Radiation protection,protease inhibition.

Radiation Protection:
Protease Inhibition.
Dmitri Popov. PhD, Radiobiology.
MD (Russia)
Advanced Medical Technology and Systems Inc. Canada.
intervaccine@gmail.com
Jeffrey Jones (USA)
Aviation Medicine Specialist at Mercy Regional Medical Center, Professor at Baylor College
of Medicine and Fleet Logistics Support Wing Surgeon at US Navy/Marine Corps
Past: NASA/JSC

Radiation Protection: Protease Inhibitors.
• File name: Radiation Protection, Protease Inhibition..pptx
DOI: 10.13140/RG.2.1.3690.3448

• Proteases are ubiquitous in all living cells. As soon as cells are
disrupted, proteases are released and can quickly degrade any
protein. This can drastically reduce the yield of protein during
isolation and purification.
• Contaminating proteases can be inhibited by protease inhibitors,
thereby protecting the protein of interest from degradation.
• http://iti.stanford.edu/content/dam/sm/iti/documents/himc/immun
oassays/ProteaseInhibitionGuide.pdf

• A protease (also called a peptidase or proteinase) is any enzyme that
performs proteolysis, that is, begins protein catabolism by
hydrolysis of the peptide bonds that link amino acids together in
a polypeptide chain.
• Proteases have evolved multiple times, and different classes of
protease can perform the same reaction by completely
different catalytic mechanisms. Proteases can be found in
animals, plants, bacteria, archaea and viruses.
• https://en.wikipedia.org/wiki/Protease

• Proteases can be classified into seven scientific groups:
• Serine proteases - using a serine alcohol
• Cysteine proteases - using a cysteine thiol
• Aspartate proteases - using an aspartate carboxylic acid
• Threonine proteases - using a threonine secondary alcohol
• Glutamic acid proteases - using a glutamate carboxylic acid
• Metalloproteases - using a metal, usually zinc
• Asparagine peptide lyases - involve asparagine, but they are a type of
proteolytic enzymes different from those above.

• The mechanism used to cleave a peptide bond involves making an
amino acid residue that has the cysteine and threonine (proteases) or
a water molecule (aspartic acid, metallo- and glutamic acid proteases)
nucleophilic so that it can attack the peptide carboxyl group. One way
to make a nucleophile is by a catalytic triad, where a histidine residue
is used to activate serine, cysteine, or threonine as a nucleophile. This
is not an evolutionary grouping, however, as the nucleophile types
have evolved convergently in different superfamilies, and some
superfamilies show divergent evolution to multiple different
nucleophiles.

• Proteases occur in all organisms,
from prokaryotes to eukaryotes to viruses. These enzymes are involved in a
multitude of physiological reactions from simple digestion of food proteins
to highly regulated cascades (e.g., the blood-clotting cascade,
the complement system, apoptosis pathways, and the invertebrate
prophenoloxidase-activating cascade). Proteases can either break specific
peptide bonds (limited proteolysis), depending on the amino acid sequence
of a protein, or break down a complete peptide to amino acids (unlimited
proteolysis). The activity can be a destructive change (abolishing a protein's
function or digesting it to its principal components), it can be an activation
of a function, or it can be a signal in a signalling pathway.

• Proteases are used throughout an organism for various metabolic processes.
Proteases present in blood serum (thrombin, plasmin, Hageman factor, etc.) play
important role in blood-clotting, as well as lysis of the clots, and the correct
action of the immune system. Other proteases are present in leukocytes
(elastase, cathepsin G) and play several different roles in metabolic control.
Some snake venoms are also proteases, such as pit viper haemotoxin and
interfere with the victim's blood clotting cascade. Proteases determine the
lifetime of other proteins playing important physiological role like hormones,
antibodies, or other enzymes. This is one of the fastest "switching on" and
"switching off" regulatory mechanisms in the physiology of an organism.
• By complex cooperative action the proteases may proceed as cascade reactions,
which result in rapid and efficient amplification of an organism's response to a
physiological signal.

• The activity of proteases is inhibited by protease inhibitors. One example of
protease inhibitors is the serpin superfamily, which includes alpha 1-
antitrypsin, C1-inhibitor,antithrombin, alpha 1-
antichymotrypsin, plasminogen activator inhibitor-1, and neuroserpin.
• Natural protease inhibitors include the family of lipocalin proteins, which
play a role in cell regulation and differentiation. Lipophilic ligands, attached
to lipocalin proteins, have been found to possess tumor protease inhibiting
properties. The natural protease inhibitors are not to be confused with
the protease inhibitors used in antiretroviral therapy.
Some viruses, with HIV/AIDS among them, depend on proteases in their
reproductive cycle.
Thus, protease inhibitors are developed as antiviral means.

• Venoms.
• Certain types of venom, such as those produced by venomous snakes,
can also cause proteolysis. These venoms are, in fact, complex
digestive fluids that begin their work outside of the body. Proteolytic
venoms cause a wide range of toxic effects, including effects that are:
• cytotoxic (cell-destroying)
• hemotoxic (blood-destroying)
• myotoxic (muscle-destroying)
• hemorrhagic (bleeding)
https://en.wikipedia.org/wiki/Proteolysis#Venoms

• Inhibitors of Serine Protease.
• Inhibitors of Cysteine Protease.
• Inhibitors of Metallo-Proteases.
• Inhibitors of Aspartic Proteases.

• Radiats Biol Radioecol. 2011 May-Jun;51(3):328-36.
• [Effect of gamma-radiation on the activity of proteases associated with spleen and brain nuclear
histones of young and old rats].
• Kutsyĭ MP.
It has been found that proteases specifically splitting histones are associated with histones from
spleen and brain nuclei of 4- and 26-month-old rats. The activity ofproteases isolated together with
histones increases after irradiation of rats with 10 Gy The activation degree of these proteases
depends on the animal age and postradiation period.
Activation of histone-associated proteases by means of gamma-radiation is more pronounced in
spleen nuclei from old rats than from the young ones. Irradiation of animals has been found to
reduce histone H1 and core histone contents in the spleen and brain nuclei of both young and old
rats. The radiation-induced proteolysis ofhistone H1 and core histones in spleen and brain nuclei
leads to chromatin deconden-sation and DNA degradation by nucleases.
The activity of histone-associated proteases is substantially higher in the nuclei of intensively
proliferating spleen cells than in the brain nuclei. The experimental data indicate that histone-
associated proteases participate in the regulation of DNA transcription, replication, and
degradation.

• THE intravenous injection of a vital dye (pontamine sky blue, 1.2 ml. of 5 per cent
solution/kg body-weight) immediately after local irradiation of a rabbit's skin (1,000 r.,
60–140 kV) is followed within a few minutes by an intense blueing of the irradiated area,
allowing the investigation of tissue events hitherto unnoticed in the so-called latent
period. This circumscribed leakage of dye in the early phase of inflammation is abolished
if the animals are pretreated with total or partial body irradiation (400 r., 230 kV, half-
value layer = 1.8 mm Cu) or systemically with alkylating agents, and also with softer
radiations used in order to avoid effects on deep-seated organs. This unresponsiveness is
temporary, as the restoration of local reactivity occurs within six days in the experimental
conditions described
• Proteases and the Depletion and Restoration of Skin Responsiveness to Radiation
• B. JOLLES & R. G. HARRISON
• Department of Radiotherapy, General Hospital, Northampton.

• LITTLE is known about the mechanisms of carcinogenesis. The fact that most carcinogens are
mutagenic has led to speculation that the primary step in cancer induction may be mutational; there is
evidence from both in vivo and in vitro studies that a strong correlation exists between the
mutagenicity and carcinogenicity of an agent. Mutagenic and carcinogenic agents, both physical and
chemical, also produce similar kinds of DNA damage and repair.
• Radiation-induced mutagenesis in some bacterial cells requires an error-prone DNA repair system, and
there is now some evidence that error-prone DNA repair may be involved in the malignant
transformation of cells by radiation. Protease inhibitors have been shown to suppress specifically both
error-prone repair and mutagenesis in bacterial cells , as well as to inhibit carcinogenesis in vivo .
• We report here that the protease inhibitors antipain and leupeptin will suppress radiation-induced
transformation in vitro as well as inhibit two-stage transformation in vitro using radiation and the
promoting agent, 12-O-tetradecanoyl-phorbol-13-acetate (TPA).
• Protease inhibitors suppress radiation-induced malignant transformation in vitro
• ANN R. KENNEDY & JOHN B. LITTLE
• Laboratory of Radiobiology, Department of Physiology, Harvard University, School of Public Health,
Boston, Massachusetts 02115

• A lysosome (derived from the Greek words lysis, meaning "to loosen",
and soma, "body") is a membrane-bound cell organelle found in most
animal cells (they are absent in red blood cells). Structurally and
chemically, they are spherical vesicles
containing hydrolytic enzymes capable of breaking down virtually all
kinds of biomolecules, including proteins, nucleic acids,
carbohydrates, lipids, and cellular debris. They are known to contain
more than 50 different enzymes, which are all optimally active at an
acidic environment of about pH 5.
http://en.wikipedia.org/wiki/Lysosome

• Cysteine proteases, also known as thiol proteases, are enzymes that
degrade proteins. These proteases share a common catalytic
mechanism that involves a nucleophilic cysteine thiol in a catalytic
triad or dyad. The first step in the reaction mechanism by which
cysteine proteases catalyze the hydrolysis of peptide bonds is de
protonation of a thiol in the enzyme's active site by an
adjacent amino acid with a basic side chain, usually a histidine
residue.

• Example of Inhibitors of proteases.
• Classes of Protease Inhibitors available from Roche Applied Science

• When isolating or purifying proteins, benefit from the ultimate in
convenience – use complete Protease Inhibitor Cocktail Tablets and
eliminate the time consuming search for the right protease inhibitor.
complete is a proprietary blend of protease inhibitors, formulated as
a ready-to-use water soluble tablet.
• Simply add the convenient tablet to your homogenization buffer, and
instantly protect your proteins against a broad range of proteases.

• Consistently inhibit a multitude of protease classes, including serine
proteases, cysteine proteases, and metalloproteases.
• Inhibit proteolytic activity in extracts from almost any tissue or cell
type, including animals, plants, yeast, bacteria, and fungi.

• Deliver consistent doses of protease inhibition.
• Obtain stable, non-toxic protection in aqueous buffers.
• Maintain the stability of metal-dependent proteins, and function of
purification techniques (i.e., IMAC [immobilized metal affinity
chromatography] for isolation of Poly-His-tagged proteins) by using
EDTA-free complete Protease Inhibitor Tablets.

• Ewing's sarcoma (ES) cells express high levels of poly(ADP-ribose) polymerase (PADPRP)
and are responsive to killing by ionizing radiation. We have determined that ionizing
radiation induced a pronounced but reversible G2-M phase cell cycle arrest that was
maximum by 24 h after exposure. Following the release from this block, floating cells
began to appear. We found that apoptosis is a significant component of radiation-
induced death in ES cells and that this is accomplished in conjunction with proteolytic
cleavage of PADPRP. Two fragments of M(r) 25,000 and M(r) 29,000 containing the
PADPRP DNA-binding domain were identified in floating (apoptotic) cells, whereas only
the full-length M(r) 116,000 native protein was detected in adherent cells that retained
DNA intact. These data are consistent with PADPRP cleavage being an early step in the
apoptotic cascade of biochemical events in ES cells after ionizing radiation exposure.
Cancer Res. 1995 Oct 1;55(19):4240-2.
• Radiation-induced apoptosis of Ewing's sarcoma cells: DNA fragmentation and
proteolysis of poly(ADP-ribose) polymerase.
• Soldatenkov VA1, Prasad S, Notario V, Dritschilo A.

• Successful management of brain tumors prolongs life, raising the risk of delayed injury
secondary to the treatment. Radiation therapy, a mainstay of brain tumor treatment, can
damage the cerebral blood vessels. Acutely a breakdown of the blood–brain barrier
(BBB) may be seen, but fibrosis complicates radiation injury in the chronic phase. Matrix
metalloproteinases (MMPs) and plasminogen activators are two matrix-degrading
proteolytic enzymes, which are induced by radiation. They disrupt the basal lamina
around cerebral capillaries and open the BBB. We report a patient with an astrocytoma
managed by partial resection and external beam irradiation to maximal tolerable doses.
The patient later developed malignant brain edema shortly after stereotactic
radiosurgery.
• “Radiation-induced Blood–brain Barrier Damage in Astrocytoma: Relation to Elevated
Gelatinase B and Urokinase” Article Journal of Neuro-Oncology
• September 1999, Volume 44, Issue 3, pp 283-289.
• John C. Adair et al. Affiliated withNeurology Service, Albuquerque Veterans Medical
CenterDepartment of Neurology, University of New Mexico School of Medicine

• Tissue obtained during surgical debulking to control the edema showed very high
levels of gelatinase B (92 kDa type IV collagenase) and urokinase-type
plasminogen activator (uPA). Tumor cells were absent from the biopsy and
subsequent autopsy specimens, but necrosis with fibrosis of the blood vessels
was seen. If abnormal matrix enzyme function participates in the expression of
radiation injury, then inhibitors to such enzymes may provide one strategy for
controlling cerebrovascular damage after therapeutic brain radiation.
• “Radiation-induced Blood–brain Barrier Damage in Astrocytoma: Relation to
Elevated Gelatinase B and Urokinase” Article Journal of Neuro-Oncology
• September 1999, Volume 44, Issue 3, pp 283-289.
• John C. Adair et al. Affiliated withNeurology Service, Albuquerque Veterans
Medical CenterDepartment of Neurology, University of New Mexico School of
Medicine

• Radiation necrosis, a focal structural lesion that usually occurs at the
original tumor site, is a potential long-term central nervous system
(CNS) complication of radiotherapy or radiosurgery. Edema and the
presence of tumor render the CNS parenchyma in the tumor bed
more susceptible to radiation necrosis. Radiation necrosis can occur
when radiotherapy is used to treat primary CNS tumors, metastatic
disease, or head and neck malignancies. It can occur secondary to any
form of radiotherapy modality or regimen.
• http://emedicine.medscape.com/article/1157533-overview

• In the clinical situation of a recurrent astrocytoma (postradiation
therapy), radiation necrosis presents a diagnostic dilemma.
• Astrocytic tumors can mutate to the more malignant glioblastoma
multiforme.
• Glioblastoma multiforme's hallmark histology of pseudopalisading
necrosis makes it difficult to differentiate radiation necrosis from
recurrent astrocytoma using MRI.
• See Medscape Reference articles Neurologic Manifestations of
Glioblastoma Multiforme and Low-Grade Astrocytoma.
• http://emedicine.medscape.com/article/1157533-overview

• Cerebral radiation necrosis refers to necrotic degradation of brain tissue
following intracranial or regional radiation.
• The brain is exposed to therapeutic radiation both for treatment of intracranial
pathology (e.g. astrocytoma, cerebral arteriovenous malformation) and is also a
bystander in irradiation of head and neck tumours (e.g. nasopharyngeal
carcinoma). Post-radiation treatment effects can be divided into 3:
• Pseudoprogression: It is related to endothelial damage and consequent
tissue hypoxia observed several weeks up to months after radiation treatment
• Radiation necrosis: may appear months to several years after radiation therapy
and involves a space occupying necrotic lesion with mass effect and neurological
dysfunction
• http://radiopaedia.org/articles/cerebral-radiation-necrosis-1

• Pathology of Radiation Induced Necrosis.
• There are numerous potential pathways to radiation necrosis which include:
• vascular injury
• acutely endothelial damage can lead to vasogenic oedema
• chronically fibrosis, hyalinization and stenosis can occur with eventual thrombosis and infarction
• vascular ectasia, and telangiectasia are also seen frequently, with capillary telangiectasias and cavernous malformations
common findings post whole brain irradiation.
• oligodendrocytes and white matter damage
• oligodendrocytes are sensitive to radiation
• loss of white matter accounts for the majority of volume loss
• effects on the fibrinolytic enzyme system
• increase in urokinase plasminogen activator and simultaneous decrease in tissue plasminogen activator may contribute to
cytotoxic oedema and tissue necrosis
• immune mechanisms
• http://radiopaedia.org/articles/cerebral-radiation-necrosis-1

• Some examples of cells, tissues, and organisms in which protease
activity has been successfully inhibited with complete tablets — as
reported in scientific literature:
• ■ Acintobacillus actinomycetemcomitans ■ Adipocytes (mouse, rat) ■
Adrenal gland (PC-12, rat) ■ Bladder carcinoma cells (T24, human) ■
Bone marrow cells (mouse, human) ■ Bone osteosarcoma (U-2 OS,
SaOs-2, human) ■ Brain neuroblastoma cells (SK-N-BE(2), human) ■
Brain tissue (bovine, mouse, rat, human) ■ Breast cancer cells (BT20,
MCF7, human) ■ Bronchial Alveolar Lavage Fluid (mouse, rat)

• Bronchial Biopsies (human) ■ Bronchial epithelial cell line (BZR,
human) ■ Cardiomyocytes (mouse, rat) ■ Cervix adenocarcinoma
(HeLa, human) ■ Cochlea (rat) ■ Colon carcinoma cells (T84, human)
■ Colorectal adenocarcinoma cells (CaCo-2) (human) ■ Colorectal and
duodenal adenomas ■ Colorectal carcinoma cells (HCT-116, human) ■
Cortex (rat). ■ Dictyostelium (amoeba) ■ E. coli ■ Endothelial cell line
■ Epidermis (human) ■ Epithelial cell lines (human, bovine) ■ Fat
(mouse) ■ Fibroblasts (human; NIH-3T3, MDTF, mouse) ■
Fibrosarcoma cell line (HT1080, human) ■ Fruit (tomato) ■
Glioblastoma cell line (U87MG) ■ Head (Drosophila).

• ■ Heart (human, mouse, chicken) ■ Hematopoietic cell lines (mouse,
human) ■ Immature seed (soy) ■ Insect cell lines (Sf2, Sf21, Sf9, Tn5)
■ Keratinocytes (human) ■ Kidney (dog, human, mouse, rat, monkey,
Xenopus) ■ Leaf (Arabidopsis) ■ Liver carcinoma cells (HepG2, Hep3B,
human) ■ Liver tissue (mouse, rat, Xenopus) ■ Lung carcinoma cells
(A549, human) ■ Lung homogenates (mouse, Xenopus) ■ Lung lavage
fluid (mouse) ■ Luteal tissue (bovine) ■ Lymph nodes (mouse) ■
Lymphoblastoids (human) ■ Lymphocytes (Jurkat, human; WEHI 3b D,
mouse; monkey)

• ■ Peripheral blood cells (BA/F3, mouse; CEM, HL-60, human) ■ Pichia
pastoris ■ Placental labyrinth (mouse, rat) ■ Platelets (human) ■ Primary
chondrocytes (human) ■ Primary lung cancer cells ■ Primary mast cells
(mouse) ■ Primary neuronal cultures (mouse) ■ Prostate adenocarcinoma
cells (PC-3, human) ■ Prostate carcinoma cells (DU-145 and LNCaP, human)
■ Pseudomonas ■ Rectal tissue (rabbit).
• Renal cell carcinomas (human) ■ Reticulocyte lysate (rabbit) ■ Retina
(mammalian) ■ Saccharomyces cerevisiae ■ Salivary gland (mouse) ■
Salmonella typhimurium ■ Seed (Arabidopsis) ■ Skin (human) ■
Spermatogenic cells (mouse) ■ Spinal cord (rat) ■ Spleen (mouse, rat,
Xenopus) ■ Staphylococcus aureus ■ Streptococcus pneumoniae ■
Superior cervical ganglion (mouse) ■ Toxoplasma gondii ■ Umbilical vein
endothelial cells (HUVEC, human) ■ Whole plant tissue

• Mammary carcinoma cells (MDA468, human) ■ Mammary epithelial
cells (HMEC) ■ Mammary gland (mouse) ■ Mast cell line (human) ■
Monocyte cells (THP-1, human) ■ Muscle (Drosophila, human,
mouse, rat, rabbit, Xenopus) ■ Neisseria gonorrhoeae ■ Neurons (rat)
■ Ovarian cancer (OVCAR-3, human) ■ Ovary cells (CHO, hamster) ■
Pancreas (mouse) ■ Parathyroid tissue (bovine)

• USING PROTEASE BIOMARKERS TO MEASURE VIABILITY AND
CYTOTOXICITY ANDREW NILES, MICHAEL SCURRIA2, LAURENT
BERNAD, BRIAN MCNAMARA, KAY RASHKA, DEBORAH LANGE, PAM
GUTHMILLER1 AND TERRY RISS , PROMEGA CORPORATION,
PROMEGA BIOSCIENCES, INC.

• Diagnosis of acute radiation disease by Enzyme Immune-Assay (EIA)
• International Nuclear Information System (INIS)
• Diagnosis of the acute radiation disease by the method of immune enzyme
assay is a simple and efficient tool of evaluating and biological dosimetry
and forecasting of development of the acute radiation defeats as at group
of population so at individuals locating in the zone polluted by
the radiation. We use as biological markers the group of essential
radiotoxins - high molecular mass glycoprotein ( molecular mass - 200 - 250
kDa ) - radiation antigens (S.D.R. - specific radiation determinant )
accumulated in the lymphoid system, with epitopes specific to each form
of radiation syndrome, after animals have been irradiated in doses inducing
the development of the cerebral (1), toxic ( 2), gastrointestinal ( 3 ) and
typical ( 4 ) forms of acute radiation sickness.

• These two phenomena allowed us to develop a technologies for diagnosis,
prophylaxis and therapy of radiation disease - enzyme immune assay ( EIA ),
anti radiation vaccine, anti radiation serum, method of immune - lymph - plasma-
sorption. The important first step in effectiveness of therapy is an accurate
assessment of severity of disease in early period after irradiation. The ideal
markers for early and accurate assessment is high weight glycoprotein with
specifics radiation induced features (S.D.R.) mentioned above. This biology active
substance isolated from lymph can induct the symptoms of radiationsyndrome
without previously radiation when it is administrated intra-muscularly or
intravenously to healthy animals. Enzyme immune assay (EIA) allowed
researchers to indicate the significant levels of different forms of S.D.R. in
peripheral blood of animals in first 24 hours after radiation. Indication of high
level of S.D.R. -1 allowed to forecast a fast development of cerebral form
of acute radiation disease. Determination of high levels of S.D.R.-2, S.D.R.-3 and
S.D.R.-4 in peripheral blood allowed to recognize early periods of toxic,
gastrointestinal and typical forms of acute radiation sickness.)

• Recognition of significantly high levels of S.D.R.-4 is important for
assessing radiation risks of mild typical lradiation diseases (S.D.R.-4/1), of moderate
typical radiation diseases (S.D.R. -4/2), of severe typical radiation diseases (S.D.R.-4/3), of
extremely severe typical radiation diseases (S.D.R.-4/4).
• The important goal of early assessment with enzyme immune assay is the accurate
description of started disease and most effectively managed therapy. The S.D.R. EIA kit is
a complete kit for the quantitative determination of different forms and levels of S.D.R. -
1, S.D.R.-2, S.D.R.-3, S.D.R.-4 in a serum. This kit is a solid phase sandwich ELISA using 4
kinds of high specific antibodies. Visual assessment utilizes a 4 point scale (++++). The
test was considered positive if the assessment was (++) or higher. Positive test allowed us
to detect the presence and severity of radiation injury by identifying S.D.R. forms and
each from them was specific for different radiation energy and depended on a volume of
absorbed doses of radiation. (authors)
• http://worldwidescience.org/topicpages/a/acute+radiation+disease.html

Radiation protection,protease inhibition.

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