Plant Proteases Advantages

Plant Proteases Advantages
Proteases are the most important type of enzymes because they represent ca. 60% of all
commercialized enzymes in the world and occupy the most relevant position among industrial
enzymes. Plant proteases have various applications such as in detergent manufacturing, in medicine,
and food science for many years (Siota and Villa, 2010). The most frequently employed plant
proteases are papain, bromelain and ficin, although new proteases with new and more appealing
physicochemical properties for industry are still emerging. Plant enzymes (such as papain,
bromelain, and ficin) have been extensively used as meat tenderizers. New plant proteases such as
actinidin and zingibain and microbial enzyme preparations have been of recent interest due to
controlled meat tenderization and other advantages.
2.1 Sources of proteases: Proteases occur naturally in all living organisms and play important role in
their functioning. They have been isolated and studied from various sources.
2.1.1. Plants: Proteases occur in all plants and play important roles throughout their lifecycle such as
plant growth, development, senescence and plant defense as well as plant death. They are
responsible for maintaining strict protein quality control and degrading specific proteins ... Show
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The most familiar proteases of animal origin are trypsin, rennin, chymotrypsin, pepsin all having
important role in metabolic processes. These are prepared in bulk in pure form. But the production
of proteases from animals has certain issues such as availability of livestock for slaughter which is
further governed by other policies and religional beliefs. Also the chances of transfer of diseases
from animals to humans are quiet high. These reasons have led to an increase in the demand of
microbial
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Effects Of Hiv-1 Pathogenesis
In the absence of a cure for HIV Type 1 (HIV–1) pathogenesis, highly active antiretroviral therapy
(HAART) are designed to suppress viral replication and maintain it at low to undetectable levels
(Prabu–Jeyabalan et al, 2002). Due to their high intrinsic antiviral activity, the introduction of HIV–
1 protease inhibitors (PIs) have led to a dramatic reduction in morbidity and mortality rates of HIV–
1 infected patients (Codoner et al, 2017). HIV–1 protease inhibitors are peptidomimetics, or
substrate or transition state analogs that mimic natural peptides or proteins and retain the ability to
interact with the original protein's biological target, that competitively targets the hydrophobic P2–
P2' domain in the active site (Prabu–Jeyabalan et ... Show more content on Helpwriting.net ...
Proteolytic processing of the Gag polyprotein results mature HIV–1 structural proteins: p17 matrix
(MA), p24 capsid (CA), p7 nucleocapsid (NC), p6 domains, and two spacer peptides, SP1 and SP2.
Each of these HIV–1 structural proteins play a crucial role in the HIV–1 retroviral replication cycle.
MA is responsible for targeting Gag to the plasma membrane and promoting the incorporation of
viral envelop glycoproteins into the forming virions; CA drives Gag multimerization during
assembly to form the Gag lattice that encapsidates the viral RNA genome; NC recruits the viral
RNA genome into the virions; and the p6 domain recruits the endosomal sorting complex required
for transport (ESCRT) apparatus that catalyzes the membrane fission step responsible for the
budding off of new virions from the infected cell (Freed, 2015).
Due to the infidelity of HIV–1 reverse transcriptase, HIV–1 protease, as with all other HIV proteins,
undergoes frequent mutation (Prabu–Jeyabalan et al, 2002). Despite the high genetic barrier, or
sufficient number of critical drug–resistance mutations to overcome the anti–HIV activity of a drug
regimen, of PI, selective pressure induced by PIs have been seen to be associated with drug
resistance mutations at both the active site of HIV–1 protease and Gag polyprotein (Codoner et al,

The Effects Of Salt Stress On Protein Metabolism
GLYCINE–BETAINE COUNTERACTS THE ANTAGONISTIC EFFECTS OF SALT STRESS
ON PROTEIN METABOLISM
Navneet Krishna Varshney and N.B. Singha
Research Scholar, Faculty of Science
Pacific Academy of Higher Education and Research University, Udaipur (Rajasthan) aDepartment
of Chemistry, Bareilly College, Bareilly – 243005 INDIA
ABSTRACT Glycine betaine is a compatible organic metabolite which is synthesized and
accumulated in pulses, cereals, grasses and many microbes in response to different types of stress. It
has been shown to exhibit enhanced tolerance of plants to salt stress. In this investigation we
examined the effects of salt stress and synthesis of glycine betaine on leaf protease activity in
Lucerne plants. Sodium chloride stress was ... Show more content on Helpwriting.net ...
The present work, therefore, was conducted to evaluate the role of glycine betaine in protecting the
proteins inleucerne plants through protease enzyme activity from being damaged by salt stress. On
the basis of our results we discuss the response of leucerne plants to salt stress and glycine betaine.
MATERIALS AND METHODS
Purification of Protease – The leucerne plant leaves are procured from 70 days old plants and
allowed to stand for two days in a humidity controlled dark growth chamber at 340C. The leaves
were homogenized with 0.1M phosphate buffer (pH 7.0) containing 0.4M sucrose and 0.05M
sodium ascorbate in a waring blender. Filtered homogenate was (I in Table 1) was squeezed through
a gauze and centrifuged at 2000 rpm for 20 mins. The supernatant (II in Table 1) was adjusted to pH
5.0 by the addition of 1N acetic acid and allowed to stand overnight. The precipitate was removed
by centrifugation and the supernatant (III in Table 1) was subjected to ammonium sulphate
fractionation between 0.25 and 0.9 saturation. The ammonium sulphate precipitate was suspended in
0.0025M Mc llvain buffer (pH 5.5) containing 2.5 × 10–3 M sodium ascorbate and dialyzed against
the same buffer at 40C for 20 hrs. The dialysate (IV in Table 1) was centrifuged and the supernatant
was fractionated by the addition of ethanol at –50C. A fraction precipitating between 20 and 60%
ethanol was collected and dialyzed in the same way.

Testing And Treatment Of Hiv 1
RESULTS:
A wide variety of articles were reviewed that adopted a number of different study designs for their
research. A number of different experiments were also looked at which demonstrated how
resistances to certain HIV–1 anti–retrovirals are arising. As presented in the appendix, this specific
section will evaluate the results from many randomized control trials (RCTs), case control studies,
prospective cohort studies with and without controls, retrospective cohort studies with and without
controls and cross–sectional studies.
HIV–1 integrase inhibitors:
HIV–1 integrase is one of the key enzymes required for the successful replication of HIV–1 and is
often a common therapeutic target to help treat HIV–1 infections. Integrase is a ... Show more
content on Helpwriting.net ...
"They looked at multiple coding sequences of the HIV–1 integrase enzyme form blood plasma
samples of 61 patients. They categorized the main integrase region sequences as subtype B, with
minor subtypes being C (CRF01–AE, CRF02–AG and CRF13–cpx), D and G. No major integrase
drug resistance mutations have been observed in new patients beginning with integrase inhibitor
treatment, however in 30 cases, polymorphic variations with the E157Q mutation were observed.
This mutation was more common among subtype B (26 cases) than with non–B subtypes (5 cases).
Major integrase inhibitor drug resistance mutations (G140S, Q148H, N155H, V151I, E92EQ,
V151I, G163R) were notable in four of these cases. Time to the development of drug resistance
ranged from 3 to 16 months with a mean increase of HIV viral load of 4.34 HIV–RNA copies/ml at
the time of emergence of the major mutations." (11) Figure 1 demonstrates four cases in which
resistance to integrase inhibitors were observed.
In a study conducted by Piralla et al., HIV–1 integrase variability was analyzed using stored plasma
samples from 95 patients infected with HIV–1, within a one year period from 2008 to 2009. Patients
with no available plasma samples or viral load less than 1,000 HIV–RNA copies/ml plasma were
excluded from the analysis. Their results demonstrate that primary mutations associated with
resistance to integrase inhibitors were not detected in patients that have previously been treated with
integrase

Isolation and Identification of Thermophilic Fungi
We isolated thirty thermophilic fungi with ability to grow at 45 °C on medium containing wheat
bran and casein as carbon sources and produced proteases in both SSF and SmF (data not shown).
The fungus strain F.2.1.4 presented the highest protease activity and was sub–jected to taxonomic
characterization.
Analyses of macro and micro–morphological characteristics accommodated the isolate F.2.1.4 in the
genus Myceliophthora due to the presence of spreading colonies with dense aerial mycelium and
blastic conidia often borne in ampulliform swellings (Van Oorschot, 1980). Particularly, this isolate
produced pulverulent pale mycelium, obovoid conida measuring 5.65–7.91 x 3.39–4.52 μm as well
as conidia walls finely ornamented. Based on such ... Show more content on Helpwriting.net ...
Alkaline proteases have many applications in the industry such as laundry detergents, leather
processing, brewing, food and pharmaceutical industries therefore the alkalophilic properties
exhibited by proteases from Myceliophthora sp. in SSF are an important aspect to be explored.
The maximum activity for both crude enzymes was 50ºC but the crude enzyme from SSF
maintained 95% of the maximum activity at 60ºC while for the protease from SmF the activity was
79% (Figure 3b). This property is consistent with the dates reported for extracellular enzymes from
thermophiilic fungi. Similar results were reported for proteases from Thermoascus aurantiacus var
levisporus (55oC) (Marcy et al., 1984). Proteases from thermophilic Scytalidium (Hasbay and Ögel,
2002) and from thermotolerant Aspergillus fumigatus (Santos et al., 1996) showed optima activities
at 45 oC.
Since the protease activity was higher in crude enzyme solution from SSF and addi–tionally, it
showed alkaline optimum pH and activity at higher temperature range (50–65 ºC), it was used for
the immobilization procedure.
Entrapment of the protease in calcium alginate beads
Among the quantities of sodium alginate (2, 2.5, 3, 3.5 % (w/v)) and CaCl2 concentrations (0.04,
0.06, 0.08, 0.1 M), the best condition for immobilization of the protease was 2.5 % alginate and 0.08
mM CaCl2 (data not shown), obtaining 2 mm (average diameter) beads.
The immobilized enzyme was reevaluated

Potential Therapy For Human Inflammatory Disorders
Elafin: Potential Therapy for Human Inflammatory Disorders
by
Obiageli C. Okpala
Supervisor: Dr Sukhvinder S. Bansal
In partial fulfilment of the requirements for the MSc Biopharmaceuticals degree, King's College
London
August 2015
Abstract
Elafin is an anti–inflammatory peptide which specifically inhibits neutrophil elastase and
proteinase–3 that would otherwise result in excessive inflammation and tissue damage. It has 57
amino acid residues and two functional domains; the C–terminus which is a globular whey acid
protein (WAP) and the N–terminus which binds to transglutaminase enzyme. Its structure is
characterised by a planar spiral shape with an exposed external loop connected by four disulphides
to the internal B–sheath ... Show more content on Helpwriting.net ...
It is an alarm anti–protease which is expressed locally to provide protection within tissues, against
the excessive inflammatory action of these proteases (Verrier, 2012). It is encoded in humans, as
trappin–2 (9.9 kDa), by a 2kb gene located on chromosome 20 (Verrier, 2012). The proteolytic
cleavage of trappin–2 or pre–elafin by the enzyme tryptase, results in the release of its derivative
molecule, elafin (Guyot, 2005) (Figure 2) by the loss of cementoinThe smaller peptide molecule
would therefore have different properties from its parent molecule that would increase its
effectiveness in inhibition of inflammation. This alarm anti–protease is secreted at the site of injury
in response to primary cytokines like interleukin–1 (IL–1) and tumour necrosis factor (TNF),
making them possibly the first line of defence in the anti–proteinase network (Sallenave, 2000).
Elafin is produced mainly by epithelia in the skin, upper gastrointestinal tract, female reproductive
tract, lungs and in low levels in serum. Its secretion is upregulated in the presence of inflammatory
stimuli or conditions such as psoriasis and acute respiratory distress syndrome (Shaw, 2011),
although some research have shown that uncontrolled protease activity may lead to its proteolytic
cleavage (Small, 2015). This was confirmed in a study by Guyot et al which demonstrated that the
involvement of purified NE in

The Effect Of Pediatric Dosages On Human Immunodeficiency...
Reyataz (atazanavir sulfate)
Antiretroviral
MW = 809.9 g mol–1 (Free Base = 704.9 g mol–1) pKa = 4.7
Chemical Formula: C38H52N6O7 ● H2SO4
IUPAC name: (3S,8S,9S,12S)–3,12–Bis(1,1–dimethylethyl)
–8–hydroxy–4,11–dioxo–9–(phenylmethyl)–6–[[4–(2–pyridinyl)
phenyl]methyl]–2,5,6,10,13–pentaazatetradecanedioic acid dimethyl ester, sulfate Description
Atazanavir sulfate is a HIV–1 aza–dipeptide protease inhibitor that has been approved and is
indicated for co–administration as part of therapy for Human Immunodeficiency Virus–1 (HIV–1)
infection.1,2 The therapy is available in 100 mg, 150 mg, 200 mg, and 300 mg capsules. Prescribed
doses in adults are taken once daily and include either 300 mg in combination with 100 mg of
ritonavir or a single therapy 400 mg dose. Pediatric dosages are determined by patient body weight.
Atazanavir is sparingly soluble in water due to large hydrophobic projections from its structure but
exhibits increasing solubility with decreasing pH.1
Although the introduction of highly active antiretroviral therapies (HAART) marked a seminal
development in HIV–1 treatment, the therapies have since been characterized by poor patient
adherence, a high incidence of acquired antiretroviral resistance in HIV–1, as well as endocrine and
metabolic imbalances resulting in irregular fat redistribution (lipodystrophy), insulin resistance, and
abnormal blood glucose (dysglycemia), cholesterol and lipids (dyslipidemia), and bone resorption
and deposition.8 Atazanavir has

Na-Aspartic Protease-1 Summary
SUMMARY
Researchers at George Washington University have been working on a vaccine to prevent infection
from the hookworm parasite for years and sought out volunteers that would let these parasitic
worms burrow into their skin. In order to effectively study the hookworm, the researchers needed to
find healthy volunteers that would allow the hookworm parasites to infiltrate their bodies and live in
there for several months and also diseased participants to compare the data that they would collect.
However, people weren't interested in doing the clinical trial when they found out they would have
parasites sucking on their blood through their intestines. After searching for months, the GW team
found a small group of volunteers to take on the task ... Show more content on Helpwriting.net ...
In children, chronic hookworm disease affects physical growth retardation and has been seen to
have a correlation between hookworm burden and reduction in intelligence quotient (Loukas et. al
2006). The development of a hookworm vaccine would benefit the future generations in places
where the hookworm infection thrives. I do find it disturbing that people would volunteer to have
hookworm parasites burrow into their skin (some of which have other reasons besides furthering
medicine) but it is assisting this clinical study in order to benefit people (especially children)
exposed to the hookworm environment. Although they are no longer taking in volunteers, I probably
would not want to volunteer for this study because of the possible side effects the parasites could
cause. Overall, this is a great study that will benefit the people of tropical and subtropical locations
by designing the low–cost hookworm

Advantages And Disadvantages Of Arbutin
Arbutin Arbutin, from the dried leaves of bearberry, blueberry, cranberry, and pear trees, is a
naturally β–D–glucopyranoside and derivative of hydroquinone. (20, 21, 24) The mechanism of
suppressing melanin production is to competitively inhibit tyrosinase enzyme at binding site and
DHICA (5,6–dihydroxyindole–2–caboxylic acid) and also inhibit melanosome maturation. (21, 24,
25) Arbutin can cause paradoxical hyperpigmentation if high concentration is used.(26, 27)
Deoxyarbutin has shown a promising skin lightener.(25) Alpha–arbutin are widely use, 20–fold
more potent and stronger inhibitory activity than arbutin.(21, 25) Aloesin Aloesin is a derivative of
aloe vera plant.(21) Competitive inhibitor at binding site of tyrosinase and DOPA (3,4–
dihydroxyphenylalanine) oxidase is its major mechanism in dose–dependent manner.(21, 24) Ellagic
acid Ellagic acid is . It is tyrosinase copper chelation.(22) The study by A. Dahl et al.(28) was
compared 0.5% ellagic acid and 0.1% salicylic acid with 4% hydroquinone in hyperpigmenatation
and dark spots. They have shown that no adverse ... Show more content on Helpwriting.net ...
synthesized N–nicotinoyl dopamine, a newly niacinamide derivative, which significantly reduce
skin pigmentation.(34) Licorice extracts (Available concentration of glabridin: 0.5%) Licorice
extracts are derived from Glycyrrhiza Glabra Linnera and Glycyrrhiza uralensis.(24, 26) Licorice
extracts properties are like two–face of coin, there are many active compounds that may inhibit or
stimulate melanogenesis. Glabridin, a polyphenolic isoflavonoid, is the main ingredient in licorice
extract which is tyrosinase inhibitor and anti–inflammatory compound.(21) In addition, it has a role
in dispersing the melanin by liquiritin and decrease free radical formation.(20, 24, 26) Mulberry
Mulberroside F, as the active compound, is derived from dried mulberry root bark (Morus alba L)
which can inhibit tyrosinase activity and scavenge superoxide in melanogenesis process.(21,

How Enzymes Work in the Home and in Industry Essay
How Enzymes work in the home and in industry essay
This essay aims to explore the ways on how enzymes are used in home and in industry, and it aims
to explain the advantages and disadvantages of using enzymes in the home and industry. An enzyme
is a protein that is formed by the body that acts as a catalyst to cause a certain desired reaction.
Enzymes are very specific. Each enzyme is designed to initiate a specific response with a specific
result.
Firstly, the AQA Science Biology textbook published in 2011 by Nelson Thornes Ltd suggests on
page 172 that "in the past, people boiled and scrubbed their clothes to get them clean – by hand!
Now we have washing machines and enzymes ready and waiting to digest the stains." This is telling
... Show more content on Helpwriting.net ...
Protease enzymes are used to make baby foods. In the book 'Chemistry at a glance' published by
Roger Owen and Sue King it says on page 116 "proteins in baby foods may be pre–digested by
proteases (enzymes which digest proteins). Babies are not very good at digesting the food when they
first begin to eat solid foods. By using protease enzymes in the food, makes it easier for a baby's
digestive system to cope with it and then the babies can also get the amino acids that need from their
food more easily. There are more enzymes used in industry like carbohydrases to convert starch into
sugar (glucose syrup).
There are many advantages and disadvantages of using enzymes. Enzymes can be good to use. In
the AQA Science Biology textbook published in 2011 by Nelson Thornes Ltd suggests on page 173
that "in industrial processes, many o the reactions need high temperatures and pressures to make
them go fast enough to produce the products needed. This needs expensive equipment and requires a
lot of energy." The good thing is that by using enzymes it would solve industrial problems like
these. On the same page, it says the enzymes "catalyze at relatively low temperatures and normal
pressures. This is why the enzyme–based processes are cheap to run. This is an advantage showing
that by using enzymes it is cheaper and easier.
There are also disadvantages of using enzymes. This is evident in the same book on the same page
as it mentions, "one

How Does Protease Inhibitors Work?
What is a disease
What is protease
How does a protease inhibitors work
What is a protease inhibitor
Protease Inhibitors and HIV
Protease inhibitors and Rheumatoid Arthritis
Anticancer activity of protease inhibitors
Protease Inhibitors and Herpes
Protease Inhibitors and Disease
Fig. 1 Showing a Diagrammatic Representation of the Enzyme Protease.
A disease is the destruction in the function of a human which leads to specific symptoms which in
turn affects specific sites and are not results of physical injury. Protease cleaves longer proteins into
shorter core proteins via proteolysis this is the process whereby hydrolysis of the peptide bonds
occur that links subsequent amino acids to a polypeptide chain (Kohei Oda 2012). It is therefore
crucial for the effectiveness of an infectious virus. Protease Inhibitors bind to the active sites of
these proteases which in turn inhibits the passage of the protein from becoming shorter units
(virions) in order to remain non–infectious. One such example is seen in the Human Immuno–
defiency Virus commonly known HIV. In a video Dr. Rufus Rajadurai extensively explained the
invasion process of the HIV virus into the uninfected cell of a human to the point of infection
.
Fig. 2 Showing a Diagrammatic Representation of a Virion. Fig.3 Showing a Diagrammatic
Representation of a Protease Inhibitor.
A protease inhibitor is therefore a drug used to interfere with the key stage of viral replication and
used to stop this fatal process.

Drug Discovery And Development Of New Medicine
Describe and discuss recent developments in drug discovery. As our society is evolving so is
diseases many drugs that were previously effective to cure certain diseases are simply not as
effective any more or on the other hand there isn't sufficient medicine to battle the current new
health problems that are arising with our evolving society for example obesity and a range of
cardiovascular diseases. In addition many of the medicine available today either only treats the
symptom or cures the disease with the addition of unwanted side effects. Hence, this is why drug
discovery and development is vital. It is through drug discovery and development that new medicine
is found.
Currently a lot of effort is going into Binuclear metallohydrolases as a target in drug development.
This is due to the fact this enzyme group has the potential to find solutions to health problems such
as erectile dysfunctions, osteoporosis, cardiovascular dieses and also even become the seed in
possible eradication of some antibiotic resistance.
The main enzymes in the binuclear metallohydrolases group that are most vital us are purple acid
phosphatases, metallo–β–lactamases and arginases. The homologous feature in all these enzymes is
the existence of two very nearly touching metal ions that hold the ability to attract water molecules
that will then act as a nucleophile in a very particular hydrolytic reaction the importance of this is
that it means that binuclear acids will have a powerful ionic

Noroviruses Research Paper
Noroviruses (NoV) are important human pathogens and a major public health threat. Noroviruses
are non–enveloped, single–stranded, positive sense RNA viruses that cause acute gastroenteritis in
humans (1–4). Noroviruses have been referred to as 'cruise ship viruses' from well–publicized cruise
ship outbreaks although these outbreaks account for less than 1% of all outbreaks worldwide (5).
The symptoms and illness associated with Norovirus infections is often referred to as 'stomach flu',
despite little resemblance to the influenza virus. Noroviruses are responsible for 21 million
infections and over 70,000 hospitalizations annually in the USA, and account for 10–15% of severe
cases of gastroenteritis in children less than 5 years old (6). In ... Show more content on
Helpwriting.net ...
The overall structure of NoV Pro is that of a cysteine protease with a chymotrypsin–like fold and a
catalytic triad consisting of His–30, Glu–54, and Cys–139 (71). The structures of Norovirus
proteases share a high degree of structural similarity to picornaviral 3C proteases (75, 76), making
picornaviral proteases an excellent model for comparison. The mechanism of catalysis for the
Norovirus proteases is similar to the serine proteases that contain a Ser–His–Asp triad in the active
site. The mechanism of catalysis of serine proteases is shown in Figure 2. The catalysis reaction
occurs in two halves, acylation and deacylation. First, the carbonyl of the peptide substrate is
attacked by the Serine and His residues of the catalytic triad (77). The result is the first of two
tetrahedral intermediates which collapses to form the acylenzyme intermediate (77). The second half
of the reaction is very similar where a water molecule and His residue attack the acylenzyme
intermediate to yield the second "transition–state" intermediate which collapses to release the
carboxylic acid product (77). Similar to the serine proteases, the Cys–139 residue of Norovirus
proteases functions similarly to the Ser residue and the Glu–54 functions as the Asp residue of the
serine proteases while the His residue function is

Sigma Erdrich Lab
Washing procedure. Different settings can be used to clean the needles, dispense wash solutions,
rinse the wells and wash off the planktonic cells in the wells of microplates. Before washing the 96–
well microplates, maintenance cycles were performed in the plate washer and the needles were
rinsed first by 10% bleach (2 times) followed by de–ionized water (3 times) and Tris buffer (2
times) to remove any deposits or contaminants. After the rinses, microplates were washed with Tris
buffer for various numbers of times (3 times, 9 times, and 15 times) using shake option in Magna
Plate Washer (Fig. 3) so that optimal wash time could be determined and used in subsequent assays.
In this research we investigated the use of enzymes such as pronase ... Show more content on
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Bax protease (DuPont Qualicon, Wilmington, DE) was obtained as a solution and used as per
manufacturer's guideline [12.5 µl in 1ml Tris (0.05M, pH 7.4)] (Gamble & Muriana, 2007). The
specific protease is undisclosed so we used it at the working strength recommended by the
manufacturer.
Papain. Papain, Carica papaya (Cat: 5125, EMD Millipore Corp., Billerica, MA) had listed activity
of 31850 U/mg and stock solution was prepared by adding 0.31 gram in 10 ml Tris (0.05 M, pH 7.4)
i.e. concentration of 1000 KU/ml.
Cellulase. Cellulase from Aspergillus niger (Cat: C1184, Sigma–Aldrich) was used in same
concentration as pronase E i.e. 100 enzyme units (U) per 200 µl. The activity for cellulase was
marked as 1.3 units/ mg solid and hence 1.92 grams of cellulase powder was added to 5 ml Tris
(0.05 M, pH 7.4) to get desired stock solution concentration of 500 U/ml.
Lipase. Lipase from Candida rugosa (Cat: L1754, Sigma–Aldrich) had activity of 1170 U/mg solid.
Lipase powder weighing 2.14 mg was dissolved in 5 ml of Tris (0.05 M, pH 7.4) to get a
concentration of 500 U/ml. It was then filter sterilized before use.
The mass (weight) of powdered enzymes needed to get the desired stock solution concentration in
U/ml, were calculated using following

How Proteins May Be Refined By Controlled Enzymatic...
The physicochemical and organoleptic qualities of proteins may be refined by controlled enzymatic
hydrolysis, which generates free amino acids and abundant short peptides with less salt and
carcinogenic compounds (Weir, 1992). Significantly milder conditions are employed: The pH is
typically maintained at pH 5–7 corresponding to optimum enzyme activity and the hydrolytic
process occurs at 50–60°C for 10–24 h, which minimizes unwanted side reactions (Clemente,
2000). Proteins are only partially hydrolysed due to the inability of most proteases to cleave
glycoproteins, phosphoproteins and protein domains containing numerous covalent–linked disulfide
bridges (Gibbs et al., 2004).
The size of the peptide, position of amino acids within the peptide and resistance of the peptide
bond to further hydrolysis are crucial in flavour formation (Izzo & Ho, 1992). The resulting
hydrolysate is comparatively lighter in colour with significantly less pronounced meaty or savoury
characteristics than acid hydrolysates, and exhibits stronger vegetable–like notes (Aaslyng et al.,
1998b). A major drawback of enzymatic hydrolysates is the generation of bitter peptides as natural
degradation products during proteolysis. Many bitter peptides in casein (Bumberger & Belitz, 1993;
Sohn & Lee, 1988) and soybean (Fujimaki et al., 1970; Lovšin–Kukman et al., 1995) protein
hydrolysates have been isolated and characterised. Nevertheless, enzymatic hydrolysis provides
numerous advantages over acid

Reaction Paper On Flagella
Summary #3
Flagella are commonly known as organelles that help with the movement and sometimes adhesions
of certain types of prokaryotic and eukaryotic cells. Proteolysis is the process in which proteins are
broken down and enzymes speed up chemical reactions. In this paper, researchers have been able to
discover enzymatic flagellins that form flagella and are able break down proteins. According to
researchers, adding a cofactor like metallopeptidase, which has flagellin, to a pathogen like
Clostridium haemolyticum, had made 74 species of metallopeptidase become available. Through
this experiment, researchers have used different assays to test if metallopeptidase within the
hypervariable region of Clostridium haemolyticum provides ... Show more content on
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Figure 2 shows a the distribution of proteolytic flagellins amongst different types of lineages for C.
haemolyticum. This tree shows the four major lineages of proteolytic flagellins which are
Gammaprotebacteria, Betaproteobacteria, Deltaproteobacteria, and Chloroflexi. Each lineage is
numbered to show the size of the lineage. The clustered red lines refer to the species that can do
lateral gene transfer. The purpose of this figure is to show that the proteolytic flagellins have
evolved from four different lineages.
Figure 3 show amino acid sequencing and structural modeling of the metalloprotease flagellin
family. Figure 3a is an assay for amino acid sequencing analysis. 86 flagellin sequences from 74
species were analyzed by multiple sequence alignment. Three sections within the alignment are
highlighted to indicate the functional motifs of the hypervariable region for metalloprotease. Figure
3b shows sequence logos for the functional motifs, which is comprised of the putative Ca++ ion
binding site, the zinc–binding HExxH motif, and the third zinc–binding residue, which are the three
highlighted sections in figure 3a. Sequence logos show the frequency of an amino acid. The x–axis
refers to the position of the amino acid within the peptide, while the y–axis refers to the size or
frequency of the amino acid, measured in bits. The

Three Unknown Enzymes
Introduction In the lab, we were provided with three unknown enzymes that can potentially be
identified as amylase, protease, or none of the two. The goal established for this experiment is to
discover the identities of the three enzymes by conducting two specific diagnostic arrays (Ninhydrin
and Benedict's Test) upon polysaccharide and protease solutions that contain one of the three
unknown solution. Through these experiments, we searched for the presence of monomers to
determine if one of the three enzymes catalyzed exergonic reactions that would help indicate its
identification.
If we conduct a Ninhydrin Test upon a mixture between a protein solution and an unknown enzyme
and the results display the presence of free amino acids, then the enzyme would be protease. If we
conduct a Benedict's Test upon a mixture between a polysaccharide solution and an unknown
enzyme and the results display the presence of monosaccharides, then the enzyme would be amylase
Experiment To determine if any of the unknown enzymes was amylase, we set up five tubes that
each contained polysaccharide solution. Three vials contained one of the three ... Show more
This was surprising due to our prior tests indicating that Enzyme B was an amylase and our inquiry
of Enzyme A being neither of the two enzymes. Because we understand that enzymes contain R
groups that are consisted of amine groups, and that the three enzymes are partially pure, we decided
to conduct the extra experiment of the enzymes themselves in order to test for unusual levels of free
amino acids within the enzymes. Our results display that there was a higher abundance of free
amino acids in Enzyme B, which would affect the results in the Ninhydrin test that was conducted
with the protein and protease

Group A Streptococcus (GAS) Essay
Streptococcus pyogenes, also known as Group A streptococcus (GAS), is a β–hemolytic, Gram–
positive bacterium that most commonly causes respiratory disease, including pharyngitis or
tonsillitis, as well as skin infections such as impetigo and cellulitis. The organism is transmitted via
respiratory droplets or by contact with fomites, and commonly infects young children. In addition to
the common clinical presentations associated with S. pyogenes, some individuals develop the
postinfectious sequelae of rheumatic fever and glomerulonephritis. Due to the severity of these
medical consequences, prophylactic antibiotic use is often recommended for any patients with
otherwise mild S. pyogenes infections (21).
In addition to its traditional ... Show more content on Helpwriting.net ...
Antibiotic use, particularly with clindamycin and linezolid, may be useful in treating GAS–
associated necrotizing fasciitis, as they target both M–proteins and exotoxins (5). Some cases have
been treated with hyperbaric oxygen therapy, with recent data suggesting prompt surgical therapy
instead is vital for preventing amputations (18). Even after treatment, however, necrotizing fasciitis
mortality has been estimated to range from 24%–60% (10).
In S. pyogenes–associated necrotizing fasciitis, the bacteria first invade host fascia before spreading
and causing more invasive damage. Initial GAS invasion can occur through three primary pathways:
1) preceding superficial infection; 2) direct inoculation; or 3) hematogenous seeding from a
previous injury site. After colonization, GAS spread through the typically sterile fascial planes that
separate muscle groups, which are only loosely held together by connective tissue. The streptococci
then release several proteases and other virulence factors that cause localized tissue damage.
Simultaneously, this dissemination prompts the arrival of acute inflammatory host cells; the
response of polymorphouclear leukocytes (PMNs), in particular, can also contribute to the tissue
damage characteristic of necrotizing fasciitis. Once the

The Distribution Of PI-Induced Mutational Changes In Gag...
To investigate the distribution of PI–induced mutational changes in HIV–1 across Gag and protease
regions, Codoner et al. sequenced the HIV–1 Gag–protease coding region from longitudinal plasma
samples from four patients receiving PI treatment over a 9–year period. Sequence analysis
confirmed that HIV–1, in response to PIs, demonstrates a stepwise accumulation of Gag cleavage
site mutations (CSM) at the following positions: V128I in the p17/p24 junction; S373P and I376V at
the p2/NC junction; A431V at the NC/p1 junction; K436R at the NC/p1 junction and P453A at the
p1/p6 junction (Codoner et al, 2017). Of these Gag CSM, A431V and K436R have been previously
associated with drug resistance mutations in the protease and V128I, A431V, K436R ... Show more
To understand how coevolving Gag residues can alter binding interactions between Gag polyprotein
and HIV–1 protease, Ozen et al. studied the interactions between wildtype and mutant I50V/A71V
protease with Gag polyprotein with mutated p1 to p6 cleavage sites. While the overall backbone
conformation of the Gag–protease complex is conserved n all coevolved structures, when
superposed onto the wildtype Gag–protease complex, minor structural changes were found at
contact surfaces between the Gag polyprotein and HIV–1 surfaces. This coincides with how
Deshmukh et al. found regions of high mutational propensities at short–lived contacts between
surface–exposed residues on the Gag polyprotein and HIV–1 proteases (Deshmukh et al, 2016;
Ozen et al, 2014).
To determine if HIV–1 protease recognition of Gag polyprotein was affected by mutations in the
Gag–protease complexes, Ozen et al. measured the substrate volume within the HIV–1 protease
envelope. While substrate volume within the envelope was the highest for the wildtype Gag–
protease complex, the I50V/A71V HIV–1 protease had decreased fit of the wildtype Gag
polyprotein. P1–mutated Gag polyprotein, however, partially restored the loss of fit and fill the
substrate envelope greater than the wildtype Gag polyprotein. Since coevolved substrates, overall,
maintained a comparable fit within the substrate envelope of both wildtype and mutant HIV–1
protease, this suggests that the substrate

What Can Studies Of Serine Proteases Tell Us About The...
What can studies of serine proteases tell us about the relationship between protein structure,
function and evolution?
Serine proteases are a group of important proteases which can fracture the peptide bond in the
macromolecules and proteins. The serine proteases take a great part in mammal lives especially in
digestion, coagulation and the complement system appears. The activation of serine proteases are all
because of the change of a set of amino acid residues which includes at least one serine and that's
why the name became. Although there are about one third of the known proteolytic enzymes are
serine proteases, the principle of interactions are really same: a complicate combined interaction in
the catalytic triad––Ser–His–Asp. This essay will firstly talk about the mechanisms of the serine
proteases and secondly mention the relationship of the protein structure, function, and evolution
during the serine proteases interact. In the first part, the chymotrypsin interaction will be analyzed
as an example to explain the mechanism of serine proteases interact.
Figure 1 the pymol figure of Trypsin
Figure 2 structure of the active center of chymotrypsin
Figure 3 the specific interaction of chymotrypsin
The Pymol figure shows a crystal structure of Trypsin, a typical kind of serine protease. In the
middle of the Trypsin, there is a Ser–His–Asp. The mechanisms of all serine proteases including
Trypsin are: firstly the serine residues in the active center are activated by

Disscusion Case Study
Disscusion The 2016 Guidelines provide recommendations on the preferred and alternative DAA
regimens by HCV genotype [40]. Therefore knowing patient genotype is still important for
determining the most appropriate treatment regimen. Ahmed Nagaty Plos one In the present study,
HCV genotyping of the 20 HCV isolates was tried using the full length NS3/4A, NS5B, and the two
third of the carboxy terminal region (including ISDR) of NS5A gene sequences. Full concordance
was observed in 18 out of the 20 isolates of which 16 were diagnosed as subgenotype 4a while 2
(10%) were of subgenotype 4n. The remaining 2 isolates were successfully sequenced only in the
NS5b region and were diagnosed as subgenotype 4a. Schnell et al (4) conducted a ... Show more
(12) Only few studies report the prevalence of DAA–naive patients with viral populations
predominantly resistant to DAAs. Thereby, it is important to assess the prevalence of HCV viral
populations containing natural variants resistant to DAAs in DAA–naive patients. (13, 14) The aim
of this study was to detect the presence of NS3, NS5A, or NS5B polymerase inhibitor–resistant
variants as the majority viral population in 20 HCV patients who are naive to DAA treatment
regimen. NS3 protease gene was successfully sequenced in 18 (90%) out of the 20 HCV isolates.
The catalytic site on NS3 protease consists of the amino acid triad serine–histidine–aspartate and is
located in a shallow substrate binding groove with solvent features which does not facilitate tight
binding to the inhibitors. (17) Therefore, inhibitors depend on few interactions with the enzyme and
only a few critical mutations in the enzyme may be enough to confer significant resistance to these
drugs. (18) Several mutations in different positions at the NS3 protease have been associated with
loss in susceptibility to Protease Inhibitors. (19) In the present study, 2 types of mutations were
observed with different frequencies. Amino acid frequencies at each position in the NS3 protease
sequence were determined with the VESPA software program. 24 Genotype 4 –specific amino acid
signatures were present in almost all of our

Genetically Induced Chronic Obstructive Pulmonary Disease
Genetically Induced Chronic Obstructive Pulmonary Disease Chronic Obstructive pulmonary
disease is a disease of the lungs that damages the tubes that allow for respiration, thereby resulting
in breathing trouble. This disease has a major effect on smokers. The purpose of the study was to
focus not only on the effects smoking has on the prevalence of COPD, but also how it can be
genetically induced. About 30 percent of people who are diagnosed with COPD have never even
smoked. In this paper, the role cigarette smoking plays in the life of a COPD patient and what role
genetics plays in the prevalence of the disease will be addressed. Also, the nature–nurture
interactions regarding the smoking behavior of an individual will equally be addressed and
emotional distress. It is hypothesized in one of the literatures, that the disease only develops in a
person that has inherited the susceptible genes for the condition. Clancy and Nobes (2012). The
subsequent five literature review will make an effort to prove and support this hypothesis. In a
research article by John Clancy and Christopher Turner (2013), various relationships leading the
cause of the disease were addressed, such as the role of smoking in accordance with genetics, and
what role the health care plays in the prevention mechanism on how to reduce the mortality rate as a
result of the development of COPD. The investigation started of with the nature–nurture
interactions, which was explained according to Clancy and

Kaposi's Sarcoma/HIV Research Paper
Block A: Kaposi's Sarcoma, HIV, ARC, Western Blot, Immunotherapy, ELISA, Protease, PI, B cells,
T cells, PCP, Opportunistic Infection
HIV (human immunodeficiency virus), is an infection that is sexually transmitted. It can be spread
by contact with any blood that carries the infection or from mother to child through birth. Also, If a
mother is infected with HIV after the birth of her child, the infection can still be passed on through
breast feeding. HIV attacks your immune system, mainly focusing on the T–cells. T–cells (T
lymphocytes) are a type of white blood cell that are very essential for human immunity. There are
many different kinds of T–cells but can be put into two main groups; helper T–cells and killer T–
cells. Killer T–cells use X–Ray vision ... Show more content on Helpwriting.net ...
Retrieved January 2016 from
http://onlinelibrary.wiley.com/doi/10.1046/j.14321033.2003.03533.x/abstract
Elisa (n.d.) (2016). Retrieved January 2016 from https://www.abdserotec.com/an–introduction–to–
elisa.html Immunotherapy in HIV Infection by Poonia B. (2013). Retrieved January 2016 from
http://www.esciencecentral.org/journals/immunotherapy–in–hiv–infection–2332–
0877.1000102.php?aid=11848
What is HIV/AIDS? (n.d.)
(2015). Retrieved January 2016 from https://www.aids.gov/hiv–aids–basics/hiv–aids–101/what–is–
hiv–aids/
Western blot. (n.d.) Segen's Medical Dictionary. (2011). Retrieved January 2016 from
http://medicaldictionary.thefreedictionary.com/Western+blot
T cells. (n.d.) Beginners Guide to T cells (2009). Retrieved January 2016 from
http://www.tcells.org/beginners/tcells/ Opportunistic infections. (n.d.) Living with HIV. (2015).
Retrieved January 2016 from
http://www.cdc.gov/hiv/basics/livingwithhiv/opportunisticinfections.html AIDS, HIV, and
Pneumocystis Pneumonia (PCP). (2015). Retrieved January 2016 from

Cys-Dependent Asp-Specific Protease
Apoptosis is a programmed cell death which is close related to all the stages of an organism, such as
the development of the embryo, eliminating the unnecessary cells as well as the maintenance of the
physiological balance. Caspases (short for Cys–dependent Asp–specific protease) are a group of Cys
proteases which are the main executioner of substrate cleavage. Two key pathways exist to activate
caspases in vertebrate: 1) the intrinsic and 2) the exogenous apoptosis pathway. 8 In the intrinsic
apoptotic signaling pathway, mitochondrial pathway, DNA damage, metabolic stress or the presence
of unfold proteins can all be stimuli. These stimuli can pass a signal to the mitochondria system, and
finally lead to the formation of the apoptosome. The latter can sequentially activate caspase–9 to
cleave the key apoptotic factors caspase–3 and ... Show more content on Helpwriting.net ...
An increase of peroxidase activity is one of the key points, which contributes to the mitochondrial
synthesis of reactive oxygen species (ROS) for example CL oxidation. Since ROSs are normally
regarded as harmful factors to the cell, the increase level of ROS has a significant role in inducing
apoptosis. Thus, the oxidation of CL might be necessary to induce the mitochondria membrane
leakage, and release of pro–apoptotic substances, such as protein tyrosine phosphatases (PTP) into
the cytosol.Additionally, the released cyt c can play some other roles. It can interact with and
oxidize another phospholipid phosphatidylserine (PS) with negative charges, which is another
important function of cyt c in apoptosis.12 It is also able to bind to the apoptotic protease–activation
factor–1 (APAF1), and stimulate its heptamerization to finally activate the caspase based
apoptosis.13 Lots of detailed studies have been done on the cyt c–lipid interaction which will be
discussed later in the

Investigating the effect of temperature on the action of a...
Investigating the effect of temperature on the action of a protease enzyme on exposed developed
film
Plan
Aim: The aim of the experiment is to find out what effect temperature has on the action of a
protease enzyme on exposed developed film.
Enzymes are biological catalysts. They are made in livings things built up by amino acids to make
protein. Enzymes are able to speed up reactions and can repeat reactions.
There are various factors that affect the activity of enzymes they are:
"Y Temperature
"Y pH
"Y Specificity
"Y Concentration of enzyme or substrate
Enzymes are specific, this means that they only work on one substrate molecule. A substrate
molecule is what the enzyme actually works on.
The factors I have chosen to ... Show more content on Helpwriting.net ...
This equipment is suitable for this experiment because it is easily available, it is easy to set up and
use and it is easy to collect results with.
This is how the experiment will be set up
I will firstly measure the volume of protease solution by using a syringe, which will be 10cm3 and
then put it into a test tube. I will then get two developed films and hook wire onto each so I am able
to get them out of the tube easily. The wire will be labelled so it is easy to see which film is which. I
will then put the test tube into an electric water bath, which is at a specific temperature for example
300C. I will leave it in the bath for three minutes and then put the two films into the test tube. Every

30 seconds I will check to see if the film has become transparent. When the two films have become
transparent I take them out of the test tube. I then check the pH of the protease solution by getting a
glass rod and dipping it into the solution and then put the solution onto pH paper.
Preliminary experiment:
For my preliminary experiment I set up the apparatus as above. As it was only preliminary I used
one film. I chose two temperatures to put two test tubes of protease into, they were 600C and 300C.
I put the two test tubes into the two different electric water baths and then after three minutes put
film in each. This is how the results turned out:
Temperature of water bath/0CTest tube in water bath with no

Hiv And Its Effects On Hiv Essay
As much as there are good happenings in life, there are bad happenings as well. Overtime there have
been new diseases and viruses that strike us unexpectedly and for the worst. One of the many
epidemic viruses that shook our world was the virus known as HIV. When the virus HIV first hit the
globe it was horrifying and everyone around the world was petrified for his or her life. For instance,
folks believed it would be another plaque sequence where many people died like the drop of a dime.
In fact, the virus was fatal in certain areas such as San Francisco and most common amongst
homosexual males. Men were living with no hope for the future and had to plan to live their lives in
the moment. However, all the doubts and unfaithful thinking stopped when a major break through
and advancement was developed for HIV. For years the medical field did not know how to help
people infected with the virus but after research and experiments were conducted they found their
first advancement against HIV. The advancement is best known as protease inhibitors, specifically
Ritonavir. Although this was not a cure, the protease inhibitor Ritonavir was an advancement that
prolonged people's lives. With this advancement folks could look towards and plan for the future
since there was a better chance of them living longer with this kind of treatment opportunity. With
that being said, there was a lot of information that had to be researched and studied in order to
transpire this advancement. This

Proper Cell Behavior : The Effects Of Natural Biological...
Proper cell behavior is maintained using a finite pool of processing resources, such as the limited
pool of enzymes required for gene transcription and protein translation1–2. Natural biological
circuits are largely thought to have evolved to buffer against the effects of limited resources, but we
are beginning to understand how processing machinery can form a bottleneck that is in fact
leveraged as a control or signaling mechanism3–4. Proteolytic (protein degrading) pathways, in
particular, have been found to form functional bottlenecks in a native E. coli network regulating the
stationary phase sigma factor S (S). The protein S is degraded by the ClpXP proteolysis system
(ClpXP protease and its chaperones) much faster during ... Show more content on Helpwriting.net ...
1A)8. Queueing theory has since been adopted to describe how competition between substrates for a
particular protease can lead to pronounced coupling and statistical correlation8, 14–16. The impact
of proteolytic queueing competition leads to a rewiring of natural and synthetic circuits to include
mutual modulation of substrate degradation rates17. This effect is due to targeting multiple species
of protein to a common protease ClpXP, and it applies to all but one existing bacterial synthetic
oscillator ClpXP18. The exception is the recently modified repressilator19, where active
degradation by protease was systematically removed to produce a more robust growth–dependent
(dilution–dependent) oscillator, which interestingly was predicted based on a prior analysis of
proteolytic competition17.
The single protease crosstalk picture is too simplistic for native circuits, and the reliance on a single
degradation pathway for bacterial synthetic oscillators presents a scalability problem that limits the
complexity of circuits that can be developed. To address this issue, we investigated the crosstalk
between multiple native degradation pathways in E. coli. This study extends a prior investigation of
computational models that suggested a multi–protease proteolytic bottleneck may still contribute
substantially to crosstalk in simple and complex (oscillatory) networks20. The influence of

The Adherence Of Taking Antiretroviral
The adherence in the consumption of taking Antiretroviral (ARV) medication in adolescents and
young adults with HIV/AIDS are often disturbed by different factors. Studies suggest that
adolescents respond poorly to highly active antiviral therapy. (Nachega et al., 2009). Factors such as
daily routine, personal mental health, negligence, and understanding of the illness shall be
addressed. Alongside the assistance of research on this topic, this essay will outline the elements
that affect the adherence to ARV treatment in young adults with HIV/AIDS.
The treatment of ARV medication in the lives of people living with HIV/AIDS is one that is the
most commonly used. The goal of the ARV therapy (ART) is to constantly suppress the replication
of HIV cells in the body, in order for the person to live a maintainable healthy life. An adolescent is
a person who is in the current development from a child to an adult. In this stage of development
adolescents are described as being in a stage of mental, physical, and emotional maturation. Some
problems they are faced with are; risk taking, an increased amount of sexual relationships,
recreational drug use, alcohol, behavioral experimentation and identity formation. (Kim, Gerver,
Fidler, & Ward, 2014). Thus adolescents are by nature still 'finding their feet' and still developing
their maturity, this increases the influence of external factors that affects their ART and therefore
increases the adherence in the consumption of their ARV's.

Introduction Of Antiviral Drugs
Antiviral Drugs
 Introduction: These are the agents which are used in the treatment and prevention of an infectious
disease caused by certain viruses. They are antimicrobial compounds like antibiotics, antifungal, ant
parasitic, etc., but they have their own different mode of action because viruses differ from bacteria
and other organisms in a way that they lack cell wall and cell membrane. They contain a core of
nucleic acid that may be DNA or RNA, surrounded by the protein coat.
Viruses are obligate intracellular parasites, which means that they cannot grow outside the host cell,
they always require a host cell for their multiplication. They use the host machinery for its
multiplication. They can ... Show more content on Helpwriting.net ...
It prevents the entry of the virus by binding to the CCR5 coreceptors, these coreceptors work
together with gp41 to promote the entry of HIV through the plasma membrane of the cell.
2. Integrase Inhibitor for HIV:
Mechanism of Action: The integrase inhibitors are also called Integrase Strand Transfer Inhibitors
(INSTIs). It inhibits the incorporation of viral DNA into the host DNA.
An enzyme called integrase help in the insertion of viral DNA into the host DNA by making a cut in
the host DNA at some points and then inserting the viral DNA into it. This enzyme has active sites
through which it makes a cut in host DNA. The inhibition process occurs when INSTIs attach to the
active sites of the enzyme, preventing the enzyme from making a cut in host DNA, therefore,
inhibiting insertion.
Dolutegravir and Raltegravir: It is an Integrase Inhibitor that works by binding to the active sites of
the enzyme and displaces one end of the DNA strand, making it impossible for the viral DNA to be
integrated into the host DNA.
Antimicrobial Spectrum: These drugs are effective against a wide range of wild–type and drug
resistance HIV, including the strains that use CCR5 or CXCR4 coreceptors for entry into the host
cell.
Drug

What Is Highly Active Antiretroviral Therapy?
Highly active antiretroviral therapy (HAART) are designed to suppress viral replication and
maintain viral load at low to undetectable levels (Prabu–Jeyabalan et al, 2002). Due to their high
intrinsic antiviral activity, the introduction of HIV–1 protease inhibitors (PIs) have led to a dramatic
decrease in morbidity and mortality rates of HIV–1 infected patients (Codoner et al, 2017). HIV–1
PIs are competitive peptidomimetic inhibitors, mimicking the natural substrate of viral HIV–1
protease, Gag and Gag–Pol polyproteins, in order to proteolytic cleavage (Fun et al, 2012).
The HIV–1 retroviral replication cycle can be divided into two phases: the early and late phases.
The early phase, which constitutes the events that occur from when the ... Show more content on
Helpwriting.net ...
Proteolytic processing of the Gag polyprotein results mature HIV–1 structural proteins: p17 matrix
(MA), p24 capsid (CA), p7 nucleocapsid (NC), p6 domains, and two spacer peptides, SP1 and SP2.
Each of these HIV–1 structural proteins play a crucial role in the HIV–1 retroviral replication cycle.
MA is responsible for targeting Gag to the plasma membrane and promoting the incorporation of
viral envelop glycoproteins into the forming virions; CA drives Gag multimerization during
assembly to form the Gag lattice that encapsidates the viral RNA genome; NC recruits the viral
RNA genome into the virions; and the p6 domain recruits the endosomal sorting complex required
for transport (ESCRT) apparatus that catalyzes the membrane fission step responsible for the
budding off of new virions from the infected cell (Freed, 2015). In HIV–1 pathogenesis, HIV–1
matrix protein p17 participates in several processes, including RNA targeting to the plasma
membrane, incorporation of the envelope into virions, and particle assembly. Furthermore, p17,
acting as a viral cytokine, can act on reactivated T–cells, promoting viral proliferation (Florentini et
al, 2006).
Due to the infidelity of HIV–1 reverse transcriptase, HIV–1 protease, as with all other HIV

Overview Of Artificial Neural Networks
Introduction Artificial neural networks are a class of computational structures (Lesk, 2013) made up
of several processing elements, called artificial neurons that are connected and organized in layers
(Larder et al., 2007). They are capable of generating models for the detection of non linear
functions(..). Their algorithms are extensively applied in biology and medicine to solve complex
problems, more specifically for prediction or classification of solutions or to refine methodological
aspects. (Florence and Balasubramanie, 2010). Human immunodeficiency virus (HIV) is a
retrovirus that can lead to acquired immunodeficiency syndrome (AIDS). (Kim et al., 2010). It is a
disease in which the body immune system weakens progressively, ... Show more content on
Helpwriting.net ...
Artificial neural networks have been used extensively as a complimentary bioinformatics tool to
make approximations of the cleavage site activity and specificity. First uses of ANNs to solve the
problem The aim of first research study was to develop a classification model that, given a sequence
of eight amino acids, could discriminate between sequences which are either cleavable or
uncleavable by the HIV– 1 protease. (Kim et al., 2010). The neural learning algorithms used most
frequently was back–propagation neural networks (BPNNs) (Thomson et al., 2003) because it
performs well on prediction problems. (Sibanda and Pretorius, 2012). When BBNN was used for the
prediction of the HIV–1 protease cleavage site, it gave a prediction accuracy 92%(Thomson et al.,
2010) However, one of the major disadvantages of using ANNs to analyse biological data referred
to the impossibility of most ANNs of recognizing non– numerical features like amino acids. Hence
an encoding process to model the amino acids was preferable. (Thomson et al., 2003) The advantage
of the Bio basic functional neural networks The peculiarity of this algorithm relates to its ability to
recognise amino acids directly. Thus, avoiding the use of 20 binary bits to represent each amino acid
is advantageous. (2003) The prediction accuracy of BBFNN was proved in a research study using
362 HIV protease sequences, where 114 were with cleavage sites and

Essay On Proteolytic Degradation Tagss
Results and Discussion
Multiple synthetic amino acid sequence tags target fluorescent proteins for degradation by different
proteases. To study proteolytic degradation by different proteases, fluorescent proteins were
engineered with different potential degradation tags on either the N–terminus or C–terminus (Table
S2). Previously tested degradation tags could not be compared directly to one another because they
were characterized in different E. coli strains and under different conditions. To our knowledge, this
is the first systematic investigation of multiple degradation tags in E. coli. We tested the previously
determined degradation tags and several newly designed tags (Table 1A–B). Degradation tags were
fused to multiple fluorescent ... Show more content on Helpwriting.net ...
The fluorescence of RepA70–YFP increased as more RepA70–CFP was produced (Fig. 3C), and
this indicated that ClpAP protease could be overloaded and a proteolytic–queue forms similar to
what was observed with the LAA tagged proteins targeted to ClpXP8. We also tested two other tags,
MarA and MarAn20 (20 amino acids from the N–terminal of MarA), which target proteins to be
degraded by the Lon protease. The Lon protease was weakly overloaded by MarA tagged proteins
but was overloaded more by MarAn20 tagged proteins (Fig. 3C–D). This made us wonder if Lon
could be overloaded when both MarA and MarAn20 were co–produced. Indeed, this was the case
(Fig. 3E).
The main proteases of E. coli can exhibit different levels of crosstalk depending on the degradation
tags used. We have demonstrated that ClpXP, ClpAP, and Lon can be overloaded using two proteins
engineered with identical degradation tags targeted for a specific protease. We have hypothesized
that crosstalk between proteases may occur through shared information (Fig. 1B). To test this
hypothesis in a synthetic system, we monitored the level of a fluorescent protein (YFP) targeted to
one protease while producing another protein (CFP) targeted to a different protease. There was
strong crosstalk when proteins with the LAA degradation tag (target to ClpXP) were co–produced
with proteins with all other tags (RepA70, MarA, MarAn20, and HipB; Fig.

Causes That Lead To PI Resistance In HIV-1
The multi–step inhibition mechanism of PIs can result in complex mutation pathways that lead to PI
resistance in HIV–1. The cause of virological failure in patients taking PIs are drug resistance
mutations outside the active site of HIV–1 protease, but rather throughout the matrix and capsid
proteins of the Gag polyprotein in HIV–1. While the mutational pathway that leads to resistance to
PIs in HIV–1 is not yet fully understood, mutations in Gag polyproteins have been shown to be
associated with changes in PI susceptibility with or without mutations in HIV–1 protease. Mutations
in Gag L449F mutation, for example, has been seen to rescue HIV–1 protease activity and increase
overall efficiency of catalytic Gag polyprotein cleavage, ... Show more content on Helpwriting.net
...
Analyzing the three–dimensional distribution of coevolving Gag residues, Codoner et al found that
the correlated residues in the matrix are clustered on the inner surface on the trimer. As these
surfaces were previously associated with interactions between gp41, a subunit of HIV–1 envelope
protein, and the matrix, Codoner et al. concluded that these mutated residues are representative of
novel interprotein interactions that modifies matrix stability in order to affect viral fusogenicity. This
is further supported by several coevolving p17 residues (residues 66, 69, and 98) were also
previously shown to be contributed to matrix stabilization and the incorporation of viral envelope
proteins into immature viral particles. Thus, Codoner et al hypothesized that coevolving residues in
HIV–1 matrix may affect HIV–1 resistance to PIs by modifying particle maturation and viral
fusogenicity (Codoner et al, 2017).
Studying the molecular basis of Gag–protease coevolution, Ozen et al. found that coevolved Gag–
protease complexes were also able to restore protease active site dynamics by increasing Gag–
protease interactions. Under selective pressures of PIs, Ozen et al. found that

Aneurysm Essay
Introduction
Despite considerable advances in surgical treatment, the ruptured human abdominal aortic aneurysm
(AAA) is still associated with a mortality rate of 65–85% [1, 2] . A diameter is currently the only
reliable determinant of the imminent rupture of an AAA and patients exceeding 5.5 cm generally
undergo surgical or endovascular intervention [2, 3] . However, the outcome of rupture is poor with
less than half of the patients being delivered to the hospital alive [1, 4] . It is well known that
inflammation and proteolytic degradation markedly contributes to the formation and rupture of an
AAA [5, 6] . During inflammation, the aortic wall is weakened by loss of smooth muscle cells
(SMCs) and destruction of the extracellular ... Show more content on Helpwriting.net ...
Hence, another study described increased expression of ADAM–17, also known as TACE (TNF– –
converting enzyme), in human AAA [18] . Especially in the transition zone, high levels of ADAM–
17 and TNF– have been detected , implementing a certain role in the development of AAA.
Therefore, the aim of the present study was to conduct an expression analysis of ADAMs with
proteolytic function [19] such as ADAMs 8, 9, 10, 12, 15 and 17, and their tissue inhibitors of
metalloprotease (TIMP)–1 and TIMP– 3 [20] in human AAA by quantitative PCR. The localisation
of the corresponding ADAMs was determined by means of immunohistochemistry.
Discussion
The development of AAA is closely associated with alternation of connective tissue in the aortic
wall, especially fragmentation of elastic fibres and collagen degradation through various proteolytic
enzymes, particularly MMPs [8, 9, 22] . Some promising drugs inhibiting MMPs have already been
shown to be successful in experimental models [23] . However, no positive results have been
observed in clinical trials. This phenomenon may be based on the fact that MMPs are not the only
proteinases able to degrade connective tissue. There is a plethora of proteolytic enzymes belonging
to the family of metalloproteinases such as ADAMs with multiple biological roles including cell–
matrix interaction, zymogen activation (shedding) and cell adhesion. These

��1at Essay
Physiological role of α1AT α1AT is an acute–phase glycoprotein that is mostly synthesized by
hepatocytes and to some extent by other cells like macrophages, lung epithelium, and monocytes. It
reaches the lungs by diffusion during systemic circulation. It provides more than 90% of the defence
against the elastolytic activity in the lower respiratory tract posed by neutrophil–elastase (NE).
Human α1AT is a hydrophilic and tissue diffusible, medium–sized (6.7×3.2 nm) circulating
glycoprotein. It has a blood half–life of 4–5 days. The human body is capable of producing
approximately 34mg kg–1 day–1 of α1AT, resulting in a high plasma concentration of 1–2 gL–1.
During acute–phase response, its levels increase up to fourfold. From circulation, ... Show more
[52–54]. Pathologically, lungs of the emphysema patients show the destruction of alveolar–wall,
diffuse–inflammation of respiratory–tract and degradation of the lung parenchyma. Investigations
point that chronic inflammation and increased oxidative stress contribute to impaired lung
maintenance and repair in emphysema. The inflammation triggered emphysema has revealed
activation of innate and acquired immune responses. The accumulation of inflammatory effectors of
the immune response contributes to lung injury in COPD patients. The recruitment of inflammatory
cells to the lungs triggers the release of inflammatory cytokines and proteases. Together, they
directly contribute to parenchymal tissue destruction and its remodelling [55]. Ordinarily, all the
proteases released during the course of inflammation in the lungs are inhibited by antiproteases
released either locally by the lung epithelium or present in the circulation. The chief inhibitors of
serine proteases are α1AT in lung parenchyma and airway–epithelium–derived secretory
leukoprotease inhibitor. At least three tissue inhibitors of matrix metalloproteinases (called TIMP–1,
TIMP–2, and TIMP–3) counteract matrix metalloproteinases. Cigarette smoking may induce
increased release of proteases that are counteracted by antiproteases to prevent lung parenchymal

Functions of Ubiquitin Specific Proteases Essay
Eukaryotic cells rely on the ubiquitination of proteins for the proper control of their internal
processes. Adding multiple ubiquitin groups aids in the breakdown of proteins, whereas adding only
one ubiquitin serves regulatory roles other than degradation. The enzymatic activity of two protease
classes frees ubiquitin groups from associated proteins: Ubiquitin specific proteases (USP) and
Ubiquitin C–terminal hydrolases (UCH). USPs are strongly involved in eukaryotic cellular
functions and are found in copious amounts. Certain protein ligases attach ubiquitin to proteins,
which degrades them and interferes with normal cellular functions. The discovery of a specific de–
ubiquitinating enzyme (HAUSP/USP7) found in humans was due to its ... Show more content on
Helpwriting.net ...
The labels associated with the resulting proteins from previous steps were rinsed and removed.
USP7 was purified and utilized in sedimentation trials in varying concentrations. Absorbance
analysis was carried out after the samples were centrifuged. EBNA1 was purified, ubiquitinated, and
combined with USP7, which was purified in previous steps. SDS–PAGE was used to disrupt the
protein interaction and separate them. The separated fragments were then used in a western blot, and
analyzed after probing. The affinity of USP7 for EBNA1 and p53 was tested using a stepwise
combination of titration, incubation, and fluorescence analysis. Lastly, amino acid residues of
EBNA1and USP7 were studied by gel filtration and size–exclusion chromatography. USP7 was
found as hexahistidine fusion after being inserted (through a vector) in insect cells. The de–
ubiquitinating capacity of USP7 for conjugated ubiquitin groups was observed on EBNA1, and was
shown to an efficient process. The capacity of USP7 to de–ubiquitinate specific proteins in various
environments (acidity, presence of inhibitors, and salt concentration) was studied. As seen in figure
2, USP7 sensitivity to salt concentration and acidity showed a steep slope followed by a plateau at
lower salt concentrations and higher pH levels, while lower pH and higher salt concentrations
rendered a steeper, less plateau–like graph. USP7 was found to be a monomer with multiple
domains, each involved

Ubiquitin Specific Protease 7 Case Study
Introduction:
The ongoing search for new anticancer and antiviral treatments has led to many new discoveries.
Ubiquitin–specific protease 7 (USP7) is an example of a protein involved in different cancer
development pathways and is also a target of many viruses belonging to the Herpesviridae family.
While ubiquitin proteins play an essential role in signaling cascades and protein degradation,
deubiquinating enzymes like USP7 interfere in these processes by cleaving the bond between the
substrate and ubiquitin. USP7 has a variety of targets including p53 (a tumor suppressing gene),
different transcription factors, and ICP0 – a protein that forms an integral part of the herpes simplex
virus–1. The aim of this study is to investigate the ... Show more content on Helpwriting.net ...
Lentiviruses that expressed shRNA were used to to infect HFF–1 cells, either in the presence of
USP7 or GFP (used as a control). These cells were then infected with HSV–1 and stained in order to
compare viral growth rates. The results showcased a 100–fold decrease in viral growth in cells that
lacked USP7 compared to those lacking GFP. Another set of experiments were done in order to
specifically determine which UBL domains played a central role in the USPL–ICP0 interaction. For
this, different UBL domains were isolated and labelled using radioactive nitrogen. These domains
were then titrated with ICP0 and an NMR spectra was used to look at binding–induced changes at
the peaks. The results showcased no changes for UBL1 and UBL3 domains, but changes in the
intensities and positions of many peaks in the labelled UBL12 spectrum. Taking the movement of
the peaks into account, it was noted that the UBL12 domain plays a pivotal role in the USPL–ICP0
interaction and the process is slow on the NMR time scale. To further locate the UBL12–ICP0
binding site, the 1H– 15N HSQC spectra of free and ICP0–bound UBL12 were compared. While the
two spectra were similar, there were many residues missing in the free UBL12 spectra that only
appeared after the binding of ICP0. This helped reveal the various conformational changes
associated with the binding of USPL with its substrate.
Discussion:
While previous studies had already characterized the structures

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