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LTA4H

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Dorland's Illustrated Medical Dictionary :
chemotactic factor,   a substance that induces chemotaxis. Called also chemoattractant and chemotaxin
 

chemotactic (che·mo·tac·tic) (ke²mo-tak¢tik)  of or pertaining to chemotaxis.

chemotaxin (che·mo·tax·in) (ke²mo-tak¢sin)  chemotactic factor.

chemotaxis (che·mo·tax·is) (ke²mo-tak¢sis) [chemo- + -taxis]  orientation of a cell along a chemical concentration gradient or movement in the direction of the gradient, either toward (positive chemotaxis) or away from (negative chemotaxis) the greater concentration of the substance, referred to as a chemotactic factor, chemotactin, or chemoattractant. Macrophages, neutrophils, eosinophils, and lymphocytes exhibit chemotaxis in response to a wide variety of substances released at sites of inflammatory reactions, including lymphokines, mediators released by basophils and mast cells, bacterial products, and C5a and other activated complement components. Cf. chemokinesis.

chemokinesis (che·mo·ki·ne·sis) (ke²mo-k[ibreve]-ne¢sis) [chemo- + -kinesis]  increased nondirectional activity of cells due to presence of a chemical substance. Cf. chemotaxis.

chemokinetic (che·mo·ki·net·ic) (ke²mo-k[ibreve]-net¢ik)  pertaining to or exhibiting chemokinesis.

chemotaxis: http://cancerweb.ncl.ac.uk/cgi-bin/omd?chemotactic

A response of motile cells or organisms in which the direction of movement is affected by the gradient of a diffusible substance. Differs from chemokinesis in that the gradient alters probability of motion in one direction only, rather than rate or frequency of random motion

Cells of the Immune System
Drawing of cells of the immune system

Cytokines

http://press2.nci.nih.gov/sciencebehind/immune/immune15.htm

Cytokines are diverse and potent chemical messengers secreted by the cells of the immune system—and the chief tool of T cells.

Lymphocytes, including both T cells and B cells, secrete lymphokines, while monocytes and macrophages secrete monokines.

Binding to specific receptors on target cells, cytokines recruit many other cells and substances to the field of action. Cytokines encourage cell growth, promote cell activation, direct cellular traffic, and destroy target cells—including cancer cells. Because they serve as a messenger between white cells, or leukocytes, many cytokines are also known as interleukins

At least two types of lymphocytes are killer cells—cytotoxic T cells and natural killer cells.

To attack, cytotoxic T cells need to recognize a specific antigen, whereas natural killer or NK cells do not. Both types contain granules filled with potent chemicals, and both types kill on contact. The killer binds to its target, aims its weapons, and delivers a burst of lethal chemicals.

Phagocytes and Granulocytes

Phagocytes are large white cells that can engulf and digest foreign invaders.

They include monocytes, which circulate in the blood, and macrophages, which are found in tissues throughout the body, as well as neutrophils, cells that circulate in the blood but move into tissues where they are needed. Macrophages are versatile cells; they act as scavengers, they secrete a wide variety of powerful chemicals, and they play an essential role in activating T cells.

Neutrophils are not only phagocytes but also granulocytes: they contain granules filled with potent chemicals. These chemicals, in addition to destroying microorganisms, play a key role in acute inflammatory reactions. Other types of granulocytes are eosinophils and basophils. Mast cells are granule-containing cells in tissue.

Phagocytes in the Body :

Drawing of the human body, showing specialized phagocytes in various organs

Organs of the Immune System:

Drawing of the human body, showing the location of the organs of the immune system

http://www.hon.ch/Library/Theme/Allergy/Glossary/leukotriene.html

Leukotrienes

MeSH definition: A family of biologically active compounds derived from arachidonic acid by oxidative metabolism through the 5-lipoxygenase pathway. They participate in host defense reactions and pathophysiological conditions such as immediate hypersensitivity and inflammation. They have potent actions on many essential organs and systems, including the cardiovascular, pulmonary, and central nervous system as well as the gastrointestinal tract and the immune system.
Leukotriene B4
MeSH definition: The major metabolite in neutrophil polymorphonuclear leukocytes. It stimulates polymorphonuclear cell function (degranulation, formation of oxygen-centered free radicals, arachidonic acid release, and metabolism). (From Dictionary of Prostaglandins and Related Compounds,
 
Leukotrienes and prostaglandins are derivatives of arachidonic acid (AA) an unsaturated fatty acid produced from membrane phospholipids. The principal pathways of arachidonic acid metabolism are :
  • the 5-lipoxygenase pathway, which produces a collection of leukotrienes (LT) and
  • the cyclooxygenase pathway, which yields a number of prostaglandins (PG) and thromboxanes (Tx).
All three are synthesized by monocytes and macrophages. Mast cells and basophils generate a mixture of leukotrienes. The products of both pathways act in concert to cause inflammation with prostaglandins producing fever and pain. Aspirin, ibuprofen, and certain other nonsteroidal anti-inflammatory drugs (NSAIDs) achieve their effects (fever and pain reduction) by blocking the activity of cyclooxygenase. [Discussion

leukotriene:  http://cancerweb.ncl.ac.uk/cgi-bin/omd?leukotriene+a4

Any of a group of hormonesderived from arachidonate (arachidonic acid).They are thought to mediate the allergic response that causes lungconstriction and muscle contraction in asthma

leukotriene b4: <chemical> (s-(r*,s*-(e,z,e,z)))-5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid. The major metabolite in neutrophil polymorphonuclear leukocytes. It stimulates polymorphonuclear cell function (degranulation, formation of oxygen-centreed free radicals, arachidonic acid release, and metabolism).

Chemical name: 6,8,10,14-Eicosatetraenoic acid, 5,12-dihydroxy-, (S-(R*,S*-(E,Z,E,Z)))-

leukotriene a4 ; Its biological actions are determined primarily by its metabolites, i.e., leukotriene b4 and cysteinyl-leukotrienes. Alternatively, leukotriene a4 is converted into leukotriene c4 by glutathione-s-transferase or into 5,6-di-hete by the epoxide-hydrolase

Macrophages are involved at all stages of the immune response. First, as already outlined, they act as rapid protective mechanism which can respond before T cell-mediated amplification has taken place
Monocytes lose their myeoloperoxidase activity during conversion to tissue macrophages
However, macrophages may acquire MPO from their environment by pinocytosis or from ingested neutrophils. In this way, especially macrophages in inflammatory site with the intensive cell destruction, can gain myeloperoxidase (or other peroxidase). Such peroxidase then participates in cytotoxic mechanisms of macrophages.

Macrophages are important producers of arachidonic acid and its metabolites. Upon phagocytosis macrophages release up to 50% of their arachidonic acid from membranous esterified glycerol phospholipid. It is immediately metabolized into different types of prostanoids. From them prostaglandins, especially PGE, and prostacyclin (PGI) are characterized as pro-inflammatory agents: they induce vasodilatation, act synergeticly with complement component C5a and LTB, mediate fever and myalgia response to IL-1, in the combination with bradykinin and histamine they contribute to erythema, oedema, and pain induction. Tromboxan TXA is considered as an inflammatory mediator; it facilitates platelet aggregation and triggers vasoconstriction. LTB is the efficient chemoatractant substance. A mixture of LTC, LTD and LTE became known as slow-reacting substance of anaphylaxis (SRS-A). These leukotrienes are important mediators of bronchial asthma, since they provoke long-term contractions of bronchial smooth muscl

A. Mast Cells [5] http://www.outlinemed.com/demo/allergy/4850.htm
See outline "Hypersensitivity Reactions"
  1. Mainly found in tissues, low numbers in blood of normal persons
  2. Usually found beneath epithelial surfaces and near blood vessels
  3. Long lived cells (weeks to months)
  4. Derived from CD34+ bone marrow progenitor cells
    1. Disbursed to tissue as precursor cells, probably related to basophils
    2. Mast cell precursors fully mature in specific tissues
    3. Development requires mast-cell growth factor, the ligand for the c-kit gene
  5. Preformed granules in cytoplasm, released on activation of cells
    1. Have immunoglobulin (Ig) Fc receptors, specific primarily for IgE
    2. Responsible for a variety of IgE dependent and some independent reactions
    3. Mast cell disease usually occurs in skin; also skeleton, bone marrow, GI tract, CNS
  6. Mast Cell Mediators
    1. Histamine and metabolites
    2. Tryptase
    3. Prostaglandin D2
    4. Heparin
  7. No humans with absence of mast cells have been reported

Curr Cancer Drug Targets. 2004 May;4(3):267-83.  

Leukotriene A4 hydrolase as a target for cancer prevention and therapy.

Chen X, Wang S, Wu N, Yang CS.

Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, 164 Frelinghuysen Road, Piscataway, New Jersey 08854, USA. xiaochen@rci.rutgers.edu

Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc enzyme with the activities of epoxide hydrolase and aminopeptidase. As an epoxide hydrolase, LTA4H catalyzes the hydrolysis of the epoxide LTA4 to the diol, leukotriene B4 (LTB4), which mainly functions as a chemoattractant and an activator of inflammatory cells. As an aminopeptidase, LTA4H may process peptides related to inflammation and host defense. In a chronic inflammation-associated animal model of esophageal adenocarcinoma, we have shown that LTA4H was overexpressed in tumor as compared to normal tissues. Bestatin, an LTA4H inhibitor, suppresses tumorigenesis in this animal model. Since LTA4H has long been regarded as an anti-inflammatory target, we propose LTA4H as a target for prevention and therapy of cancers, especially those associated with chronic inflammation. Here we review the gene structure, expression, regulation and functions of LTA4H, as well as its involvement in carcinogenesis. We believe LTA4H/LTB4 may play an important role in chronic inflammation associated carcinogenesis by at least two mechanisms: a) the inflammation-augmenting effect on inflammatory cells through positive feedback mediated by its receptors and downstream signaling molecules; and b) the autocrine (Secretion of a substance, such as a growth factor, that stimulates the secretory cell itself. One route to independence of growth control is by autocrine growth factor production. ) growth-stimulatory effect of LTB4 produced by epithelial cells, and the paracrine ( Form of signalling in which the target cell is close to the signal releasing cell. Neurotransmitters and neurohormones are usually considered to fall into this category. ) growth-stimulatory effect of LTB4 produced by inflammatory cells, on precancerous and cancer cells. Based on our present knowledge, inhibitors of LTA4H or antagonists of LTB4 receptors may be used alone or in combination with other agents (e.g., cyclooxygenase 2 inhibitors) in cancer prevention and treatment trials to test their effectiveness.

Gene. 1995 Aug 19;161(2):249-51.
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Amino-acid sequence and tissue distribution of guinea-pig leukotriene A4 hydrolase.

Minami M, Mutoh H, Ohishi N, Honda Z, Bito H, Shimizu T.

Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.

The guinea-pig leukotriene A4 hydrolase (LTA4H)-encoding cDNA was isolated from a guinea-pig lung cDNA library by cross-hybridization using a human probe. The deduced amino acid (aa) sequence consists of 611 aa (68 756 Da) and contains all twelve internal peptide and N-terminal sequences determined from the purified enzyme from guinea-pig intestine. The aa identity of the guinea-pig enzyme with its human, mouse and rat counterparts was 92.9, 90.5 and 90.4%, respectively. The previously characterized zinc-binding motif and a putative active site were highly conserved, supporting the aminopeptidase activity described for this enzyme. RNA blot analysis demonstrated ubiquitous (Present everywhere. The small protein called ubiquitin was so-named because it is present in all types of cells and its amino acid sequence is identical in all creatures from insects to humans.) expression of the LTA4H mRNA.

Translocation of 5-lipoxygenase to the membrane in human leukocytes challenged with ionophore A23187

reference 25 in co-localization article/Brock

CA Rouzer and S Kargman
Merck Frosst Canada Inc., Pointe Claire-Dorval, Quebec, Canada.

Challenge of human peripheral blood leukocytes with ionophore A23187 resulted in leukotriene (LT) synthesis, a decrease in total cellular 5- lipoxygenase activity, and a change in the subcellular localization of the enzyme. In homogenates from control cells, greater than 90% of the 5-lipoxygenase activity and protein was localized in the cytosol (100,000 X g supernatant=The soluble liquid fraction of a sample after centrifugation or precipitation of insoluble solids). Ionophore challenge (2 microM) resulted in a loss of approximately 55% of the enzymatic activity and 35% of the enzyme protein from the cytosol. Concomitantly, there was an accumulation of inactive 5-lipoxygenase in the membrane (100,000 X g pellets) which accounted for at least 45% of the lost cytosolic protein. There was a good correlation between the quantities of LT synthesized and 5-lipoxygenase recovered in the membrane over an ionophore concentration range of 0.1-6 microM. The time course of the membrane association was similar to that of LT synthesis. Furthermore, although the pellet-associated enzyme recovered from ionophore-treated leukocytes was inactive, an irreversible, Ca2+-dependent membrane association of active 5-lipoxygenase could be demonstrated in cell-free systems. To determine whether ionophore treatment induced proteolytic degradation of 5-lipoxygenase, the total activity and protein content of 10,000 X g supernatants from control and ionophore-treated cells were examined. These supernatants, which included both cytosolic and membrane-associated enzyme, showed a 35% loss of 5-lipoxygenase activity but only an 8% loss of enzyme protein as a result of ionophore challenge (2 microM). Therefore, the majority of the loss of 5- lipoxygenase activity was most likely due to suicide inactivation during the LT synthesis, rather than to proteolytic degradation. Together these results are consistent with the hypothesis that ionophore treatment results in a Ca2+-dependent translocation of 5- lipoxygenase from the cytosol to a membrane-bound site, that the membrane-associated enzyme is preferentially utilized for LT synthesis, and that it is consequently inactivated. Thus, membrane translocation of 5-lipoxygenase may be an important initial step in the chain of events leading to full activation of this enzyme in the intact leukocyte.

LTA4 serves as the precursor for the potent

chemotactic factor, LTB4 as well as for the smooth muscle

contracting agonists LTC4, LTD4 and LTE4. These products

are secreted by leukocytes after exposure to various immunologic

and inflammatory stimuli, and their biological activities

suggest that they may play a role in immediate hypersensitivity

reactions and inflammation (4). Therefore, the regulation

of the 5-lipoxygenase may be of great importance to

the pathophysiology of these processes.

Calcium ionophore:

Ionophores are compounds that increase the permeability of cellular membrane barriers to ions by functioning as mobile ion carriers or channel formers. They contain hydrophobic regions conferring lipid solubility and hydrophilic ion-binding regions which delocalise the charge of the ion to shield it from the hydrophobic regions of the membrane lipid bilayer.


formula of A23187


Calcium ionophoreA23187 is an artificial mobile iron carrier that normally acts as an ion-exchange shuttle molecule transporting one divalent calcium ion into the cell in exchange of two H +. As intracellular calcium levels can be monitored by a variety of fluorescent probes use of A23187 provides information about the involvement of elevated levels of cytosolic free calcium (and indirect information about associated secondary messenger systems) following cytokine receptor mediated signal transduction processes. A postulated participation of calcium in the process under study can be confirmed by employing calcium-specific chelators such as EGTA to produce very low intracellular calcium levels which should then block the response.

A23187 can cause cell activation , differentiation, or proliferation and thus mimics cellular processes normally observed in response to cytokines . It can be used, therefore, to probe functional capacities of cells and to dissect complex processes into a series of discrete stages at the molecular level. Responses can include production and release cytokines , expression of an ERG (Early response gene ), an Oncogene , differentiation antigens (see also: CD antigens ), and intracellular adhesion molecules, cell death by apoptosis , progression through the Cell cycle , or inhibition of any of these processes.

Simultaneous treatment of cells with calcium ionophores and other agents (cytokines , drugs, hormones) can be used to investigate whether any of these agents affect (enhance or reverse) any of the elicited responses.

For other agents used to dissect signal transduction pathways mediated by cytokines see: Bryostatins , Calphostin C , Genistein , H8 , Herbimycin A , K-252a , Lavendustin A , Phorbol esters , Okadaic acid , Staurosporine , Suramin , Tyrphostins , Vanadate .

Biotinylated Antibodies
Biotinylated antigen-purified antibodies are developed for use as detection antibodies in ELISA, immunohistochemistry, and western blot applications  http://www.rndsystems.com/asp/c_biotinylated_add.asp
 
 is widely used for DNA/RNA detection/isolation due to the extremely high affinity of the biotin-streptavidin interaction (association constant 1015/M). Biotin moieties can be incorporated within an oligo on any place and in any number. We have long and super-long tethering arms covalently attached to biotin for improved binding kinetics, increased binding capacity of large DNA fragments, and for accessibility to enzymatic events occurring at the solid-phase surface.

Blocking kit reagents may be used to block nonspecific binding of Biotin/Avidin System reagents:

http://www.vectorlabs.com/Protocols/SP2001.pdf

Principle:

Some tissues may bind avidin, biotinylated horseradish peroxidase or other Biotin/Avidin System components

without prior addition of biotinylated antibody. This binding may be due to endogenous biotin or biotin-binding

proteins, lectins, or nonspecific binding substances present in the section. If a high background is present using the

ABC reagents (or other avidin conjugate) in the absence of biotinylated secondary antibody, pre-treatment of the

tissue with avidin, followed by biotin (to block the remaining biotin binding sites on the avidin), may be required.

The blocking kit consists of an Avidin D solution and a biotin solution. Pre-treatment of the section with the

Avidin D solution should always be followed by incubation with the biotin solution. The Avidin D and biotin

solutions should be used directly as supplied.

Suggested Protocol for Tissue Sections:

After incubation with normal serum, incubate section with Avidin D solution for 15 minutes. Rinse briefly

with buffer, then incubate for 15 minutes with the biotin solution. These steps should be performed prior to

the addition of primary antibody or lectin.

 

Biotin-(Strept)avidin Systems:

http://www.vectorlabs.com/products.asp?catID=28&locID=0

Anti-Avidin Reagents
Anti-Biotin Antibodies
Anti-Streptavidin Conjugates
Avidin Reagents
Avidin/Biotin Blocking Kit
Biotinylated Reagents

Biotinylating Reagents
Streptavidin Reagents
Streptavidin/Biotin Blocking Kit
VECTASTAIN ABC Systems
Vector Mouse on Mouse (M.O.M.™ ) Kits

    

Previous Methods
Before we offered the Biotin-Avidin System, which now also includes the use of streptavidin, researchers used directly-labeled, enzyme-tagged primary and secondary antibodies for detection of tissues, blots, and microtiter plates (Figs. 1 and 2). These direct/indirect methods were followed by the PAP method (Fig. 3), which provided some amplified signal.
The Biotin-Avidin System
When we introduced the Biotin-Avidin System a substantial amplification over the earlier methods was achieved. Avidin is an egg-white derived glycoprotein with an extraordinarily high affinity (affinity constant > 1015 M-1) for biotin. Streptavidin is similar in properties to avidin but has a lower affinity for biotin. Many biotin molecules can be coupled to a protein, enabling the biotinylated protein to bind more than one molecule of avidin. If biotinylation is performed under gentle conditions, the biological activity of the protein can be preserved. By covalently linking avidin with different ligands such as fluorochromes, enzymes or EM markers, what we have termed the Biotin-Avidin System can be utilized to study a wide variety of biological structures and processes. The Biotin-Avidin System has proven to be particularly useful in the detection and localization of antigens, glycoconjugates, and nucleic acids by employing biotinylated antibodies, lectins, or nucleic acid probes.
Various Biotin-Avidin Methods
Over the years, several basic biotin-avidin techniques have evolved. One of the earliest and currently least used has been termed the “sandwich” or “bridge” technique and relies on the multiple biotin binding sites on avidin. Following the application of a biotinylated antibody, avidin is added, and then a biotinylated enzyme or EM marker. (This technique is not shown.)
The next technique that evolved has been called the “covalent conjugate” or “labeled avidin” (or “labeled streptavidin”) procedure (Fig. 4). Following the addition of a biotinylated primary or secondary reagent, a covalent conjugate between (strept)avidin and an enzyme, fluorochrome, or EM marker is added.
One example might be staining a histological section with monoclonal mouse primary antibody directed toward a specific determinant on the cells. After the primary antibody is applied, biotinylated anti-mouse IgG is added, followed by peroxidase conjugated avidin.
Development is accomplished by adding an appropriate substrate for peroxidase.
The most recent and the most widely used technique is our patented(U.S. Patent No. 4,684,609) procedure called the “ABC” or “preformed complex” method. After application of a biotinylated secondary or primary antibody, a preformed complex between avidin or streptavidin and a biotinylated enzyme is added (Figs. 5 and 6). This latest technique appears to be the most sensitive in many applications and is discussed more fully in the VECTASTAIN® ABC section of this catalog.
Although each technique has its merit, it is now widely appreciated that the Biotin-Avidin System provides the highest sensitivity in fluorescence and enzyme-based detection, versatility that allows easy interchange or introduction of multiple markers, and the ability to localize or detect antigens which are difficult or impossible to see or measure with other systems.
Advantages of the Biotin-Avidin System
The Biotin-Avidin System has several advantages over direct coupling of the marker to an antibody, a lectin or a nucleic acid probe.
  • The Biotin-Avidin System can improve sensitivity because of the potential for amplification due to multiple site binding.
  • Avidin can be prepared with high fluorochrome to protein ratios and avidin conjugates are very stable.
  • Only a single labeled conjugate, namely avidin or streptavidin, need be kept on hand since it can be used with a variety of biotinylated lectins, antibodies or probes.
  • Biotin-Avidin System reagents can overcome the problem of background fluorescence sometimes encountered in the use of heavily fluorescein-labeled or rhodamine-labeled antibody. These conjugates are sometimes “sticky” and adsorb nonspecifically to tissues, while fluorochrome-conjugated Avidin D does not.
  • The extraordinarily high affinity between avidin or streptavidin and biotin assures the user of a rapidly formed and stable complex between the (strept)avidin conjugate and the biotin-labeled protein.
  • Simultaneously localizing more than one antigen in the same tissue section can be performed even with two or three primary antibodies from the same species. By using either separate enzyme systems, two different substrates for the same enzyme, or assorted fluorochrome conjugates, more than one antigen can be localized in the same tissue section.
  • Applications of the Biotin-Avidin System
    Biotin-Avidin System reagents have been found to be superior substitutes for many conventional, less sensitive methods. Just a few of these uses include:
  • Immunohistochemical staining
  • Introducing multiple labels into tissues
  • Localizing hormone binding sites
  • Flow Cytometry
  • Nitrocellulose and nylon transfer blot detection
  • In situ hybridization
  • Radio-, enzyme-, and fluorescent immunoassays
  • Neuronal tracing
  • Genetic mapping
  • Hybridoma screening
  • Purification of cell surface antigens
  • Coupling of antibodies and antigens to agarose
  • Examination of membrane vesicle orientation
  • *

    adenocarcinoma:

    <oncology, tumour> A form of cancer that involves cells from the lining of the walls of many different organs of the body. Breast cancer is a type of adenocarcinoma.

    Esophageal adenocarcinoma is the faster growing cancer in the western world. Major risk factors for this cancer are Gastroesophageal Reflux Disease (GERD) and Barrett's esophagus.   http://heartburn.about.com/od/esophagealcancer/

    anastomosis:<surgery> An opening created by surgical, traumatic or pathological means between two normally separate spaces or organs.

    esophagogastroduodenal anastomosis: anastomosing the duodenum to the gastroesophageal junction

    dysplasia:<embryology> Abnormality of development, in pathology, alteration in size, shape and organisation of adult cells.

    columnar-lined esophagus:

    columnar:1:of, relating to, resembling, or characterized by columns/ 2:of, relating to, being, or composed of tall narrow somewhat cylindrical or prismatic epithelial cells

    lined: to place or form a line along

    epithelium:<pathology> The covering of internal and external surfaces of the body, including the lining of vessels and other small cavities. It consists of cells joined by small amounts of cementing substances. Epithelium is classified into types on the basis of the number of layers deep and the shape of the superficial cells.

     

    Mutat Res. 2003 Feb-Mar;523-524:137-44.
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    Mechanisms and applications of non-steroidal anti-inflammatory drugs in the chemoprevention of cancer.

    Steele VE, Hawk ET, Viner JL, Lubet RA.

    Division of Cancer Prevention, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-7322, USA. vsly@nih.gov

    Biological and chemical irritants can be the cause of irritation in a variety of organ sites. It is becoming well understood that chronic irritation in any form can initiate and accelerate the cancer process in these same organs. This understanding comes in part from the many epidemiologic studies which point out that chronic inflammation correlates with increased risk of developing cancer in that organ which is affected. One of the hallmarks of chronic irritation is the increased activity in the arachidonic acid pathway which provides many of the necessary inflammatory biochemical mediators to this process. Arachidonic acid metabolism diverges down two main pathways, the cyclooxygenase (COX) and the lipoxygenase (LOX) pathways. The COX pathway leads to prostaglandin and thromboxane production and the LOX pathway leads to the leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). These classes of inflammatory molecules exert profound biological effects which enhance the development and progression of human cancers. A large number of synthetic drugs and natural products have been discovered that block many of these key pathways. Much experimental evidence in animals has shown that inhibition of the key enzymes which drive these pathways can, in fact, prevent, slow or reverse the cancer process. The data are convincing in a number of organ sites including colon, breast, lung, bladder and skin. More recently, double-blinded randomize clinical trials in humans have shown the prevention of colonic polyps by anti-inflammatory agents. These studies have primarily used non-steroidal anti-inflammatory drugs (NSAIDS) which block the COX pathways. Recent preclinical studies indicate that the LOX pathway also may be an important target for cancer prevention strategy. The expression of high levels of these enzymes in cancerous tissues make them an obvious first target for cancer prevention strategies. As newer more specific drugs are developed with few adverse effects this important prevention strategy may become a reality.

    Clinical Cancer Research Vol. 10, 6703-6709, October 1, 2004
    © 2004
    American Association for Cancer Research

    Experimental Therapeutics, Preclinical Pharmacology

    Overexpression of 5-Lipoxygenase in Rat and Human Esophageal Adenocarcinoma and Inhibitory Effects of Zileuton and Celecoxib on Carcinogenesis

    Xiaoxin Chen1, Su Wang1, Nan Wu1, Sandeep Sood1, Peng Wang1, Zhe Jin1, David G. Beer2, Thomas J. Giordano3, Yong Lin4, Wei-chung J. Shih4, Ronald A. Lubet5 and Chung S. Yang1

    1 Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey; 2 Section of General Thoracic Surgery, Department of Surgery and 3 Department of Pathology, University of Michigan, Ann Arbor, Michigan; 4 Division of Biometrics, University of Medicine and Dentistry of New Jersey School of Public Health, New Brunswick, New Jersey; and 5 Chemoprevention Branch, Division of Cancer Prevention, National Cancer Institute, Bethesda, Maryland

    Purpose: Aberrant arachidonic acid (AA) metabolism, especially through the cyclooxygenase (Cox) and 5-lipoxygenase (5-Lox) pathways, has been suggested to play an important role in the development of esophageal adenocarcinoma (EAC). The purpose of this study was to investigate the expression of 5-Lox in EAC of a rat model and in human samples as well as the chemopreventive effects of zileuton (a specific 5-Lox inhibitor) and celecoxib (a specific Cox2 inhibitor) in the rat EAC model.

    Experimental Design: 5-Lox expression in EAC of a rat esophagogastroduodenal anastomosis model and of humans was examined with immunohistochemistry. A chemoprevention study was designed to test whether zileuton and celecoxib could suppress aberrant AA metabolism and esophageal adenocarcinogenesis.

    Results: With immunohistochemistry, we found that 5-Lox was overexpressed during esophageal adenocarcinogenesis in our rat model and in humans. In the chemoprevention study, EAC incidence was reduced in a dose-dependent manner from 68.8% (11 of 16) to 44.4% (8 of 18; P > 0.05) and 31.3% (5 of 16; P < 0.05) by 500 and 1,000 ppm zileuton, respectively, and to 33.3% (7 of 21; P < 0.05) and 20% (3 of 15; P < 0.05) by 500 and 1,000 ppm celecoxib, respectively. With isobolographic analysis, zileuton and celecoxib, both at a dose of 500 ppm, had an additive effect by reducing the tumor incidence to 16.7% (3 of 18, P < 0.01). Leukotriene B4 and prostaglandin E2 levels in the esophageal tissues were also significantly reduced by zileuton and celecoxib.

    Conclusions: This study clearly demonstrated that 5-Lox and Cox2 play important roles in the development of EAC. Both zileuton and celecoxib had inhibitory effects on esophageal adenocarcinogenesis through inhibition on their respective enzymes of AA metabolism.


    Copyright © 2002 Elsevier Science Inc. All rights reserved.

    Leukotriene A4 hydrolase

    Jesper Z. HaeggströmCorresponding Author Contact Information, E-mail The Corresponding Author, a, Filippa Kulla, Peter C. Rudberga, Fredrik Tholandera and Marjolein M. G. M. Thunnissenb

    a Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institutet, S-171 77, Stockholm, Sweden
    b Department of Biochemistry, Arrhenius Laboratories A4, University of Stockholm, S-106 91, Stockholm, Sweden

    Available online 6 September 2002.


    Abstract

    The leukotrienes (LTs) are a family of lipid mediators involved in inflammation and allergy. Leukotriene B4 is a classical chemoattractant, which triggers adherence and aggregation of leukocytes to the endothelium at only nanomolar concentrations. In addition, leukotriene B4 modulates immune responses, participates in the host-defense against infections, and is a key mediator of PAF-induced lethal shock. Because of these powerful biological effects, leukotriene B4 is implicated in a variety of acute and chronic inflammatory diseases, e.g. nephritis, arthritis, dermatitis, and chronic obstructive pulmonary disease. The final step in the biosynthesis of leukotriene B4 is catalyzed by leukotriene A4 hydrolase, a unique bi-functional zinc metalloenzyme with an anion-dependent aminopeptidase activity. Here we describe the most recent developments regarding our understanding of the structure, function, and catalytic mechanisms of leukotriene A4 hydrolase.

    Author Keywords: Leukotriene; Aminopeptidase; Inflammation; Crystal structure; Epoxide hydrolase

    Copyright © 1998 Elsevier Science Ltd. All rights reserved

    Molecules in focus

    Leukotriene B4

    S. W. Crooks* and R. A. Stockley

    Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH UK

    Received 19 July 1997; accepted 19 September 1997. Available online 22 July 1998.


    Abstract

    Leukotriene B4 is a pro-inflammatory mediator synthesised in myeloid cells from arachidonic acid. Synthesis is catalysed by 5-lipoxygenase and leukotriene A4 hydrolase and is increased by inflammatory mediators including endotoxin, complement fragments, tumor necrosis factor and interleukins. A nuclear membrane protein, 5-lipoxygenase activating protein, is an essential co-factor for 5-lipoxygenase. Leukotriene B4 induces recruitment and activation of neutrophils, monocytes and eosinophils. It also stimulates the production of a number of proinflammatory cytokines and mediators indicating an ability to augment and prolong tissue inflammation. Elevated levels of leukotriene B4 have been found in a number of inflammatory diseases and levels are related to disease activity in some of these. Initial data from pharmacological inhibition studies support a role for leukotriene B4 in the pathogenesis of neutrophil mediated tissue damage, and treatments which reduce its production or block its effects may prove beneficial in neutrophil mediated inflammatory diseases.

    Leukotriene A4-hydrolase expression and leukotriene B4 levels in chronic inflammation of bacterial origin
    Immunohistochemistry and reverse-phase high-performance liquid chromatography analysis of oral mucosal epithelium
    Virchows Archiv
    Publisher: Springer-Verlag Heidelberg
    ISSN: 0945-6317 (Paper) 1432-2307 (Online)
    DOI: 10.1007/s00428-001-0597-2
    Issue: Volume 440, Number 6
    Date:  June 2002
    Pages: 627 - 634

    Jörg Eberhard, Søren Jepsen, Markus Tiemann, Ralph Krause, Yahya Açil, Hans-Karl Albers

    A1 Department of Operative Dentistry and Periodontology, University of Kiel, Arnold-Heller-Strasse 16, 24105 Kiel, Germany
    A2 Department of Hematopathology and Lymph Node Registry, University of Kiel, Germany
    A3 Department of Oral and Maxillofacial Surgery, University of Kiel, Germany

    Abstract:

    Abstract. Chronic inflammation of the oral epithelium of bacterial origin is associated with elevated leukotriene B4 (LTB4) levels. We investigated leukotriene A4 (LTA4)-hydrolase expression and LTB4 levels in oral epithelium in relation to the clinical disease manifestation and immunohistopathology and LTA4-hydrolase expression in cultured oral keratinocytes. In 11 patients, three different types of biopsy specimens of the oral mucosa tissues were examined . Each sample was divided, and one-half was analysed using immunohistochemistry with antibodies to LTA4-hydrolase, CD1a, CD3, CD19, macrophages/monocytes and granulocytes. The other half of the sample was homogenised and analysed using reverse-phase high-performance liquid chromatography to determine LTB4 levels. We found strong LTA4-hydrolase expression in basal cells of the oral epithelium from tissue samples that appeared clinically healthy; however, histologically a mild chronic inflammation was observed. In contrast, patients with symptoms of an inflammation of the oral mucosa showed only weak LTA4-hydrolase staining of the epithelial cell layers, but strong immunoreactivity in endothelial and invading inflammatory cells. LTB4 levels were elevated in inflamed tissues compared with non-inflamed controls. Most significantly, there was a strong association between the immunohistochemical detection of the enzyme, LTB4 levels, cellular infiltration and the clinical disease manifestations. In vitro experiments indicated that LTA4-hydrolase expression may be induced by bacterial contamination. This study suggests that LTA4-hydrolase expression and elevated LTB4 levels in oral mucosal epithelium are integral parts of the induction and progression of chronic inflammatory reactions. Epithelial cells may participate in early stages of inflammation as a source of LTB4.

    Curr Pharm Des. 2001 Feb;7(3):163-79.

    Inhibitors of leukotriene A4 (LTA4) hydrolase as potential anti-inflammatory agents.

    Penning TD.

    Pharmacia, Corp., 4901 Searle Parkway, Skokie, IL 60077, USA. thomas.d.penning@monsanto.com

    Leukotriene A4 (LTA4) hydrolase is a zinc-containing enzyme which stereospecifically catalyzes the hydrolysis of the epoxide LTA4 to the diol leukotriene B4 (LTB4). There is substantial evidence that LTB4 plays a significant role in the amplification of many inflammatory disease states. Therapeutic agents which selectively inhibit LTA4 hydrolase would block the formation of LTB4 and thus be potentially useful for the treatment of inflammation. Numerous inhibitors of LTA4 hydrolase have been reported over the past 15-20 years. Several early inhibitors were based on the structure of the natural substrate, LTA4. Later approaches utilized known inhibitors of related zinc-containing metalloproteinases and led to the identification of captopril, bestatin and kelatorphan as potent inhibitors of LTA4 hydrolase. This led to the design of a number of peptide and non-peptide analogs which contained potential zinc-chelating moieties, including thiols, hydroxamates and norstatines. A more recent series of non-peptidic, non-zinc chelating inhibitors of LTA4 hydrolase has been reported. This work led to the identification of several novel classes of analogs, including imidazopyridines, amidines and cyclic and acyclic amino acid derivatives, ultimately resulting in the identification of two potential clinical candidates SC-56938 and SC-57461A.

    Copyright © 1994 Published by Elsevier Science Inc.

    Modulation of pulmonary leukotriene formation and perfusion pressure by bestatin, an inhibitor of leukotriene A4 hydrolase

    Danny T. Muskardin, Norbert F. Voelkel and F. A. FitzpatrickCorresponding Author Contact Information

    Departments of Pharmacology and Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, U.S.A.

    Received 2 September 1993;  accepted 23 February 1994.  Available online 8 November 2002.


    Abstract

    We investigated the effects of bestatin, a prototype leukotriene A4 (LTA4 hydrolase inhibitor, on leukotriene (LT) formation and pulmonary artery perfusion pressure (Ppa) in isolated, perfused rat lungs. In lung parenchymal strips stimulated with a 10 small mu, GreekM concentration of the Ca2+ ionophore A23187, bestatin inhibited LTB4 formation with an IC50 = 10.4 ± 3.0 small mu, GreekM (mean ± SD, N = 4). It did not alter cysteinyl LT formation, confirming that it inhibited LTA4 hydrolase selectively, without inhibiting phospholipase, 5-lipoxygenase, or LTC4 synthase. In isolated, perfused lungs stimulated with 10 small mu, GreekM A23187, 300 small mu, GreekM bestatin inhibited LTB4 release by 72.2 ± 10.6% (mean ± SEM, N = 6, P < 0.01) but had no significant effect on LTE4 formation (P > 0.5). In these perfused lungs, bestatin did not alter the change in Ppa following stimulation with A23187. This effect is consistent with the insubstantial re-direction of LTA4 toward formation of vasospactic cysteinyl LTs. Separate experiments used lungs from rats treated with lipopolysaccharide endotoxin in vivo, prior to isolation, perfusion, and stimulation with 5 small mu, Greekm formyl-methionyl-leucyl-phenylalanine, in vitro. In these inflamed lungs, 750 small mu, GreekM bestatin inhibited LTB4 formation (P < 0.05) and increased LTE4 formation (P < 0.05), compatible with selective inhibition of LTA4 hydrolase. The re-direction of LTA4 metabolism toward formation of cysteinyl LTs by inflamed, perfused lungs did not cause an increase in Ppa.

    enucleation (-noo-kl-shn)
    1. Removal of an entire structure (such as an eyeball or tumor), without rupture, as one shells the kernel of a nut.
    2. Removal or destruction of the nucleus of a cell.

    enucleation (enu·cle·a·tion) (e-noo²kle-a¢sh[schwa]n) [L. e out + nucleus kernel]  the removal of an organ, of a tumor, or of another body in such a way that it comes out clean and whole, like a nut from its shell. Used in connection with the eye, it denotes removal of the eyeball after the eye muscles and optic nerve have been severed.

    ficoll

    This biochemically inert sucrose polymer is used as athickening additive in solutions and gradients

    ficoll gradient

    A density gradient of ficoll (synthetic sucrose polymer) in solution, where concentration of the ficoll varies continuously through the solution. It is often used to separate different types of cells from each other during the process of sedimentation.

    Ficoll-Hypaque technique

    A density-gradient centrifugation technique for separating lymphocytes from other formed elements in the blood; the sample is layered onto a Ficoll-sodium metrizoate gradient of specific density; following centrifugation, lymphocytes are collected from the plasma-Ficoll interface

    ficoll http://www.med.upenn.edu/bmcrc/immune/Ficcoll_info.pdf

    FicollPM 70 and Ficoll PM 400 are high molecular weight

    sucrose-polymers formed by copolymerization of sucrose

    with epichlorohydrin. The molecules are highly branched and

    the high content of hydroxyl groups leads to very good

    solubility in aqueous media. Ficoll PM 70 and PM 400 are

    supplied as spray-dried powders. Ficoll behaves as an ideal

    neutral sphere and has been proposed as the molecule of

    choice for studying pore size distribution and the permeability

    of membranes. Ficoll PM 70 and Ficoll PM 400 have

    analogous structures, but differ in molecular weight, and are

    therefore appropriate for different applications.

    Stability

    The stability of Ficoll is chiefly determined by the glycosidic

    bonds in the sucrose residues. Ficoll does not contain any

    ionized groups, so the structure does not react under

    physiological conditions. It is stable in alkaline and neutral

    solutions, but is rapidly hydrolyzed in solution at pH 3,

    especially at elevated temperature. Ficoll can be sterilized by

    autoclaving at 110 ºC for 30 minutes in neutral solutions.

    Strong oxidizing and reducing agents should be avoided.

    Shipping and storage are at ambient temperatures.

    Chemical and physical properties

    Ficoll is provided as a dry powder and is extremely

    hydrophilic. Solutions are best prepared by slowly stirring

    Ficoll into aqueous buffer. Gentle heating may be required for

    complete solubilization.

    Centrifugation

    In centrifugation methods, the density and viscosity of the

    medium are adjusted to allow particle sedimentation with a

    convenient speed. With sucrose, the high osmotic pressure,

    which results from the concentrations used, often damages

    the cells. If, instead, you add a high molecular weight

    polymer such as Ficoll, you obtain the required density

    without significantly increasing the osmotic pressure. This

    preserves cells intact and retains their viability. Ficoll is

    therefore preferred to sucrose for forming density gradients,

    and is primarily used in this way for the routine separation of

    cells (10, 11, 12).

    Ficoll PM 400 can be used for gradient centrifugation in all

    types of centrifuge rotors and for separation at unit gravity.

    For centrifugation, both discontinuous and continuous

    gradients are possible. Discontinuous gradients offer two

    main advantages: First, the abrupt changes in Ficoll PM 400

    density mean that isolated cells are found in sharp bands at

    the interface between layers of different density. This allows

    for easy removal of the purified sample with a pipette.

    Second, cells with great differences in density can easily be

    isolated with as few as two density layers. This is achieved by

    choosing densities that will prevent one or more type of cell

    from entering the lower phase, banding these cell types at the

    interface. To estimate the densities required for a particular

    application, refer to Table 1.

    Discontinuous gradients are established as follows:

    1. Using Table 1 as a guide, dissolve Ficoll PM 400 in buffer

    or isotonic (0.25 M) sucrose solution at various

    concentrations (generally differing by 5-10% w/v), which

    should separate the cells of interest. Most cells and organelles

    have a buoyant density between 1.0 and 1.2 g/ml in

    Ficoll PM 400. Often, a simple two-layer gradient is sufficient.

    You may store these fractions in a refrigerator, but ensure

    that they reach room temperature before use.

    2. In normal centrifuge tubes, make layers (approx. 1 cm deep)

    of decreasing density with the most dense solution at the bottom.

    3. Keep the tubes at room temperature for a few hours to

    allow diffusion across the interfaces, and thereby even out the

    sharp borders between fractions.

    4. Layer the suspension to be fractionated carefully on top.

    Stir the sample and upper Ficoll layer gently with a glass rod

    to eliminate the interface between them before centrifugation.

    During centrifugation, particles collect either in or between

    the various Ficoll layers, depending on the density of the

    layers. The cells/organelles collect at a lower density than on

    sucrose gradients of equivalent concentration, as Ficoll does

    not penetrate cell membranes. After centrifugation, pipette

    off the various phases, and remove the Ficoll from the

    required fraction by repeatedly diluting with buffer, and

    centrifuging to sediment the particles. Residual amounts of

    Ficoll PM 400 in the sample can be estimated with the

    anthrone reaction (1).

    La centrifugation est une méthode couramment utilisée en biochimie pour séparer ou analyser des fractions ou des structures cellulaires, des macromolécules, etc.

    On peut accentuer ou raffiner les méthodes de séparation en faisant cette centrifugation dans un gradient de concentration. En effet, un des facteurs qui influence la vitesse de sédimentation est la différence entre la densité de la particule et celle du solvant. On peut donc moduler cette vitesse en faisant varier de façon continue ou par étape (discontinue) cette différence de densité en créant un gradient de concentration.

    Si la densité de la particule est plus grande que celle du milieu, elle sédimentera. Plus la différence de densité est grande plus la sédimentation est rapide. S'il n'y a aucune différence de densité, il n'y aura aucune sédimentation, quelle que soit l'accélération. Si la particule est moins dense que celle du milieu, celle-ci s'élèvera dans le tube jusqu'à atteindre un niveau de densité égal à la sienne ou, le cas échéant, jusqu'à flotter à la surface.


     

    MÉTHODES ET APPAREILLAGE

    Fabrication des gradients

    Les variations de densités sont obtenues en faisant varier la concentration d'un produit chimique dans la solution. Divers produits peuvent être utilisés pour faire ces gradients. Ils doivent être très solubles en solution aqueuse, ce qui permet d'obtenir des densités suffisantes. On recherche aussi des produits qui sont relativement inertes, peu coûteux, faciles à manipuler, non toxiques, etc. Évidemment aucun produit ne réunit toutes ces qualités et on doit choisir en tenant compte des contraintes expérimentales

    Le saccharose ("sucrose") est très souvent employé. Il permet d'atteindre des densités assez élevées, de l'ordre de 1.3 g/mL avec du saccharose 2.5 M. Ce produit a l'avantage d'être peu coûteux, électriquement neutre et plutôt inerte pour la plupart des fractions cellulaires. Son principal défaut est sa viscosité à forte concentration, ce qui rend son utilisation plus difficile. Il est aussi à déconseiller si la pression osmotique est un facteur important, par exemple dans l'isolement de cellules entières.

    http://www.chimie-biochimie.umoncton.ca/bch/dg/siitub/centgradient.html

    Au début de la centrifugation, le mélange contenant la substance a isoler est déposée à la surface du liquide. Au cours de la centrifugation, la molécule descend en fonction de sa densité. La vitesse de sédimentation est exprimée dans une unité indépendante des densitées du milieu et de l'accélération : le Svedberg (S). Plus la valeur en Svedberg est élevée, plus elle arrivera vite au fond du tube. A la fin de la centrifugation, le tube contient deux phases distinctes : un dépot plus ou moins solide au fond du tube qui correspond au molécules ayant reussi à sédimenter jusqu'au bout et appelé culot, un phase liquide qui contient toute les molécules n'ayant pas atteind le fond du tube, le surnageant. Selon les cas, la molécules désirée est dans le culot, le surnageant ou repartie entre les deux (dans ce dernier cas, cela signifie que les paramêtres de centrifugation ont été mal choisis).

    http://webiologie.free.fr/techniques/purification/centrifugation.html

     

    cytologie methodes:

    http://www.fmed.ulaval.ca/bio-11134/Cytol-RP-Notes-p1.pdf

    nucleoplasm

    <cell biology> By analogy with cytoplasm, that part of the nuclear contents other than the nucleolus

    nucleolus

    <cell biology> A small dense body (sub organelle) within the nucleus of eukaryotic cells, visible by phase contrast and interference microscopy in live cells throughout interphase. Contains RNA and protein and is the site of synthesis of ribosomal RNA. The nucleolus surrounds a region of one or more chromosomes (the nucleolar organiser) in which are repeated copies of the DNA coding for ribosomal RNA.

    cy·to·plast The intact cytoplasm of a single cell.

    cytochalasin

    A group of fungal metabolites that inhibit the addition of G actin to a nucleation site and therefore perturb labile microfilament arrays. Cytochalasin B inhibits at around 1 microgram/ml but at about 5 _g/ml begins to inhibit glucose transport. Cytochalasin D affects only the microfilament system and is therefore preferable.

    alpha tubulin marker :

    Tubulin is the protein that polymerizes into long chains or filaments that form microtubules, hollow fibers which serve as a skeletal system for living cells. Microtubules have the ability to shift through various formations which is what enables a cell to undergo mitosis or to regulate intracellular transport. The formation-shifting of microtubules is made possible by the flexibility of tubulin which is why scientists have sought to understand the protein's atomic structure since its discovery in the 1950s.

    mouse monoclonal anti–alpha-tubulin antibody (A11126) in combination with Alexa Fluor 546 goat anti–mouse IgG antibody

    Moving boundary/Zone Centrifugation :

    http://ntri.tamuk.edu/centrifuge/centrifugation.html

    A third method of defining centrifugation is by the way the samples are applied to the centrifuge tube. In moving boundary (or differential centrifugation), the entire tube is filled with sample and centrifuged. Through centrifugation, one obtains a separation of two particles but any particle in the mixture may end up in the supernatant or in the pellet or it may be distributed in both fractions, depending upon it size, shape, density, and conditions of centrifugation. The pellet is a mixture of all of the sedimented components, and it is contaminated with whatever unsedimented particles were in the bottom of the tube initially. The only component which is purified is the slowest sedimenting one, but its yield is often very low. The two fractions are recovered by decanting the supernatant solution from the pellet. The supernatant can be recentrifuged at higher speed to obtain further purification, with the formation of a new pellet and supernatant

    In rate zonal centrifugation, the sample is applied in a thin zone at the top of the centrifuge tube on a density gradient. Under centrifugal force, the particles will begin sedimenting through the gradient in separate zones according to their size shape and density. The run must be terminated before any of the separated particles reach the bottom of the tube.

    Zone Velocity Centrifugation:

    http://bioweb.wku.edu/courses/Biol502/centrifugation.html

    A method that results in yields greater than differential centrifugation and allows greater resolution of all particles sizes is zone velocity centrifugation. A sample is layered on top of a shallow density gradient and then centrifuged. Each particle size will migrate as a zone or band at a characteristic velocity. If the velocities of the particles are sufficiently different then the zones of the particles will resolve. The gradient through which the particles are centrifuged is used to stablilze the zones during recovery and helps prevent mixing of resolved zones. The density of the gradient material should be less than the components being sedimented.

     

    centrifugation.jpg

    sonicate

    To expose a suspension of cells or microbes to the disruptive effect of the energy of high frequency sound waves.

    minimal essential medium =MEM

    Horseradish peroxidase
     is a heme protein found in the roots of the horseradish and has a molecular weight of about 40,000 daltons. we see in the microscope a reaction product produced by incubation with an appropriate substrate and hydrogen peroxide. In consequence, a relatively small number of enzyme molecules can create an easily visualized precipitate. Interestingly, the sequence of reactions involved is similar to the reactions that produce melanins.

    horseradish (horse·rad·ish) (h[omacr]rs¢rad-ish)  1. Armoracia lapathifolia.  2. any of several other plants of the family Cruciferae resembling A. lapathifolia.  3. the pungent root of one of these plants, used as a condiment and appetite stimulant; in the past it was used as a rubefacient and plaster like mustard, because it contains sinigrin.

    horseradish peroxidase (horse·rad·ish per·ox·i·dase) (hors¢rad-ish p[schwa]r-ok¢s[ibreve]-d[amacr]s²)  peroxidase isolated from horseradish (Armoracia lapathifolia); used as a reagent in biochemical assays. Abbreviated HRP .

    The western blotting technique for the immunochemical detection and analysis of proteins can be performed with radioisotopic, colorimetric, fluorescent, and chemiluminescent modes of detection1. The scope and flexibility of the fluorescence detection modes are now significantly enhanced by the addition of a specific substrate for peroxidase-based western blots that allows for both chemiluminescent and fluorescent detection. The new Amersham Pharmacia Biotech ECL Plus substrate used in the detection of horseradish peroxidase (HRP)-based westerns is compatible with direct image analysis using the Molecular Dynamics Storm gel and blot imaging system. Storm system imaging of western blots developed with ECL Plus results in detection limits that match those obtained with film-based exposures and may  complement, or offer advantages to, the previously described ECF chemifluorescence approach2. Storm system image analysis of western blots using ECL Plus can be applied to current HRPdetection protocols for fast and sensitive protein immune detection. http://www.link.med.ed.ac.uk/ELEGI/downloads/ECL%20plus.pdf

    The chemiluminescent signal was captured on Hyperfilm™ (Amersham Pharmacia Biotech) using a 30 sec exposure for optimal contrast.

    ps/figure1.gif

    http://www4.amershambiosciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&parentid=577914&moduleid=46852#content

     

    Speed and Sensitivity

    Lumigen PS-atto is the newest substrate for the chemiluminescent detection of HRP conjugates. Reaction of the substrate with an HRP label rapidly generates sustained high-intensity luminescence for maximum detection sensitivity in solution assays. Lumigen PS-atto replaces Lumigen PS-1 for solution applications using peroxidase detection.

    reaction scheme
     

    Sites of Leukotriene Biosynthesis

    http://www.jfmed.uniba.sk/patfyz/separatky/Role_of_Leukotrienes_in_Inflammation/t.html
    The locations at which the leukotrienes are synthesized are determined by the cellular distribution of the enzymes controlling each stage of the biosynthetic pathway. Because 5-lipoxygenase is only found in cells of myeloid lineage, the synthesis of LTA4 is limited to these cells [17]. However, the enzymes determining the next step in the arachidonic acid cascade, either to LTB4 or to the sulfidopeptide leukotrienes, are more widely distributed; thus, metabolism of LTA4 may occur in an equally wide range of cell types. The export of LTA4 from cells that can actively synthesize it enables a much broader range of cells to act as leukotriene secretors.

     

    Figure 2.   Model of cellular leukotriene biosynthesis. Stimulation of the cell leads to mobilization of Ca2+, which triggers activation and translocation of cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase (5-LO) to the nuclear envelope. Together with five lipoxygenase-activating protein (FLAP) these enzymes constitute a biosynthetic complex that produces LTA4 for further biosynthesis of LTB4 and LTC4 via the soluble LTA4 hydrolase and membrane-bound LTC4 synthase, respectively. http://ajrccm.atsjournals.org/cgi/content/full/161/2/S1/S25

    NOUVEAU REACTIF CHIMILUMINESCENT,

    La luminescence est le phénomène par lequel certaines molécules portées à un état excité retournent à l'état fondamental en restituant une partie de l'énergie sous forme d'émission de lumière. On distingue plusieurs types de luminescence selon la source d'énergie impliquée dans le processus d'excitation.

    Lorsque l'énergie qui permet aux molécules d'atteindre l'état excité provient d'une réaction chimique, il s'agit du phénomène de chimiluminescence. La bioluminescence, qui est le phénomène d'émission de lumière observé chez certains organismes vivants, peut être considérée comme un cas particulier de chimiluminescence pour lequel une protéine, le plus souvent enzymatique, est impliquée dans la réaction génératrice de lumière.

    La chimiluminescence est à l'origine de signaux utilisables en immunoanalyse, soit pour un dosage, soit pour une recherche qualitative. Deux réactions d'émission de lumière sont particulièrement exploitées dans ce domaine :

    - la chimiluminescence des 1,2-dioxétanes,

    - la chimiluminescence du luminol.

    REACTION DE CHIMILUMINESCENCE DES 1,2-DIOXETANES.

    Les 1,2-dioxétanes utilisés pour les mesures par luminescence sont des composés stables à température ambiante et qui, sous l'action de la chaleur, se décomposent en deux produits carbonylés dont l'un est à l'état excité et est donc susceptible d'émettre de la lumière. La stabilité des dioxétanes à température ambiante dépend de la nature des substituants présents sur l'hétérocyclobutane;

    Il est possible de synthétiser des dérivés stables non luminescents qui, sous l'action d'un enzyme, produiront un intermédiaire instable se décomposant en émettant de la lumière. C'est le cas de l'AMPPD (3-(2'-spiroadamantane)-4-méthoxy-4-(3"-phosphoryloxy)phényl-1,2-dioxétane). L'enzyme déclenchant est la phosphatase alcaline. L'hydrolyse enzymatique de cet ester phosphorique génére de l'AMPD-, instable, qui se scinde en deux produits dont l'un, l'anion méthyl m-oxybenzoate émet de la lumière .

    Un conjugué marqué par la phosphatase alcaline peut ainsi être détecté par cette méthode. L'addition de micelles constituées de molécules fluorescentes et de bromure de cétyltriméthylammonium permet d'amplifier le signal luminescent produit au cours de cette réaction.

    REACTION DE CHIMILUMINESCENCE DU LUMINOL

    En milieu alcalin, l'oxydation du luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) produit une émission lumineuse. L'agent oxydant le plus utilisé est le peroxyde d'hydrogène. Cette réaction de chimiluminescence peut être catalysée par la peroxydase de raifort et la quantité de lumière émise est proportionnelle à la quantité de catalyseur si les substrats de la réaction sont en excès.

    http://www.gazettelabo.fr/2002archives/pratic/1996/5reactif.htm