Cytokinesis

Cytokinesis () is the part of the cell division process during which the cytoplasm of a single eukaryotic cell divides into two daughter cells. Cytoplasmic division begins during or after the late stages of nuclear division in mitosis and meiosis. During cytokinesis the spindle apparatus partitions and transports duplicated chromatids into the cytoplasm of the separating daughter cells. It thereby ensures that chromosome number and complement are maintained from one generation to the next and that, except in special cases, the daughter cells will be functional copies of the parent cell. After the completion of the telophase and cytokinesis, each daughter cell enters the interphase of the cell cycle.
Particular functions demand various deviations from the process of symmetrical cytokinesis; for example in oogenesis in animals the ovum takes almost all the cytoplasm and organelles. This leaves very little for the resulting polar bodies, which in most species die without function, though they do take on various special functions in other species.
Another form of mitosis occurs in tissues such as liver and skeletal muscle; it omits cytokinesis, thereby yielding multinucleate cells.
Plant cytokinesis differs from animal cytokinesis, partly because of the rigidity of plant cell walls. Instead of plant cells forming a cleavage furrow such as develops between animal daughter cells, a dividing structure known as the cell plate forms in the cytoplasm and grows into a new, doubled cell wall between plant daughter cells. It divides the cell into two daughter cells.
Cytokinesis largely resembles the prokaryotic process of binary fission, but because of differences between prokaryotic and eukaryotic cell structures and functions, the mechanisms differ. For instance, a bacterial cell has only a single chromosome in the form of a closed loop, in contrast to the linear, usually multiple, chromosomes of eukaryote. Accordingly, bacteria construct no mitotic spindle in cell division. Also, duplication of prokaryotic DNA takes place during the actual separation of chromosomes; in mitosis, duplication takes place during the interphase before mitosis begins, though the daughter chromatids do not separate completely before the anaphase.
Etymology and pronunciation.
The word "cytokinesis" () uses combining forms of "cyto-" + "kine-" + "-sis", New Latin from Classical Latin and Ancient Greek, reflecting "cell" and "kinesis" ("motion, movement"). It was coined by Charles Otis Whitman in 1887.
Origin of this term is from Greek (', a hollow), Latin derivative ' (cellular), Greek ("", movement).
Animal cell.
Animal cell cytokinesis begins shortly after the onset of sister chromatid separation in the anaphase of mitosis. The process can be divided to the following distinct steps: anaphase spindle reorganization, division plane specification, actin-myosin ring assembly and contraction, and abscission. Faithful partitioning of the genome to emerging daughter cells is ensured through the tight temporal coordination of the above individual events by molecular signaling pathways.
Anaphase spindle reorganization.
Animal cell cytokinesis starts with the stabilization of microtubules and reorganization of the mitotic spindle to form the central spindle. The central spindle (or "spindle midzone") forms when non-kinetochore microtubule fibers are bundled between the spindle poles. A number of different species including "H. sapiens", "D. melanogaster" and "C. elegans" require the central spindle in order to efficiently undergo cytokinesis, although the specific phenotype associated with its absence varies from one species to the next (for example, certain Drosophila cell types are incapable of forming a cleavage furrow without the central spindle, whereas in both "C. elegans" embryos and human tissue culture cells a cleavage furrow is observed to form and ingress, but then regress before cytokinesis is complete).
The process of mitotic spindle reorganization and central spindle formation is caused by the decline of CDK1 activity during anaphase. The decline of CDK1 activity at the metaphase-anaphase transition leads to dephosphorylating of inhibitory sites on multiple central spindle components. First of all, the removal of a CDK1 phosphorylation from a subunit of the CPC (the chromosomal passenger complex) allows its translocalization to the central spindle from the centromeres, where it is located during metaphase. Besides being a structural component of the central spindle itself, CPC also plays a role in the phosphoregulation of other central spindle components, including PRC1 (microtubule-bundling protein required for cytokinesis 1) and MKLP1 (a kinesin motor protein). Originally inhibited by CDK1-mediated phosphorylation, PRC1 is now able to form a homodimer that selectively binds to the interface between antiparallel microtubules, facilitating spatial organization of the microtubules of the central spindle. MKLP1, together with the Rho-family GTPase activating protein CYK-4 (also termed MgcRacGAP), forms the centralspindlin complex. Centralspindlin binds to the central spindle as higher-order clusters. The centralspindlin cluster formation is promoted by phosphorylation of MLKP1 by Aurora B, a component of CPC. In short, the self-assembly of central spindle is initiated through the phosphoregulation of multiple central spindle components by the decline of CDK1 activity, either directly or indirectly, at the metaphase-anaphase transition. The central spindle may have multiple functions in cytokinesis including the control of cleavage furrow positioning, the delivery of membrane vesicles to the cleavage furrow, and the formation of the midbody structure that is required for the final steps of division.
Division plane specification.
The second step of animal cell cytokinesis involves division plane specification and cytokinetic furrow formation. Precise positioning of the division plane between the two masses of segregated chromosomes is essential to prevent chromosome loss. Meanwhile, the mechanism by which the spindle determines the division plane in animal cells is perhaps the most enduring mystery in cytokinesis and a matter of intense debate. There exist three hypotheses of furrow induction. The first is the astral stimulation hypothesis, which postulates that astral microtubules from the spindle poles carry a furrow-inducing signal to the cell cortex, where signals from two poles are somehow focused into a ring at the spindle. A second possibility, called the central spindle hypothesis, is that the cleavage furrow is induced by a positive stimulus that originates in the central spindle equator. The central spindle may contribute to the specification of the division plane by promoting concentration and activation of the small GTPase RhoA at the equatorial cortex. A third hypothesis is the astral relaxation hypothesis. It postulates that active actin-myosin bundles are distributed throughout the cell cortex, and inhibition of their contraction near the spindle poles results in a gradient of contractile activity that is highest at the midpoint between poles. In other words, astral microtubules generate a negative signal that increases cortical relaxation close to the poles. Genetic and laser-micromanipulation studies in C. elegans embryos have shown that the spindle sends two redundant signals to the cell cortex, one originating from the central spindle, and a second signal deriving from the spindle aster, suggesting the involvement of multiple mechanisms combined in the positioning of the cleavage furrow. The predominance of one particular signal varies between cell types and organisms. And the multitude and partial redundancy of signals may be required to make the system robust and to increase spatial precision.
Actin-myosin ring assembly and contraction.
At the cytokinesis furrow, it is the actin-myosin contractile ring that drives the cleavage process, during which cell membrane and wall grow inward, which eventually pinches the mother cell in two. The key components of this ring are the filamentous protein actin and the motor protein myosin II. The contractile ring assembles equatorially (in the middle of the cell) at the cell cortex (adjacent to the cell membrane). Rho protein family (RhoA protein in mammalian cells) is a key regulator of contractile ring formation and contraction in animal cells. The RhoA pathway promotes assembly of the actin-myosin ring by two main effectors. First, RhoA stimulates nucleation of unbranched actin filaments by activation of Diaphanous-related formins. This local generation of new actin filaments is important for the contractile ring formation. This actin filament formation process also requires a protein called profilin, which binds to actin monomers and helps load them onto the filament end. Second, RhoA promotes myosin II activation by the kinase ROCK, which activates myosin II directly by phosphorylation of the myosin light chain and also inhibits myosin phosphatase by phosphorylation of the phosphatase-targeting subunit MYPT. Besides actin and myosin II, the contractile ring contains the scaffolding protein anillin. Anillin binds to actin, myosin, RhoA, and CYK-4, and thereby links the equatorial cortex with the signals from the central spindle. It also contributes to the linkage of the actin-myosin ring to the plasma membrane. Another protein, septin, has also been speculated to serve as a structural scaffold on which the cytokinesis apparatus is organized. Following its assembly, contraction of the actin-myosin ring leads to ingression of the attached plasma membrane, which partitions the cytoplasm into two domains of emerging sister cells. The force for the contractile processes is generated by movements along actin by the motor protein myosin II. Myosin II uses the free energy released when ATP is hydrolyzed to move along these actin filaments, constricting the cell membrane to form a cleavage furrow. Continued hydrolysis causes this cleavage furrow to ingress (move inwards), a striking process that is clearly visible through a light microscope.
Abscission.
The cytokinetic furrow ingresses until a midbody structure (composed of electron-dense, proteinaceous material) is formed, where the actin-myosin ring has reached a diameter of about 1–2 μm. Most animal cell types remain connected by an intercellular cytokinetic bridge for up to several hours until they are split by an actin-independent process termed abscission, the last step of cytokinesis. The process of abscission physically cleaves the midbody into two. Abscission proceeds by removal of cytoskeletal structures from the cytokinetic bridge, constriction of the cell cortex, and plasma membrane fission. The intercellular bridge is filled with dense bundles of antiparallel microtubules that derive from the central spindle. These microtubules overlap at the midbody, which is generally thought to be a targeting platform for the abscission machinery. Microtubule severing protein spastin is largely responsible for the disassembly of microtubule bundles inside the intercellular bridge. Complete cortical constriction also requires removal of the underlying cytoskeletal structures. Actin filament disassembly during late cytokinesis depends on the PKCε–14-3-3 complex, which inactivates RhoA after furrow ingression. Actin disassembly is further controlled by the GTPase Rab35 and its effector, the phosphatidylinositol-4,5-bisphosphate 5-phosphatase OCRL. Understanding the mechanism by which the plasma membrane ultimately splits requires further investigation.
Timing cytokinesis.
Cytokinesis must be temporally controlled to ensure that it occurs only after sister chromatids separate during the anaphase portion of normal proliferative cell divisions. To achieve this, many components of the cytokinesis machinery are highly regulated to ensure that they are able to perform a particular function at only a particular stage of the cell cycle. Cytokinesis happens only after APC binds with CDC20. This allows for the separation of chromosomes and myosin to work simultaneously.
After cytokinesis, non-kinetochore microtubules reorganize and disappear into a new cytoskeleton as the cell cycle returns to interphase (see also cell cycle).
Plant cell.
Due to the presence of a cell wall, cytokinesis in plant cells is significantly different from that in animal cells, Rather than forming a contractile ring, plant cells construct a cell plate in the middle of the cell. The stages of cell plate formation include (1) creation of the phragmoplast, an array of microtubules that guides and supports the formation of the cell plate; (2) trafficking of vesicles to the division plane and their fusion to generate a tubular-vesicular network; (3) continued fusion of membrane tubules and their transformation into membrane sheets upon the deposition of callose, followed by deposition of cellulose and other cell wall components; (4) recycling of excess membrane and other material from the cell plate; and (5) fusion with the parental cell wall
The phragmoplast is assembled from the remnants of the mitotic spindle, and serves as a track for the trafficking of vesicles to the phragmoplast midzone. These vesicles contain lipids, proteins and carbohydrates needed for the formation of a new cell boundary. Electron tomographic studies have identified the Golgi apparatus as the source of these vesicles, but other studies have suggested that they contain endocytosed material as well.
These tubules then widen and fuse laterally with each other, eventually forming a planar, fenestrated sheet [8]. As the cell plate matures, large amounts of membrane material are removed via clathrin-mediated endocytosis [7] Eventually, the edges of the cell plate fuse with the parental plasma membrane, often in an asymmetrical fashion, thus completing cytokinesis. The remaining fenestrae contain strands of endoplasmic reticulum passing through them, and are thought to be the precursors of plasmodesmata [8].
The construction of the new cell wall begins within the lumen of the narrow tubules of the young cell plate. The order in which different cell wall components are deposited has been determined largely by immuno-electron microscopy. The first components to arrive are pectins, hemicelluloses, and arabinogalactan proteins carried by the secretory vesicles that fuse to form the cell plate. The next component to be added is callose, which is polymerized directly at the cell plate by callose synthases. As the cell plate continues to mature and fuses with the parental plasma membrane, the callose is slowly replaced with cellulose, the primary component of a mature cell wall [6]. The middle lamella (a glue-like layer containing pectin) develops from the cell plate, serving to bind the cell walls of adjoining cells together.
Forces.
Animal cells.
Cytokinetic furrow ingression is powered by Type II Myosin ATPase. Since Myosins are recruited to the medial region, the contractile forces acting on the cortex resemble a 'purse string' constriction pulling inwards. This leads to the inward constriction. The plasma membrane by virtue of its close association with the cortex via crosslinker proteins


Smallpox epidemic


Camille (Red Dwarf)

"Camille" is the first episode of science fiction sitcom "Red Dwarf" Series IV, and the nineteenth episode in the series run. It was first broadcast on the British television channel BBC2 on 14 February 1991. The episode was planned to be shown third, but was moved forward in the schedule to be shown on Valentine's Day. Written by Rob Grant and Doug Naylor, and directed by Ed Bye. The episode, a parody of the film "Casablanca", sees Kryten rescue and fall in love with an android who appears to be the same model as himself.
Plot.
Dave Lister does his best to teach Kryten to lie, stating he needs it as a natural form of defence. Although Kryten has some success, he finds he cannot properly lie in the presence of anyone else, and is forced to abandon his lessons when Arnold Rimmer requires him to pilot a "Starbug" for asteroid-spotting. When they receive a distress call from a doomed ship, Kryten manages to make use of Lister's insubordination training to go search for survivors despite Rimmer's orders against this. The pair swiftly encounter and rescue Camille, the only survivor and become attracted her – Kryten sees her as another mechanoid like himself, yet Rimmer sees her as a hologram with similar interests that he has. When they bring her back to "Red Dwarf", Lister meets her and finds her attractive – to him, she appears as a human female with interests matching his own. However, he soon suspects something is not right with her, when Rimmer arrives, questions her over viewing one of his collections and hears a different response to what Lister gets from her. When Cat meets her, he sees Camille as a mirror version of himself.
Lister informs the crew that Camille is actually a pleasure GELF – a Genetically Engineered Life Form – designed to appear to each individual as the object of their desire, and is in reality a slimy green blob with tentacles. Although hurt, Kryten compliments Camille's true appearance and opts to date her in her actual form, including showing her the film "Casablanca" that Lister used as inspiration for Kryten's lessons. When Camille finds that her husband has turned up looking for her, Kryten advises her, in a similar manner to the film's ending, to leave with him rather than stay, and stoically waves goodbye. When Lister learns that Kryten lied to her to spare her feelings, he smiles at knowing he has learnt well, especially when Kryten insults him for being responsible for what happened.
Production.
For Series IV, recording of the show moved from the studios in Manchester to Shepperton Studios due to the old studio undergoing refurbishment. Shepperton turned out to be a blessing to the show as it allowed for use of the sets for rehearsals in addition to recording. Production starting with Series IV also permanently shifted to Grant Naylor Productions.
The droid version of the GELF was played by Judy Pascoe, Robert Llewellyn's then girlfriend (they have since married). Robert has often joked how he used to complain to Judy about the amount of make-up he had to endure, and yet when Judy wore it she had no complaints. She also provided the voice of the blob Camille, which was controlled from inside by effects artist Mike Tucker.
The initial plan was for Camille to appear as Kristine Kochanski in Lister's eyes; while this later changed, Suzanne Rhatigan was still credited as "Kochanski Camille". Like Pascoe and Llewellyn, Rhatigan and Craig Charles were in a relationship at the time of the recording. The Hologram Camille was played by Francesca Folan. Rupert Bates voiced the Hector Blob.
This episode was not originally planned to be aired first, but was moved forward when "Meltdown" was held back due to the ongoing hostilities in the Gulf War. It was decided to move this episode up to the first in the run due to the fact that it seemed an appropriate one to air on Valentine's Day.
Cultural references.
"Casablanca", Lister's favourite film, was used for the main plot of the episode and is mentioned and referenced throughout. Kryten and Camille even watch the film in the episode. When Lister explains to Kryten why it is necessary to lie he mentions examples of Humphrey Bogart in the final scene of "Casablanca" and Nelson's "I see no ships." The ending of "Casablanca" is also parodied in the Kryten and Camille farewell scene.
Lister watches a vid recording of the television show "Tales of the Riverbank: The Next Generation", a parody of the "Tales of the Riverbank" show and "". Lister compares the main character Hammy Hamster the rodent equivalent of Marlon Brando. The television show "St. Elsewhere" is named by Lister as a tongue-in-cheek defense of his credentials to be ship's surgeon ("I've seen every episode"). His Spider-Man costume is also mentioned.
Steve McQueen and the film "The Blob" are referenced by Lister after Kryten takes the true form of Camille to dinner. Kryten refers to the actor Karl Malden as an example of a human less attractive than Camille in her blob-like form.
The concept of an alien whom each person sees differently is also a reference to the first-aired "Star Trek" episode, "The Man Trap".
Reception.
The episode was first broadcast on the British television channel BBC2 on 14 February 1991 in the 9:00pm evening time slot, although it was originally planned to be shown as the third episode of Series IV as shown in the repeat runs in both 1992 and 1994. The series' transmission order was changed as it was felt more appropriate to run the episode on Valentine's Day and so it went out first. Further changes to the series' running order came about because of the outbreak of the Gulf War and the subject matter of some of the other episodes, notably "Dimension Jump" featuring the war-hero Ace Rimmer and the anti-war-themed "Meltdown". The episode received a mixed response from fans.


Mechanotaxis

Mechanotaxis refers to the directed movement of cell motility via mechanical cues (e.g., fluidic shear stress, substrate stiffness gradients, etc.). In response to fluidic shear stress, for example, cells have been shown to migrate in the direction of the fluid flow.
A subset of mechanotaxis - termed durotaxis - refers specifically to cell migration guided by gradients in substrate rigidity (i.e. stiffness). The observation that certain cell types seeded on a substrate rigidity gradient migrate up the gradient (i.e. in the direction of increasing substrate stiffness) was first reported by Lo et al. The primary method for creating rigidity gradients for cells (e.g., in biomaterials) consists of altering the degree of cross-linking in polymers to adjust substrate stiffness. Alternative substrate rigidity gradients include micropost array gradients, where the stiffness of individual microposts is increased in a single, designed direction.


Milica Radisic

Milica Radisic is a Serbian Canadian tissue engineer, academic and researcher. She is a Professor at the University of Toronto’s Institute of Biomaterials and Biomedical Engineering, and the Department of Chemical Engineering and Applied Chemistry. She co-founded TARA Biosystems and is a senior scientist at the Toronto General Hospital Research Institute.
Radisic is known for creating beating heart tissue in a dish using human-induced pluripotent stem cells. Her research has led the replication of diseased human heart tissue that can be used for drug screening to help create treatment therapies for patients with heart injury.
Radisic was recognized as a YWCA Toronto Woman of Distinction in 2018, and received the Women in Science and Engineering Breaking the Glass Ceiling Award. She is a Former Chair of the Membership Committee for the Tissue Engineering and Regenerative Medicine International Society. Radisic is a Fellow of the Royal Society of Canada, Academy of Sciences, Canadian Academy of Engineering, American Institute for Medical and Biological Engineering as well as Tissue Engineering and Regenerative Medicine Society.
Education.
Radisic attended the University of Novi Sad for her freshman year and transferred to McMaster University in 1996. She graduated with a bachelor's degree in chemical engineering in 1999 and earned her Ph.D. in chemical engineering from Massachusetts Institute of Technology in 2004. She then completed a postdoctoral fellowship at Harvard-MIT Division of Health Sciences and Technology in 2005.
Career.
Radisic started her career as a Research Assistant at McMaster University and MIT before working as a Post-doctoral Associate at Harvard-MIT Division of Health Science and Technology. In 2005, Radisic joined Institute of Biomaterials and Biomedical Engineering at the University of Toronto as an Assistant Professor at the Department of Chemical Engineering and Applied Chemistry. She was promoted to Associate Professor in 2010 and to Professor, in 2014.
Research.
Radisic's general research focuses on treatments for myocardial infarction and drug cardio-toxicity screening. Her work centers on using human embryonic and induced pluripotent stem cells to develop a heart patch that could be used to study the safety and efficacy of new drugs as well as its compatibility with various cell lines. Radisic was the first author of a highly cited paper in PNAS where she successfully generated beating heart tissues from embryonic stem cells via electrical stimulation.
Tissue engineering of cardiac patches.
Radisic has worked on designing advanced bioreactors for cardiac tissue engineering capable of integrating mechanical and electrical stimuli with perfusion. She has also conducted research on developing strategies to engineer vascularized myocardium based on the tri-culture of key heart cell types using the engineered cardiac tissue as a model system for cardiac cell therapy or drug testing.
Organ-on-a-chip engineering.
Radisic's lab works on the development of injectable hydrogels with specific peptides such as QHREDGS peptide–modified hydrogel. She also researched on the biometric cues in vitro and developed an engineered oriented cardiac tissue. Radisic has also worked on other biomaterials such as moldable elastomeric polyester-carbon nanotube scaffolds for cardiac tissue engineering that will promote survival and localization of the cardio-myocytes injected into the infarcted myocardium.
Radisic developed a flexible shape-memory scaffold for minimally invasive delivery of functional tissues. The scaffold utilizes a biodegradable polymer and a micro-fabricated lattice design to allow its shape memory property. Her lab designed micro-fabricated cell culture systems with built-in electrodes and defined groove and ridge heights for simultaneous application of field stimulation and contact guidance cues, in order to understand interactive effects of multiple physical stimuli.
Radisic and her team developed a microfabricated system for generating 3D, aligned beating cardiac tissue (Bio-wire) from human pluripotent stem cells (hPSC) derived cardiomyocytes. The system utilized electrical stimulation to increase the maturation of hPSC derived cardiomyocytes. Bio-wire is the first system combining electrical stimulation with geometry control of 3D tissue assembly to improve the electrical and ultra-structural properties of human cardiac tissue.
Bibliography.
Book.
• Cardiac Tissue Engineering Methods and Protocols (2014)


MRNA guanylyltransferase

In enzymology, a mRNA guanylyltransferase () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are GTP and (5')ppPur-mRNA, whereas its two products are diphosphate and G(5')pppPur-mRNA.
This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing nucleotide groups (nucleotidyltransferases). The systematic name of this enzyme class is GTP:mRNA guanylyltransferase. Other names in common use include mRNA capping enzyme, messenger RNA guanylyltransferase, and Protein 2.
Structural studies.
As of late 2007, 5 structures have been solved for this class of enzymes, with PDB accession codes , , , , and .


Alpha cell

Alpha cells (α-cells) are endocrine cells in the pancreatic islets of the pancreas. They make up to 20% of the human islet cells synthesizing and secreting the peptide hormone glucagon, which elevates the glucose levels in the blood.
Function.
To elevate glucose levels, glucagon binds to receptors on hepatocytes (liver cells) and some other cells (e.g. kidney cells). This activates an enzyme, glycogen phosphorylase, inside the hepatocyte to hydrolyse glycogen into glucose. This process is called glycogenolysis.
In rodents, alpha cells are located in the periphery of the islets, however in humans, the islet architecture is generally less organized and alpha cells are frequently observed inside the islets as well. When viewed from an electron microscope, alpha cells can be identified by their characteristic granules with a large dense core and a small white halo.
Alpha cells also generate GLP-1 and may have protective and regenerative effect on beta cells. They possibly can transdifferentiate into beta cells to replace lost beta cells.


Hemogenic endothelium

Hemogenic endothelium is a special subset of endothelial cells scattered within blood vessels that can differentiate into haematopoietic cells.
The development of hematopoietic cells in the embryo proceeds sequentially from mesoderm through the hemangioblast to the hemogenic endothelium and hematopoietic progenitors.


Heat Biologics

Heat Biologics Inc. is a US biotechnology company focused on the field of immunotherapy. Heat Biologics was founded by serial bio-tech entrepreneur Jeff Wolf and scientific cofounder Eckhard Podack, based upon Dr. Podack's novel work in the development of gp96 and other approaches to modulate the immune system, with multiple programs for indications in cancer, COVID-19, infectious disease, and others. The company is based in Morrisville, North Carolina.
"gp96".
Heat Biologics’ proprietary gp96 platform leverages gp96’s role as a natural molecular warning system, engineered to secrete antigens bound to gp96, with broad applications in cancer treatment and vaccines for infectious diseases. Their lead product is in a Phase 2 trial for treatment of non-small cell lung cancer (NSCLC).
Pipeline products.
HS-110.
HS-110, also called viagenpumatucel-L, is in Phase II in NSCLC, in combination with cyclophosphamide. Another Phase I study is combining HS 110 with nivolumab and other checkpoint inhibitors.
HS-130.
HS-130 is in Phase I clinical trial for patients with solid tumors. HS-130 is an allogeneic (“off-the-shelf”) cell line engineered to express OX40 ligand fusion protein (OX40L-Fc). OX40 ligand is a key co-stimulator of T cells that augments antigen-specific CD8+ T cell responses.
COVID-19 Vaccine.
The company initiated a COVID-19 vaccine collaboration with the University of Miami in March 2020 using their gp96 platform which activates the human immune system to combat infectious diseases with the potential of generating long-term immune responses.
PTX-35.
PTX-35 is in Phase I clinical trial for patients with solid tumors. PTX-35 is a potential first-in-class T cell co-stimulator targeting TNFRSF25 (Death Receptor 3). Favorable safety profile was demonstrated in mice and non-human primates. PTX-35’s development plan focuses on cancer immunotherapy.
Heat Biologics stock.
Heat Biologics stock has been traded on the NASDAQ under ticker symbol HTBX since 24 July 2013.
Main people.
Heat Biologics' Chairman and CEO is Jeffrey Wolf, a serial Life Sciences entrepreneur who founded the company in 2008.


Muscular layer

The muscular layer (muscular coat, muscular fibers, muscularis propria, muscularis externa) is a region of muscle in many organs in the vertebrate body, adjacent to the submucosa. It is responsible for gut movement such as peristalsis. The Latin, tunica muscularis, may also be used.
Structure.
It usually has two layers of smooth muscle:
However, there are some exceptions to this pattern.
The inner layer of the muscularis externa forms a sphincter at two locations of the gastrointestinal tract:


KcsA potassium channel

KcsA (K channel of streptomyces A) is a prokaryotic potassium channel from the soil bacteria "Streptomyces lividans" that has been studied extensively in ion channel research. The pH activated protein possesses two transmembrane segments and a highly selective pore region, responsible for the gating and shuttling of K+ ions out of the cell. The amino acid sequence found in the selectivity filter of KcsA is highly conserved among both prokaryotic and eukaryotic K+ voltage channels; as a result, research on KcsA has provided important structural and mechanistic insight on the molecular basis for K+ ion selection and conduction. As one of the most studied ion channels to this day, KcsA is a template for research on K+ channel function and its elucidated structure underlies computational modeling of channel dynamics for both prokaryotic and eukaryotic species.
History.
KcsA was the first potassium ion channel to be characterized using x-ray crystallography by Roderick MacKinnon and his colleagues in 1998. In the years leading up to this, research on the structure of K+ channels was centered on the use of small toxin binding to reveal the location of the pore and selectivity filter among channel residues. MacKinnon’s group theorized the tetrameric arrangement of the transmembrane segments, and even suggested presence of pore-forming “loops” in the filter region made of short segments of amino acids that interacted with K+ ions passing through the channel The discovery of strong sequence homology between KcsA and other channels in the Kv family, including the "Shaker" protein, attracted the attention of the scientific community especially as the K+ channel signature sequence began to appear in other prokaryotic genes. The simplicity of the two transmembrane helices in KcsA, as opposed to the six in many eukaryotic ion channels, also provided a method to understand the mechanisms of K+ channels conduction at a more rudimentary level, thereby providing even great impetus for the study of KcsA.
The crystal structure of KcsA was solved by the MacKinnon group in 1998 after discovery that removal of the C-terminus cytoplasmic domain of the native protein (residues 126-158) increases the stability of crystallized samples. A model of KcsA at the 3.2A resolution was produced that confirmed the tetrameric arrangement of the protein around a center pore, with one helix of each subunit facing the inside axis and the other facing outwards. Three years later, a higher resolution model was produced by Morais-Cabral and Zhou after monoclonal Fab fragments were attached to KcsA crystals to further stabilize the channel. In the early 2000s, evidence for the occupation of the selectivity filter by two K+ atom during the transport process emerged, based on energy and electrostatic calculations made to model the pore region. Continued investigation of the various opened and closed, inactive and active conformations of KcsA by other imaging methods such as ssNMR and EPR have since provided even more insight into channel structure and the forces gating the switch from channel inactivation to conduction.
In 2007, Riek et. Al. showed that the channel opening that results from titrating the ion channel from pH 7 to pH 4, corresponds to conformational changes in two regions: transition to the ion-exchanging state of the selectivity filter, and the opening of the arrangement of TM2 at the C-terminus. This model explains the ability of KcsA to simultaneous select for K+ ions while also gating electrical conductance. In 2011, the crystal structure of full length KcsA was resolved to reveal that hindrance by the previously truncated residues permits only straightforward expansion of the intercellular ion passage region of the protein. This research provides a more detailed look into the motion of separate channel regions during ion conduction. In the present day, KcsA studies are focused on using the prokaryotic channel as a model for the channel dynamics of larger eukaryotic K+ channels, including hERG.
Structure.
The structure of KcsA is that of an inverted cone, with a central pore running down the center made up of two transmembrane helices (the outer-helix M1 and the inner-helix M2), which span the lipid bilayer. The channel itself is a tetramer composed of four identical, single-domain subunits (each with two α-helices) arranged so that one M2 helix faces the central pore, while the other M1 helix faces the lipid membrane. The inner helices are tilted by about 25° in relation to the lipid membrane and are slightly kinked, opening up to face the outside of the cell like a flower. These two TM helices are linked by a reentrant loop, dispersed symmetrically around a common axis corresponding to the central pore. The pore region spans approximately 30 amino acid residues and can be divided into three parts: a selectivity filter near the extracellular side, a dilated water-filled cavity at the center, and a closed gate near the cytoplasmic side formed by four packed M2 helices. This architecture is found to be highly conserved in the potassium channel family in both eukaryotes and prokaryotes.
The overall length of the pore is 45 Å, and its diameter varies considerably within the distinct regions of the inner tunnel. Travelling from the intracellular region outwards (bottom to top in the picture) the pore begins with a gate region formed by M2 helices at 18 Å in diameter, and then opens into a wide cavity (∼10 Å across) near the middle of the membrane. In these regions, K+ ions are in contact with surrounding water molecules but when they enter the channel from the selectivity filter at the top, the cavity is so narrow that K+ ions must shed any hydrating waters in order to enter the cell. In regards to the amino acid composition of the pore-lining residues within KcsA, the side chains lining the internal pore and cavity are predominantly hydrophobic, but within the selectivity filter polar amino acids are present that contact the dehydrated K+ ions.
Selectivity filter.
The wider end of the cone corresponds to the extracellular mouth of the channel made up of pore helices, plus a selectivity filter that is formed by a TVGYG sequence, (Threonine, Valine, Glycine, Tyrosine, Glycine), characteristic of potassium channels. Within this region, coordination between the TVGYG amino acids and incoming K+ ions allows for conduction of ions through the channel. The selectivity filter of KcsA contains four ion binding sites, although it is proposed that only two of these four positions are occupied at one time. The selectivity filter is about 3 Å in diameter. though molecular dynamics simulations suggest the filter is flexible. The presence of TVGYG in the filter region of KcsA is conserved even in more complex eukaryotic channels, thus making KcsA an optimal system for studying K+ channel conductance across species.
Function.
The KcsA channel is considered a model channel because the KcsA structure provides a framework for understanding K+ channel conduction, which has three parts: Potassium selectivity, channel gating by pH sensitivity, and voltage-gated channel inactivation. K+ ion permeation occurs at the upper selectivity filter region of the pore, while pH gating rises from the protonation of transmembrane helices at the end of the pore. At low pH, the M2 helix is protonated, shifting the ion channel from closed to open conformation. As ions flow through the channel, voltage gating mechanisms are thought to induce interactions between Glu71 and Asp80 in the selectivity filter, which destabilize the conductive conformation and facilitate entry into a long-lived nonconducting state that resembles the C-type–inactivation of voltage-dependent channels.
In the nonconducting conformation of KcsA at pH 7, K+ is bound tightly to coordinating oxygens of the selectivity filter and the four TM2 helices converge near the cytoplasmic junction to block the passage of any potassium ions. At pH 4 however, KcsA undergoes millisecond-timescale conformational exchanges filter permeating and nonpermeating states and between the open and closed conformations of the M2 helices. While these distinct conformational changes occur in separate regions of the channel, the molecular behavior of each region is linked by both electrostatic interactions and allostery. The dynamics of this exchange stereochemical configurations in the filter provides the physical basis for simultaneous K+ conductance and gating.
K+selectivity.
The sequence TVGYG is especially important for maintaining the potassium specificity of KcsA. The glycines in this selectivity filter sequence have dihedral angles that allow carbonyl oxygen atoms in the protein backbone of the filter to point in one direction, toward the ions along the pore. The glycines and threonine coordinate with the K+ ion, while the side-chains of valine and tyrosine are directed into the protein core to impose geometric constraint on the filter. As a result, the KcsA tetramer harbors four equal spaced K+ binding sites, with each side composed of a cage formed by eight oxygen atoms that sit on the vertices of a cube. The oxygen atoms that surround K+ ions in the filter are arranged like the water molecules that encircle hydrated K+ ions in the cavity of the channel; this suggests that oxygen coordination and binding sites in the selectivity filter are paying for the energetic cost of K+ dehydration. Because the Na+ ion is too small for these K+-sized binding sites, dehydration energy is not compensated and thus, the filter selects against other extraneous ions. Additionally, the KcsA channel is blocked by Cs+ ions and gating requires the presence of Mg2+ ions.
pH Sensitivity.
The pH-dependent conductance of KcsA indicates that the opening of the ion channel occurs when the protein is exposed to a more acidic environment. NMR studies performed by the Riek group show that pH sensitivity occurs in both the C-terminal TM2 region of the protein as well as with Tyr78 and Gly79 residues in the selectivity filter. There is evidence to suggest that the main pH sensor is in the cytoplasmic domain. Exchanging negatively charged amino acids for neutral ones made the KcsA channel insensitive to pH even though there were no amino-acid changes at the transmembrane region. In addition, between the pH of 6 and 7, histidine is one of the few titratable side chains of histidines; they are absent in the transmembrane and extracellular segments of TM2 but present at KcsA's C-terminus. This highlights a possible mechanism for the slow opening of KcsA which is particularly pH sensitive, especially as the conformational propagation of channel opening signal from the C-terminus to the selectivity filter could be important in coordinating the structural changes needed for conductance along the entire pore.
NMR studies also suggest that a complex hydrogen bond network between Tyr78, Gly79, Glu71 and Asp80 exists in the KcsA filter region, and further acts as a pH-sensitive trigger for conductance. The mutation of key residues in the region, including E71A, results in a large energy cost of 4 kcal mol−1, equivalent to the loss of the hydrogen bond between Glu71 and Tyr78 and the water-mediated hydrogen bond between Glu71 and Asp80 in KcsA(E71A). These studies further highlight the role of pH gating in KcsA channel function.
Voltage Gating.
In 2006, the Perozo group proposed a mechanistic explanation for the effects of voltage fields on KcsA gating. After adding a depolarizing current to the channel, the reorientation of Glu71 towards the intracellular pore occurs, thereby disrupting the Glu71-Asp80 carboxyl-carboxylate pair that initially stabilizes the selectivity filter. The collapse of the filter region prevents entry into or facilitate exit from the inactivated state. Glu71, a key part of the selectivity filter signature sequence that is conserved among K+ ion channels, plays a pivotal role in gating as its ability to reorient itself in the direction of the transmembrane voltage field is able to provide an explanation for voltage gating events in KcsA. The orientation of amino acids in the filter region might play significant physiological role in modulating potassium fluxes in eukaryotes and prokaryotes under steady-state conditions.
Research.
Function.
The precise mechanism of potassium channel selectivity continues to be studied and debated and multiple models are used to describe different aspects of the selectivity. Models explaining selectivity based on field strength concept developed by George Eisenman based on Coulomb's law have been applied to KcsA. An alternative explanation for the selectivity of KcsA is based on the close-fit model (also known as the snug-fit model) developed by Francisco Bezanilla and Armstrong. The main chain carbonyl oxygen atoms that make up the selectivity filter are held at a precise position that allows them to substitute for water molecules in the hydrated shell of the potassium ion, but they are too far from a sodium ion. Further work has studied thermodynamic differences in ion binding, topological considerations, and the number of continuous ion binding sites.
In addition, a major limitation of crystal structure study and simulations has yet to be discussed: the best resolved and most applied crystal structure of KcsA appears to be that of the ‘closed' form of the channel. This is reasonable as the closed state of the channel is favored at neutral pH, at which the crystal structure was solved by X-ray crystallography. However, the dynamic behavior of KcsA makes analysis of the channel difficult as a crystal structure inevitably provides a static, spatially and temporally averaged image of a channel. To bridge the gap between molecular structure and physiological behavior, an understanding of the atomic resolution dynamics of potassium channels is required.
Applications.
Due to the high sequence similarity between the pore of KcsA and other eukaryotic K+ ion channel proteins, KcsA has provided important insight into the behavior of other important voltage conducting proteins such as the drosophilla-derived "Shaker" and the human "hERG" potassium channel. KcsA has been used in mutagenesis studies to model the interactions between hERG and various drug compounds. Such tests can screen for drug-hERG channel interactions that cause acquired long QT syndrome, are essential for determining the cardiac safety of new medications. In addition, homology models based on the closed state KcsA crystal structure have been generated computationally to construct a multiple state representation of the hERG cardiac K+ channel. Such models reveal the flexibility of the hERG channel and can consistently predict the binding affinity of a set of diverse ion channel-interacting ligands. Analysis of the complex ligand-hERG structures can be used to guide the synthesis of drug analogs with reduced hERG liability, based on drug structure and docking potential.


4-HO-MPMI

4-HO-MPMI (also known as 4-Hydroxy-"N"-methyl-(α,"N"-trimethylene)-tryptamine or lucigenol) is a tryptamine derivative that is a psychedelic drug. It was developed by the team led by David Nichols from Purdue University in the late 1990s. This compound produces hallucinogen-appropriate responding in animal tests with a similar potency to the amphetamine-derived psychedelic DOI, and has two enantiomers, with only the ("R")-enantiomer being active.
The binding affinity for 5-HT2A receptor is 13 ± 2 nM (Ki [125I]DOI). It is reported at doses starting at 0.5 mg and 1.0-1.5 mg seem to be psychedelic doses. The duration it is reported between six and eight hours. The effects, still not too documented, are OEV/CEV, sedation and anxiety.


Cirazoline

Cirazoline is a full agonist at the α1A adrenergic receptor, a partial agonist at both the α1B and α1D adrenergic receptors, and a nonselective antagonist to the α2 adrenergic receptor. It is believed that this combination of properties could make cirazoline an effective vasoconstricting agent.
Cirazoline has also been shown to decrease food intake in rats, purportedly through activation of α1 adrenoceptors in the paraventricular nucleus in the hypothalamus of the brain.


Arthrobacter roseus

Arthrobacter roseus is a species of red-pigmented psychrophilic bacteria first isolated from a cyanobacterial mat. Its type strain is CMS 90r(T) (= MTCC 3712T = DSM 14508T).


Kassinin

Kassinin is a peptide derived from the Kassina frog. It belongs to tachykinin family of neuropeptides. It is secreted as a defense response, and is involved in neuropeptide signalling.
The amino acid sequence is H-Asp-Val-Pro-Lys-Ser-Asp-Gln-Phe-Val-Gly-Leu-Met-NH2.
Ion Transportation in Frog Skin.
In frog skin, tachykinins are responsible for ion transportation. Kassinin is one of the tachykinin peptides, which interacts with NK2 receptor to increase short circuit current (SCC), resulting in an ion transportation in frog skin. Another peptide that belongs to tachykinin family is Eledoisin that interacts with NK3 receptor for stimulation of SCC, but can be reduced by either NK1 or NK2 antagonist. Whereas Kassinin can't be reduced by neither of these NK1, NK2 or NK3 antagonists. Kassinin is very effective in increasing short circuit current to its maximum within 10 minutes with increase of 26.13 ± 1.53μΑ/cm2.
There are some requirements that must be accomplished to gain short circuit stimulation. One of them is to have Phe-X-Gly-Leu-Met-NH2 sequence at C-terminal where X could be Val or Ile. The other requirement is to have 2 or 1 proline residue at N-terminal. If only one proline residue is present, then there must be one basic amino acid present in the sequence. For instance kassinin has one proline residue and one basic amino acid (Lys). Another example would be of enterokassinin that does have one proline reside but doesn't have any basic amino acid in the sequence. Thus it doesn't increase SCC and therefore is ineffective in ion transportation. While kassinin plays an effiecient role in ion transportaion in frog skin.


Oryzorictes


HITS-CLIP

High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP, also known as CLIP-Seq) is a genome-wide means of mapping protein–RNA binding sites or RNA modification sites in vivo. HITS-CLIP was originally used to generate genome-wide protein-RNA interaction maps for the neuron-specific RNA-binding protein and splicing factor NOVA1 and NOVA2; since then a number of other splicing factor maps have been generated, including those for PTB, RbFox2, SFRS1, hnRNP C, and even N6-Methyladenosine (m6A) mRNA modifications.
HITS-CLIP of the RNA-binding protein Argonaute has been performed for the identification of microRNA targets by decoding microRNA-mRNA and protein-RNA interaction maps in mouse brain, and subsequently in "Caenorhabditis elegans", embryonic stem cells and tissue culture cells.
As a novel modification of HITS-CLIP, m6A-CLIP was developed to precisely map N6-Methyladenosine(m6A) locations in mRNA by UV-crosslinking m6A antibody to the target RNA. Recently, improved bioinformatics applied to Argonaute HITS-CLIP enables identification of binding sites with single nucleotide resolution.


Anti-Vaccination League of America


Alpha-adrenergic agonist

Alpha-adrenergic agonists are a class of sympathomimetic agents that selectively stimulates alpha adrenergic receptors. The alpha-adrenergic receptor has two subclasses α1 and α2. Alpha 2 receptors are associated with sympatholytic properties. Alpha-adrenergic agonists have the opposite function of alpha blockers. Alpha adrenoreceptor ligands mimic the action of epinephrine and norepinephrine signaling in the heart, smooth muscle and central nervous system, with norepinephrine being the highest affinity. The activation of α1 stimulates the membrane bound enzyme phospholipase C, and activation of α2 inhibits the enzyme adenylate cyclase. Inactivation of adenylate cyclase in turn leads to the inactivation of the secondary messenger cyclic adenosine monophosphate and induces smooth muscle and blood vessel constriction.
Classes.
Although complete selectivity between receptor agonism is rarely achieved, some agents have partial selectivity. NB: the inclusion of a drug in each category just indicates the activity of the drug at that receptor, not necessarily the selectivity of the drug (unless otherwise noted).
α1 agonist.
α1 agonist: stimulates phospholipase C activity. (vasoconstriction and mydriasis; used as vasopressors, nasal decongestants and during eye exams). Selected examples are:
α2 agonist.
α2 agonist: inhibits adenylyl cyclase activity, reduces brainstem vasomotor center-mediated CNS activation; used as antihypertensive, sedative & treatment of opiate dependence and alcohol withdrawal symptoms). Selected examples are:
Nonspecific agonist.
Nonspecific agonists act as agonists at both alpha-1 and alpha-2 receptors.
Undetermined/unsorted.
The following agents are also listed as agonists by MeSH.
Clinical significance.
Alpha-adrenergic agonists, more specifically the auto receptors of alpha 2 neurons, are used in the treatment of glaucoma by decreasing the production of aqueous fluid by the ciliary bodies of the eye and also by increasing uveoscleral outflow. Medications such as clonidine and dexmedetomidine target pre-synaptic auto receptors, therefore leading to an overall decrease in norepinephrine which clinically can cause effects such as sedation, analgesia, lowering of blood pressure and bradycardia. There is also low quality evidence that they can reduce shivering post operatively
The reduction of the stress response caused by alpha 2 agonists were theorised to be beneficial peri operatively by reducing cardiac complications, however this has shown not to be clinically effective as there was no reduction in cardiac events or mortality but there was an increased incidence of hypotension and bradycardia


Unnatural Exposure (novel)

Unnatural Exposure is a crime fiction novel by Patricia Cornwell. It is the eighth book in the Dr. Kay Scarpetta series. The story is set in Richmond, Virginia and Ireland.
Plot summary.
Virginia Chief Medical Examiner Kay Scarpetta has a bloody puzzle on her hands: five headless, limbless cadavers in Ireland, plus four similar victims in a landfill back home. Is a serial butcher loose in Virginia? That's what the panicked public thinks, thanks to a local TV reporter who got the leaked news from Scarpetta's rival, Investigator Percy Ring. But this is no run-of-the-mill serial killer. A shadowy figure has plans involving mutant smallpox, mass murder, and messing with Scarpetta's mind by e-mailing her hot naked photos of the murder scenes, along with cryptic AOL chat-room messages. Central to the plot is the case of Janet Parker, the last person known to have died of smallpox, which she contracted in 1978 due to a lab accident in Birmingham, England, after the disease was eradicated in the wild. Cornwell makes the villain a junior employee of the lab at the time who was made a scapegoat for the accident and whose career was blighted as a result. This provides the plot with a credible source for the virus and a motive for the central crime.
The emergency response is complicated by a Federal budget freeze, when all "non-essential" government employees are sent home. Cornwall says in an introduction to a 2008 reprint that a decade ago the idea of a deliberate virus threat seemed fanciful and improbable, and she got more access to government facilities like the CDC for researching the book than she would have got a decade later. She said that her "modus operandi when I write a book is to ask my characters where they are and what they are doing. Then I focus on an image. From that image comes the story ... ".


Wassim Almawi

Wassim Y. Almawi is professor in the School of Pharmacy at Lebanese American University in Byblos, Lebanon, and adjunct professor at Faculty of Sciences, El Manar University in Tunis, Tunisia. This followed appointment as professor and chairman of Department of Biochemistry at Arabian Gulf University in Bahrain from 2000–2017. Almawi is also the Chief of the Special and Molecular Diagnostics Laboratory in Bahrain.


Gorakshya Rajya Lakshmi Devi

Gorakshya Rajya Lakshmi Devi (1798–16 December 1816) was the Queen of Girvan Yuddha Bikram Shah, King of Nepal. She was the mother of Rajendra Bikram Shah. She died of smallpox.


Methallylescaline

Methallylescaline (4-methylallyloxy-3,5-dimethoxyphenethylamine) is a lesser-known psychedelic drug. It is the 4-methyl analog of allylescaline. Methallylescaline was first synthesized by Alexander Shulgin. In his book "PiHKAL", the dosage range is listed as 40–65 mg and the duration is listed as 12–16 hours. Little data exists about the pharmacological properties, metabolism, and toxicity of methallylescaline, though it is known to be an agonist of 5-HT2A receptors, and has been sold as a designer drug.
Legal status.
Methallylescaline is illegal in Sweden as of 26 January 2016.


TATA box

In molecular biology, the TATA box (also called the Goldberg-Hogness box) is a sequence of DNA found in the core promoter region of genes in archaea and eukaryotes. The bacterial homolog of the TATA box is called the Pribnow box which has a shorter consensus sequence.
The TATA box is considered a non-coding DNA sequence (also known as a cis-regulatory element). It was termed the "TATA box" as it contains a consensus sequence characterized by repeating T and A base pairs. How the term "box" originated is unclear. In the 1980s, while investigating nucleotide sequences in mouse genome loci, the Hogness box sequence was found and "boxed in" at the -31 position. When consensus nucleotides and alternative ones were compared, homologous regions were "boxed" by the researchers. The boxing in of sequences sheds light on the origin of the term "box".
The TATA box was first identified in 1978 as a component of eukaryotic promoters. Transcription is initiated at the TATA box in TATA-containing genes. The TATA box is the binding site of the TATA-binding protein (TBP) and other transcription factors in some eukaryotic genes. Gene transcription by RNA polymerase II depends on the regulation of the core promoter by long-range regulatory elements such as enhancers and silencers. Without proper regulation of transcription, eukaryotic organisms would not be able to properly respond to their environment.
Based on the sequence and mechanism of TATA box initiation, mutations such as insertions, deletions, and point mutations to this consensus sequence can result in phenotypic changes. These phenotypic changes can then turn into a disease phenotype. Some diseases associated with mutations in the TATA box include gastric cancer, spinocerebellar ataxia, Huntington's disease, blindness, β-thalassemia, immunosuppression, Gilbert's syndrome, and HIV-1. The TATA-binding protein (TBP) could also be targeted by viruses as a means of viral transcription.
History.
Discovery.
The TATA box was the first eukaryotic core promoter motif to be identified in 1978 by American biochemist David Hogness while he and his graduate student, Michael Goldberg were on sabbatical at the University of Basel in Switzerland. They first discovered the TATA sequence while analyzing 5' DNA promoter sequences in "Drosophila," mammalian, and viral genes. The TATA box was found in protein coding genes transcribed by RNA polymerase II.
Evolutionary history.
Most research on the TATA box has been conducted on yeast, human, and "Drosophila" genomes, however, similar elements have been found in archaea and ancient eukaryotes. In archaea species, the promoter contains an 8 bp AT-rich sequence located ~24 bp upstream of the transcription start site. This sequence was originally called Box A, which is now known to be the sequence that interacts with the homologue of the archaeal TATA-binding protein (TBP). Also, even though some studies have uncovered several similarities, there are others that have detected notable differences between archaeal and eukaryotic TBP. The archaea protein exhibits a greater symmetry in its primary sequence and in the distribution of electrostatic charge, which is important because the higher symmetry lowers the protein's ability to bind the TATA box in a polar manner.
Even though the TATA box is present in many eukaryotic promoters, is important to note that is not contained in the majority of promoters. One study found less than 30% of 1031 potential promoter regions contain a putative TATA box motif in humans. In "Drosophila," less than 40% of 205 core promoters contain a TATA box. When there is an absence of the TATA box and TBP is not present, the downstream promoter element (DPE) in cooperation with the initiator element (Inr) bind to the transcription factor II D (TFIID), initiating transcription in TATA-less promoters. The DPE has been identified in three "Drosophila" TATA-less promoters and in the TATA-less human IRF-1 promoter.
Features.
Location.
Promoter sequences vary between bacteria and eukaryotes. In eukaryotes, the TATA box is located 25 base pairs upstream of the start site that Rpb4/Rbp7 use to initiate transcription. In metazoans, the TATA box is located 30 base pairs upstream of the transcription start site. While in yeast, "S. cerevisiae", the TATA box has a variable position which can range from 40 to 100 bp upstream of the start site. The TATA box is also found in 40% of the core promoters of genes that code for the actin cytoskeleton and contractile apparatus in cells.
The type of core promoter affects the level of transcription and expression of a gene. TATA-binding protein (TBP) can be recruited in two ways, by SAGA, a cofactor for RNA polymerase II, or by TFIID. When promoters use the SAGA/TATA box complex to recruit RNA polymerase II, they are more highly regulated and display higher expression levels than promoters using the TFIID/TBP mode of recruitment.
Analogous sequences.
In bacteria, promoter regions may contain a Pribnow box, which serves an analogous purpose to the eukaryotic TATA box. The Pribnow box has a 6 bp region centered around the -10 position and an 8-12 bp sequence around the -35 region that are both conserved.
A CAAT box (also CAT box) is a region of nucleotides with the following consensus sequence: 5’ GGCCAATCT 3’. The CAAT box is located about 75-80 bases upstream of the transcription initiation site and about 150 bases upstream of the TATA box. It binds transcription factors (CAAT TF or CTFs) and thereby stabilizes the nearby preinitiation complex for easier binding of RNA polymerases. CAAT boxes are rarely found in genes that express proteins ubiquitous in all cell types.
Structure.
Sequence and prevalence.
The TATA box is a component of the eukaryotic core promoter and generally contains the consensus sequence 5'-TATA(A/T)A(A/T)-3'. In yeast, for example, one study found that various "Saccharomyces" genomes had the consensus sequence 5'-TATA(A/T)A(A/T)(A/G)-3', yet only about 20% of yeast genes even contained the TATA sequence. Similarly, in humans only 24% of genes have promoter regions containing the TATA box. Genes containing the TATA-box tend to be involved in stress-responses and certain types of metabolism and are more highly regulated when compared to TATA-less genes. Generally, TATA-containing genes are not involved in essential cellular functions such as cell growth, DNA replication, transcription, and translation because of their highly regulated nature.
The TATA box is usually located 25-35 base pairs upstream of the transcription start site. Genes containing the TATA box usually require additional promoter elements, including an initiator site located just upstream of the transcription start site and a downstream core element (DCE). These additional promoter regions work in conjunction with the TATA box to regulate initiation of transcription in eukaryotes.
Function.
Role in transcription initiation.
The TATA-box is the site of preinitiation complex formation, which is the first step in transcription initiation in eukaryotes. Formation of the preinitiation complex begins when the multi-subunit transcription factor II D (TFIID) binds to the TATA box at its TATA-binding protein (TBP) subunit. TBP binds to the minor groove of the TATA box via a region of antiparallel β sheets in the protein. Three types of molecular interactions contribute to TBP binding to the TATA box:
Additionally, binding of TBP is facilitated by stabilizing interactions with DNA flanking the TATA box, which consists of G-C rich sequences. These secondary interactions induce bending of the DNA and helical unwinding. The degree of DNA bending is species and sequence dependent. For example, one study used the adenovirus TATA promoter sequence (5'-CGCTATAAAAGGGC-3') as a model binding sequence and found that human TBP binding to the TATA box induced a 97° bend toward the major groove while the yeast TBP protein only induced an 82° bend. X-ray crystallography studies of TBP/TATA-box complexes generally agree that the DNA goes through an ~80° bend during the process of TBP-binding.
The conformational changes induced by TBP binding to the TATA box allows for additional transcription factors and RNA polymerase II to bind to the promoter region. TFIID first binds to the TATA box, facilitated by TFIIA binding to the upstream part of the TFIID complex. TFIIB then binds to the TFIID-TFIIA-DNA complex through interactions both upstream and downstream of the TATA box. RNA polymerase II is then recruited to this multi-protein complex with the help of TFIIF. Additional transcription factors then bind, first TFIIE and then TFIIH. This completes the assembly of the preinitiation complex for eukaryotic transcription. Generally, the TATA box is found at RNA polymerase II promoter regions, although some "in vitro" studies have demonstrated that RNA polymerase III can recognize TATA sequences.
This cluster of RNA polymerase II and various transcription factors is known as the basal transcriptional complex (BTC). In this state, it only gives a low level of transcription. Other factors must stimulate the BTC to increase transcription levels. One such example of a BTC stimulating region of DNA is the CAAT box. Additional factors, including the Mediator complex, transcriptional regulatory proteins, and nucleosome-modifying enzymes also enhance transcription "in vivo".
Interactions.
In specific cell types or on specific promoters TBP can be replaced by one of several TBP-related factors (TRF1 in Drosophila, TBPL1/TRF2 in metazoans, TBPL2/TRF3 in vertebrates), some of which interact with the TATA box similar to TBP. Interaction of TATA boxes with a variety of activators or repressors can influence the transcription of genes in many ways. Enhancers are long-range regulatory elements that increase promoter activity while silencers repress promoter activity.
Mutations.
Mutations to the TATA box can range from a deletion or insertion to a point mutation with varying effects based on the gene that has been mutated. The mutations change the binding of the TATA-binding protein (TBP) for transcription initiation. Thus, there is a resulting change in phenotype based on the gene that is not being expressed (Figure 3).
Insertions or deletions.
One of the first studies of TATA box mutations looked at a sequence of DNA from "Agrobacterium tumefaciens" for the octopine type cytokinin gene. This specific gene has three TATA boxes. A phenotype change was only observed when all three TATA boxes were deleted. An insertion of extra base pairs between the last TATA box and the transcription start site resulted in a shift in the start site; thus, resulting in a phenotypic change.  From this original mutation study, a change in transcription can be seen when there is no TATA box to promote transcription, but transcription of a gene will occur when there is an insertion to the sequence. The nature of the resulting phenotype may be affected due to the insertion.
Mutations in maize promoters affect the expression of the promoter genes in a plant-organ-specific manner. A duplication of the TATA box leads to a significant decrease in enzymatic activity in the scutellum and roots, leaving pollen enzymatic levels unaffected. A deletion of the TATA box leads to a small decrease in enzymatic activity in the scutellum and roots, but a large decrease in enzymatic levels in pollen.
Point mutations.
Point mutations to the TATA box have similar varying phenotypic changes depending on the gene that is being affected. Studies also show that the placement of the mutation in the TATA box sequence hinders the binding of TBP. For example, a mutation from TATAAAA to CATAAAA does completely hinder the binding sufficiently to change transcription, the neighboring sequences can affect if there is a change or not. However, a change can be seen in HeLa cells with a TATAAAA to TATACAA which leads to a 20 fold decrease in transcription. Some diseases that can be caused due to this insufficiency by specific gene transcription are:  Thalassemia, lung cancer, chronic hemolytic anemia, immunosuppression, hemophilia B Leyden, and thrombophlebitis and myocardial infarction.
Savinkova et al. has written a simulation to predict the "KD" value for a selected TATA box sequence and TBP. This can be used to directly predict the phenotypic traits resulting from a selected mutation based on how tightly TBP is binding to the TATA box.
Diseases.
Mutations in the TATA box region affects the binding of the TATA-binding protein (TBP) for transcription initiation, which may cause carriers to have a disease phenotype.
Gastric cancer is correlated with TATA box polymorphism. The TATA box has a binding site for the transcription factor of the PG2 gene. This gene produces PG2 serum, which is used as a biomarker for tumours in gastric cancer. Longer TATA box sequences correlates with higher levels of PG2 serum indicating gastric cancer conditions. Carriers with shorter TATA box sequences may produce lower levels of PG2 serum.
Several neurodegenerative disorders are associated TATA box mutations. Two disorders have been highlighted, spinocerebellar ataxia and Huntington's disease. In spinocerebellar ataxia, the disease phenotype is caused by expansion of the polyglutamine repeat in the TATA-binding protein (TBP). An accumulation of these polyglutamine-TBP cells will occur, as shown by protein aggregates in brain sections of patients, resulting in a loss of neuronal cells.
Blindness can be caused by excessive cataract formation when the TATA box is targeted by microRNAs to increase the level of oxidative stress genes. MicroRNAs can target the 3'-untranslated region and bind to the TATA box to activate the transcription of oxidative stress related genes.
SNPs in TATA boxes are associated with B-thalassemia, immunosuppression, and other neurological disorders. SNPs destabilize the TBP/TATA complex which significantly decreases the rate at which TATA-binding proteins (TBP) will bind to the TATA box. This leads to lower levels of transcription affecting the severity of the disease. Results from studies have shown the interaction in vitro so far, but results may be comparable to that in vivo.
Gilbert's syndrome is correlated with UTG1A1 TATA box polymorphism. This poses a risk for developing jaundice in newborns.
MicroRNAs also play a role in replicating viruses such as HIV-1. Novel HIV-1-encoded microRNA have been found to enhance the production of the virus as well as activating HIV-1 latency by targeting the TATA box region.
Clinical significance.
Technology.
Many of the studies so far have been performed in vitro, providing only a prediction of what may happen not a real-time representation of what is happening in the cells. Recent studies in 2016 have been done to demonstrate TATA-binding activity in vivo. Core promoter-specific mechanisms for transcription initiation by the canonical TBP/TFIID-dependent basal transcription machinery has recently been documented in vivo showing the activation by SRF-dependent upstream activating sequence (UAS) of the human ACTB gene involved in TATA-binding.
Cancer therapy.
Pharmaceutical companies have been designing cancer therapy drugs to target DNA in traditional methods over the years, and have proven to be successful. However, the toxicity of these drugs have pushed scientists to explore other processes related to DNA that could be targeted instead. In recent years, a collective effort has been made to find cancer-specific molecular targets, such as protein-DNA complexes, which include the TATA binding motif. Compounds that trap the protein-DNA intermediate could result in it being toxic to the cell once they encounter a DNA processing event. Example of drugs that contain such compounds include topotecan, SN-38 (topoisomerase I), doxorubicin, and mitoxantrone (topoisomerase II). Cisplatin is a compound that binds covalently to adjacent guanines in the major groove of DNA, which distorts DNA to allow access of DNA-binding proteins in the minor groove. This will destabilize the interaction between the TATA-binding protein (TBP) to the TATA box. The end result is to immobilize the TATA-binding protein (TBP) on DNA in order to down-regulate transcription initiation.
Genetic engineering.
TATA box modification.
Evolutionary changes have pushed plants to adapt to the changing environmental conditions. In the history of Earth, the development of Earth's aerobic atmosphere resulted in an iron deficiency in plants. Compared to other members of the same species, "Malus baccata" var. "xiaojinensis" has a TATA box inserted in the promoter upstream of the iron-regulated transporter 1 (IRT1) promoter. As a result, the promoter activity levels are enhanced, increasing TFIID activity and subsequently transcription initiation, resulting in a more iron-efficient phenotype.


Cholesterol 7 alpha-hydroxylase

Cholesterol 7 alpha-hydroxylase also known as cholesterol 7-alpha-monooxygenase or cytochrome P450 7A1 (CYP7A1) is an enzyme that in humans is encoded by the "CYP7A1" gene which has an important role in cholesterol metabolism. It is a cytochrome P450 enzyme, which belongs to the oxidoreductase class, and converts cholesterol to 7-alpha-hydroxycholesterol, the first and rate limiting step in bile acid synthesis.
The inhibition of cholesterol 7-alpha-hydroxylase (CYP7A1) represses bile acid biosynthesis.
Evolution.
Sequence comparisons indicated a huge similarity between cytochromes P450 identified in man and bacteria, and suggested that the superfamily cytochrome P450 first originated from a common ancestral gene some three billion years ago.
The superfamily cytochrome P450 was named in 1961, because of the 450-nm spectral peak pigment that cytochrome P450 has when reduced and bound to carbon monoxide. In the early 1960s, P450 was thought to be one enzyme, and by the mid 1960s it was associated with drug and steroid metabolism.
However, the membrane-associated and hydrophobic nature of the enzyme system impeded purification, and the number of proteins involved could not be accurately counted. Advances in mRNA purification in the early 1980s allowed to isolate the first cDNA encoding a complete cytochrome P450 (CYP) protein, and thereafter, results of many cloning studies have revealed a large number of different enzymes.
Advances in molecular biology and genomics facilitated the biochemical characterisation of individual P450 enzymes:
Molecular structure.
Cholesterol 7 alpha hydroxylase consists of 491 amino acids, which on folding forms 23 alpha helices and 26 beta sheets.
Function.
Cholesterol 7 alpha-hydroxylase is a cytochrome P450 heme enzyme that oxidizes cholesterol in the position 7 using molecular oxygen. It is an oxidoreductase. CYP7A1 is located in the endoplasmic reticulum (ER) and is important for the synthesis of bile acid and the regulation of cholesterol levels.
Synthesis of bile acid.
Cholesterol 7 alpha-hydroxylase is the rate-limiting enzyme in the synthesis of bile acid from cholesterol via the classic pathway, catalyzing the formation of 7α-hydroxycholesterol. The unique detergent properties of bile acids are essential for the digestion and intestinal absorption of hydrophobic nutrients.
Bile acids have powerful toxic properties like membrane disruption and there are a wide range of mechanisms to restrict their accumulation in tissues and blood. The discovery of farnesoid X receptor (FXR) which is located in the liver, has opened new insights. Bile acid activation of FXR represses the expression of CYP7A1 via, raising the expression of small heterodimer (SHP), a non-DNA binding protein.
The increased abundance of SHP causes it to associate with liver receptor homolog (LRH)-1, an obligate factor required for the transcription of CYP7A1. Furthermore, there is an "FXR/SHP-independent" mechanism that also represses CYP7A1 expression. This "FXR/SHP-independent" pathway involves the interaction of bile acids with liver macrophages, which finally induces the expression and secretion of cytokines. These inflammatory cytokines, which include tumor necrosis factor alpha and interleukin-1beta, act upon the liver parenchymal cells causing a rapid repression of the CYP7A1 gene.
Regulation of activity.
Regulation of CYP7A1 occurs at several levels including synthesis. Bile acids, steroid hormones, inflammatory cytokines, insulin, and growth factors inhibit CYP7A1 transcription through the 5′-upstream region of the promoter. The average life of this enzyme is between two and three hours. Activity can be regulated by phosphorylation-dephosphorylation.
CYP7A1 is upregulated by the nuclear receptor LXR (liver X receptor) when cholesterol (to be specific, oxysterol) levels are high. The effect of this upregulation is to increase the production of bile acids and reduce the level of cholesterol in hepatocytes.
It is downregulated by sterol regulatory element-binding proteins (SREBP) when plasma cholesterol levels are low.
Bile acids provide feedback inhibition of CYP7A1 by at least two different pathways, both involving the farnesoid X receptor, FXR. In the liver, bile acids bound to FXR induce small heterodimer partner, SHP which binds to LRH-1 and so inhibits the transcription of the enzyme. In the intestine, bile acids/FXR stimulate production of FGF15/19 (depending on species), which then acts as a hormone in the liver via FGFR4.
Enzymatic mechanism.
Specificity.
One feature of enzymes is their high specificity. They are specific on a singular substrate, reaction or both together, that means, that the enzymes can catalyze all reactions wherein the substrate can experience.
The enzyme cholesterol 7 alpha hydroxylase catalyzes the reaction that converts cholesterol into cholesterol 7 alpha hydroxylase reducing and oxidizing that molecule.
Clinical significance.
Deficiency of this enzyme will increase the possibility of cholesterol gallstones.
Disruption of CYP7A1 from classic bile acid synthesis in mice leads to either increased postnatal death or a milder phenotype with elevated serum cholesterol. The latter is similar to the case in humans, where CYP7A1 mutations associate with high plasma low-density lipoprotein and hepatic cholesterol content, as well as deficient bile acid excretion. There is also a synergy between plasma low-density lipoprotein cholesterol (LDL-C) and risks of coronary artery disease (CAD). Glucose signaling also induces CYP7A1 gene transcription by epigenetic regulation of the histone acetylation status. Glucose induction of bile acid synthesis have an important implication in metabolic control of glucose, lipid, and energy homeostasis under normal and diabetic conditions. CYP7A1-rs3808607 and APOE isoform are associated with the extent of reduction in circulating LDL cholesterol in response to plant sterol consumption and could serve as potential predictive genetic markers to identify individuals who would derive maximum LDL cholesterol lowering with plant sterol consumption. Genetic variations in CYP7A1 influence its expression and thus may affect the risk of gallstone disease and gallbladder cancer.
One of the many lipid lowering effects of the fibrate drug class is mediated through the inhibition of transcription of this enzyme. This inhibition leads to more cholesterol in the bile, which is the body's only route of cholesterol excretion. This also increases the risk of cholesterol gallstone formation.
Inhibition of CYP7A1 is thought to be involved in or responsible for the hepatotoxicity associated with ketoconazole. The levorotatory enantiomer of ketoconazole, levoketoconazole, shows 12-fold reduced potency in inhibition of this enzyme, and is under development for certain indications (e.g., Cushing's syndrome) as a replacement for ketoconazole with reduced toxicity and improved tolerability and safety.


Chlorobium chlorochromatii

Chlorobium chlorochromatii, originally known as "Chlorobium aggregatum", is a symbiotic green sulfur bacteria that performs anoxygenic photosynthesis and functions as an obligate photoautotroph using reduced sulfur species as electron donors. "Chlorobium chlorochromatii" can be found in stratified freshwater lakes.
Topic morphology.
"C. chlorochromatii" is a Gram-negative, non-motile bacillus, that exist in short chains. They are green in color and have a ring of chlorosomes around that line the inside of their cell wall. Within these chlorosomes contain the light harvesting pigment bacteriochlorophyll a and bacteriochlorophyll c which feed electrons into Photosystem 1.
Ecology.
Photosynthetic green sulfur bacteria such as "Chlorobium chlorochromatii" reside in freshwater, stratified lakes beneath the micro-aerophilic algal layer in anaerobic, light-exposed environments. They have been found worldwide, mostly in holomictic or meromictic stratified lakes. Lakes that support this environment have been found in Germany, Tasmania, the USA, ice-covered lakes in Antarctica, Israel and Japan. "Chlorobium chlorochromatii" prefer environments with low temperature and low sulfur concentrations.
Symbiosis.
"Chlorobium chlorochromatii", strain CaD, was originally isolated from the phototrophic microbial consortium "Chlorochromatium aggregatum". The ability of this epibiont to grow in pure culture indicates that it is not an obligately symbiotic organism. Despite this fact, "C. chlorochromatii" has never been found in a free-living state in naturally occurring bacterial communities.
Metabolism.
"C. chlorochromatii" conducts anoxygenic photosynthesis which means it does not produce oxygen as a waste product like plants and cyanobacteria, this type of photosynthesis is exclusive to Bacteria. In their electron transport chain reduced forms of sulfur, e.g., H2S. These reduced forms of sulfur are used in the electron transport chain cyclic Photosystem 1 as electron donors to reduce NADP+ to NADPH. It grows under strictly anaerobic conditions as a photolithoautotroph. They grow best at a pH of 7.0-7.3 at 25 C in continuous light and absorb light maximally at 748 and 453 nm.
Genome structure.
"C. chlorochromatii" contains a circular genome that contains 2,572,079 bp. There are a total of 2047 genes contained on its chromosome. Of these 2047 genes, there are 1999 protein coding genes and 48 RNA coding genes. There are no pseudogenes contained within the chromosome of "C. chlorochromatii". Data from DNA analysis suggests that genomes of green sulfur bacteria range between 2-3.3 Mb. From these data, it can be assumed that the total genome size of the consortium of "C. aggregatum" is less than 10 Mb in length.


AU-rich element

Adenylate-uridylate-rich elements (AU-rich elements; AREs) are found in the 3' untranslated region (UTR) of many messenger RNAs (mRNAs) that code for proto-oncogenes, nuclear transcription factors, and cytokines. AREs are one of the most common determinants of RNA stability in mammalian cells.
AREs are defined as a region with frequent adenine and uridine bases in a mRNA. They usually target the mRNA for rapid degradation.
ARE-directed mRNA degradation is influenced by many exogenous factors, including phorbol esters, calcium ionophores, cytokines, and transcription inhibitors. These observations suggest that AREs play a critical role in the regulation of gene transcription during cell growth and differentiation, and the immune response.
AREs have been divided into three classes with different sequences. The best characterised adenylate uridylate (AU)-rich Elements have a core sequence of AUUUA within U-rich sequences (for example WWWU(AUUUA)UUUW where W is A or U). This lies within a 50–150 base sequence, repeats of the core AUUUA element are often required for function.
A number of different proteins (e.g. HuA, HuB, HuC, HuD, HuR) bind to these elements and stabilise the mRNA while others (AUF1, TTP, BRF1, TIA-1, TIAR, and KSRP) destabilise the mRNA, miRNAs may also bind to some of them. HuD (also called ELAVL4) binds to AREs and increases the half-life of ARE-bearing mRNAs in neurons during brain development and plasticity.
AREsite—a database for ARE containing genes—has recently been developed with the aim to provide detailed bioinformatic characterization of AU-rich elements.
Classifications.
No real ARE consensus sequence has been determined yet, and these categories are based neither on the same biological functions, nor on the homologous proteins.
Mechanism of ARE-mediated decay.
AREs are recognized by RNA binding proteins such as tristetraprolin (TTP), AUF1, and Hu Antigen R (HuR). Although the exact mechanism is not very well understood, recent publications have attempted to propose the action of some of these proteins. AUF1, also known as hnRNP D, binds AREs through RNA recognition motifs (RRMs). AUF1 is also known to interact with the translation initiation factor eIF4G and with poly(A)-binding protein, indicating that AUF1 senses the translational status of mRNA and decays accordingly through the excision of the poly(A) tail.
TTP's expression is rapidly induced by insulin. Immunoprecipitation experiments have shown that TTP co-precipitates with an exosome, suggesting that it helps recruit exosomes to the mRNA containing AREs. Alternatively, HuR proteins have a stabilizing effect—their binding to AREs increases the half-life of mRNAs. Similar to other RNA-binding proteins, this class of proteins contain three RRMs, two of which are specific to ARE elements. A likely mechanism for HuR action relies on the idea that these proteins compete with other proteins that normally have a destabilizing effect on mRNAs. HuRs are involved in genotoxic response—they accumulate in the cytoplasm in response to UV exposure and stabilize mRNAs that encode proteins involved in DNA repair.
Disease.
Problems with mRNA stability have been identified in viral genomes, cancer cells, and various diseases. Research shows that many of these problems arise because of faulty ARE function. Some of these problems have been listed below:


Protein measurement with the Folin phenol reagent


Photoprotein

Photoproteins are a type of enzyme, made of protein, from bioluminescent organisms. They add to the function of the luciferins whose usual light-producing reaction is catalyzed by the enzyme luciferase.
History.
The term photoprotein was first used to describe the unusual chemistry of the luminescent system of "Chaetopterus" (a marine Polychaete worm). This was meant to distinguish them from other light-producing proteins because these do not exhibit the usual luciferin-luciferase reaction.
Reaction kinetics.
Photoproteins do not display typical enzyme kinetics as seen in luciferases. Instead, when mixed with luciferin, they display luminescence proportional to the amount of the photoprotein. For example, the photoprotein aequorin produces a flash of light when luciferin and calcium are added, rather than the prolonged glow that is seen for luciferases when luciferin is added. In this respect, it may appear that photoproteins are not enzymes, when in fact they do catalyze their bioluminescence reactions. This is due to a fast catalytic step, which produces the light, and a slow regeneration step, where the oxyluciferin is freed and another molecule of luciferin is then enabled to bind to the enzyme. Because of the kinetically slow step, each aequorin molecule must "recharge" with another molecule of luciferin before it can emit light again, and this makes it appear as though it is not behaving as a typical enzyme.
Photoproteins form a stable luciferin-photoprotein complex, often until the addition of another required factor such as Ca2+ in the case of aequorin.


Southwestern blot

The southwestern blot, is a lab technique that involves identifying as well as characterizing DNA-binding proteins by their ability to bind to specific oligonucleotide probes. Determination of molecular weight of proteins binding to DNA is also made possible by the technique. The name originates from a combination of ideas underlying Southern blotting and Western blotting techniques of which they detect DNA and protein respectively. Similar to other types of blotting, proteins are separated by SDS-PAGE and are subsequently transferred to nitrocellulose membranes. Thereafter southwestern blotting begins to vary with regards to procedure as since the first blotting’s, many more have been proposed and discovered with goals of enhancing results. Former protocols were hampered by the need for large amounts of proteins and their susceptibility to degradation while being isolated.
Southwestern blotting was first described by Brian Bowen, Jay Steinberg, U.K. Laemmli, and Harold Weintraub in 1979. During the time the technique was originally called "protein blotting". While there were existing techniques for purification of proteins associated with DNA, they often had to be used together to yield desired results. Thus, Bowen and colleagues sought to describe a procedure that could simplify the current methods of their time.
Method.
Original Method.
To begin, proteins of interest are prepared for the SDS-PAGE technique and subsequently loaded onto the gel for separation on the basis of molecular size. Large proteins will have difficulty navigating through the mesh-like structure of the gel as they can not fit through the pores with the ease that smaller proteins can. As a result, large proteins do not travel very far on the gel in comparison to smaller proteins that travel further. After enough time, this results in distinct bands that can be visualized from a number of post-gel electrophoresis staining procedures. The bands are at different positions on the gel relative to the well that they were loaded into.
Next, proteins are to be renatured followed by the gel being subjected to pressed between two nitrocellulose filters which rely on diffusion to transfer the proteins from the gel to the membrane filters. At this point replicas of the gel have been created of which each serves a particular purpose. One membrane filter can be stained to see the protein bands that were created from gel electrophoresis and the other is used in the actual process of hybridizing with prepared 32P radioactively labeled specific oligonucleotide probes. To detect any protein-DNA interactions, autoradiography is commonly used.
Southwestern Blot Mapping.
"Southwestern blot mapping" is a time-efficient way of identifying DNA-binding proteins and specific sites on the genomic DNA that they interact with.
Results.
After time is allowed for binding with the oligonucleotide probes, the hope is that some of the proteins on the membrane filter have bound to the probes. Any probe that was not able to bind a protein needs to be removed. Once unbound probe removal has been taken care of, to better visualize the membrane filter, it is subjected to further varying procedures. By corresponding the resulting membrane filter to the second membrane filter that the gel was sandwiched between, the position of the protein in comparison to the molecular weight ladder gives information about the weight of the protein that bound to the probe.


Ctgf/hcs24 CAESAR

ctgf/hcs24 CAESAR is the name given to the cis-acting RNA element identified in the 3' untranslated region (3'UTR) of the human connective tissue growth factor (CTGF) messenger RNA. This gene is also known as hypertrophic chondrocyte specific 24 (hcs24).
The importance of the 3'UTR in repressing ctgf gene expression was initially characacterised and subsequently the minimal RNA element responsible for repression was identified This element was predicted to form a stable secondary structure, which acts as a post-transcriptional cis-acting element of structure-anchored repression (CAESAR).
The 3'UTR of the ctgf/hcs24 gene in chicken has also been shown to be involved in repression of gene expression.


Sulochrin oxidase ((-)-bisdechlorogeodin-forming)

In enzymology, a sulochrin oxidase [(−)-bisdechlorogeodin-forming] () is an enzyme that catalyzes the chemical reaction
Thus, the two substrates of this enzyme are sulochrin and O2, whereas its two products are (−)-bisdechlorogeodin and H2O.
This enzyme belongs to the family of oxidoreductases, specifically those acting on X-H and Y-H to form an X-Y bond with oxygen as acceptor. The systematic name of this enzyme class is sulochrin:oxygen oxidoreductase (cyclizing, (−)-specific). This enzyme is also called sulochrin oxidase.


Chlorophyll d

Chlorophyll "d" is a form of chlorophyll, identified by Harold Strain and Winston Manning in 1943. It is present in cyanobacteria which use energy captured from sunlight for photosynthesis. Chlorophyll "d" absorbs far-red light, at 710 nm wavelength, just outside the optical range. An organism that contains chlorophyll "d" is adapted to an environment such as moderately deep water, where it can use far red light for photosynthesis, although there is not a lot of visible light.


Blood agent

A blood agent is a toxic chemical agent that affects the body by being absorbed into the blood. Blood agents are fast-acting, potentially lethal poisons that typically manifest at room temperature as volatile colorless gases with a faint odor. They are either cyanide- or arsenic-based.
Exposure.
Blood agents work through inhalation or ingestion. As chemical weapons, blood agents are typically disseminated as aerosols and take effect through inhalation. Due to their volatility, they are more toxic in confined areas than in open areas.
Cyanide compounds occur in small amounts in the natural environment and in cigarette smoke. They are also used in several industrial processes and as pesticides. Cyanides are released when synthetic fabrics or polyurethane burn, and may thus contribute to fire-related deaths. Arsine gas, formed when arsenic encounters an acid, is used as a pesticide and in the semiconductor industry; most exposures to it occur accidentally in the workplace.
Symptoms.
The symptoms of blood agent poisoning depend on concentration and duration.
Cyanide-based blood agents irritate the eyes and the respiratory tract, while arsine is nonirritating. Hydrogen cyanide has a faint, bitter, almond odor that only about half of all people can smell. Arsine has a very faint garlic odor detectable only at greater than fatal concentrations.
Exposure to small amounts of cyanide has no effect. Higher concentrations cause dizziness, weakness and nausea, which cease with the exposure, but long-time exposure can cause mild symptoms followed by permanent brain damage and muscle paralysis. Moderate exposure causes stronger and longer-lasting symptoms, including headache, that can be followed by convulsions and coma. Stronger or longer exposure will also lead to convulsions and coma. Very strong exposure causes severe toxic effects within seconds, and rapid death.
The blood of people killed by blood agents is bright red, because the agents inhibit the use of the oxygen in it by the body's cells. Cyanide poisoning can be detected by the presence of thiocyanate or cyanide in the blood, a smell of bitter almonds, or respiratory tract inflammations and congestions in the case of cyanogen chloride poisoning. There is no specific test for arsine poisoning, but it may leave a garlic smell on the victim's breath.
Effects.
At sufficient concentrations, blood agents can quickly saturate the blood and cause death in a matter of minutes or seconds. They cause powerful gasping for breath, violent convulsions and a painful death that can take several minutes. The immediate cause of death is usually respiratory failure.
Blood agents work at the cellular level by preventing the exchange of oxygen and carbon dioxide between the blood and the body's cells. This causes the cells to suffocate from lack of oxygen. Cyanide-based agents do so by interrupting the electron transport chain in the inner membranes of mitochondria. Arsine damages the red blood cells which deliver oxygen throughout the body.
Detection and countermeasures.
Chemical detection methods, in the form of kits or testing strips, exist for hydrogen cyanide. Ordinary clothing provides some protection, but proper protective clothing and masks are recommended. Mask filters containing only charcoal are ineffective, and effective filters are quickly saturated.
Due to their high volatility, cyanide agents generally need no decontamination. In enclosed areas, fire extinguishers spraying sodium carbonate can decontaminate hydrogen cyanide, but the resulting metal salts remain poisonous on contact. Liquid hydrogen cyanide can be flushed with water.
Cyanide poisoning can be treated with antidotes.
List of blood agents.
The information in the following table, which lists blood agents of military significance, is taken from Ledgard. The values given are on a scale from 1 to 10.
Sodium cyanide and potassium cyanide, colorless crystalline compounds similar in appearance to sugar, also act as blood agents. Carbon monoxide could technically be called a blood agent because it binds with oxygen-carrying hemoglobin in the blood (see carbon monoxide poisoning), but its high volatility makes it impractical as a chemical warfare agent.
One of the earliest proposed chemical weapons, cacodyl oxide, or Cadet's fuming liquid, also displays properties of a blood agent (as well as those of a malodorant). It was proposed as a chemical weapon in the British Empire during the Crimean War, along with the significantly more potent blood agent, cacodyl cyanide.
Use.
The most significant practical application of blood agents was the use of hydrogen cyanide (Zyklon B) in gas chambers by Nazi Germany to commit the mass murder of Jews and others in the course of the Holocaust. This resulted in the largest death toll as a result of the use of chemical agents to date.


Sphingobacterium psychroaquaticum

Sphingobacterium psychroaquaticum is a Gram-negative and psychrophilic bacterium from the genus of Sphingobacterium which has been isolated from lake water from the Lake Michigan in Wisconsin in the United States.