General purpose
One,
19C
Claude Bernard: Experimental toxicity and anesthesiology
Second, 1906 John Langley: Chemicals Quality
Third, 1921 Loewi: Discovery Chemistry (ACH)
Fourth, 1937 David Nachmansohn: Discover Nachr Super Repository (EO)
Fives,
19C
70Y separation purification Nachr
Sixth, 19Cl70Y Nachr structure reveals
Seven, Nachr's fine structure and its mechanism
Eight, 1980s u.j. McMahan: Agrin induces the mechanism of aggregation Nachr
Nine, progress
One,
19C
Claude Bernard: Experimental toxicity and anesthesiology
Bernard, Claude (1813-1878). French physiologists, born in St.julien. In 1843, 30-year-old Claude Bernard is a pharmacist and a very promising dramatic work, but in order to become a literary dream, he left his hometown and came to Paris.
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In the 19th century French literature 1789, the French revolution has formed a romantic literature that focuses on natural scenery and expressing subjective feelings.
Balzac, Aoyod: (1799-1850) French writer and realist novel laying found, he described the panoramic view of French social life in the work of the work.
Hugo "
Zhongma, Alexander: (1802-1870) French writer, writing bizarre historical romantic novel, such as the county of the Kisa Mountain and three fire guns (all in 1844). Its son Alexander (1824-1895), is known as "Xiao Zhongma" is a drama writer, and the work includes tea blossom women (1852)
)
However, after Paris, an accidental opportunity allows Bernard to continue to study medicine, and serve Madi Madi's Assistant of Madi (F. Magendie), starting research and life. His research involves the extensive field of physiology, and by the future generations are the founders of experimental medicine and ordinary physiology.
curare
Curare is a highly toxic drug taken from the tropical plant, which is used as a material that makes a poison arrow because it can paralyifen.
Bernard found that CURARE did not affect the ability of nerve conductivity to muscle during paralysis, and did not affect the ability of muscles to shrink. Thus, Bernard believes that the action of CURARE is the region (NMJ) of nerve and muscle contact.
This conclusion later was confirmed by John Langley and further complement and improved.
Second, 1906 John Langley: Chemicals Quality
Langley is currently studying the characteristics of another plant extract cigarette. He found NICOTINE to stimulate the contraction of the void skeletal muscle, but curare will fail.
In 1906, Langley proposed that the impulse is transmitted from the neurons to the muscles, not by means of a physical method, as the current flows between the two wires, but a special substance released from the end of the nerve.
He called this passing material as a chemical proposal, and believed that chemical proposal was conducted by binding to the receptor substance surfaces of the muscle cells, which in combined Nicotine and Curare were combined with this site. These arguments have been proven to be visionary. Langley's stimulation passed the neural release chemicals to the muscles of the muscular hypothesis proved in 1921 by Austrian physiologist Otto Loewi.
Third, 1921 Loewi: Discovery Chemistry (ACH)
Episode: Dream of Inspiration
Chemist Kakuler (Friedrich August Kekulé) is a story of a parent-well-known story in the dream.
In some winter in 1864, German chemist Keculer sat in front of the fireplace, the atomic and the molecules began to dance in hallucinations, and a carbon atomic chain bitbied like a snake. After sudden awakening, Kakuler's white benzene molecule is a ring - is now full of the hexagon of our organic chemistry textbook.
This most famous example is not unique. The exquisite experiment of Loewi is also from dreams.
In 1921, the night before Sunday, Austrian biologist Loywi woke up from the dream, opened the light, grabbed a piece of black and wrote something, fell down and fell asleep. . At 6 o'clock in the morning, he suddenly thought that he wrote some extremely important things last night, and quickly took the paper, but I didn't understand what ghost paints I wrote. Fortunately, 3 o'clock in the morning of the next day, the new thoughts fled were back.
It is an experiment design method that can be used to verify that a hypothesis raised correctly ago.
Roy hurriedly got up, ran to the laboratory, killed two unfortunate frogs, took out the frog heart bubble in normal saline, one of which took the vagus nerve, No. 2 without bringing. The vagulent nerve of the first heart of the first number made the heart beat slow. After a few minutes, it moved its brine to the container where the second heart was located, and the number of hearts of the second heart also slowed down.
This experiment shows that the nerve does not act directly in muscles, but by releasing chemicals, certain substances have produced certain substances when they are stimulated, they are dissolved in brine, and they have effects on the 2nd heart. Loewi refers to "VagusStoff" in a substance that carries this heartbeat of frogs.
After several years, Loewi found that the chemistry and physiological properties of this substance were identical to acetylcholine, so he concluded that the acetylcholine (ACH) was released at the end of the vagus.
The chemical transfer of nerve impulsive is discovered, it has opened a new research area and enables Rozy to win the 1936 Nobel Physiology or Medical Award.
Fourth, 1937 David Nachmansohn: Discover Nachr Super Repository (EO)
In 1937, when David Nachmansohn, the University of Sorbonne (Sorbonne) visited the Paris World Expo, he noted that there were several electric organs (EO) of ELECTIC ORGAN, EO. These 鳐 EOs can issue 40-60 voltages to kill potential food in the water.
OE: The generator includes a large number of electric sheets (up to 20,000 pieces), each of which is from a special multi-core muscle cell, and the electric sheet is separated from a glue substance, and many electric sheets are composed of a connective tissue. In the small room, the nerve is branched through the connective tissue to each of the plates. The electric plate is arranged vertically, and the discharge direction is from the abdominal side back side; the electric sheet of the electric blast is arranged horizontally, and the discharge direction is from the tail to the head.
At that time, Nachmansohn was studying acetylcholine esterase (ACHASE), ACHASE can be enzymatically released from the athletic nerve. Nachmansohn knew that the EO of this fish and skeletal muscle were homologous, so after the Expo ended, the EO was started to study.
The first experimental results for EO indicate that it is a super repository for ACHASE. This organ is also a very rich repository of Nachr. Nachr exists on the synaptic film of skeletal muscle cells, which combines with ACH molecules that are slightly released at the end of the motor god.
If an ideal system model can be found, it can be said to be invaluable for research on the specific field of cellular structure and function. This will be confirmed in the discussion, and fish's power generation organs actually a unique substance source in Nachr studies.
Fives,
19C
70Y separation purification Nachr
1, chen-yan lee: Ideal marker for Nachr
Alpha-bgt: ideal marker for nachr
Since Nachr is an integrated membrane protein until the 1970s, it has developed techniques to isolate such proteins.
A special protein is purified to detect the purity of the affected protein fragment with the proximity.
The ideal label as Nachr is a compound that can be selectively combined with it. This compound was found by the CHEN-Yuan Lee of Taiwan National University and his colleagues found that it was extracted from a poisonous venom of Taiwan, called α-silver ring snake toxin (α-BGT). α-BGT is closely bonded by NachRs of the skeletal synaptic film, while hindering the transmission of the ACH, causing muscle paralysis.
2, Nachr separation and purification
The Nachr was separated in the 1970s after the label, EO as a Nachr, as as well as a feedstock of dissolved film proteins.
In the study of separation and purification, there is a way to:
1 get a cell membrane containing Nachr
First, a portion of EO was first prepared into a uniform suspension, then centrifuged, and the plasma film in the suspension was first formed.
2 extract membrane protein
After many dissolved membrane proteins, finally found Tritonx-100 has the best effect.
Tritonx-100 is a nonionic detergent. It is the same point as the other detergent, which has a long hydrophobic group that can replace the membrane protein, and a hydrophilic end capable of dissolving the water; the difference is that most other defective agents have one A negative hydroxyl group, and it is not, so non-ionic type, which is small to the protein structure.
Thus, the membrane protein can be extracted from the platen segment with Tritonx-100.
3 separation purification Nachr
The membrane protein mixture was poured into the chromatography column equipped with a human composite-agarose bead, and the end of these agarose beads had an ACH structure analog as a surface adsorbent. (Figure)
2A
)
When these membrane proteins are laminated, there are two proteins, nachr and ache with ACH, Nachr and Ache, they are adsorbed on agar grease beads. About 90% of other membrane proteins remain directly flowing through the column directly.
Use 10
-3M
Elite Flaxedil (Trice iodide (β-diethyl amoxy)) elution, Nachr will be selectively eluted and collected, and finally ACHE is left. After this approach, the test results of the marker with α-BGT showed that ACHRS was purified by more than 150 times. This method is affinity chromatography.
Sixth, 19Cl70Y Nachr structure reveals
Arthur Karlin, University of Columbia, found that Nachr is a pentamer, which is a protein consisting of 5 subunits. Each receptor includes one alpha subunit, β, γ, Δ subunit. Sepailed separation and purification is based on the front step, and the technical method of polyacrylamide gel electrophoresis is completed, mainly by separating the size of each of the polypeptides (subunits). Another landmark study for Nachr is proved that the purified receptor protein has a cationic channel in addition to a site that is combined with the ACH.
The hypothesis of this conclusion has been previously been proposed by Jean-Pierre Changeux, the Paris Bastee Research: When the ACH is bonded to the Nachr, the conformation of the NACHR changes, and the ion channel is open. Thus, since Na ions in the cell membrane are poor, Na internal flow, membrane depolarization, and skeletal muscle contraction are caused.
In the second half of the 1970s, Changeux and his colleagues successfully added purified Nachr molecules to liposomes (artificial phospholipin). They have been labeled with different Na , K concentrations of liposome demonstrated that the binding of ACH and Nachr in the phospholipid bimolecularly flows the cation in the film.
Obviously, this receptor protein does have all structural components that deliver chemical signals: a binding site with the ACH; a gantry ion channel, and the coordination mechanism between the two.
Seven, Nachr's fine structure and its mechanism
There are many different ways to analyze the structure. One of them is a method of purifying, amino acid sequencing, and fixed-point mutagenesis, determining the transmembrane site, neurotransmitter of the neurotransmitter, and the site of the ion channel.
Another approach requires an electron microscope. The first glimpse of Nachr was a plasma film of the emitting organ cell under electrical mirror (Figures).
Nachr appears annular, with a diameter of about 8 nm and a central channel of 2 nm diameter, and it protrudes from the phospholipid dual molecule to extracellular space.
Nachr's more accurate model has been depicted by Nigel Unwin, England Medical Research Institute and his colleagues. After mathematical analysis of the EO freeze film electron microscope, Unwin describes the composition of Nachr as 5 subunits around a central channel. (Figure5)
The ion channel is a very narrow diameter of only 7 to 8 å, and the aperture is surrounded by a wall consisting of 5 subunits of α-Helix. The gateway switch of the hole is considered to be near the center of the transmembrane, here, 5 α-Helix fucking centers to form a junction. In this model, 5 α-Helix LEU (leucine) residues form a tightening ring, limiting the transmembrane flow of ions. When the ACH molecule is bonded to one site of the two alpha subunitone outer domain pocket structure, the gate switch is opened.
In order to study the changes in Nachr during the opening of the channel, Unwin made the following experiment.
The prepared Nachr-rich cell membrane is applied to a support network and allows it to flow into a container with liquid nitrozoethane that can freeze the cell membrane. When the Nachr solution reaches about 5 milliseconds (MSEC), the ACH solution is sprayed to the grid, and the ACH is bonded to the receptor and causes a substantially change to open the passage.
By comparing the Nachr channel open and closed electron microscope photos, UNWIN found that the combination of ACH triggered a change in the formation of the receptor subunit cell membrane domain, and propagated to the entire receptor protein, resulting in an α-Helix conformation surrounding the aperture change. (Figure 6) Confucius is closed due to the formation of α-Helix, which can be seen from the white and blue comparison of Figure6b.
doubt
We know that the membrane peripheral components can limit the movement of the membrane integrin. A typical example is also discovered on Nachr. In the early stages of embryos, the Nachr is evenly distributed in the surface of the muscle when the muscles is no nerve. The mature neuromuscular joint Nachr gathered high density. The synaptic region under the active nerve is less than 0.1% of the surface area of the muscle cells, but a concentrated approximately 90% of Nachr. Why is this?
Eight, 1980s u.j. McMahan: Agrin induces the mechanism of aggregation Nachr
In the 1980s, the U.j. McMahan of Stanford University and his colleagues found a protein in EO. When this protein was added in the cultured immature muscle cells, it can induce Nachrs at its cell membrane aggregation. It also found that it has a high concentration at the eo synaptic gap.
In 1987, they purified this protein and named it to agrin.
McMahan proposes Agrin to be synthesized from sports nerves, extending axons to terminal gods, and is released to extracellular space at neuromuscular joints. These Agrin molecules are combined with specific Agrin receptors adjacent to myocytes, trigger a series of cascaded reactions, bringing Nachr molecules (and other components of the synaptic film) to the vicinity of activated Agrin receptors, and To achieve the maximum concentration.
Agrin is part of a synaptic gap extracellular substance to help maintain the structure of the post-contaminated film and ensure the enrichment of the newly synthetic Nachrs in the synaptic site.
A variety of research supports this hypothesis, including research on the use of antibody and Agrin binding.
If a paragraph of the chicken embryo is allowed to carry anti-Agrin antibodies, this antibody is specifically marked with the cytoplasm of the motor nerve, which indicates that the Agrin molecule is from this. Antibodies are of course also present in a synaptic gap at the neuromuscular joint. When this antibody is added to the motor nerve of the chicken, the normal aggregation of the NACHR at the two types of cell contact sites in the cultured two types of cell contact sites will be blocked.
A more deeply in-depth problem is formed by a rat-free Agrin gene knockout experiment. That is, the nerve of the mouse cannot be produced by a method of genetic engineering. Agrin defective mice developed normal in the parent, but died during pregnancy.
These rats did not have any muscle movement before death, and in the inspection of muscle tissues, it was found to lack normal neuromuscular synaptic.
Agrin induces the mechanism of agglocking Nachr, which is still known to us, but many of the steps in this process have been defined. After adding Agrin to an immature muscle cell, one of the events occurring is phosphorylated phosphorylation of one or more tyrosine residues of the NachR beta subunit.
This phosphorylation occurs in a long time before the sign of Nachr aggregation.
TYR phosphorylation is often a key step in the process of cell membrane signal conduction. If Nachr TYR phosphorylation is blocked by Tyr kinase, Nachr will no longer aggregate, indicating that agrin induces Nachr Tyr phosphorylation in aggregation is an indispensable step.
The transmission path of the signal is activated due to phosphorylation of Tyr and often leads to the rearrangement of the cell skeleton element, particularly actin fibers. The cell skeleton is often connected to the actin skeleton (AS), and the AS plays a key role on the distribution of the membrane protein.
Recent studies have shown that the production of cell surface signals is after Agrin and its receptor binding results in AF-to-polarization, which is considered to be directly reasons for NACHR aggregation. If the formation of AF is capable of binding to the ACTIN monomer and blocks the combination of the monomer, Agrin induces the aggregation of Nachrs to be blocked.
Nine, currently recognized causes ACHR to high density gathering pathway after synaptic membrane
Agrin / MUSK signal transduction pathway
The agglin (Agrin) is mainly synthesized by motor neurons, released by the nerve endings and deposited on the base layer, triggered ACHR aggregation without increasing the number of ACHRs. Muscle-Specific Receptor Tyrosine Kinase, Musk is Agrin receptor, selectively in skeletal muscle expression, with ACHR aggregate in a synaptic film of adult neural muscle joints.
Affected by Agrin, MUSK was phosphorylated, mediated by phosphorylation of neuromuscular joint Nachr.
This process must also depend on the existence of ACETYLCHOLINE ACH receptor association protein (ACETYLCHOLINE receptor-associated protenapse, rapsyn). RapSyn is mainly synthesized in muscle tissue and is a cytoskeletal protein. It is presented in an in vivo and neuromuscular joint Nachr to be placed together.
After the above three molecules are closely connected, the nerve axon is secreted, and the MUSK is phosphorylated in the participation of RapSyn, and NachR phosphorylation is induced, thereby inducing ACHR at the final plate membrane.
Neuregulin / Erbb signal transduction pathway
Neurotrandin neuregulin (NRG) is generated by neurons, released from a synaptic gap and fixed in a neuromycmon joint.
The homozygous lack of NRG mice abortion in front of the neuromuscular joint in the formation of cardiac joints, and mice with a heterozygous lack of NRG were reduced by 50% in the ACHR of neuromuscular joints, and neurogenic neurotransmitter transmission also defects. It can be seen that NRG has ACHR-induced activity, which promotes ACHR gene expression in developing and mature muscle cells.
Erbb receptors are aggregated after neuromuscular joints, and after binding to NRG, the Erbb receptor tyrosine kinase is activated, and the signal is transmitted to the downstream Ras / RAF / MEK / ERK kinase signaling pathway through the joint SHC and GRB2. Resulting in synaptic cell nuclear specific transcription to maintain high density of ACHR in mature muscle joints.
Other polypeptides such as calcitonin gene-associated peptides as a neuropeptide, existing in the spine of motor neurons, and estimated to accompany the accumulation of post-synaptic receptors.
