LIFE227

Apoptosis

- Cell shrinkage and fragmentation.

- Nuclear condensation.

- Internucleosomal DNA fragmentation.

- Loss of phospholipid assymetry in the membrane.

- Detatchment and engulfment by phagocytosis.

Necrosis

- Cell swelling and lysis.

- Karyolysis.

- Random DNA breaks.

-  Loss of plasma membrane integrity and recruitment of inflammatory cells.

Cell culture refers to the removal of cells from a live organism and keeping these cells living in a favourable artificial environment.

Cells can be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation. They may also be derived from a cell line or cell strain which has already been established.

Cells taken from tissues are known as primary cells and will stop growing when they run out of space. This is known as contact inhibition. 

Cells taken from a tumour cell are of continous cell line and will never stop growing. 

Model Systems: Studying basic biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of ageing and nutritional studies.

Cancer Research: Study of the function of various chemicals, virus and radiation to convert normal, cultured cells to cancerous cells. 

Toxicity Testing: Study the effect of new drugs.

1. Label the plate.

2. Add PBS and prepare serial dilution.

3. Add PBS and the drug dilution.

4. Mix.

5. Incubate at 37C for 1 hour.

6. Add MTT labelling reagent.

7. Incubate at 37C for 3 hours. 

8. Add solubilisation reagent and mix.

9. Mix.

10. Incubate at 37C overnight.

1. Remove MCF-7 cells from cell culture using trypsin.

2. Add exclusion dye and mix.

3. Place solution into haemocytometer.

4. Focus with microscope. 

5. Count cells.

6. Perform calculations.

1. Make agarose gel.

2. Insert gel comb into casting tray.

3. Pour agarose gel into tray.

4. Remove sample comb once set.

5. Add TAE.

6. Add gel loading buffer to DNA samples.

7. Mak DNA ladder.

8. Load DNA samples into tray.

9. Attach gel tank to electricity.

Bacteria have limited morphological features but extensive physiological diversity which enables habitisation of almost every environment.

In order to identify bacteria, it is necessary to examine their biochemical behaviour using a range of tests. 

Simple biochemical tests are supplemented with data derived from studies of nucleic base composition and sequence.

Clinicians need to figure out which microbes are likely to be responsible.

They will take a case history which may provide important diagnostic clues. 

Antibiotic Resistance: Characterising a microbes antibiotic resistance profile is importnant. Understanding which antibiotics are effective will help clinicians avoid prescribing an inappropriate drug.

Pathogen-Specific Disesae Complications: Some diseases have serious complications that are common to a specific organism. Knowing the causative agent allows prompt treatment. 

Tracking the Spread of Disease: If in an outbreak of infection, a lab identifies the same strain of bacteria in each case, public health officials can then investigate to find a common source/link to stop further spread.

- Morphology

- Biochemical tests

- Resistance

- Molecular methods

- MALDI-TOF mass spectrometry

1. Place drop of water onto slide.

2. Add bacteria using a sterile loop.

3. Heat fix. 

4. Flood with crystal violet for 30 seconds.

5. Wash with tap water.

6. Flood with iodine for 1 minute.

7. Wash with tap water.

8. Wash with acetone for 2-3 seconds.

9. Wash with tap water.

10. Observe slide.

Arabidopsis is able to generate rapidly, self-fertilise and has a small genome. Its small genome size led to it being one of the first organisms to have a fully sequenced genome.

There are two methods which can be used to study the role of auxin:

- Identify mutants that are defective in response to auxin.

- Create transgenic plants that contain reporter genes whose expression will directly respond to auxin. 

When wild type plants are grown in the presence of auxin, the roots won't grow. 

In a mutant plant which is unable to respond to auxin, root growth won't be inhibited as strongly.

Aux1 (auxin response mutant) protein acts as an auxin carrier. Therefore, in the absence of the function of this aux1 carrier, a lower amount of auxin is transported into each cell. This reduces auxin-induced gene expression.

A reduction in gene expression signals to the roots to reduce cell elongation leading to the same scenario where auxin won't be seen in the root cells of aux1 mutants. Therefore, the roots stay long.

The second way to study the auxin response involves the stable integration of a visible reporter gene that will respond to changes in auxin concentration. 

IAA2p::GUS is an example of a synthetic transgene construct.

The transgene has two components:

     - The promoter region from the IAA2 which responds to auxin.

     - The GUS reporter gene which encodes an enzyme that produces a blue product. 

Therefore, when wild type plants are grown in the presence of auxin, there will be an increase in the blue colour that is produced. 

However, aux1 mutants carrying the same IAA2p::GUS reporter gene will not respond in the same way to auxin. This means the induction of the IAA2p::GUS reporter gene will be reduced in the aux1 mutant relative to the wild type.

- A widely used model organism.

- First plant genome to be sequenced (in 2000).

- Short life-cycle.

- Prolific seed production.

- Easily prepared in small spaces.

- Easily transformed.

- A large number of mutants available. 

Auxin binds to the auxin receptor TIR-1. This causes degredation of negative regulator proteins (Aux/IAA). This results in the promotion of auxin-induced gene expression regulated by ARFs.

Auxin increases growth of shoots and decreases growth of roots.

Auxin-related gene expression can be measured by:

- Reporter genes.

- GFP.

- IAA2p::GUS.

Cytokinins act antagonistically to auxins.

Auxin and cytokinin regulate each others function at the root apex.

- Lateral organ initiation at the shoot apical meristem.

- Patterning and vascular development.

- Maintainance of stem cell fate at the root apical meristem.

- Inhibition of branching in the shoot.

- Promotion of branching in the root.

Phytohormones regulate cellular activities (division, elongation and differentiation), pattern formation, organogenesis, reproduction, sex determination and responses to abiotic and biotic stresses.

Phytohormones include auxin, cytokinins, gibberellins, abscisic acid, ethylene, brassinosteroids, salicylates, strugolactones and jasmonates.

1. Prepare four plates each with different concentrations of auxin.

2. Add bleach and leave for 10 mintues, shaking occasionally.

3. Wash with sterile water three times.

4. Plate seeds onto the plates.

1. Add GSS into eight wells of the tissue-culture dish.

2. Put seedlings into GSS.

3. Place in a 37C oven for 30 minutes.

1. Measure using a ruler the length of each of the roots.

2. Perform a series of ethanol washes.

3. Use a dissecting microscope to look at GUS expression in the wild type and the aux1 mutant in the tissue culture dish.

4. Choose one of each seed to photograph.

Histology: the microscopic observation of fixed and stained tissues.

Most cells are colourless and transparent. Therefore, histological sections are usually stained in some way to make the cells visible.

Most histological stains lack specificity and therefore, the identification of different cell types relies on additional information such as cell shape and location. 

Histology has become a precise science as cell and molecular biological tecniques are now available to detect the products of expression of individual genes by specific cells. This can be done both in situ and in vitro.

In situ hybridisation techniques can be used to detect specific RNA molecules by means of complementary labelled probes.

The availability of antibodies directed against specific antigens permits precise identification of cells and the location of their products. 

The first approach to staining tissues in research and medical diagnosis is to use traditional histological stains. The most commonly used stains are haematoxylin and eosin (H&E).

Haematoxylin is a stain called hematein obtained from the log-wood tree. It is used in combination with aluminium ions and it stains acidic (basophilic) structures a purplish blue.

Eosin is an acidic dye. It has a negative charge and stains basic (acidophilic or eosinophilic) structures red or pink.

DNA in the nucleus and RNA in the ribosomses on the RER are acidic so are stained purple.

Some extracellular material including glycosaminoglycans are basophilic.

Amino groups in the cytoplasm and extracellular matrix are eosinophilic.

Histology: the branch of biology which studies the microscopic anatomy of biological tissues.

Histopathology: the diagnosis and study of diseases of the tissues and it involves examining tissues and/or cells under a microscope.

Histopathologists are responsible for making tissue diagnoses and helping clinicians to manage patient care.

Dissection: Specimens should be fresh to avoid cell autolysis (destruction of cells by their own enzymes). Think about the structures you wish to observe. Dissect the specimens using a sharp scalpel blade.

Fixation: Maintain the natural structure of the tissue. Prevents autolysis and allows quick penetration. Prevents tissue shrinkage.

Freezing: Fixation can msak antigen epitopes due to cross-linking. Freezing preserves enzyme function but tissue morphology can be damaged.

Embedding: Provides a solid structure. Prevents tissue damage during sectioning. Allows tissue to be orientated. Allows thinner sections to be cut.

1. Put under running water for 1 minute.

2. Put under Harris Haematoxylin for 5 minutes and breifly rinse.

3. Put under acid alcohol for 15 seconds then briefly rinse.

4. Put under Scott's tap water for 3 minutes then briefly rinse. 

5. Put under Eosin Y for 5 minutes.

6. Put under running water for 10 seconds.

7. Put in ethanol for 30 seconds.

8. Mount in glycerol.

9. Add the coverslip and view.

Immunohistochemistry: uses antibodies to stain tissue sections for the presence of specific proteins. 

Detecting specific proteins allows for precise identification of cells. 

An antibody that has high affinity for the protein that is to be detected is added. This binds to the protein if it is there. This is known as the primary antibody. If this is a monoclonal antibody, it will bind to a single epitope. 

The bound antibody has to be detected and this step also amplifies the signal. Usually, this involves a secondary antibody which detects the primary antibody. 

The secondary antibody is coupled to an enzyme which converts a colourless substrate into an insoluble coloured product. 

1. Isolate human antigen of interest. 

2. Inject antigen into mouse.

3. Mouse produces antibodies against the human antigen. 

Tissues are exposed to primary antibodies which binds to the antigen of interest. Any unbound antibodies are washed away. 

A secondary antibody is applied which binds to the primary antibody if present. The secondary antibody is conjugated with HRP enzyme. Using a secondary antibody amplifies the signal.

If the first antibody is raised in mice, then the second antibody will be raised in another animal against mouse IgG.

The first antibody can be raised in mice, rabbits, goats or sheep. 

The appropriate secondary antibody needs to be used/raised in another species which is phylogenetically dissimilar. 

The buffer most commonly used in immunohistochemistry is PBS.

No First Antibody: checks that the secondary antibody is not binding non-specifically. This is known as the negative control. 

Positive Control: use a section from a tissue that is known to express the protein that the primary antibody is directed against. 

Blocked Control: preincubate the antibody with solube antigen before adding it to the tissue section. Ensures specificity of the first antibody. 

Instead of an enzyme, a fluorescent flag can be used. This gives a good resolution and can stain more than one antigen at a time. 

- Prognostic markers in cancer.

- Identifying tumours of unknown origin.

- Confirmation of infection.

- Genetics.

- Neurodegenerative disorders and brain trauma.

- Other disease identification. 

Drug candidates can be screened using mammalian cell lines (CHO or MCF-7 cells) to assess any cytotoxic effects the compound could exert on the body's own cells.

The MTT assay uses a reduction of MTT to measure cellular metabolic activity as a measure for cell viabilty. 

Viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce the MTT reagent to formazan which has a deep purple colour. 

Formazan crystals are then dissolved using a solubilising agent and aborbance is measured at 500-600 nm using a plate-reader.

The darker the solution, the greater the number of viable, metabolically active cells and therefore, the lower the cytotoxicity of the compound. 

Manual cell viability counts are performed using a dye which selectively penetrates dead or dying cells whose cellular membrane has been damaged. Live (viable) cells remain unstained. 

The two most common viabilty stains are trypan blue and erythrosin B. 

Trypan blue is a blue dye which is chemically derived from toluene. 

Erythrosin B is a cherry pink dye composed of fluorescein salt. In some wavelengths, it can be sensitive to light. 

Cell counting is carried out using a haemocytometer. 

A haemocytometer is a modified glass slide engraved with two counting chambers of known areas. 

It is divided into 9 squares of 1mm x 1mm size. 

The four corners are further divided into 4 x 4 grids. 

The height of the chamber including the cover glass is 0.1mm.

Therefore, the volume of the chamber is 0.1mm^3.

Apoptosis: this is referred to as programmed cell death where the cell is induced to commit suicide. The contents of the cell are contained during phagocytosis.

Necrosis: where external influences damage the cell membrane and cause uncontrolled cell death. The contents of the cell are released into extracellular space and trigger an inflammatory response. 

Apoptosis is a crucial part of cell development. Development of drugs that induce apoptosis are good as they are anticancer targeted. 

Necrosis is often thought of as detrimental due to the uncontrolled release of cellular contents causing an inflammatory response. Cell death by necrosis is often seen as a side effect of paracetamol overdose and prolonged fluroquinolone antibiotic therapy. 

There are emerging therapies under investigation for their influential anticancer efficacy in breast cancer cells, where cell death occurs via necrosis. AG311 is a small molecule known for its anticancer activity. 

Apoptotic and necrotic cells have characteristic morphological features which enable them to be differentiated. 

During cell death by apoptosis, caspase-3 activated DNase (CAD) cleaves nuclear DNA into internucleosomal fragments of around 180 base pairs. 

When observed using gel electrophoresis, it is possible to differentiate between cell death via necorsis and apoptosis.