\ce = - \log_ K_\text = \log_\frac where quantities in square brackets represent the concentrations of the species at equilibrium. Theoretical background The acid dissociation constant for an acid is a direct consequence of the underlying thermodynamics of the dissociation reaction; the p''K''a v ...
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