Selnikraj-Works on Anthrax

8/14/2019 Selnikraj-Works on Anthrax

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Work of Selnikraj www.selnikraj.110mb.com

spread by eating undercooked meat from infected animals. It is rare to find infected animals in the United States

Symptoms of disease vary depending on how the disease was contracted, but symptoms usually occur within 7

days.

Cutaneous: Most (about 95%) anthrax infections occur, when the bacterial spore enters a cut or abrasion on theskin, such as when handling contaminated wool, hides, leather or hair productsof infected animals. Skin infection

begins as a raised itchy bump that resembles an insect bite but within 1-2 days develops into a vesicle and then a

painless ulcer, usually 1-3 cm in diameter, with a characteristic black necrotic (dying) area in the center. Edema

or swelling of the surrounding tissues may develop and lymph glands in the adjacent area may swell. About 20%

of untreated cases of cutaneous anthrax will result in death. Deaths are rare with appropriate antimicrobial

therapy.

Inhalation: Initial symptoms may resemble a common cold. After several days, the symptoms may progress to

severe breathing problems and shock. Inhalation anthrax is usually fatal, and even with aggressive antibiotic and

supportive therapy 45% of inhalation anthrax cases were fatal in the bioterrorist attack in the fall of 2001.

Intestinal: The intestinal disease form of anthrax may follow the consumption of contaminated meat and is

characterized by an acute inflammation of the intestinal tract. Initial signs include nausea, loss of appetite

vomiting, fever are followed by abdominal pain, vomiting of blood, and severe diarrhea. Symptoms may also

include lesions and soreness in the throat, difficulty swallowing, marked swelling of the neck and regional lymph

glands. Intestinal anthrax results in death in 25% to 60% of cases.

Structure and Targets of the Anthrax

The identification of [the receptor for the anthrax protein] now allows for a more detailed investigation of the

mechanism of uptake by cells of anthrax toxin, write John A. T. Young, of the University of Wisconsin-Madison,

and colleagues. In the second paper, researchers report the three-dimensional structure of another part of the

toxin, called lethal factor. This enzyme disrupts communication within immune cells, inhibiting a response. Cells

eventually rupture, which causes shock, a loss of blood pressure, and death. Anthrax toxin has three parts:

protective antigen (PA), a protein that binds to a receptor on the target cell surface; and two enzymes that must be

transported into the cell to cause damage. The enzymatic portions of the toxin enter the cell through a pore

created for them by PA after it binds to the cell's outer surface. PA can be seen as a bundle of seven cigar-shaped

parts, a molecular arrangement referred to as "polyvalent," meaning it displays multiple binding sites.

The inhibitor designed by Dr. Kane and his colleagues is also polyvalent. Just as a glove matches the shape of a

hand more closely than a mitten, and so fits more snugly, the polyvalent inhibitor binds the toxin at multiple sites

and is orders of magnitude more potent than an inhibitor that binds at a single site. The multiple peptides on the

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how the immune system responds toBacillus anthraces infection. Part of the immune system response, known as

adaptive immunity, consists of B and T cells that specifically recognize components of the anthrax bacterium.

The other type of immune response,innate immunity-aims more generally to combat a wide range of microbial

invaders and likely plays a key role in the bodys front-line defenses. Scientists are conducting studies of howthose two arms of the immune system act to counter infection, including how Bacillus anthraces spore

germination affects individual immune responses.

In another study, NIAID-supported scientists have discovered a potential target for developing new measures to

prevent and treat anthrax toxicity. Their study shows that a human gene called LRP6 plays a role in the delivery

of anthrax toxins into cells. Antibodies directed against LRP6 protected cell cultures from anthrax lethal toxin

These results suggest that targeting LRP6 may prove useful in developing ways to protect against the effects of

accumulated toxin.

Result

The disease anthrax was selected as of the interest and their characteristics, symptoms,

gene information, treatments were determined.



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Bacillus Anthraces

Aim

To study the organism responsible for the disease anthrax Bacillus anthraces.

Description

Anthrax is an acute disease in humans and animals caused by the bacterium bacillus anthraces which is highly

lethal in some forms, there are effective vaccines against anthrax and some forms of the disease respond well to

the antibiotic treatment.

Classification

Kingdom: Bacteria

Phylum: Firmicutes

Class: Bacilli

Order: Bacillales

Family: Bacillaceae

Genus: Bacillus

Species: B. anthraces

Table: Classification ofBacillus anthraces

Bacillus anthraces is a gram positive, facultatively anaerobic, rod shaped bacterium of the genus bacillus, an

endospore forming bacterium, Bacillus anthraces is a natural soil dwelling organism, as well as the causative

agent of anthrax, under conditions of environmental stress, Bacillus anthraces bacteria naturally produce

endospores which rest in the soil and can survive for thousands of years in this state. Bacillus anthraces may be

inoculated into a wound, inhaled or ingested. In ruminants, the bacterium causes sudden death from septicemia.For this reason any ruminants found to have died suddenly and without obvious reason should be treated as a

suspected anthrax case. In these events, a blood sample is taken, by a qualified veterinary surgeon, from a

superficial vein and subjected to the MacFaydean polychrome methylene blue staining procedure which screens

for Bacillus anthraces. Conformational diagnosis is achieved through PCR and Immunofluorescence. Horses

respond variably toBacillus anthraces depending on the site of entry. Ingestion tends to lead to a severe enteritis



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and septicemia. Inoculation in the skin tends to result in a local swelling and associated lymphadenitis. In pigs,

Bacillus anthraces again causes acute necrotising tonsillitis, or a subacute pharyngeal swelling, or the intestinal

disease described in horses. The intestinal disease carries a higher mortality. Dogs and cats seem less susceptible

toBacillus anthraces and require a relative large dose of infectious agent before they begin to show clinical signs

Bacillus anthraces

Bacillus anthraces is typically a disease of herbivores (plant-eating mammals), although it can affect other

animals as well. Among domestic animals, cattle, sheep, and goats have been the most frequent victims. In most

industrialized countries, livestock are routinely vaccinated, and cases of anthrax are rare. In developing countries,

however, where animal vaccination is not regularly practiced, anthrax in animals is a problem. This is especially

so in tropical and sub-tropical environments. In the USA, anthrax cases among animals have been generally

limited to the western plains.

Endospores can survive in the soil for years. Animals consume the spores along with grass as they graze. After

the spores enter an animal, they germinate, changing from the resistant form into the growing and dividing

vegetative form. The sporangium lyses, the spore germinates, and the bacilli multiply rapidly.

Anthrax is a very serious disease in animals, culminating in a fatal septicemia. The carcass of the animal should

be burned where it lies. The carcass should never be opened, since doing this will cause the vegetative forms,

which can be destroyed relatively easily, to form into the resistant spores that can survive for years.

Infection in humans traditionally has been much rarer than infection in animals. Anthrax occurred in people who

came in contact with animals or animal products. It was frequently an occupational disease, affecting

veterinarians, people who raised livestock, and people who prepared products from wool, hide, and hair of

animals. The ordinary citizen in the USA is most likely to encounter anthrax from imported products that have not

been treated sufficiently to destroy spores. In humans there are three possible forms of the disease anthrax

Historically, the most common form has been cutaneous anthrax, in which the organism enters through a break in

the skin. The cutaneous form begins as a papule at the entry site that progresses over several days to a vesicle and

then ulcerates. Edema, sometimes massive, surrounds the lesions, which then develop a characteristic black

eschar. The patient may have fever, malaise and headache. A small percentage of cutaneous infections become

systemic, and these can be fatal.

A more serious form is inhalation anthrax. Here the victim breathes in the organism and develops a severe

respiratory disease. Systemic infection resulting from inhalation of Bacillus anthraces has a mortality rate

approaching 100%. Initial symptoms are vague and flu-like, progressing to hypotension, shock and massive



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bacteremia and toxemia. The severe symptoms are believed to be the result of the bacillis exotoxins. Early

antibiotic treatment is an absolute necessity and should be started during the incubation period if a person has

been exposed.

The third form, intestinal anthrax, is contracted from the consumption of contaminated meat. In industrialized

countries this is not usually a risk, although rare exceptions have been described. In August 2000, the Minnesota

Department of Health was notified that Bacillus anthraces had been isolated from a steer on a farm in Roseau

County. The infected steer was one of five dead cattle found in a pasture. On the basis of identification of the

bacteria by phage typing of isolates cultured from tissues and blood samples by the North Dakota State University

Veterinary Diagnostic Laboratory, anthrax was confirmed. A report of this incident described the management of

and public health response to human exposure to meat contaminated with anthrax.

Genes Involved

In order to cause the disease anthrax,Bacillus anthraces requirestwo plasmids,pX01 andpX02,which carry toxin

and capsule genes,respectively, that are used as genetic targets in the laboratory

detection of the bacterium

presence of 10 genes (acpA, capA, capB, CapC, capR, capD, IS1627, ORF 48, ORF 61, and repA )

Diagonisation

Anthrax is diagnosed by culture and isolation of the causative bacterium, B. anthraces- by detecting the bacterial

DNA or antigens; or by measuring specific antibodies in the blood of persons with suspected cases. The bacteria

can be cultured from the blood, skin lesions, fluid from the lungs or respiratory secretions, spinal fluid, or other

affected tissues prior to the start of antibiotic treatment. Detection of the DNA or antigens of the bacteria, and

detection of antibodies in the blood of suspected cases, is important tools for diagnosis because positive culture is

unlikely after antibiotic treatment has been started.

Treatment

The antibiotics ciprofloxacin, doxycycline and penicillin can be given in high doses to treat the condition.

Ciprofloxacin and doxycyline are also used as prophylaxis (prevention) for people who have been exposed. Early

treatment is needed if inhalational anthrax is suspected. There is an immunisation against anthrax but it takes five

doses of vaccine over the course of a year to get immunity. This makes immunisation too slow to deal with

accidental or deliberate exposure. It is normally offered to those who handle infected animals, and laboratory staff

who work with the bacteria. It would be recommended for a person who had been exposed, in conjunction with

antibiotics, because of uncertainty about when the spores may germinate. The armed forces in the USA are

currently being given immunisation, but concerns have been expressed about how safe, and effective, it is for the



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general public. In the UK, the Public Health Laboratory Service controls supply of the vaccine. Their advice, as

of 15 October 2001, is that immunisation is not recommended pre-exposure to anyone except people who are in

the relevant occupational groups mentioned above. PHLS is monitoring the situation and will act according to any

outbreaks or events

Result

The organism responsible for anthrax was found to be Bacillus anthraces. The structure was described and also

the causes, diagnosis, and the treatment of the disease were determined.



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DOXYCYCLINE

Aim

To find the protein responsible for the anthrax disease this is the binding sites for the human.

Description

Doxycycline is a tetracycline antibiotic. It works by slowing the growth of bacteria in the body. Doxycycline is

used to treat many different bacterial infections, such as urinary tract infections, acne, gonorrhea, and chlamydia,

periodontitis (gum disease), and others. It may be used in combination with other medicines to treat certain

amoeba infections.

Doxycycline comes as a regular and a coated capsule, a tablet, syrup, and a suspension (liquid), all to take by

mouth. Doxycycline is usually taken once or twice a day. Drink a full glass of water with each dose of the capsule

or tablet.

CAS Number 10597-92-9

Chemical IUPAC

(2Z,4S,4aR,5S,5aS,6R,12aS)-2-(amino-hydroxy-

methylidene)-4-dimethylamino-5, 10,11,12a-tetrahydroxy-6-

methyl-4a,5,5a,6-tetrahydro-4H-tetracene-1,3,12-t rione

PubChem Drug Bank KEGG

154560 APRD00597 C06973

Table: Details of Doxycycline

The structure of doxycycline has positive changes represented in the SAR. The north western portion of

Doxycycline has subsistent on R2 thru R4 which increases activity and pharmacokinetic properties compared to

older tetracycline agents. Improvements include longer half life due to slower elimination, better stability and

tissue penetration, and good oral availability due to higher lipophilicity. There is no R1 substitute, R2 is H, R3 is

a methyl, and R4 is a hydroxyl subsistent. Typical tetracycline form nephrotoxic anhydrotetracycline in prolonged

acidic medium. Also, epimerization at C4 amine from beta position to alpha causes inactivation. In basic medium,

ring opening inactivates the antibiotic. However, the removal of C6 hydroxyl group improves acid and basic



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stability. Doxycycline is less susceptible to chelation thus has less GI disturbance, such as cramps which is

usually most common.

Fig.: Structure of Doxycycline

Interactions

Doxycycline interacts with many types of medications. It can increase the absorption of digoxin, which may lead

to digoxin toxicity. The gastrointestinal side effects (nausea, vomiting, stomach upset) of theophylline may be

increased by doxycycline. The dosage of oral anticoagulants (blood thinners, such as warfarin) may need to be

adjusted when this medication is started. Doxycycline may decrease the effectiveness of oral contraceptives (birth

control pills), and pregnancy could result. Barbiturates, carbamazepine, phenytoin, and antacids can lower the

levels of doxycycline in the blood, thus decreasing its effectiveness. Iron and antacid containing aluminum

calcium and magnesium can chelate doxycycline in the gastrointestinal tract and form an insoluble complex,

which can decrease its absorption and, therefore, its effectiveness.

Resistance

Doxycycline resistance occurs via two mechanisms. First, bacteria can produce proteins that bind to ribosome and

can inhibit drug binding or allow protein biosynthesis to continue in the presence of bound doxycycline. The

second mechanism is through active efflux of doxycycline to the outside of the cell. Because doxycycline was

once very widely used, the popularity has decreased considerably due to wide spread resistance and the

introduction of newer broad spectrum agents.

Mechanism of Action

Doxycycline works by inhibiting protein synthesis by reversibly binding to bacterial ribosome (30s subunit) at the

A site, preventing the attachment of amino acyl t-RNA and leading to the termination of translation. It is more

selective for the bacterial 70s ribosome versus mammalian 80s ribosome. Doxycycline also has selective toxicity



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against bacteria because it has high efficiently transport into bacterial cells. They enter Gram negative bacteria

through porins due to its hydrophilicity, and through their lipophilicity in Gram positive bacteria. The bacteria

mistakes doxycycline for food thus passes through the cytoplasmic membrane by an energy requiring active

transport.

Divalent ions may be significant in Doxycycline effectiveness. Magnesium ions attached to the phosphates on

RNA seem to aid in their initial binding to the ribosome, while magnesium in the cytoplasm will limit their ability

to interact with the ribosome.

Result

The ligand Doxycycline plays a significant role in curing the disease anthrax. The description, interaction

resistance, mechanism of action determines the effect of drug with respect to anthrax.



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INTERACTION STUDIES USING SWISS-PDB VIEWER

Aim

To analyze the interaction between the docked protein-ligand complex.

Swiss-PDB Viewer

Swiss-Pdb Viewer is an application that provides a user friendly interface allowing analyzing several proteins at

the same time. The proteins can be superimposed in order to deduce structural alignments and compare their

active sites or any other relevant parts. Amino acid mutations, H-bonds, angles and distances between atoms are

easy to obtain thanks to the intuitive graphic and menu interface. Swiss-Pdb Viewer has been developed since

1994 by Nicolas Guex. Swiss-Pdb Viewer is tightly linked to SWISS-MODEL, an automated homology modeling

server developed within the Swiss Institute of Bioinformatics (SIB) at the Structural Bioinformatics Group at the

Biozentrum in Basel. Working with these two programs greatly reduces the amount of work necessary to generate

models, as it is possible to thread a protein primary sequence onto a 3D template and get an immediate feedback

of how well the threaded protein will be accepted by the reference structure before submitting a request to build

missing loops and refine side chain packing. Swiss Pdb Viewer can also read electron density maps, and provides

various tools to build into the density. In addition, various modeling tools are integrated and command files for

popular energy minimization packages can be generated.

Procedure

1. Open the Swiss-PDB Viewer.

2. Load the docked complex obtained from HEX in SPDBV.

3. Go to WindowsControl panel.

4. Make the whole molecule invisible and make only the ligand molecule visible using control panel window.

5. Select the ligand using select menuselect the neighbouring amino acidfrom the dialog box appearing a

distance of 3.00A0 click ok.

6. The amino acids around 3.00A distance with respect to the ligand appear in the graphics window. Make the

labels visible using the control panel window.

7. Colorby secondary structurethe amino acid is colored based on the secondary structure.

8. ToolsCompute H bondthe H bond interaction between the ligand and the molecules within the distance of

3.00A0 is made visible.



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9. Toolsshow H bond distancesthe distance of the hbonds are made visible.

Output

Fig.: Interaction between Doxycycline with Anthrax Toxin

Result

The interaction study is done on the docked model of the anthrax toxin receptor protein with the inhibitor

doxycycline.



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CHEMSKETCH

Aim

To generate a 2D structure and SMILES notations of polar amino acids using ChemSketch.

Procedure

1. Open the ChemSketch tool.2. Select the structure mode to draw a structure.3. To start drawing click on C, CH4 appears on the screen, the same way select any desired atom from the

structure tool bar to draw, for example Cl, Br, H, N, O, S etc.

4. The atom name can be edited in the structure mode by selecting the corresponding atom and clicking theedit text button.

5. To add a double or triple bond, select the bond position with the cursor. A box will highlight it as seen to theright and then click it once for double bond and same for triple bond and again to go back to single bond.

6. To optimize the structure, especially the rings for standardizing all bond lengths and angles, trigonal andlinear for 2D by using the clean structure.

7. To generate the name for a structure click generate name from structure button.8. To generate SMILES (Simplified Molecular Input Line Entry System) for a specified molecule first select the

molecule and then go to tool and choose generate SMILES notation.

9. To save files got to file in the menu and select save as option. It automatically saves it in .sk2 file format.To save it in .mol file format select export from the file menu and give the name.



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utput

Table: Chemical Structure of Non-Polar Amino Acids Generated using ChemSketch

sult

e chemical structure and SMILES notation of polar amino acids were generated using ChemSketch 11.



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HEX

Aim

To predict the interactive molecular docking using Hex 4.2.

Description

Docking

Docking is a method which predicts the preferred orientation of one molecule to a second when bound to each

other to form a stable complex. Knowledge of the preferred orientation in turn may be used to predict the strength

of association or binding affinity between two molecules using for example scoring functions. Docking is

frequently used to predict the binding orientation of small molecule drug candidates to their protein targets in

order to in turn predict the affinity and activity of the small molecule.

Hex 4.2

Hex 4.2 is an interactive protein docking and molecular superimposition program. Currently, Hex 4.2 understands

protein and DNA structures in PDB format. Hex 4.2 was written by Davie Ritchie at the University of Aberdeen.

This is the main thing which distinguishes Hex 4.2 from other macro molecular (i.e. protein and DNA) docking

programs and molecular calculations. The graphical nature of Hex 4.2 came about largely to visualize the results

of such docking calculations in a natural and seamless way, without having to export unmanageably many and

usually quite big coordinate files to one of the many existing molecular graphics packages. The graphical

capabilities in Hex 4.2 are relatively primitive, although these days user can do quite a lot with a few calls to

OpenGL.

Procedure

Step1: Open HEX .exe file.

Step2: Go to file-> select open and select receptor to load the protein molecule.

Step3: Again go to open and select ligand to load the ligand molecule.

Step4: After loading the protein and ligand select the controls and click Docking.Step5: Docking control box will display, set default option and click Activate.

Step6: Save the HEX messages and the docked structure.

Output



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Fig.: Anthrax Receptor Toxin with Doxycycline

Fig.: Average Energy Values of Anthrax Receptor Toxin Docked with Doxycycline

Result

The Anthrax Toxin receptor was docked with Doxycyline ligand and the RMS values, the energy values and the

BMP values were observed using HEX4.2


Selnikraj-Works on Anthrax

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