Abstract reason of alertness or somewhat how the

Abstract

The instant venture of
the aesthetics and the neurosciences is to find out the reason of alertness or
somewhat how the material brain produces our in Material sense of awareness.
Some scientist says that consciousness is an operation of brain, but as consciousness
can happen when brain is not working that’s why some scientists thinks that
consciousness is not physically related to body or brain. As the brain is a
material object, consciousness can understand 
by study of science. The human brain has unbelievable abilities and it’s
a complicated mass of tissue. An 
important part in learning and remembering is played by microtubules as
they focus on neurotransmitter function. Microtubules could connect the memory
and consciousness because these two processes are related to each other. The
total brain protein consists of 15% tubulin, tubulin is protein which
polymerize and arrange in hexagonal cylinders to form microtubules. The
heterodimer tubulin subunit’s shape straight or curved tells the arrangements
of microtubules polymers . As the process by which brain functions to cause
consciousness is not known that’s why the process by which consciousness is
limited by anaesthetics is not well explained. Tubulins have the pi
electron-rich indole rings, has the distance of 2mn,  on the smaller non-polar regions . Penrose-
Hameroff Orchestrated Objective Reduction (Orch-OR) Theory explains that
entangling of  these electrons occurs
because these are close enough.The arrangement of tubulin in presence of
anesthetics is explained in this paper.

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Introduction

Consciousness could be
summarized in many aspects. A number of a researchers thought that even whenever
our brain is not working the consciousness is continued. It can be explained in
other words that the consciousness is a term that is aparted from physical body
and the brain. From scientific point of view consciousness is brain
functionality. Brain is an object or material for the study of science. The
brain of the human is a very complicated mass tissue granted with the
outstanding abilities.in higher animals like humans the brain is a centre for
controlling the CNS.  In these days
several scientists as well as researchers are busy in decoding the secret of the
consciousness. Now here a question arises that can a strong brain imaging
technology define consciousness that is an immaterial thing in the nature. the
consciousness theory explained by Penrose and Hameroff, consciousness arises
from brain and target specially on the synapses complicated computation which
will let the communication within the neurons. The Neurons contain a
well-organized pattern of the microtubules due to a type of proteins high in
number called special nonmotor microtubule (MAPs) attacted with the nerve cells.
It was said that the many organizations of MT between the dendrites and the
axons can perform a special function in signaling of neurons. Evidences shows
that a connection exists within the performance of MT and the imaginary
functions by explaining that within the interval when the accumulation of the
synapses and also the imaged information happens at the  high degree the  cortex (visual) of brain yields a very high
number of the tubulin 1.  A latest
connection within the microtubules and the cognition has studied having the patients
of the Alzheimer’s disease 2.

Methods and Materials:

Tubulin, taxol and
general tubulin Buffer are supplied by cytoskeleton Inc.Denver, Co.USA.

Preparation of Buffer
T:

100mM of GTP stock
solution is added to General Tubulin Buffer at a final concentration of GTP
that is 1mM. Buffer T will be stale for 2-4 hours on ice.

Flourescent reporter
buffer:

It contains 80mM of of
Piperazine-N.N,-bis seuisodium salt,2.0mM Magnesium chloride, 0.5mM Ethylene
glycol-bis N, N,N,N,,N,-tetra-acetic acid, pH 6.9,10 micro meter DAPI.

Tubulin Reconsitution:

1mg of lyophilized
tubulin is resuspended in 1ml of Buffer T at 0-4oC. It is as used as prepared
because it is not stable. Three concentration of Propofol were prepared. All
soloution are prepared in MilliQ water.

Kinetics study of
polymerization of bovine brain tubulin in presence of Propofol:

Spectrophotometer was
used for the polymerization of Bovine brain tubulin. Kinetics of microtubule
was monitored by Eon spectrophotometer for 1-2 hours at 350nm for 1 min at
370C.

Propofol effect on
polymerization of tubulin was studied too.It has 95-99% protein binding
affinity and life time of 30-60 min. Propofol has many mechanism action but due
to potentiation of GABA receptor activity the channel closing time is slowing
down and also it act as sodium channel blocker. It also causes reduction in the
brain information integration capacity at gamma wave frequencies.

The effect of propofol
in presence of GTP also studied.in the presence of GDP and zinc ion tubulin
protein do not polymerize into microtubules.

Circular Dichorism
Measurements:

The study of tubulin by
CD carried through a polarimeter of Applied Photohysics with a Quantum. It is
calibrated with the d-10-camphorsulphonic acid. All measurements are made at
25oC.Spectra are collected. Each spetra has an average of 10 scan. All spectra
was smooth with 25 convolution width.

TRFS measurements:

LASER was used as
source for the DAPI excitation present in fluorescent buffer.Emission was
collected at 440nmthrough monochromator. Emission decay were fit with
instrument response function that was collected by scattering solution.

Results and discussions

Eon Spectrometer was
used at 350 nm for 1-2 hours  Instrument with
the time interval of 1 minute at 37°C to monitor  the kinetics of microtubule assembly.
Polymerization of bovine brain tubulin was done by without usage and usage of
guanosine triphosphate (GTP). Graph show different effects in the presence and
absence of (GTP). In the presence of (GTP) the microtubule polymerization or
self organization was found to be stable to some extent and also less dynamic.
Microtubule was found to be less stable in the absence of  GTP.( GTP) also show other effects that are
not only enhance the of polymerization, but is also essentially required for
polymerization . Effects of taxol also seen on polymerization of tubule and
microtubule in the presence of GTP and glycerol are stabled in the presence of
taxol. The effect of propofol was seen on tubulin polymerization in the
presence of GTP and zinc ion  and
strongly affected with all three concentrations of propofol  with all three sets of experiments. It is
inferred that propofol (anesthetic) binds with hydrophobic pockets of tubulin
by weak force of  Vander waals London
dispersive force.Propofol effect is defined as dosage dependent and time . When
we remove propofol then it can be reversed. 
For the evaluation of the conformation and stability of tubulin protein
a technique is used that is called 
circular dichroism spectroscopy.Method is based on ?helix,?sheets and
random coil.By adding profol to tubulin in presence of Zinc chloride as result
a complete collapse of structure show complete change in overall
confirmation.DAPI not bind to tubulin heterodimer remains free so  it is prefer that binding of propofol with
tubulin rather  binding site than that of
DAPI and then DAPI transfer energy to neighbouring molecule.                                                                      

Conclusion                                                                                                                      

Experiments show that
polymerization of tubules or self organization of microtubles is strongly
effected by profol.By removing propofol the process can be reverse.In presence
of anesthetic propofol microtubule cannot perform collective action and do not
communicate with each other.From CD in the presence of propofol it show major
changes in overall confirmation.So, it is suggested that binding of anesthetics
to tubulin protein produce an alternation in secondary strcture.Change in
secondary strcture is due to binding with anesthetic.