Intergrated cell biology – tissue homeostatis in mammals

guiltlesscyanΒιοτεχνολογία

3 Δεκ 2012 (πριν από 4 χρόνια και 9 μήνες)

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Stuff i need to know before the exams...

NOT YET DONE IN DEPTH:

bacterial Classification (flint stuff)

PGG

Proteins

-

20 amino acids

o

A
LAnine , A
R
Ginine , ASparagi
N
e , ASPar
D
ic acid , CYSteine , GLUtamic acid , GLutamiNe ,
GLYcine , HIStidine , IsoLEucine ,
LEUcine , LYSine , METhionine , PHEnylalanine , PROline ,
SERine , THReeonine , TRyPtophan , TYRosine , VALine

-

Alpha Helixes

o

Details:



Rise 1.5 Angstrom per residue



Each turn = 3.6 residues



Proline / Glycine would destabilize an alpha helix and thus aren’t
found.

o

Functions : Interactions with other molecules, stability , rigidity (fibres)

-

Christian
Anifinsen experiment (Disproving random refolding)

‘protein structure is determined by the sequence of amino acids , tertiary structure is determined by the prima
ry structure.’

o

8M urea disrupts H
-
Bonds and hydrophobic interactions , BME disrupts disulphide bonds

o

Dialysis used to remove the urea and Beta
-
mercaptoethanol slowly.

o

Protein becomes scrambled if BME is removed before urea due to incorrect disulphide
bonds

forming , scrambled protein can be renatured by trace amounts of BME which will
allow it to break and reform slowly.

o

Protein Disulphide isomerise (PDI)


promotes shuffling of disulphide bonds and as such
renatures scrambled proteins much quicker than B
-
m
ercaptoethanol.

-

Haemoglobin

o

Binding of O
2

to Hb is cooperative , Binding of one O
2

enhances binding of another.
Similarly , release of one O
2

encourages release of more O
2
.

o

Deoxy
-
Hb is the Tense form, Oxy
-
Hb is the relaxed Form

-

BPG
(2,3
-
bisphosphoglycerate)

o

Formed from 1,3
-
bisphosphoglycerate


glycolysis

o

One molecule of BPG binds to the central cavity in Hb , negative charges of BPG interact
with positive charges in Hb. The additional interactions stabilise deoxy
-
Hb. Thus BPG
lowers

O
2

affinity of Hb.

o

Binds to Hb which is deoxygenated instead of oxy
-
hb , this tells the Hb to give off the
remaining oxygen thus enhancing the RBC’s ability to release O
2

where it is needed most.

Membranes

-

Functions:

o

Semipermeable barrier

o

Detects changes

in extracellular environment

o

Provides an alternative environment to the cytoplasm

o

Provides anchorage sites

-

Why do membranes contain different lipids

o

Act as cofactors for various proteins

o

Provide substrates for important reactions by hydrolysis

o

Interact wi
th extracellular molecules

-

Integral membrane proteins (go through the membrane and stay like that)
-
|
-

o

Cross the membrane at least once and have transmembrane , cytoplasmic and
exoplasmic domains. The topology is maintained by hydrophobic and electrostatic

interactions. Exoplasmic region may be glycosylated or contain disulfide bonds.

-

Peripheral membrane proteins

o

Do not interact with hydrophobic core of the membrane , can be exoplasmic or
cytoplasmic but does not change between the two.

-

Detergents work by
turning hydrophobic clumps of molecules into water soluble complexes

-

Membrane fluidity

o

Lipids with short fatty acid chains increase fluidity


allow few interactions btwn lipids

o

Lipids with unsaturated chains increase fluidity


kinked chains are hard to p
ack together

o

Cholesterol minimizes changes in membrane fluidity :



Increases fluidity in the middle of the membrane / decreases at edge of membrane

o

Some organisms can regulate lipid composition , mechanisms are usually temperature dependant.



Enzymes synthes
izing fatty acids , enzymes introducing C=C bonds and solubility of
oxygen are temperature dependant.

-

Membrane transporters (channels/carriers)

o

They are usually specific or selective, only transporting one or a few solutes.

o

Transporters are regulated



Volta
ge gated


respond to membrane potential



Ligand gated


respond to the binding of ligands (neurotransmitters/2
nd

msngers)



Mechanically gated


Respond to touch/sound/stretch

o

Co
-
transport systems :



Symport : taking another compound with it down the conc gra
dient



Antiport : Swapping with a compound on either side of conc gradient

PA stuff

-

Mendel’s Peas


chose peas to work with as they produce lots of offspring , short generation
time , self/cross fertilization possible , contrasting features

-

Lifecycle of T2
Phage

o

Attachment of phage to E.Coli and injection of phage chromosome.

o

Complete breakdown of bacterial chromosome by phage
-
specific enzyme

o

Replication of phage chromosome using bacterial materials and phage enzymes.

o

Phage genes produce structural component
s (tail fibre/base plate/heads/sheath)

o

Assembly of progeny phage particles

o

Release of progeny phages by lysis of bacterial cell wall

-

DNA is transferred by bacteriophage , not protein

o

Hershey and Chase did experiment, labelling protein and DNA in bacterioph
age
experiments. DNA labels remained after cell lysis , protein labels not really found.

-

DNA structure discovered by Rosalind franklin + Maurice Wilkins via Xray crystalography

o

DNA is a right handed double helix (goes clockwise when viewed from below and c
lose)

-

DNA replication models

o

Semi
-
Conservative : 1 parent strand kept for each offspring , 1 new strand generated.

o

Conservative replication : turns into intermediate , generates 1 completely new strand.

o

Dispersive replication : parent disappears , 2 new st
rands formed.

o

Proved to be semi
-
conservative and Watson
-
crick double helix model by meselson
-
stahl
experiment.

-

DNA Synthesis occurs at a replication fork

o

DNA polymerase has 3’
-
5’ exonuclease activity required for proofreading/editing.

o

Okazaki fragments




DNA Helicase seperates the strands of the DNA ahead of the DNA polymerase so
that single stranded DNA can be used as the template



DNA primase synthesizes an RNA primer to initiate DNA synthesis on the lagging
strand , this happens once for every okazaki fr
agment



single stranded binding proteins attach to prevent the strands from reannealing



DNA polymerase 3 then adds to the RNA primer to create the okazaki fragment.



More RNA primer is laid so more can be transcribed from the template strand.



DNA polymerase
1 replaces the primer with DNA and then DNA ligase links the
fragments together.

o

Supercoiling at the front of the replication fork is relieved by ‘topoisomerases’

o

DNA polymerase does not stick to the DNA easily so a ‘sliding clamp’ is used to help it.



This

needs a ‘Clamp loader’ complex to get the two halves of the sliding clamp onto
the DNA. This uses ATP.

-

PCR

(Polymerase chain reaction)

o

Primers are added in excess to bind with strands.



94deg , separation of 2 strands



45
-
64deg , annealing of
primers to
the

template



72deg extension of taq polymerase

o

Applications : Altering DNA sequence , amplifying tiny amounts of DNA to obtain enough
for analysis


medical tests ,forensics etc.

-

Sanger’s Dideoxy sequencing method

o

Use single stranded template DNA and primer a
long with pool of radioactive labelled
dideoxynucleotide’s with normal deoxynucleotides.

This is used in 4 reactions , one for
each base.

o

Generates fragments of different length which can be separated by electrophoresis to
determine the genetic sequence.


-

Transcription (Prokaryotic)

o

Sigma factor is the initiator ,
sigma factor destabilizes non
-
specific interaction between
DNA and RNA polymerase ,

allows the binding of RNA polymerase to gene promoters.
RNA opens the double helix. With the DNA unwound , one o
f the strands acts as a
template for complementary base pairing , reaction from 5’ to 3’ and is driven by
hydrolysis of incoming nucleotides. (RNA polymerase does not need a primer). RNA
polymerase then breaks from promoter and sigma factor
and
moves down
the DNA
symthesizing RNA


the loss of sigma factor increases the affinity of RNA polymerase for
DNA. Termination occurs by either nucleotides forming a hairpin structure which
destabilizes the RNA
-
DNA hybrid , or through Rho factor which breaks the hydrog
en
bonds between template DNA and RNA

-

Transcription (Eukaryotic)

o

TATA box is at the top of the transcription region , TFIID binds to the TATA box by the
TBP

(tatabox binding protein)

on TFIID , TFIIA and TFIIB then join. RNA polymerase
II
then
joins

onto the transcription factors along with TFIIF , E and H.

o

TFIIH unwinds DNA (like DNA helicase) and phosphorylates the carboxyl terminal domain
of the RNApolII. This phosphorylation causes the transition from initiation to elongation ,
initiation factor
s then detatch and RNA polymerase transcribes.

o

(then Poly A Tail , 5’ Cap)

o

General transcription factors (initiation of eukaryotic RNA polymerases)



Position the RNA polymerase , separate the DNA strands , release RNA polymerase
into elongation mode after
initiation.

-

Processing of pre
-
mRNA to make mature mRNA

o

5’ Cap : One of the Terminal phosphate group is removed by phosphatase leaving two
terminal phosphates. GTP added to terminal phosphates , this results in 5’
-
5’
triphosphate linkage. The 7
-
nitrogen of
guanine is methylated.

o

Poly A Tail :
Cleavage signal is on the template DNA (3’ end) , endonuclease cleaves it
and tail is added by Poly(a) polymerase. Around 200 A’s are added after cleaving.

o

GU at 5’ , pyrimidine rich region and AG , these determine wher
e the intron is.

2 complexes of the spliceosome
(U1 and U2)
then attach to the GU , RNA is then looped
and 3 other complexes then bind
(U4 , U5 and U6)


this is the spliceosome in its true
form , it then undergoes a conformational change and the intron is

cleaved at the 5’ end
and forms a branch with the A branch site forming a lariat , the 3’ end is then cleaved
and the exons are ligated.

-

Differences between prokaryotes/eukaryotes

Prokaryotes

Eukaryotes

mRNA transcript is mature

mRNA transcript needs to
be processed before
translation can occur.

Translation can be coupled to transcription

Translation cannot be coupled to transcription
because of the nucleus.

mRNA are polycistronic containing amino acid
coding info for more than one gene

mRNA are
monocistronic , containing amino acid
sequences for just one gene

-

4 kinds of introns

o

Group 1


self splicing (organelles , nuclear rRNA)

o

Group 2


self
-
splicing (organelles in fungi and plants)

o

Spliceosome dependent (nuclear mRNA)

o

Nuclear tRNA

-

tRNA synthe
tases attach an amino acid to the 3’ end of the corresponding tRNA.

o

The tRNA synthetase also proofreads the amino acid


-

Translation (Prokaryotes)

o

Initiation : IF3 prevents 30S and 50S ribosome complexes from joining if no mRNA.

IF1 + IF2 ensure that fMet
-
tRNA binds to P Site of the ribosome complex.

o

Termination : release factors promote release of synthesized polypeptide from ribosome


RF1 or RF2 binds to UAA , RF3 mediates interaction between RF1/RF2 & ribosome.

-

Translation

(eukaryotes)

o

Initiation :
Begins with AUG (Met) codon. There is a shine dalgarno sequence upstream
of the Met codon

o

Initation factors , 40S subunit and the initiator tRNA bind to the 5’ cap of mRNA to form
the initiation complex. Initiation complex moves in 3’ direction using
energy from ATP
until first AUG is found.

-

(Translation) tRNA function and structure along with ribosomes

o

Small ribosomal subunit binds to the start codon , tRNA then attaches to the codon with
its anti
-
codon sequence. The large ribosomal subunit now binds
over the top and forms
the P and A site , the P site holds the current protein and the A site is where new arriving
proteins enter , the amino acid connected to the tRNA is then transferred to the end of
the amino acid on the A site , the chain moves along

and the first tRNA is ejected , the
2
nd

tRNA is moved to the P site and the next tRNA enters the A site. At the end of the
chain , when the stop codon is reached , the release factor enters the A site and the
ribosome dissociates.

o

When fMet
-
tRNA enters
the p
-
site , a conformational change occurs and activates the A
site allowing new aminoacyl tRNA to bind. Peptide bonds forming between the amino
acids are catalyzed by peptidytransferase

o


-

Puromycin : resembles 3’ end of aminoacylated tRNA , enters the A
-
s
ite of the ribosome and
transfers to the growing chain leading to premature termination.

-

Restriction enzymes , hydrolyze a phosphodiester bond in each strand.

o

Named by 3 letter abbrev of the organism in which they are found in nature followed by
a strain
designation (if needed) then a roman numeral if more than 1 restriction enzyme
from the same strain has been identified.

o

Biological function :bacterial defence against viruses by cutting viral DNA restriction sites

Flint’s Stuff

-

Auxotroph : impaired in som
e metabolic capabilities , cannot synthesize certain things.

-

Temperature sensitive mutants grow at LOW temperatures , cold sens grow at HIGH temps.

-

Mutations:

o

Lamarckian evolution : drive towards complexity, shaping power of environment.

o

Darwinian evolutio
n : genetic change is random , survival of the fittest

-

Experiments to prove theories (Lamarckian/Darwinian) :

o

Newcombe experiment : Left cells
to replicate and some not to replicate and sprayed
them both with ton
r

, the ones which had replicated had more g
rowth (Darwinian)

o

Lederberg and Lederberg experiment : developed replica plating. Proved Darwinian by
using Ton
r

on only one of the replica plates and showing that that bacteria was present
before exposure to the Ton
r

-

The effects of point mutations

o

Point
mutations can vary in magnitude of damage :



Nonsense mutations


Code for a stop (premature termination)



Missense mutations


different amino acid , can change shape of protein



Silent mutations


code for the same/different amino acid with no functional
ch
ange in protein



Frameshift mutation


Addition/deletion shifts the AA’s so that the triplets are read
differently.



[[intragenic suppression , double mutations can sometimes fix the original mutation
(eg. 1 addition 1 deletion) ]]

-

Mutagens

o

Deamination of b
ases (amino groups drop off of bases , resulting in base mispairing)

o

Reactive oxygen species

(cause DNA lesions)

o

Alkylating agents (such as EMS)

add alkyl groups to DNA which alters base pairing

o

UV Light (dimerisation of adjacent pyramidine residues)

-

Pheno
typic Lag



mutation which takes several generations to express itself (eg. Resistance to
phage T1 arises through loss of a particular protein , through mutation and replication the
amount of proteins will decrease until it is eventually resistant.

-

Isolati
on of histidine auxotroph

o

Mutagenise cells

o

Allow expression of mutations in complex medium

o

Penicillin enrichment


resuspend all cells in minimal medium with glucose and penicillin.
This kills all cells able to grow. Auxotrophs don’t grow so able to surviv
e

o

Incubate and obtain survivors

o

Replica plate to minimal medium plus and minus histidine. Pick colonies which grow in
presence of histidine but not in absence.

-

Cross feeding : Bacterial strains with mutations affecting pathways will sometimes
overaccumula
te an intermediate , this diffuses out and can be used by other bacteria.

-

Lac operon

o

Lac operon is the part of the bacterial dna which encodes for the production of B
galactosidase and Permease , to increase permeability of membrane to allow lactose in
for

the B galactosidase to break down.

Inducer joins to the repressor protein which is upstream of the lac operon , this removes
the repressor and the B galactosidase enzyme is transcribed.

-

Genetic exchange between bacteria (compare the 3 processes of genetic

exchange which occur
in bacteria)

o

Transformation , Transduction , Conjugation

o

Transformation : bacterial cell
takes up

and then expresses
foreign DNA

, may be due to
taking up Naked DNA (DNA without associated cells or proteins) or by competence.

o

DNA
taken up should have its own ‘origin of replication’ to allow itself to replicate



Natural Competence occurs naturally in about 1% of all species and allows the
bacteria to take up DNA under lab conditions



Artificial Competence , induced by chilling cells i
n the presence of divalent cations
such as Ca
2+

(to increase membrane permeability) and then heat shocked which
causes the DNA to enter the cell

o

Transduction : DNA transferred from one bacterium to another
through a

virus

, also
counts if a viral vector is

used.



Generalized Transduction , if bacterial DNA manages to get into the viral capsid , or
if the virus replicates using
he
a
dful packaging

and there is spare space , bacterial
genetic material sometimes gets taken up. This genetic material is then passed

on
to the next bacteria the virus attacks.




The transfer from a donor cell with approximately equal frequency of any gene to
a recipient cell via a phage , carried out by both lytic and temperate phages when
they go through the lytic cycle.’



When DNA is i
nserted into recipient cell , may be absorbed and recycled



If it was a plasmid , may re
-
circularize inside and live on



If DNA matches with homologous region of recipient cell’s chromosome ,
may
recombine with the host DNA by
undergo
ing

a similar process to

conjugation.



Specialized transduction , if virus removes itself from the chromosome incorrectly ,
bacterial DNA can be packaged into the virion



‘The transfer via a phage of only specific genes from a donor to a recipient carried
out by temperate phages th
at have entered the lysogenic state’



Results , DNA absorbed and recycled



DNA can match with homologous DNA in recipient cell and exchange it.
Recipient now has own DNA and DNA from other bacterial cell.



DNA can insert itself into genome of recipient cell r
esulting in double
bacterial genes.

o

Conjugation : F+ Donor transfers genetic information to the F
-

Recipient via a Pilus
. The
plasmid replicates by rolling circle replication.

-

Infection of host cell with a lytic phage leads to lysis of cell, infection of h
ost cell with temperate
phage can lead to lysis OR a stable relationship between phage and host.

-

Holliday model of recombination (when 2 sequences have similar base pairs)

o

Alignment : Homologous regions of the 2 duplexes become aligned

o

Cleavage : one stran
d of each duplex is cleaved

o

Invasion : the two freed 3’ single strand ends produced must invade the homologous
double stranded DNA to form a synapse. In E.Coli this is catalysed by the recA protein, a
Holliday junction is formed , if the two duplex molecul
es are not identical then a
heteroduplex is formed.

o

Branch migration : branches migrate to increase the hetereoduplex region.

o

Isomerisation : crosses and uncrosses the strands of a holliday junction.

o

Cleavage : The two crossed strands of the holliday
junction are cleaved by RuvC.
Recombination depends on the configuration of the junction at the time of cleavage.

Results are either :



Two duplex molecules with a region of heterduplex



Two recombinant duplex molecules with a region of heteroduplex

Enzymes

and that

-

K
cat

= number of molecules of substrate an
enzyme molecule can convert in 1 second.

-

Double reciprocal/lineweaver
-
burk plot of
normal enzyme vs inhibited

-

Km and Vmax (Enzyme Kinetics)

o

Km is the Michaelis constant , is
[S] at (Vmax/2)

o

Vmax is the
maximum reaction
rate

o

With competitive inhibitor , same
Vmax

, raises Km as more [S]
needed to obtain Vmax

o

With Non
-
competitive inhibitor ,
lowered Vmax , same Km as enzymes disabled and thus cannot obtain original Vmax

-



-

Chromosomal composition

-

Differences between Prokaryotic and Eukaryotic Translation
*

o

Initiator : Eukaryotic ‘Methionine’ , prokaryotic ‘N
-
formylmethionine’ (or f
-
Met)

o

Initiation : Eukaryotic ‘AUG’ , Prokaryotic ‘Shine
-
Dalgarno Sequence’

Bacterial RNA polymerase uses 1 sigma factor

, eukaryotic polymerases require
additional proteins called the general transcription factors.next

o


-

Alpha helixes

-

Origin of life

-

M
eiosis

-

Pedigrees / recessive/dominant etc

o



-

Comparison of translation in mammals and that in e.coli

-

Capping slicing and
polyadenylation in the production of mRNA

o

Methylated Cap is added to the 5’ end of the mRNA shortly after translation starts ,
Specific sequences in the mRNA attract ‘cleavage factors’ the cleavage factors then
move the mRNA into the correct position for c
leaving (looped over) , poly A polymerase
then cleaves the 3’ end , poly A polymerase then begins synthesizing adenine to give the
poly A tail. Proteins then bind to the tail increasing the rate at which it grows.

Genetics and Shit

-

Definitions:

o

Autosome :
non
-
sex determining chromosome

o

Hemizygote : Half a chromosome (male)

o

Chiasma : Overlap point (chiasmata is plural)

o

2n Gamete : Disomic

o

0n Gamete : nullisomic

o

Triple chromosomes : Trisomy

-

Prophase I


in depth

o

Leptotene (thin thread) : Chromosomes condense
, homolog pairing begins.

o

Zygotene (paired threads) : homologous chromosomes pair , synaptonemal complex
begins to form between homologs (synapsis), paired homologs now referred to as
bivalents.

o

Pachytene (thick thread) : coiling/shortening of chromosomes
continues , synaptonemal
complex assembly completes , chiasma visible

o

Diplotene (2 thread) : Synaptonemal complex disassembles ,

o

Diakinesis (moving apart) : Chromosomes repel each other , non
-
sister chromatids
remain loosely associated by chiasmata , nucl
ear membrane and nucleolus disappear ,
centromeres attach to spindle fibres.

-

Non
-
disjunction of autosomal chromosomes

o

Chromosome 21


Down’s syndrome (also linked to Alzheimers)

o

Chromosome 18


Edward’s syndrome

o

Chromosome 13


Patau’s Syndrome

-

Non
-
Disjunction of sex chromosomes

o

XXY


Klinefelter syndrome Male : Sterile , tall, low IQ , some female phenotypic traits.

o

X


Turner syndrome female : Short , webbed neck, lack of menstruation/tits , low IQ.

o

Others : XXX (fertile female , premature meno
pause), XYY (v.tall male)

-

Histocompatability genetics (HLA stuff)

o

HLA antigens are found at one locus on chromosome 6.

o

Organ transplants work well if the donor and recipient have identical alleles at the major
HLA loci.

-

Blood groups

o

Blood group O has no an
tigens on the cells , but has Anti
-
A and Anti
-
B antibodies in the
blood plasma. This Makes Blood Group O the Universal Donor.

o

Blood group AB has both antigens on cells and no antibodies in the plasma , Blood group
AB is the universal recipient.

o

Bombay phen
otype : Caused by homozygous recessive ‘hh’ , is epistatic to I
A
/A
B
/I
O
.

o

Rhesus Blood groups : Triple mutant is Rh negative (cde/cde)



Can cause problems in childbirth , first child is unaffected then mother becomes
immunised


begins producting Anti
-
Rh.
Future foetuses may be stillborn.

-

Gonads

o

If the gonad cells are XY ,TDF gene will be switched on, gonads differentiate to form:



[
SRY Gene , homologous to SOX9 = Testes determining Factor (TDF)
]



Sertoli cells
-
> AMH (
Anti
-
Müllerian hormone
,)



Leydig Cells
-
>

Secrete testosterone
-
> development of Wolffian ducts and cock.

o

In lack of Y chromosome , gonads differentiate into ovaries which contain:



Oocytes
-
> Eggs



Follicular cells
-
> Secrete oestrogen and promote development of
Müllerian

ducts
and genetalia and degeneration of Wolffian ducts.

o

Mutation of androgen receptor causes ‘default’ female sex organs.


Intergrated cell biology


tissue homeostatis in mammals

-

Tissue structure is maintained by cells attached to each other via cell
-
cel
l adhesion or by
attaching to proteins on the ECM (extracellular matrix)

-

4 Main tissue types , Epithelium , muscle , nervous and connective

-

Epithelium cells are polarised with apical (outside) , lateral (side) and basal (inside) surfaces.
These surfaces en
sure that special structures like cillia are on the right side and that
secretion/absorbtion is going the right way.

-

Muscle cells are arranged into fibres and then arranged into bundles surrounded by connective
tissue.

-

Collagen is very strong , forms fibro
us coils and becomes part of the basal membrane as a mesh.

-

Skin : Epidermis is outermost layer , underlying connective tissue is called the dermis


these two
layers are separated by basement membrane. underneath this is layer of adipose tissue called
the
hypodermis.

o

Cells multiply in the basal layer and the progeny move outwards , in outer layers the
cells do not divide , instead they synthesize keratin

-

Cell proliferation (multiplication) is controlled by diffusible polypeptide growth factors which act
by
cell surface receptors.

o

When these GF’s bind to a receptor , a signal is transmitted across the membrane and
picked up by a signalling cascade.

-

EGF Signalling

o

Polypeptide growth factor receptors have an extracellular ligand binding domain and an
intracellu
lar protein tyrosine kinase domain ; upon ligand binding the receptor
dimerises , tyrosine kinase is activated and other proteins including P21
ras

associate with
it , this becomes the ‘transduction complex’ and P21
ras

activation.

o

Activation of P21
ras

passe
s the signal to a series of serine threonine kinases.

o

Intracellular signalling molecules , these are 3 kinases

o

Gene Activation



The targets of the MAPK cascade are transcription factors , particularly of the ETS
family. These interact with factors such as A
P
-
1 to activate transcription of
“immediate early” genes.



The targets of DAG are protein kinase C’s, which also activate transcription of
“immediate early genes”



Inside of IP3 are intracellular Ca
++

stores leading to further gene activation.

-

Cell death
(apoptosis)

o

Final death mechanism is activation of caspases , these are proteases with Cysteine at
the active site acting on proteins at ASPartic acid residues.



Some Caspases act on other caspases and activate them

cascade.(initator caspase)


eg. Caspase
8 & 9



Others demolish the cell. Eg. Caspase 3 & 7



Caspases target intraceullular proteins , usually cleavage by caspases inactivate
proteins, occasionally though they activate them by removing inhibitors.

Physiology

Renal

-

Functions of the renal system

o

Regu
lation of water and inorganic ion balance

o

Removal from the blood and excretion of metabolic waste products (eg. Urea , creatine)

o

Removal from the blood and exctretion of foreign substances (eg. Drugs)

o

Gluconeogenesis

o

Production of hormones (vit D) and enzy
mes (renin)

-

Structures of the renal system

o

Glomerulus : tuft of interconnected renal capilary loops

o

Bowmans Capsule : A fluid filled capsule , the blind end of the nephron

o

Juxtaglomerular Apparatus : Comprises the macula densa of the distal tubule , granul
ar
cells of the afferent arteriole and extraglomerular matrix

-

Regulation of renal blood flow

o

Autoregulation : renal blood flow is held nearly constant over the systemic mean arterial
pressure range , 90
-
200mm Hg

o

Physiological regulation : increases in symp
hathetic nerve activity increases renal
vascular resistance and can decrease renal blood flow and GFR.

-

Renal Clearance


the clearance of a substance is the rate at which
plasma is completely cleared of that substance in order to yield the
substance at the

rate at which it appears in the urine.

o

Clearance =( ‘Concentration of X in Urine’ x ‘urine volume per unit time’ ) / Concentration
of X in plasma

o

If net tubular reabsorption , clearance < GFR , and vice versa (secretion)

o

Measurement of GFR =
(‘Concentration of X in urine’ x ‘Urine volume per unit time’) /
Concentration of X in plasma

o

X transported by tubule = (Concentration of X in plasma x GFR)


(‘Concentration of X in urine’ x ‘Urine volume per unit time’)

-

Sodium reabsorption : Na+/K+ ATPas
e pump on the basolateral membrane pumps Na+ out of the proximal
tubular cell and into the interstitial fluid in return for K+. Na+ moves passively from the lumen into the cell
due to the lowered Na+ concentration.


-

Chloride Reabsorption : Diffuses with Na
+ through tight junction , and then is transported with Na+ into
the tubular cells by the parallel action of the Na
+
-
H
+

and Cl
-

anion exchanges. Cl
-

exits
the cell via Cl
-

channels.

-

Glucose Reabsorption : Cotransported with Na+ at the luminal membrane by a

Na+/glucose cotransporter.

-

Bicarbonate Reabsorption : H
+

enters the tubule lumen in exchange for Na
+

, HCO
3
-

and
H
+

are converted to CO
2

and H
2
O , CO
2

diffuses into the cell. CO
2

and H
2
O are converted
back into HCO
3

and H
+

, HCO
3

then exits the cell.

-

Mor
e ADH (Vasopression) = more water reabsorption from the collecting duct.

-

Renin

o

Secretion is stimulated by increased renal sympathetic activity , decreased renal perfusion
pressure and decreased NaCl delivery to the macula densa.

o

Renin is an enzyme which c
hanges angiotensinogen to angiotensin I.

(From angiotensin I, converting enzyme changes it to II , this causes secretion of aldosterone
from adrenal cortex.)

-

Angiotensin

II

o

Causes vasoconstriction of arterioles in the systemic circulation.

o

Stimulates Na+
reabsorption in the proximal tubule (Cl
-

and water follow passively)

o

Stimulates Vasopression secretion from posterior pituitary gland and stimulates thirst



Vasopressin stimulates thirst as well.

-

Aldosterone

o

Is a hormone

secreted by the adrenal cortex
(due
to Angiotensin II)
, it stimulates Na+ and hence
H2O reabsorption in the collecting duct.

-

Arginine Vasopressin (ADH)

o

Released from the posterior pituitary gland

o

The main stimuli are decrease in arterial blood pressure or increase in plasma osmolarity.

o

Actions: Causes vasoconstriction of arterioles of systemic circulation , increases renal water
reabsorption by increasing water permeability of collecting duct.

-

Atrial Natriuretic Peptide (ANP)

o

Synthesized and released by myocardial cells of the atrium , s
timulus is the stretch of the atrium.

o

Inhibits renin , aldosterone and vasopressin secretion.

o

Increases GFR by afferent arteriole dilation and efferent constriction.

o

Decreases Na+ and water reabsorption.

-

Parathyroid Hormone (PTH)

o

Is secreted by the parath
yroid gland , secretion stimulated by decrease in plasma concentration
of Ca
2+

o

Stimulates the production of active Vitamin D
3

, Stimulates Ca
2+

reabsorption, inhibits tubular
reabsorption of phosphate

Respiratory

-

Functions of the respiratory zone of the ai
rways

o

Provide oxygen / Remove Carbon Dioxide

o

Regulates the blood’s pH in coordination with the kidneys

o

Influences arterial concentrations of chemical messengers

o

Traps and dissolves blood clots arising from systemic veins.

-

Dieseases

o

Emphysema



Destruction of

alveolar walls and collapse of lower airways by proteolytic enzymes
secreted by leukocytes.



As a result , nearby alveoli fuse to form larger alveoli , this reduces total surface
area.

o

Chronic Obstructive pulmonary Disease (COPD)


refers to bronchitis or

emphysema

o

Asthma



Diesease in which the bronchioles get inflamed and the airway smooth muscle
contracts strongly increasing airway resistance.



Inflammatiion causes airway smooth muscle to be hyper
-
responsive and to contract
in response to triggers , eg.
Exercise or allergens.

o

Pneumothorax (collapsed lung)



Perforation of the lung/chest wall allows air to enter the intrapleural space , lung
cannot increase in size as no transpulmonary pressure


elastic recoil collapses lung.

-

Defense of the airways

o

Cilia


hair like projections from epithelial cells which line the airways , wave upwards
towards the pharynx (throat)

o

Mucus


secreted by glands and epithelial cells lining airways , used with cilia (mucus
escalator). The airway epithelium also secretes a watery
fluid [impaired in cystic fibrosis]

o

Macrophages


Phagocytic cells present in the airways and alveoli,engulf particles/bac~.

-

The alveoli

o

Each alveoli is surrounded by many pulmonary capilaries , ventilated air is brought into
close proximity to the blood
-
> gas exchange

o

The alveolar wall


composed of alveolar type I and II cells , capilaries , interstitial space.

o

Gas exchange optimized by



Thinness of the barrier between blood capilary and the air in the alveolus.



Surface of the alveoli in contact with the

capilaries



Moist surface of the alveolar cells

-

Ventilation

o

For air flow into and out of the lungs:

Flow = (Atmospheric pressure


Alveolar pressure) / Resistance

o

Alveolar pressure is altered to achieve air flow into/out of the lungs , Alveolar pressure
de
creases when alveoli size increases , making air enter the lungs. (Boyles Law that
volume is related to pressure)

o

Transpulmonary pressure = Alveolar pressure
-

Intraplural fluid pressure

o

Transpulmonary pressure (the force which expands the lungs) is oppose
d by Elastic
recoil (the tendancy of an elastic structure to oppose stretching)

o

When Elastic pressure >Transpulmonary pressure the lungs recoil to normal size (exhale)

o

Surface tension within alveoli



Surface of alveolar cells is moist , the surface tension
at the air
-
water interface
resists stretching.



Surface tension is lowered and lung compliance is increased by pulmonary
surfactant.



Secreted by type II alveolar cells , secretion increased by deep breaths
(stretching type II cells)



Mixture of phospholipids

and protein



Makes lungs easier to expand

-

Bohr Shift/Effect


large amount of CO2 causes bohr shift and O2 binds easier to haemoglobin

-

Lack of Oxygen (Hypoxia) / Excess of Carbondioxide (Hypercapnia)

o

Low arterial O
2

is sensed by peripheral chemoreceptors.

Peripheral chemoreceptors
stimulate the respiratory rhythm generator in the medulla , this increases the
contraction of the respiratory muscles.

o

Central and peripheral chemoreceptors can detect high CO2 , stimulates more
contractions.

-

Pulmonary Stretch Re
ceptors

o

Slowly adapting stretch receptors



Located in the smooth muscles of the bronchi and trachea , and stimulated by the
stretch in these muscles. Signal to the brain the volume of the lungs at any moment.

Inhibit inspiration and lengthen expiration and
are important in resetting
respiratory rhythm.



Located in the larnyx , trachea and airways and are stimlated by alterations in the
rate of change of lung volume. Accelerate breathing by shortening expiration.

Cardiovascular

-

Cardiac output = stroke volume x

heart rate

o

The average human stroke volume is 70ml per beat, resting heart rate is 70bpm.

-

Starling’s Law of the Heart



The greater the volume of blood entering the heart during diastole,
the greater the volume of blood ejected during systolic contraction
.


(stroke volume is determind by filling pressure)

-

Heartbeat

o

Is myogenic


initiated within the heart itself

o

Begins in the sinoatrial node , travels through the atrial muscle to the atrioventricular
node then travels to the ventricles through the purkinje

fibres in the bundles of His and
finally spreads through the myocardium.

-

Starling Hypothesis : Filtration pressure = hydrostatic pressure


oncotic pressure

o

Hydrostatic pressure in blood will force fluids out of the capilary

o

Oncotic pressure will draw flu
id back into capilaries

-

Mean arterial blood pressure = Cardiac output x Total peripheral resistance

-

Baroreceptor Reflex (response to change in blood pressure)

o

Baroreceptors in carotid sinus and aortic arch send impulses to vasomotor centres in
medulla. The

medulla then acts on the (para)sympathetic systems accordingly.

-

Haemorrhage


dangerously rapid loss of blood

o

Fall in blood volume
-
> fall in venous pressure (pressure which fills the heart)
-
>
reduction in stroke volume (by starlings law)
-
> reduction in

Cardiac output.

o

Response by baroreceptor reflex : raises total peripheral resistance , this increases
venous pressure at the expense of blood flow to metabolising tissues.

o

Correction from secretion of rennin , begins with the increased frequency of impuls
es
from sympathetic system.



Short term : Vasoconstriction of arterioles increases Total peripheral resistance

Constriction of Veins helps restore filling pressure and reinforces symp nervous sys.



Long Term : Secretion of aldosterone , restoring Extracellul
ar fluid volume

-

Exercise


Adrenaline acts on β
2

receptors which have a vasodilator action , anaerobic respiration
which results in lactate production also leads to vasodilation via the change in pH.

o

Movements of skeletal muscle will increase venous return



and subsequently pressure.

o

Heart rate has to increase after stroke volume hits max ,by the sympathetic systems .

-

Pulmonary circulation

o

Hydrostatic pressure is less than oncotic pressure , this means that fluid is not pushed
out of the arterioles and no
tissue fluid is formed. Instead reabsorption.

o

Pulmonary vessels have little smooth muscle in their walls , they are highly compliant
and will adapt to large changes in cardiac output without much change in hydrostatic
pressure.

o

Principal regulator is oxyge
n , low O2 results in vasoconstriction in the pulmonary
circulation , switching blood away from poorly ventilated areas.

Ear

-

Basilar membrane is the mechanical analyzer of sound , it is a coil which varies in width along its
length , The membrane is thin a
nd floppy at end and thicker at the base. It detects vibrations in
the air at different frequencies at different thickness of the membrane.

-

Inner hair cells transduce sound into electrical signals , they are present on the basilar
membrane

-

Outer hair cells

expand and contract upon sound.

-

Hair cells can amplify the vibration of the membrane by changing shape

-

The tips of stereocillia are linked together.

-

Medial Superior Olive

:
directional sound means sound reaches ears at different times , the MSO
gets input

from both ears and relays to the brain telling the horizontal angle at which the sound
is. [sound positioning in space]

-

All transduction of sound to electrical activity occurs in cochlea , it mechanically amplifies the
distortions of the basilar membrane.

Mechanosensitive ion channels transduce distortion of
stereocillia into graded changes of membrane potential.

G
-
Forces and
Antigravity

-

High G
-
forces result in (in order of severity) making standing up impossible , loss of sight , cannot
raise arms or head

, and finally unconsciousness.

-

This is because the blood in the body is forced into legs and the heart cannot pump effectively
enough , diaphragm is pulled down making expiration difficult and blood perfusion to upper
lungs is less. This results in less b
loodflow to the brain and less oxygenation of blood in general.

-

Low gravity conditions:

o

RBC production is reduced and total cell number drops after ~4 days and continues for 2
months



This is due to erythropoietin levels falling , erythropoietin prevents ap
optosis
(programmed cell death) and cooperates with growth factors which produce RBC’s.

o

Muscles waste away , particuarly leg muscles. Heart has a lower workload and so
decreases cardiac muscles.

o

Slight loss of bone mass (1%) per month , demineralisation an
d loss of Ca
2+

-

Bone remodels (adapts) in response to mechanical loading due to osteoblasts and osteoclasts

o

Osteoblasts produce osteoid and mineralize osteoid matrix , they build bone.

o

Osteoclasts absorb bone .

-

Vestibular system

o

Semicircular canals detect b
alance/acceleration

-

Hypoxia (Lack of oxygen at high altitudes/low atmos pressure)

o

100% O
2

at 40k feet only gets you 188mbar (normal sea level O
2

= 212mbar)

o

People living at 4000m have reduced mental performance , reduced attention span.

Night vision decrea
sed by 50% at 5000m

o

Hyperventilation reduces PCO
2

of blood , this results in blood becoming more alkaline.

Neurobiology

-

What does the nervous system do

o

Receive and interpret information about the internal and external environment of the
body [Sensory syste
m}



Includes : touch/pain , proprioception/balance , vision , sound , taste , smell, time...

o

To make decisions about this information [Intergrating system]



From sensory information and previous experiences..

o

To organize and carry out action [Motor System]



Motor neurones , muscles , glands..

-

How do things get to and from axon terminals (from cell body to synapse)

o

Anterograde transport


from soma down axon to terminals (v.fast/slow)

o

Retrograde transport
-

From terminals to soma (fast)

-

Complexity of nervous
system : Nerve nets
-
> Nerve Ring
-
> Ganglia
-
> Fusion of Ganglia

-

Further complexity of NS : Ganglia
-
> Brain and Nerve cord
-
> Vertebrates encephalization(brain)

-

Vertebrate nervous system is divided into:

o

Central Nervous System (CNS), which is divided int
o the brain and spinal cord

o

Peripheral Nervous System (PNS) which is divided into autonomic (involuntary) and
somatic (voluntary) nervous systems.

-

Diencephalon (region of the brain) contains the

o

Thalamus : integrates sensory information

o

Hypothalamus : aut
onomic control , appetitive drives , reproductive behaviour ,
homeostasis , endocrine control

-

Resting Membrane potential

o

Depends on intact cell membrane , ionic concentration gradients and ionic
permeabilities and metabolic processes

o

The mem
brane is not af
fected by Sodium ions

o

Concentration gradient wants to move K+ out , Electrical gradient wants to bring K+ in.

-

The balance points (equilibrium E
k
) can be calculated using the Nernst equation

o

E
k

= (RT / ZF) Log
10

([K+] Out / [K+] in)

o

RT/ZF ~ 58
at room temp.

o

Membrane potential =
-
58mV

-

In reality , membrane potential is usually less negative than E
k

o

Ek =
-
80mV , Em =
-
70mV

o

This is due to cell membrane not being completely impermeable to Na+ (Na+ moves in)

o

There is K+ leakage (K+ moves out)

-

Summary of action po
tential characteristics

o

Triggered by depolarisation

o

Threshold level of depolarisation must be reached in order to trigger an action potential

o

Action potentials are All or nothing events

o

Action potential propagates without decrement throughout a neuron

o

At i
ts peak the membrane potential reverses becoming positive

o

After firing of an action potential there is refractory period.

o

Larger diameter of axon = faster conduction

o

Energy is not immideately needed for action potentials to occur , however over time the
me
mbrane potential will not be able to regenerate as no pumps.

-

Synapses (the junction where information is passed from one neurone to another)

o

Can be electrical (2
-
way , no delay) or chemical (one
-
way , small delay)

o

Neurotransmitters for chemical synapse are

packaged in vesicles

-

Neurotransmitter Release

o

1. Docking


SNAP and SNARE proteins anchor vesicles to presynaptic membrane.

o

2. Ca+ influx


Action potential opens voltage gated Ca2+ channels , Ca2+ floods into
presynaptic area.

o

3. Fusion


Ca2+ causes
vesicle fusion and release of transmitter by exocytosis

o

4. Recycling


Vesicle buds off and is recycled

-

Acetylcholine


released from nervous system to muscles to activate them

o

Acts at 2 types of receptor :



Nicotinic (activated by nicotine) : ionotropic ,
permeable to Na+/K+ , fast signalling



Muscarinic (“ muscarine): Metabotropic , 2
nd

messenger cascade , slower signalling

o

After postsynaptic action , acetylcholine is broken down into Choline + Acetate by AChE
(acetylcholinesterase)

-

Two branches of the Aut
onomic Nervous system

o

Sympathetic : Preganglionic transmitter = acetylcholine , postGT = noradrenaline



Fight or flight

o

Parasympathetic : Pre and postganglionic transmitter = acetylcholine



Rest and digest

-

Autonomic reflexes modulated by higher centres

o

Brain

stem : Urination reflex , defecation

o

Hypothalamus : Regulation of homeostasis , Motivation emotional behaviour


Animal and Plant

Animal


-

Features of embryogenesis important in classification
(distinguishing protostomes from deuterostomes)

o

Prenote: 3 germ
layers , Ectoderm (outer) , mesoderm (middle) and endoderm (inner)

o

Patterns of cleavage



Ra
d
ial (often with regulative development) , symetrical stacked layers of cells.

Typical of
d
euterostomes.



Spiral (often with mosaic development) , intercalating cell l
ayers

Typical of Protostomes

o

Speed of Cellular Determination



Regulative Development , cell fate is determined slowly. Typical of Deuterostomes



Mosaic Development , cell fate is determined early, often through maternal
determinants. Typical of protostomes

o

Fate of the blastopore



Becomes mouth in protostomes and the anus in deuterostomes.

o

Origin of the mesoderm and coelom



Protostomes : mesoderm forms from accumulation of cells between the endoderm
and the ectoderm. Coelom forms by splitting.
Schizocoelous



Deu
terostomes : mesoderm forms from outpocketing of the early gut and coelom is
formed directly.
Enterocoelous

o

Taxonomic groupings



Monophyletic (aka. Clade)


most recent common ancestor and all descendants



Paraphyletic


Most recent common ancestor and some
of its descendants



Polyphyletic


Not containing most recently common ancestor, multiple origins.

-

Chordate Hallmarks

o

Notocord

o

Dorsal Tubular nerve cord

o

Pharyngeal pouches

o

Endostyl

o

Postanal tail

-

Neoteny : the process where an animal becomes sexually mature
at larval stage of their life cycle.

-

Evolution of primates ,Primates
-
> prosimians/simians,simians
-
> new world monkeys/old world

o

Prosimians : Binocular vision , nocturnal , arboreality (living in trees)

o

New world vs Old world : Grasping tail , nostrils ,
thumbs , teeth

-

Fish

o

Gill slits from pharngyeal slits , in mammals these become jaws/ear bones

o

Gill supports (arch)

o

Adaptations of the fish



Locomotion (fins/body) , neutral buoyancy (swim bladder)



Osmotic regulation , temperature regulation, respiration ,
feeding , migration



Stiffer body = more posterior force , drag reduced by body shape



Fins provide stability , some fins are used for lift generation (keeping horizontal)

Some fish also use them for walking on land and others for manoeuvrability.

o

Swim bladd
er


large sac of gas , in primitive fish filled by swallowing air but cannot
adjust to pressure as fish dives. In advanced fish is filled by secretion of gas from blood.

o

Ventilation


Resting fish


buccal muscles move H
2
O through gills , Swimming fish us
e
‘ram jet’ ventilation (swim with mouth open)

-

Anura (Frogs)


adaptations to jumping

o

Skull


flattened and widened

o

Pelvic girdle


Ilium long

o

Vertebrae


reduced in number

o

Limbs


Hind limbs elongated , Tibia , fibia , radius and ulna fused to take stress

off on
jumping and landing.

o

Ribs


usually lost

o

Pectoral girdle strengthened and fused up in advanced jumpers to withstand landing.

-

Snakes


feeding adaptations

o

Girdles (shoulders) reduced and incomplete

o

End of tracheae (air pipe) at front of mouth,
allows swallowing and breathing.

o

2
nd

Lung halfway down tracheae , breathes as food collapses main lung.

o

Bones of skull can move extensively and jaws detatch.


mouth stretching.

-

Pit organs


respond to infrared radiant energy

-

Human face genes

o

Hoxa2 gene e
xpressed in the jaw , inactivation results in jaw bone turning into another
hyoid arch.

o

Bcl3 : cleft palate

-

Evolution of homo sapiens

o

Multiregional model : parallel evolution occurred at different areas at approximately the
same time. (deep genetic root)

o

O
ut of Africa model : Global takeover of humans starting from Africa (small genetic root)

Plant

-

Communication between cells


neighbouring cells via direct connections ,distant cells via chems

o

Animals : Tight junctions , anchoring junctions and gap junction
s

o

Plants : Plasmodesmata


pass small molecules and water , allow them to communicate.

o

Signalling molecules can be hydrophobic or hydrophilic , hydrophilic molecules have cell
surface receptors and hydrophobic molecules have intracellular receptors.

o

Hormon
es : travel in blood in animals , travel in vascular system in plants.



Diffus or are transported into the cell and bind to a receptor



Activate specific genes.

-

Aquaporins


a protein which facilitate’s water movement

o

Increases hydraulic conductivity of
tissues.

o

Regulate symplastic water movement

-

Transpiration (also moves water up plant)

o

Governed by ‘guard cells’ which open/close the stomata. Change shape (open) when
water goes into them , making them turgid and push against each other.

-

Cohesion
-
tension t
heory and column of water


you know this shit.~


-

Recalcitrant seeds : Seeds which do not survive drying (eg. Avocado, mango)

-

Vivipary : seeds germinate while still attached to mother plant

-

Seeds store starch and oils to support germination

-

Seed dispersal


o

Self dispersal : wind , wings , plumes

o

By animals : hook/barbs/spines , covered in sticky substances , eaten and dispersed later

-

Fruit types (fruit develop from ovary walls)

o

Berry : fruit wall is soft and fleshy (any berry)

o

Drupe : fruit wall is soft and

fleshy , inner part is hard and stony (peach)

o

Capsule : dry and splits open along several seams (cotton)

o

Legume : Dry and splits open along two seams (peas)

o

Grain : dry , contains single seed, seed coat is fused to fruit wall (wheat)

o

Nut : single seeded f
ruit in which ovary wall and seed coat remain separate (walnut)


-

Photosynthesis recap

o

Light reactions on the hylakoid membrane



Input : Light /// output : NADPH and ATP

o

Carbon fixation (dark reactions) in the stroma



Calvin cycle



Input : RUBP and CO2 , NADPH

and ATP



Output : Trioses
-
> to starch and/or sucrose

-

C4 Photosynthesis

o

Physical separation of CO2 fixation (mesophyll) and calvin cycle (bundle sheath)

o

CO2 is fixed by PEP carboxylase in mesophyll cells to form Malate

o

C4 compounds (mostly malate) carry CO
2 from mesophyll to bundle sheath cells.



C4 decarboxylation feeds CO2 into calvin cycle.

o

PEP
-
carboxylase is saturated at atmospheric levels of CO2 , O2 does not compete.

o

Plants can minimize stomatal opening , conserving water.

o

ATP must be used to regenerat
e PEP , less efficient in their use of light

o

C4 plants benefit less from increases in atmospheric CO
2

but they make better use of
water and nitrogen than normal C3 plants.

-

CAM Photosynthesis (best water usage, lose 2.5
-
5 times less water than C3 or C4 plan
ts)

o

C4
-
C3 seperation is temporal



C fixing at night , calvin cycle in the day.

-

Nitrogen fixing organisms : Cyanobacteria , free living soil bacteria , leguminous plants

o

Problem : Nitrogenase is extremely sensitive to O
2

o

Root Nodules : symbiosis between bact
eria and plant , roots attract rhizobia.

Bacteria produces Nod factors , plant expresses nodulin genes and the bacteria then
modify root hairs. Bacteria move in to the plant by dividing force


-

Plastids (major organelle , differentiate into chloroplasts ,
amyloplasts , statoliths.. etc etc)

o

Perform many important metabolic functions

o

Double membrane + thylakoids

o

Plastid type and number differs among cell types.

o

Differentiate into Proplastids , etioplasts , chloroplasts , chromoplasts...

-

Thylakoid biogenesis

o

Lamellae develop first , followed by grana stacks

o

Thylakoid membranes have unique lipid composition



Rich in galactolipids



Asymetrically distributed

(proteins also distributed like this)

-

Vacuoles

o

Provacuoles are vesicles from the er/golgi

o

(LV) Lytic

vacuol
es contain a dilute solution of ions and organic acids for turgor ,

Secondary metabolites (pigments) , hydrolytic enzymes , metabolic waste products

o

(PSV)
Specialised protein storage vacuoles

store proteins in developing seeds


-

Cell Walls

(made out of 2 w
alls and a layer of pectin)

o

Network of polysaccharides and proteins

o

Functions : Determines cell shape and volume, confers mechanical strength , determines
relationship between volume and turgot pressure , forms vascular tissue , acts as
diffusion barrier/d
efense , cell wall fragments can act as signalling molecules.

o

Primary wall : Cellulose microfibrils extruded from plasma membrane and orientated by
microtubules. Matrix of pectin , hemicelluloses and structural proteins.

o

Cell walls move by ‘polymer creep’
, pressure expands the wall and the matrix yields
allowing cellulose microfibrils to expand the wall slightly

o

Secondary wall (inner): variable structure , inextensible , may contain lignin.


AIDS

-

Glycopeeptide antibiotics

o

Bacteriicidal by binding to termin
al D
-
ala at the end of pentapeptide chains which are
part of the growing cell wall. This inhibits transglycosylation reaction and prevents
incorporation of new subunits into the growing wall

-

The names of the organisms which cause ‘plague’ , ‘tularemia’ , ‘
botulism’ ‘anthrax’

o

Yersinia pestis ,
Franciscella Tularensis , Cloistridium botulinum
, Bacillus Anthracis

o

Plague referred to
as
black death due
gangrene

, called bubonic plague due to formation
of buboes on body.

o

Botulism = due to neurotoxins

o

Anthrax =

due to exo
-
toxins

o

Plague = due to Yersinia murine toxin

-

Gram negative and gram positive distinction

-

Functions of polysaccharides in prokaryotes

-

Bacterial motility

-

% similarities of bacteria (eg. GC
ratio comparison , DNA DNA hybridization , Multi
-
locus
se
quence typing)

o

If DNA
-
DNA hybr
idization , when hybridization >70% = same species , <10% different
genus , ~25% same genus

o

>5% difference in GC ratio means very unlikely to have common sequences

-

Importance of 16S rRNA molecule in phylogenetic studies

-

What
is streptomycin

o

Streptomycin is a bactericidal antibiotic which binds to the 16S rRNA of the bacterial
ribosome , interfering with the binding of f
-
Met so that initiation cannot take place.

-

B lactamase

o


-

Differences between intrinsic antimicrobial
resistance and acquired resistance

-

Advantages of using phylogenetic methods over traditional methods for bacterial identification

-

2 examp[es of infectious diseases (host , vectors, mode of transmission , method of control)

-

Forms of Anthrax

o

Cutaneous anthra
x

o

Gastrointestinal Anthrax

o

Pulmonary Anthrax

-

Desirable properties of a microbial agent (antimicrobial properties)

o

Selectivity for microbial rather than mammalian targets

o

Cidal activities rather than static activities

o

Slow emergence of resistance

o

Narrow spe
ctrum of activity

-

Aminoglycoside antibiotics

o

Streotyomycin , gentamycin

o

Work by binding to 30s ribosomal subunit and preventing fMet
-
tRNA from binding.

-

Tetracyclines

o

Work by binding to the small ribosomal subunit in a way which prevents tRNA from
entering
the receptor sites on the ribosome.

-

Chloramphenicol

o

Affinity for large 50S subunit where it blocks peptidyl transferase

-

Quinolones

o

Inhibits DNA gyrase and topoisomerases so DNA replication undergoes supercoiling...

-

Rifamycin

o

Binds to RNA polymerase and blo
cks synthesis of mRNA

-

Beta
-
lactams

o

Inhibits cross linking in bacterial cell walls

o

Does not work against organisms with no cell walls , like mycoplasma/rickettsia


Microbiology

-

Hallmarks of cellular life

o

Metabolism via taking chemicals from the cells extern
al environment and excretion of
wastes into the environment.

o

Chemicals are turned into new cells through direction of pre
-
existing cells.

o

Differentiation via formation of new cell structures (spores)

o

Communication by cells through chemicals taken up.

o

Livi
ng organisms are often capable of self
-
propulsion

o

Evolution.

-

Uses of microorganisms

o

Agriculture : N2 fixation , nutrient recycling

o

Enviroment : Biofuels (via fermentation) , microbial mining (CuS to Cu)

o

Biotechnology : Genetically modified organisms , gene

therapy, pharmaceuticals

o

Food: Preservation , food additives (e.g MSG)

o

Diesease : Treatment/cure , Prevention , Adaptation

-

Koch’s Postulates


Pathogen present in all cases of disease must be isolated and grown in pure
culture. Pure culture of suspected o
rganism must cause disease to healthy animal


organism
should be re
-
isolated and shown to be the same as the original.

-

Gram staining [gram +ve cells are purple , Gram

ve cells are pink/red]

o

Heat fix the smear and then flood with crystal violet to dye all

cells purple

o

Add iodine solution

o

Decolourize with alcohol , gram +ve cells are purple , gram
-
ve cells are colourless

This is because the stain affects the peptidoglycan , gram +ve have lots of peptidoglycan.

o

Gram positive cell wall has a large layer of p
eptidoglycan , on the peptidoglycan there
are Teichoic acid chains along with lipoteichoic acids.

o

Gram negative cell wall has small layer of peptidoglycan followed by outer phosphoslipid
membrane which contains alot of lipopolysaccharides/other polysacchar
ides.

o


-

Peptidoglycan : gives structural strength to cell wall and counteracts osmotic pressure of the
cytoplasm (thicker in gram positive bacteria). In gram positive , is important for attachment and
stereotyping purposes.

-

Lipopolysacharides (gram

ve onl
y) contributes to structural integrity and protects the
membrane from some types of chemical attack.

-

Bacterial Capsules

o

Functions : Virulence factor , protection from bacteriophage attack ,
minimize effect of
dehydration , attachment to other cell

o

Glycocalyx : 95% water, polypeptides

-

Bacterial motility


modes of flagellation (how the bacteria move)

o

Monotrichous


single polar flagellum (on one side of the
bacteria)

o

Amphitrichous


single flagellum at each pole (each side of
the bacteria)

o

Polytrich
ous


Tuft of flagellum on one pole (
or both ends)

o

Peri
trichous


Flagellum all over the bacterial surface (all
sides)

o

Motor proteins
(
working
via protein motive force )
drive
the S and M rings in the
Motor complex , this spins the
flagellum.

The protons are
extruded into the periplasm

and the electrons
recaptured by
iron
-
sulphur proteins
, these are used to
reduce ubiquinone
,
releasing two protons

and
transferring electrons

to the
cytochrome system
.

-

Bacteria respond to environment (tactic re
sponses)

o

Aerotaxis


movement [towards/away/towards optimum] oxygen

o

Chemotaxis


movement towards source of nutrients



A biased random walk , transmembrane proteins detect and measure changes in
concentration in the environment.
Methyl accepting chemotaxis
proteins detect
and measure changes in the environment. These then

interact with cytoplasmic
proteins called che proteins which interact with flagellum rings telling it whether to
tumble or continue

o

Magnetotaxis


movement along lines of magnetism


o

Gliding
Motility


Some bacteria can ‘glide’ , these bacteria have no flaggelum , they
produce lots of capsular material and their outer membranes are rich in sulphonolipids.
Areas of outer membrane move relative to each other to achieve movement.

-

Strategies for c
ontrol of infectious diseases

o

General : Water purification , sewage disposal , improved nutrition/housing

o

Food : Cold storage , pasteurization , food inspection , adequate cooking

o

Others : Control of vectors , vaccines, chemotherapy , changes in personal h
abits
(condoms/hygene) ,control of drugs , inspection of transfused blood/organs

-

Stuff which inhibits bacterial growth

o

Low temperatures , very high temperatures , acidic pH/alkaline pH , high osmotic
pressures (salt/sugar) , dry environments.

-

Physical , ch
emical and anatomical barriers to infection

o

Lysozyme in tears and other secretions dissolves cell walls

o

Cillia , flora and mucus trap and remove microorganisms from lungs/throat

o

Undesirable pH in gut inhibits microbial growth

o

Flushing of urinary tract prev
ents colonization , phagocytes in lungs prevent colonization.

-

Molecular Mugging


bacterium seizes hold of transferrin (transports iron around the body) and
steals the iron from it.

-

FAME (fatty acid methyl ester) analysis

o

Involves growing the cells under
standard conditions isolating the fatty acids and then
changing them to their methyl ester form.

-

Other Genotypic analysis

o

DNA
-
DNA hybridisation


distinguishes how alike two DNA’s are

o

DNA profiling


rapid method to distinguish between strains

o

MLST


strai
n typing using different genes , distinguishes very highly related strains

o

GC ratio


poor resolution

Virology

-

Different modes of transmission used by viruses

o

Host to Host transmission (Person to Person)



Airborne (influenza , common cold)



Faecal


Oral ( p
oliovirus , rotavirus)



‘Fomites’
-

infected materials , such as bedding hepatitis A)



Contact


including sexual transmission (herpes , HIV , ebola)



Blood exchange (Hep B , Hep C , HIV)

o

Vector transmission



Almost always arthropods


‘Arthropod borne’ (aka.

Arbo
-
viruses)



Eg. West Nile Virus


from birds to birds and then to humans via mosquitos

-

Classification schemes for viruses

o

Baltimore

classification



‘Positive sense’ refers to a version of the strand which can be directly translated to
make protein , this

also applies to DNA as if it were RNA it could be translated to
protein directly. Negative sense strands have to first be converted to positive sense
strands before they can be turned into protein.



Advantages : relies upon fundamental unchanging character
istics of the
virus , predicts many aspects of the molecular biology of the virus.



Disadvantages :
Nature of genome may be hard to determine , group IV and
VI cannot be distinguished on genome alone , group I and VII cannot be
distinguished on genome alone

(but not usually an issue)

o

ICTV


developed a latinized family naming system widely used for animal viruses, uses
the taxon structure



Alot of Viruses have yet to been classified

o

Holmes classification


tried to throw everything under 3 groups , bacteria a
ttacking ,
plant attacking and human attacking.




-

Plaque assay

o

Get a single cell monolayer under liquid media , add virus then add semi
-
solid media on
top of this and incubate.

o

You need to dilute the virus stock solution until you find a dilution which give
s a low
number of viruses. Stain with crystal violet , live cells will stain and plaques show as
cleared areas (virus)

o

This will result in plaques per ml
-
> plaque forming units per ml.

-


-

Virus replication , a generic overview

o

Attachment , penetration , unc
oating , biosynthesis (genome replication and gene
expression) , assembly and release

-

Measuring virus replication

o

Single step growth experiment (analysis of single replication cycle in an infected culture)



Latent Phase = Eclipse Phase (minimal replication)

+ Maturation Phase (replication)

-

Lysogeny / Lysogenic Cycle

o

The intergration of the bacteriophage nucleic acid into the host bacterium’s genome.
The newly integrated genetic material called a prophage , can be transmitted to
daughter cells at each subsequ
ent cell division.

-

Viral Tropism

o

A virus will only infect cells which have the correct receptor to which it can bind to.



If many cell types have the viral receptor , the virus will infect widely (such as the
receptor for measles , this results in all human

cells being infected)



If only one or a few cell types have the receptor , the infection will be restricted
(such as the HIV receptor only present on white blood cells)

-

Mechanism of dissemination (spreading after replicating at the site of infection)

o

Virae
mia = when viruses enter the bloodstream

o

Virus progeny spread to the circulatory system , ‘
Primary vira
emia’

o

more distant cells are affected , wave of ‘Secondary
Vira
emia’ occurs

o

Virus progreny spreads mechanically or in infected migating cells to the circ
ulatory
system

-

Polarised release of virus (2 types of viral release)

o

Apical release favours spread to other hosts
-
> tries to release viruses as external as
possible

o

Basolateral release favours spread within the host (viremia)

-

Phage Lambda

o

Integration



Occ
urs at a single location (att lambda locus) adjacent to gal operon



Phage integrase protein catalyses homologous recombination between ‘att lambda’
and identical 15bp sequence in phage ‘att P’ located near int gene



E.Coli cellular response induces lysogeni
sed phage to enter the lytic replication cycle.



Stimulates host RecA protein activity to change from a role in recombination to
function as a protease targetting LexA repressor and the phage Cl protein.

Continued RecA inactivates any Cl so only lytic cycl
e can occur.

o

Replication



Stage 1
-

Theta Replication



Starts from origin and proceeds in both directions



Replication forks



Generates interlinked double stranded DNA circles , thisi s resolved by DNA
gyrase activity.



Stage 2
-

Rolling Circle replication



Continuous DNA synthesis causes the outer strand to continue extending
eventually leading to a σ shape.

o

Processing



Concatamers cleaved by the terminase protein complex , this cleaves double
stranded cos sites.


-

Pathogenicity


nature and severity of the d
iesease caused

-

Virulence


Refers to the differing pathogenicity of different strains of the same virus.

-

Infection outcomes:



Acute infection
-
> Death



Acute infection
-
> recovery by immune response



Persistant infection
-
> continuous viral production with or

without chronic disease.



Chronic persistance


Constant virus production



Latent persistance


usually undetectable but production may reactivate

o

Majority of symptoms from disease is from immune response , eg. Inflammation , fever
etc.



‘Bystander effect’ m
eans uninfected cells may also be damaged in the purging of
viral cells.

o


Immunology

o

When bacteria bind to macrophage receptors , cytokines are released and small lipid
meiadtors of inflammation.

o

Basic Innate Immunity



Physical Barriers : Skin
, mucus ,
epithelia



Cellular systems :



Phagocytosis by neutrophils and macrophages


detects foreign material by
bacterial carbohydrates recognized by receptors or by coating with opsonin.



Cytotoxicity by eosinophils and large granular lymphocytes , kill by release
of free radicals onto the surface of the cell (Oxidative Burst) or by insertion
of a pore structure into the cell membrane which results in osmotic bursting
by Na+ Influx



Inflammation , caused by basophils and mast cells which degranulate. This
releases va
soamines like histamine which have roles in increasing blood
flow , blood vessel permeability and increasing adhesion



Extracellular molecules



Lysozyme


bathes the cells and destroys peptidoglycan on bac cell walls

by
splitting amino sugars.



Complement


activated directly by bacteria or via antibody antigen
complexes, consists of a cascade of proteases which results in massive amp
of ‘membrane attack complex’



Interferons / other cytokines


acts as chemoattractants / local hormones

o

Inhibits viral replicat
ion, stimulatory role for many aspects of
immune system

o

Basic Adaptive Immunity



Carried out by the small lymphocytes , T and B
-
Cells and aided by dentritic and
macrophage cells.



Small lymphocytes produced for the arms of immunte response

, 2 types:

o

T Cells

help other cells or kill infected host cells

o

B Cells produce antibodies

(needs to be activated by T cell)



Specificity of receptor for epitope (ligand present on antigen)



Selection by antigen



Clonal Expansion



(Clonal Selection ) Following infection , indiv
idual clones are selected by
antigen based on the fit of the antigen and receptor



(Clonal Expansion ) Clones of cells with receptors complimentary to antigen
divide



(Clonal Deletion ) Immune cells which react inappropriately with self are
deleted

o

ADCC


also known as Killer cells

o

Antibodies binding to antigens
can:



Enhance the Innate Immune system



Helping with phagocytosis ,Opsonisation , involvement in ADDC cytotoxicity ,
triggering the activation of complement, triggering inflammatory response



Act direc
tly with pathogens



Binding to toxins produced by pathogens can neutralise them , binding to
antigen can change the confirmation of the antigen


altering its function ,
bind to virus directly may neutralise it.

o

Immunoglobulins , Ig
M , G , A , D and E (Met
al Gear Acid : Double ender)



IgG


exists as subclasses , classic Ig structure

(Y)

of 2H 2L chains. Fundamentally
important as part of 2ndary response. Performs toxin neutralisation , binding to
microorganisms and inducing their opsonisation and activates
complement.



IgM


Pentamer , is an agglutinator and is produced in primary response.



IgA


Exists as a polymer of 2 Y units , principal cell for mucosal immunity



IgD


Trace Ig , important as an indicator of developmental stage.



IgE


responsible for peps
in digestion and triggering allergic response by binding to
Fc receptor on the surface of mast cells and basophils and so cause degranulation
and release of histadine.

Epidemology

o

Epidemic : Occurance in a community or region of a group of illnesses simila
r in nature in
excess of normal expectancy.

Pandemic : Outbreak of widespread illness/death of exceptional proportions.

Endemic : stable equilibrium of illness

-

How can an infectious agent persist in a population when the susceptible are depleted

o

Reservoir
or transmission stages outside the human host.

o

“Carrier states” , clinincally recovered remain infectious

o

Long incubatiopn period with low host mortality

-

Vector Control

o

Human bait traps (insectiside nets)

o

Non
-
Human bait traps (sponging cows with
insectiside)

o

Urban breeding site source reduction

o

Rural drainage of breeding sites

-

Eradication and elimination definitions , examples of each.

o

Eradication = Zero incidence worldwide

o

Elimination = Zero incidence locally

-

Critical community size , Extrinsic i
ncubation period definitions

o

Minimum host population size required for the pathogen to persist

-

Prevalence / incidence of an infectious agent in a host population

-

R
0

, R (effective R)

[microparasites]

o

R
0

= Avg new cases arising from one infectious case int
roduced into wholly susceptible
individuals

o

Effective R = R
0
x Susceptibles

o

S* = 1/R
0

, eg if R
0

is 5, then S* is 0.2 , which means 20% pop susceptibility results in
parasite persistance.

-

R0 of macroparasites

o

R0 = Average number of female worm daughters
produced by a single female worm
during her lifetime in a completely susceptible population

-

Density dependence in microparasites : largely governed by lack of susceptibles

-

Vaccination coverage / herd immunity

o

To protect the population , need to vaccinate ‘
1


(1/R
0
)’ , so eg R
0

is 5 , need to
vaccinate 0.8 , or 80%

o

The 20% unvaccinated gain protection by everyone else being vaccinated , this is Herd
Immunity

-

Transmission cycle , epidemiology , control of vector borne diseases

-

S
-
I
-
R compartmental models of
infectious agents

o

Susceptible , Infected , recovered box plot thingy with arrows

-

Why was smallpox suited to eradication

o

Distinctive and easily recognised disease , incubation of 12
-
14 days

o

Infectious 4
-
6 dayd during rash

o

Patients usually transmitted to few

others, R
0

<6

o

Strong seasonality , low incidence period when small number of cases can be dealt with
easily.

-

Example : control without eradication


measles

o

Shares some of the properties of smallpox

o

No animal reservoir

o

Safe, cheap , effective vaccine avai
lable

o

High disease related morbidity / mortality

o

But higher R0 than smallpox

-

Culling : mass killing
of animal to prevent spread of disease.

-


Case Studies

Badgers / bovine tuberculosis

-

Krebs trial

o

Tried 3 treatments , proactive (cull regularly) , reactive (
cull when outbreak) and a survey
group for comparison.

o

Reactive cull areas experienced increased risk of infection

o

Proactive cull areas had decreased risk of infection but outside the culling area the risk
increased.