This document discusses subcellular fractionation, which is the process of separating intact organelles from homogenized cells and tissues using differential centrifugation. It begins by introducing the cell and its organelles. It then covers the history of the technique, methods for homogenizing cells, and the two main centrifugation methods - differential and density gradient centrifugation. Marker enzymes are also discussed as a way to identify isolated organelles. The summary provides an overview of the multi-step centrifugation process and identifies marker enzymes for different organelle fractions.
Centrifugation principle and types by Dr. Anurag YadavDr Anurag Yadav
concept of cnetrifugation,
basic Principle
centrifugal force
types of centrifugation based on use and rotor type
application of the each type of centrifuge
Ultracentrifuge in detail
application in general
wo-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell and Klose in 1975.
Centrifugation principle and types by Dr. Anurag YadavDr Anurag Yadav
concept of cnetrifugation,
basic Principle
centrifugal force
types of centrifugation based on use and rotor type
application of the each type of centrifuge
Ultracentrifuge in detail
application in general
wo-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell and Klose in 1975.
PAGE is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels use as the support matrix.
widely used and has very much importance.
COMPLETE PROCEDURE & USES are described in the slide.
Active sites of the enzyme is that point where substrate molecule bind for the chemical reaction. It is generally found on the surface of enzyme and in some enzyme it is a “Pit” like structure
The active site is a three-dimensional cleft formed by groups that come from different parts of the amino acid sequence
The active site takes up a relatively small part of the total volume of an enzyme
Active sites are clefts or crevices
Substrates are bound to enzymes by multiple weak attractions.
The specificity of binding depends on the precisely defined arrangement of atoms in an active site.
Isolation of organelles is accomplished by cell membrane lysis and density gradient centrifugation to separate organelles from contaminating cellular structures. Intact nuclei and organelles have distinctive sizes in mammalian cells, enabling them to be separated by this method.
PAGE is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels use as the support matrix.
widely used and has very much importance.
COMPLETE PROCEDURE & USES are described in the slide.
Active sites of the enzyme is that point where substrate molecule bind for the chemical reaction. It is generally found on the surface of enzyme and in some enzyme it is a “Pit” like structure
The active site is a three-dimensional cleft formed by groups that come from different parts of the amino acid sequence
The active site takes up a relatively small part of the total volume of an enzyme
Active sites are clefts or crevices
Substrates are bound to enzymes by multiple weak attractions.
The specificity of binding depends on the precisely defined arrangement of atoms in an active site.
Isolation of organelles is accomplished by cell membrane lysis and density gradient centrifugation to separate organelles from contaminating cellular structures. Intact nuclei and organelles have distinctive sizes in mammalian cells, enabling them to be separated by this method.
Common Approaches for Centrifugation in the Laboratory.pdfRWDLifeScience
Cell centrifugation is an indispensable step in common cell experiments. It not only allows adherent samples to settle completely at the bottom of the tube, but also helps confirm cell concentration. The centrifuged samples can be used for subsequent experiments such as primary cell culture, cell passage culture, single cell sequencing, etc.What are the common approaches for cell centrifugation? Check out the detail steps for centrifugal separation and learn more.
Gene Therapy, Somatic cell gene therapy, germ line gene therapy, classical gene therapy, non-classical gene therapy, targets of gene therapy, barriers of gene therapy, ex vivo gene therapy, in vivo gene therapy, vectors for gene delivery, antisense therapy
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
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Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
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Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
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Testicular torsion refers to twisting of the spermatic cord, causing ischaemia of the testicle.
Testicular torsion results from inadequate fixation of the testis to the tunica vaginalis producing ischemia from reduced arterial inflow and venous outflow obstruction.
The prevalence of testicular torsion in adult patients hospitalized with acute scrotal pain is approximately 25 to 50 percent
3. I. Introduction
Cell is the structural and functional unit of life.
Cell contain organelles which perform a variety of specific
functions.
Electron micrographs explains only the structure but not
functions of the cell organelles.
To obtain precise information about the cell organelles, it is
necessary to isolate them free from contaminating organelles.
4. Contd
Hence subcellular fractionation using centrifugation technique
is used.
Individual organelle is identified using specific markers.
This information is used to study potential cellular
irregularities and methods to correct them.
5. II. History
Albert Claude in 1930 developed the
technique of cell fractionation &
identified the different organelles using
the technique of centrifugation. He
received Nobel Prize for the same in
1974.
7. Methods of homogenization
Homogenizers fall broadly into two categories on the basis of
disruptive forces:
Type 1 homogenizers: The material is subjected to the
disruptive force once.
Eg: The French press is commonly used in the laboratory
for the homogenization of microorganisms particularly
those with a tough outer wall.
8. Type 2 homogenizers: The material is repeatedly or
continuously exposed to the disruptive force.
1. Liquid Shear
2. Mechanical Shear
3. Sonication
4. Osmotic lysis
5. Other abrasive methods
9. Homogenization of cells and
tissues
Homogenization just means to “break open”.
Homogenization is concerned only with rupture of the
surface membrane.
The principal aim is to achieve highest degree of cell
breakage using the minimum of disruptive forces without
damage to any of the organelles of interest.
11. 1. Bead Mill
• It consists of a jacketed chamber with
a rotating shaft, running in its centre.
• Agitators are fitted with the shaft
which provide kinetic energy to the
small beads present in the chamber.
• Glass or ceramic beads often used to
crack open cells.
• The choice of bead size and weight is
greatly dependent on the type of cells.
12. •Disruption takes place due to the grinding action of the rolling beads and
the impact resulting from the cascading ones.
•Bead milling can generate enormous amounts of heat.
•Cryogenic bead milling : Liquid nitrogen or glycol cooled unit.
•Application: Yeast, animal and plant tissue.
•Small scale: Few kilograms of yeast cells per hour.
•Large scale: Hundreds of kilograms per hour.
13. 2. Ultra Sonication
•Ultrasonic homogenizers work by inducing
vibration in a titanium probe that is
immersed in the cell suspension.
• A process called cavitation occurs, in which
tiny bubbles are formed and explode,
producing a local shockwave and disrupting
cell walls by pressure change.
• This method is very popular for plant and
fungal cells but comes at a disadvantage: It’s
very loud and has to be performed in an
extra room.
•Used in conjunction with chemical methods
14. 3. French Press
•Primary mechanism: High shear
rates within the orifice
•Secondary mechanism:
Impingement
•Operating pressure: 10,000 to
50,000 psi
•Application: Small-scale recovery
of intracellular proteins and DNA
from bacterial and plant cells
15. 4. Thermolysis
More common method in large scale release of proteins from
cells.
Cells are exposed to high temperature shocks for short
durations immediately followed by longer exposure to lower
values in presence of buffer (1 mM MgCI2, 10 mM Tris-HCI pH
7.4)
Periplasmic proteins in Gram Negative bacteria are released
when the cells are heated up to 50ºC.
Cytoplasmic proteins can be released from E.coli within 10min
at 90 ºC.
17. By changing the solute concentration of the liquid surrounding
the cell.
Through the process of osmosis, water can be moved into/out
the cell causing its volume to increase/decrease to the point
that results in cell bursts.
Note that this method can only work with animal cells and
protozoa, since they do not have cell walls.
18. 6. Chemical Solvents
Often used with plant cells, organic solvents such as toluene, ether,
benzene, methanol, surfactants, and phenyl ethyl alcohol, DMSO (Dimethyl
sulphoxide) can be used to permeate cell walls.
EDTA can be used specifically to disrupt the cell walls of gram negative
bacteria, whose cell walls contain lipopolysaccharides that are stabilized by
cations like Mg2+ and Ca2+. EDTA will chelate the cations leaving holes in
the cell walls.
This method can be used with wide range of production organisms but the
problem can be that some proteins are denatured.
19. 7. Detergents
• Directly damage the cell wall or
membrane, and this will lead to
release of intracellular content.
• Its mechanism of action is to
solubilize membrane proteins.
• Detergents can be anionic, cationic
and non-ionic detergents.
• Most commonly used anionic
detergent is sodium dodecyl sulfate
(SDS) which reorganizes the cell
membrane by disturbing protein-
protein interactions.
20. 8. Enzymes
• Enzymes degrade the cell wall
components which will lead to
release of intracellular compounds.
• Enzymes that are commonly used
for degradation of cell wall of plants,
yeast and fungi include cellulases,
pectinases, xylanases and
chitinases.
• The enzyme’s high price and
limited availability limits their
utilization in large scale processes.
22. The aim of subcellular fractionation is to separate organelles
with as little damage as possible.
The methods of separation of cell organelles differ from tissue
to tissue.
Approaches of subcellular fractionation:
1. Size: Difference in size result in differences in rate of
sedimentation.
Problem: Individual mitochondria and microsomes derived from
different membrane systems are similar in size.
2. Surface Charge – Very limited use in practice
3. Density – Currently the most useful property for separation of
cell organelles.
23. Factors affecting organelle density
and size
Density = Mass___
Volume
Neither mass nor volume of cell organelle are necessarily
constant.
The easiest method for separating cell organelles according to
their density is to form a concentration gradient of some suitable
material, known as density gradient solute.
A particle suspended in a liquid of its own density neither floats
nor sinks whatever the centrifugal field is applied.
25. Principle of centrifugation
Particles suspended in a solution are pulled downward by
Earth’s gravitational force.
The centrifuge works using the sedimentation principle,
where the centrifugal acceleration causes denser substances
and particles to move outward in the radial direction.
In a solution , particles whose mass or density is higher than
that of the solvent sink or sediment and particles that are
lighter than it float on the top.
Centrifugal force = mω2r
ω is the angular velocity
r is the distance from the centre of rotation
26. Centrifugal methods for the
separation of organelles
1. Separation by size – Differential centrifugation
2. Separation by density – Density gradient
centrifugation
27. Differential Centrifugation
Differential centrifugation separates particles based on
difference in sedimentation rate, which reflect
differences in sizes and densities.
Steps:
1. Preparation of broken cells is poured in a centrifuge tube.
2. The preparation is initially centrifuged at low speeds to
completely sediment the largest and heaviest sub-cellular
component.
3. The supernatent is carefully decanted and is again
centrifuged at a higher speeds till the desired portion of cell
lysate is obtained.
28.
29. Density Gradient Centrifugation
Density gradient centrifugation is a variation of
differential centrifugation in which the sample is
centrifuged in a medium that gradually increases in
density from top to bottom.
Two types of density gradient centrifugation are:
1. Rate Zonal Centrifugation
2. Isopycnic Centrifugation
30. 1. Rate Zonal Centrifugation
The particles are separated according to their size, shape, and
density or the sedimentation coefficient(s).
Methods used for preparation of density gradients are
sucrose, glycerol, ficoll etc. 5-20% sucrose solution is
commonly used to form density gradient.
The sample is applied in a thin zone at the top of the
centrifuge tube on a density gradient.
If the density of the particle at any point in the gradient is
same to that of the gradient, then these particles will stop
otherwise it will move downward towards more denser region.
Separation of particles depends on the duration of
centrifugation.***
31.
32. 2. Isopycnic Centrifugation
Isopycnic centrifugation separates the particles solely on
the basis of buoyant density.
Cesium chloride is used as a density gradient.
This technique is used to separate particles of similar size
but different densities.
It is also independent of time of centrifugation.
Sedimentation of particles occur until the buoyant
density of particle is similar to the density of the
gradient.
35. An enzyme that is known to be localized exclusively in the
particular organelle.
Examples-Acid phosphatase in lysosomes; Succinate
dehydrogenase in mitochondria.
By monitoring where each enzyme activity is found during a
cell fractionation protocol, one can monitor the fractionation
of organelle protocol.
Marker enzymes also provide information on the biochemical
purity of the fractionated organelles. The presence of
unwanted marker enzyme activity in the preparation indicates
the level of contamination by other organelles.
36.
37.
38. Summary
Homogenate
[in isotonic buffer]
(Centrifuge at
2000*g for 10 min)
Pellet
[Nuclei]
(Marker Enzyme – DNA
Polymerase)
Supernatent
(Centrifuge at 10,000*g
for 20 min)
Pellet
[Mitochondria]
(Marker - Succinate
dehydrogenase and
Monoamine oxidase)
Supernatent
[Post Mitochondrial
Supernatent]
(Centrifuge at
1,05,000*g for 4 hrs)
Pellet
[Microsome]
(Plasma membrane, ER,
Golgi, Lysosome,
Peroxisome)
Supernatent
[Cytosol containing
ribosomes and other
macromolecules]
39. References
Subcellular fractionation - A Cellular Approach by J.M. Graham
and D. Rickwood, Oxford University Press
Wilson & Walker - Principles and Techniques of Biochemistry
and Molecular Biology, 7th Edition
Stryer Biochemistry, 8th Edition
Textbook of Biochemistry – DM Vasudevan, 8th Edition