Studying Genes the Ophthalmic Route by MRI, and That too in Living Subjects

It is said that the eyes are the windows to the soul, though science is yet to prove that given the elusive nature of ‘soul’. But researchers has now been able to probe genes in a traumatized brain using the eyes as a gateway.

The brain is normally ‘secured’ from the circulating blood directly, so that endogenous and exogenous toxic substances, macromolecules can not gain entry easily into (and out of) the brain. More importantly, this ‘firewall’ like barrier, called the ‘blood brain barrier’ maintains the constancy of ions inside the brain such as K+, H+, Mg++, Ca++, which is vitally important for the neurons to function normally.

The ‘blood brain barrier’ (BBB: see picture) results from the ‘relative’ impermeability of both the capillaries supplying the brain as well as that of the ‘choroid plexus’ covering the brain. Actually, the endothelial cells of the capillaries are tightly packed (tight junctions) and they are non-fenestrated too. In addition, end feet of astrocytes, a type of glial cells (cells that support and aid neurons), cover these capillaries.

But there are disease conditions in which the BBB becomes leaky. For example, in traumatic brain injury, cardiac arrest, stroke and multiple sclerosis the blood brain barrier is breached, to different extents. In Alzheimer’s disease too, there is thinning of the capillaries as the disease progresses. As expected, the supporting glial cells, particularly the astrocytes, jump into action to seal the leaks. They proliferate, resulting in ‘gliosis’. Gliosis is also found in a tumorous condition of the glial cells called ‘glioma’.

These glial cells contain a protein in them called the glial fibrillary acidic protein (GFAP). Naturally, there is an mRNA for it that ‘translates’ its formation in the cytoplasm. Scientists target this mRNA molecule because tagging it will track the GFAP and consequently the astrocytes in whom GFAP is expressed.

Previously scientists had to inject MR contrast agents intra-cerebro-ventricularly or by other invasive techniques to map these leaking areas. Scientists at Harvard embarked on a novel idea. They produced a short cDNA sequence ‘complementary’ to the mRNA of GFAP. This short stretch of this ‘antisense’ oligodeoxynucleotide (ODN-gfap) would latch onto the GFAP mRNA just as a lock would to its key. They then tagged it with a paramagnetic molecule that they designed, called superparamagnetic iron oxide nanoparticles or SPION, a magnetic resonance susceptibility contrast agent. The SPION-ODN ‘report’ any inhomogeneity in transverse magnetization in ‘T2 star’weighted MRI scan, due to the paramagnetic properties of iron oxide. Liu et al also used a sequence complementary to the mRNA of beta-actin as well (actin is the most abundant protein in mammalian cells and its mRNA is found in all types of cells) to act as a ‘control probe’.

They then anesthetized the mice, the animal model they selected; and caused BBB leakage by inflicting a small puncture or by performing bilateral carotid artery occlusion (BCAO) for 60 minutes. They also tried other methods (see reference). They subjected another group of mice to a sham (=false) operation (no puncture or vessel occlusion but the same operation) at the same time. BBB leakage was checked by T1 weighted Gadolinium-DTPA contrast MRI scan. Gd-DTPA was injected into the jugular veins of the mice. Leakage would show up as enhanced areas on T1 weighted scan (normally Gd-DTPA does not cross the BBB). Due to repair process to seal the leak, glial cells would be recruited and gliosis would result.

The telltale signature of gliosis (and BBB breach) may be found in postmortem tissue samples of the brain. Previously, the GFAP antigen was detected by immunohistochemical methods. But the Harvard team was looking for a non invasive method to detect GFAP. They instilled ‘SPION-ODN gfap’ reporter into the conjunctival sac of the mice by means of eyedrops. They then measured the ‘T2 star’ values in MRI scan and transformed the values to ‘R2 star’ maps (R2 star = 1/T2 star). Areas of leakage showed up as elevated (hyperintense) signals in R2 star maps. It corroborated well with Gd-DTPA scans and also on post mortem examination. SPION-beta actin, the control probe, got bound to the endothelial cells of the vasculature as expected.

The eye drop was absorbed by the lymphatics draining the palpebral (eyelid) and bulbar conjunctiva. The lymphatics then transferred the reporter probe into the veins which finally found their way into the brain. Since the BBB was breached, it finally came out of the circulation into the brain parenchyma. As the probe is detecting mRNA which is ‘transcribed’ from the DNA of the cell, it may be said that they are, in a sense, detecting the genes for GFAP.

Thus we may hope to detect gliosis, a pathology that occurs in a variety of diseases already mentioned, non invasively, the ophthalmic way.

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Reference: Liu, C., You, Z., Ren, J., Kim, Y., Eikermann-Haerter, K., & Liu, P. (2007). Noninvasive delivery of gene targeting probes to live brains for transcription MRI The FASEB Journal, 22 (4), 1193-1203 DOI: 10.1096/fj.07-9557com

A Tale of a Microprocessor, RISC and a Few Loops of miRNA

The word ‘microprocessor’ is generally used to designate VLSI and SLSI (Very/Super Large Scale Integrated circuits) devices which accept, decode and execute instructions presented in binary coded forms. They may be called the heart of the computer. RISC (Reduced Instruction Set Computer), on the other hand, is a type of microprocessor architecture that uses a simplified, yet highly-optimized set of instructions to deliver good performance. However, like ‘cell’ and ‘nucleus’, they too have been adopted in biology, and not without reason!

Proteins are essential for cells as they perform various functions as enzymes, ion channels, receptors and so on. They are manufactured in the ribosomes, organelles present in the cytoplasm, under the instruction of messenger RNA (mRNA). This instruction code is encoded in the sequence of nucleotides that make the mRNA molecule. However, the sequence of nucleotides in mRNA is dictated in turn by the DNA that is present in the nucleus. Messenger RNA carries this message from the nucleus into the protein production units. But what would happen if we interfered with the ‘message’?

RNA interference (RNAi) would occur affecting the regulation of gene expression. Micro RNAs (miRNA) are one of the small RNAs that regulate the expression of protein-encoding-genes, after the mRNA strand has formed. miRNAs have partly or fully complementary sequence to one or more mRNAs. This enables them to latch on to the mRNA molecule masking the ‘instruction codes’ in the mRNA strand, interfering with protein formation (translation). In other words, the gene has been silenced!

miRNAs are first transcribed from DNA by the enzyme RNA polymerase II into primary miRNA (pri-miRNA). pri miRNA is then cleaved by another enzyme, RNAse III, called Drosha, into precursor miRNA (pre miRNA) (see the picture on the left). However, Drosha (an RNAase III endonuclease) is assisted by Pasha (partner of Drosha), another enzyme, in this task. Later, it was found out that these two resided in a 500 kilo Dalton complex, called the microprocessor (micro RNA processor). So far, all these have been happening in the nucleus of the cell. The pre miRNA then moves into the cytoplasm through the exportin 5 pathway. Next, Dicer, another RNase III endonuclease, makes a mature miRNA duplex, which is then ‘uploaded’ into a complex called RISC (RNA induced silencing complex). RISC then prevents translation of the mRNA strand, as the ‘partially’ complementary miRNA strand interferes with the translation of the mRNA molecule into specified amino acid sequences can not occur. We can compare complementarity of nucleotide bases in terms of a pair of gloves and its corresponding fingers. The information of the gloves' coordinates gets obliterated by the occupying fingers. This RISC dependent mechanism occurs in parts of the cytoplasm, called P bodies (‘p’ for processing).

RNAi is very important for plants as they lack an immune system. Invading organisms can not dictate foreign protein formations as their RNAs are destroyed, not merely inhibited, as is usually seen in higher animals (animal miRNAs exhibit only imperfect homology to the mRNA in contrast to plants, and thus they only inhibit translation). Some of the tumor suppressor genes inhibit tumor formation by the action of miRNAs and not through protein formation. In humans, exploiting RNAi may be a useful tool in combating diseases such as cancer, AIDS etc. So it remains to be seen whether the microprocessor can bring a revolution in medicine and research as its counterpart in electronics did in the field of computing.

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Reference: Saumet, A., & Lecellier, C. (2006). Anti-viral RNA silencing: do we look like plants ? Retrovirology, 3 (1) DOI: 10.1186/1742-4690-3-3
Processing of primary microRNAs by the Microprocessor complex. doi:10.1038/nature03049
Wikipedia
The Macro World of MicroRNA (pdf)

Of Twinkling Nanostars and the Possible Application of Stroboscopes in Biological Imaging

Imagine a strong crowd, as you see in a Manchester United versus Liverpool football match and you wished to concentrate on a particular person. How would you do it? Make him wear a fluorescent shirt and dye his hair (don’t do it in the middle of the crowd, I can’t guarantee your safety).

Purdue University researchers have been successful in focusing at the cell of interest among a background of equally noisy and boisterous biomolecules and other metabolically active cells. Currently, researchers use immunological techniques to create an antibody to a molecule and then visualize the ‘molecule of interest’ by tagging the antibody to a radioisotope or a fluorescent dye; and flow cytometry can sort out different types of cells.

The Purdue University team used gold coated nanoparticles with an iron oxide core that was impregnated in the cell they wished to see. They then subjected the specimen to a periodically changing magnetic field. The superparamagnetic cores (superparamagnetic nanoparticles have no net magnetization, but an external magnetic field can magnetize them) responded by rotating as the magnetic field rotated around them. The rotation could be seen in the ‘near infra-red’ light spectrum, as the incident light bounced off (scattered) the specially designed arms of the gold nanostar as it revolved. The rate (rpm) of this gyromagnetic (gyros means to rotate) twinkling could be externally controlled by varying the rate of the externally applied field. You now could identify the cell by its characteristic ‘twinkling’ (lighthouse type) effect.

I am tempted to go beyond what’s been achieved so far. Here I go. I guess you are all familiar what happens to the rotating ceiling fan blades when you turn on a fluorescent lamp. Don’t you see a momentary snapshot of the three blades (some have 4)? That’s what where stroboscope comes in. It consists of a Xenon lamp (ordinary fluorescent lamps could do, but incandescent lamps won’t work as the glowing filament takes time to extinguish) flashing at a controllable rate. The electronic circuitry may be had here.

Suppose that the fan is revolving at 1200 RPM and it is not changing. Set your stroboscope to flash at this rate. You’ll ‘see’ that the fan blades are absolutely not moving, which is certainly not true! But be there any mechanical defect in the fan, it will stand out as the centrifugal force widens it (provided that the fault is more or less tangential to the axis of rotation). Here also we are looking at our object of interest, aren’t we?

Now lets look what implication it might have in biological imaging. We now know that the gamma subunit of mitochondrial F type ATP Synthase ‘actually’ rotates when it is synthesizing ATP (reverse rotation occurs when ATP is hydrolyzed). There are other locomotive units within the cell as well. They comprise of actin and myosin based molecular motors. Could we study them using an externally adjustable stroboscope? The optical (electromagnetic) signals so obtained may then be similarly broken down into simpler trigonometric (sine and cosine) functions by Fourier analysis (Fourier transform) as was done in the ‘twinkling nanostars’ experiment. At least, we expect to get rid of some 'noise' and some good still photos. But if we wanted better resolution and used higher frequency (electromagnetic) for it, some extraneous error will be introduced. It's a trade-off!

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Reference: hyper-links, unless specifically mentioned
Principles of Biochemistry, Lehninger, 4th ed
http://en.wikipedia.org/wiki/ATP_synthase
Wei, Q., Song, H., Leonov, A., Hale, J., Oh, D., Ong, Q., Ritchie, K., & Wei, A. (2009). Gyromagnetic Imaging: Dynamic Optical Contrast Using Gold Nanostars with Magnetic Cores Journal of the American Chemical Society, 131 (28), 9728-9734 DOI: 10.1021/ja901562j

An Overview of Gene Therapy

Ashanthi, a four year old girl, was suffering from an immune deficiency disorder called SCID (Severe Combined Immune Deficiency). Due to the lack of a healthy immune system, she was susceptible to infections even from germs which otherwise would not affect healthy persons. She was confined to her room, met no one outside her family, and had to take heavy doses of antibiotics to fight the microbes on behalf of her dilapidated immune system. A team of doctors from the National Institutes of Health, in the United States, drew blood from the patient’s body, and separated the WBCs (white blood cells; cells which fight infections). They then cultured the WBCs, inserted the missing gene and then infused the blood back into Ashanthi’s bloodstream. The girl survived, she no longer lived a recluse life and antibiotics were no longer a ritual. That was the first approved gene therapy (ex vivo, as the engineering was done outside the body) procedure carried out in a human.

Gene therapy is the procedure of replacement of faulty genes (nucleic acid sequences) by healthy ones. Frequently, a normal gene is added to an existing faulty allele, rather than a replacement of the gene at fault. Genes consist of stretches of deoxy-ribonucleic acids (DNA). The nucleic acid sequences in the DNA dictate the formation of proteins via the mediation of ribonucleic acids (RNA). Information contained in the DNA is passed on to the RNA by a process called ‘transcription’, which occur in the nucleus of the cell. RNA then goes to the cytoplasm of the cell where it forms a protein, in a process called ‘translation’; the functional product of that gene, its spokesman! The DNA sequence determines the sequence of amino acids in the protein, which is important in that any mistake in having the right amino acid in the right place may yield a non-functional protein with an abnormal configuration. Thus, an abnormal DNA sequence might (not always) produce a non functioning enzyme (a protein), causing diseases of immunity, metabolism and cancer.

We can ‘insert’ a normal functional gene into the genome containing an abnormal one; exchange an abnormal gene for its normal counterpart by homologous recombination; we could even ‘regulate’ the ‘expression’ of a particular gene. Inherited genetic diseases like thalassaemia, sickle cell anemia and cystic fibrosis could best be tackled by manipulating the ‘germ cells’ (sperms and ova) and this not only would ensure that the progeny was healthy but would also be passed (this new gene) onto the next progeny. Such heritable ‘germ line therapy’ despite sounding promising, is prohibited due to ethical concerns and the lack of expert technical knowhow. ‘Somatic cell gene therapy’, the gene therapy practiced these days, however, is not heritable.

Now that we know the basics, we should find a suitable carrier (vector) to deliver the goods inside the cell. Viral vectors are the most commonly used. Retroviruses, for example, take with them 2 identical copies of single stranded RNA (ssRNA); an enzyme called ‘reverse transcriptase’ and ‘integrase’, another enzyme, when it enters a cell. Reverse transcriptase or RNA dependent DNA polymerase converts the RNA sequences into DNA. The double stranded DNA then integrates with the host genome by the mediation of ‘integrase’. A therapeutic gene could now express itself in the form of a usable protein, via the integrated viral genome. Since viruses may cause disease, researchers must ensure that the disease causing genes of the virus are deleted. For example, AIDS is caused by a retrovirus (HIV). Another cause for concern is that retroviruses integrate randomly in the human genome. If they sat close to a proto-oncogene, or in the middle of a tumor suppressor gene (this might disable the suppressor gene), it might cause cancer.

Adenovirus is another option. A double stranded DNA (dsDNA) virus, adenovirus, does NOT integrate with the host cell, hangs free in the nucleus and just carries out transcription. Frequent administration is necessary, as the gene does not replicate with the host cell. Adeno-associated virus (AAV), an ssDNA virus, may also be used as a vector. The recombinant type (rAAV) carries NO viral gene & does NOT integrate. But they can infect quiescent (non-dividing) cells, hence may prove useful in neural/neurodegenerative diseases.

Non viral vectors include:
Naked DNA: Transfection (using phosphate-DNA mixture), Electroporation (use of electrical pulse for better membrane permeability), Sonoporation (using ultrasound for facilitation of DNA delivery), gene gun (DNA coated gold nanoparticles ejecting out along with high velocity gas) are some techniques for delivering DNA fragments.
Oligonucleotides: Antisense nucleotide sequences for the target gene. Being antisense, the nucleotides will latch onto the sense strand, just like the opposite poles of a magnet, thus preventing its translation. Fomivirsen is one such drug which is used in cytomegalo virus (CMV) retinitis. 
Short interfering RNA (siRNA); Small nucleotide sequences which tell the cell to cleave faulty mRNA.
DNA-lipid complexes (lipoplexes): here the DNA molecule is covered with an arrangement of lipids in the form of a micelle. Using a nonionic surfactant such as Tween 80 in addition, gave a better yield.

The challenges are still great. Our immune system and the genome do not take these pieces of DNA easily. For example, the gene transfer frequency (in hematopoietic stem cells of dogs and monkeys) for adenosine deaminase, the deficiency of which causes SCID, was only 3%. Still scientists hoped that the healthy cells would outgrow diseased cells as they had distinct survival advantages. But the efficacy of delivery didn't improve.
As of today, most major trials on gene therapy are on pluripotent hematopoietic stem cells (PHSC) and cancer cells. It is only natural to assume that genetic manipulations on blood stem cells (PHSC) would cure a variety of diseases affecting the blood cell lineages. And the quest goes on.

1. Mark A. Kay*,, 2. Dexi Liu, and, 3. Peter M. Hoogerbrugge (1997). Gene therapy PNAS , 94
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References: Gene therapy
Gene therapy PNAS November 25, 1997 vol. 94 no. 24 12744-12746

Molecular Basis of Genetic Switch In The Circadian Clock

It is said that the early bird gets the worm. So what is it that makes them rise early? Scientists have questioned it for long. It was in 1995, David Welsh, then a graduate student, discovered that individual cells dissected out from the 'suprachiasmatic nucleus' of rats' hypothalamus showed spontaneous oscillations. And this set the ball rolling!

All organisms from simple unicellular to humans have their own clock mechanisms. We for example, have not one but many oscillators. The master clock that oversees all the other clocks is located in a part of the brain called hypothalamus, the suprachiasmatic Nucleus or SCN for short. The clock circuit is based on transcription and translation of a genetic switch that resides in the SCN. In the nucleus, a gene, called the Per1 gene,  produces a protein called PER (for period). Like other proteins, its production is regulated by a promoter sequence of DNA, which is known as E-box. A heterodimer (dimer because it consists of two molecules; hetero because the molecular weights/size is different) consisting of proteins BMAL1 (also known as MOP 3) and CLOCK sit atop the E-box sequence. Together they regulate the Per1 gene (other clock genes like AVP or arginine-vasopressin genes are also regulated)  resulting in the production of PER1 protein. So, in a way the E-box may be considered as the genetic switch and the heterodimer of BMAL1 and CLOCK the regulator.

Lets suppose that Per1 gene is producing PER1 protein. So, the concentration of this protein in the cytoplasm will rise. This PER1 protein will now combine with other clock proteins namely, PER2 protein, CRY 1 and 2 proteins (CRY for cryptochrome) in the cytoplasm; and will finally reach the nucleus. In the nucleus, they inhibit the Bmal1 and Clock heterodimer transcription factor, which will lead to a drop in PER production. Thus, the positive feedback of BMAL1 and CLOCK on Per1 gene; and negative feedback of PER and CRY protein on the BMAL1 and CLOCK heterodimer keep the clock running. See the adjoining figure. Other proteins like TIM (timeless) and CK1e (casein kinase 1 epsilon; it degrades PER proteins) may also play some role. New research however suggests that CRY proteins, particularly CRY1 protein is a stronger repressor of the said heterodimer.

Research by Leloup et al showed that the mRNA of Bmal1 was in antiphase with that of Per and Cry. This was expected, because they are negatively correlated. Else both the proteins would peak at the same time and the periodicity would be lost. They also observed that the phase of the spontaneous circadian rhythm did not lock. This is because, circadian rhythm is very flexible. In humans, the cycle repeats about every 24.2 hours. The circadian clock is reset by light and our circadian apparatus is exquisitively sensitive to lights falling on the retina. The retina sends this light (for synchronization) to the SCN via the retino-hypothalamic tract. This synchronization or entrainment can now 'phase lock' the circadian rhythm.

Clinical implication of circadian (circa=about; dian=day) rhythm is enormous. Our sleep-wake cycle, growth hormone and cortisol secretion are only a few example. A person in whom the circadian period is short will rise early (early bird?) and a 'night owl' will have his/her circadian period short. Curiously, our sleepiness, tendency to sleep and occurrence of REM sleep peaks (resulting from endogenous circadian rhythm) when we are about to rise; and our endogenous clock reaches its peak about 1-3 hrs before our habitual bedtime. They say that it is a natural homeostatic mechanism, so that we fell less sleepy as daytime passes on and etc. But I not convinced.

But one thing I am sure to abide by is this that I won't deprive my SCN its daily dose of sunlight. I will also not expose myself to undue light (from computer monitor etc) at night and go to bed at a reasonably fixed time. Fiddling with these may result in insomnia or excessive somnolence as in night shift workers and in jet lag (due to latitude/time-zone changes).


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Reference: BMC Molecular Biology 2008, 9:41 doi:10.1186/1471-2199-9-J.-C. Leloup (2003). Toward a detailed computational model for the mammalian circadian clock Proceedings of the National Academy of Sciences, 100 (12), 7051-7056 DOI: 10.1073/pnas.1132112100

Learning Memory Lessons From Aplysia

While you are reading this on your computer, there may be many distractions in the background. Your mom may be shouting at someone or your daughter may be receiving her piano lessons. But after you finished reading this, no memory trace of this background remains. They are not registered with your memory. We seem to ignore this 'negative memory' by a process called 'habituation'. We remember by consolidating memories by another process known as 'sensitization'. But what is this that we call memory? Memory helps us to store, retain and retrieve information. To start it all, learning is needed.

Now you might ask why Aplysia out of all animals?

Aplysia californica, called 'sea hare' by the ancient Greeks, is a marine snail which has some resemblance to a rabbit. A hermaphrodite by sexual orientation, Aplysia feeds on marine algae, and often takes the color of the algae that it eats. When threatened, it liberates a colored, irritant compound to blind the attacker, as seen in the left. It was Eric Kandel who used this phenomenon to study how neural transmission occurred through synapses. He was awarded the Nobel Prize in Physiology or Medicine in 2000 for his pioneering work on Aplysia. Aplysia offers a distinct advantage by: 1. eliciting a visible (measurable) response (siphon-mediated gill withdrawal reflex) to a stimulus, that can be studied directly; 2. the response is triggered by several electrical synapses firing simultaneously, exemplifying several output in response to a single input; 3. Smaller number of neurons, about 20,000; 4. BIG neurons. Hence this animal is considered a role model in neurobiology.

The organism can be stimulated by the application of a minor electric shock to one side of the siphon, and the magnitude of response can be measured by a force transducer, a device, usually piezoelectric, which converts mechanical pull in terms of electricity. Applying a more intense shock to the tail, or administration of chemicals into its abdominal ganglion are other ways of stimulating the mollusk. After careful observations, scientists postulated that habituation, sensitization and classical conditioning were responsible for the learning that occurred in Aplysia.

In the picture on the left, you can see a presynaptic neuron on the left side, a postsynaptic neuron on the right and another neuron (facilitator terminal) on top left stimulating the presynaptic neuron. The flow of impulse propagation is from left to right, that is, from presynaptic to postsynaptic neuron. When the presynaptic neuron was stimulated alone, the post-synaptic neuron responded in a ‘what is it’ response. When the stimulus was given repeatedly, then the response of the post-synaptic neuron became less and less. This implies that the post synaptic neuron became as if ‘habituated’. On the other hand, when a noxious stimulus was applied at the ‘facilitator terminal’ at the same time the presynaptic neuron was stimulated, the response became stronger and stronger. They called this ‘sensitization’.

In the case of habituation, it was found that the release of neurotransmitter diminished in the presynaptic neuron, possibly due to progressive inactivation of calcium ion channels. It is the calcium ion channel which allows degranulation and release of neurotransmitters. Thus inactivation means blockage of impulse. Structurally, both the presynaptic vesicle number and size are seen to decrease in habituation.

In sensitization, both the presynaptic vesicle number and size increase. Here the ‘noxious stimulus’ delivered alongside, causes release of serotonin from the facilitator terminal. This chemical, also called 5HT, binds with 5HT receptors in the presynaptic terminal. This is followed by activation of an enzyme known as adenylyl cyclase, which in turn produces cyclic AMP from ATP. cAMP then activates another enzyme, protein kinase A (PKA), which phosphorylates potassium ion (K+) channels. As a result K+ channels get blocked. When an imulsee arrives at the synapse, the membrane gets depolarized, i.e., the inside of the cell becomes positive with respect to the outside. For the cell voltage to return to its normal polarized state, the potassium ions must diffuse out, through K+ channels. But the exit route is blocked now. The action potential remains longer; more Ca++ enters into the presynaptic terminal.

Recent evidence points at the role of post synaptic neurons in habituation, sensitization and classical conditioning. Glanzman et al have proposed that activation of postsynaptic glutamate receptors might play a critical role in mediating long-lasting habituation of gill withdrawal reflex in Aplysia. A whole new range of activity starting at the synaptic knob to the nucleus and thence back to the knob again has been proposed for memory storage.

Short-term memory, which usually lasts for a few minutes, involves covalent bonding of pre-existing proteins leading to alterations in the strength of already existing connections. By contrast, long-term memory requires mitogen activated protein kinase (MAPK), CREB and new mRNA and protein synthesis; in addition to PKA.

MAPK migrates into the nucleus where it phosphorylates cAMP-responsive element binding protein (CREB). This then regulates gene expression, resulting in transcription and translation. Moreover, long-term memory is associated with the growth of new synaptic connections, phosphorylation of post synaptic densities (PSD is like a scaffold on which proteins are assembled) and many other processes. Thus it seems post synaptic mechanisms might have a bigger role than imagined.

So far, we discussed about only one form of memory: implicit or non-associative memory. Declarative or explicit memory consists of semantic (book) and episodic (related with places, memory of events) memory, which are consciously stored. Humans are perhaps, the best or only known subject in this regard. We certainly can't expect Aplysia to speak out.

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References:
Prolonged Habituation of the Gill-Withdrawal Reflex inAplysia Depends on Protein Synthesis, Protein Phosphatase Activity, and Postsynaptic Glutamate Receptors
Youssef Ezzeddine, David L. Glanzman

Molecular Mechanisms of Memory Storage in Aplysia
Robert D. Hawkins, Eric R. Kandel and Craig H. Bailey

C. Bailey, M Chen (1983). Morphological basis of long-term habituation and sensitization in Aplysia Science, 220 (4592), 91-93 DOI: 10.1126/science.6828885

Generation Of Cardiac Rhythm

The heart is vital to our bodies, since it pumps blood into various parts of our bodies, that provide us oxygen, nutrients and takes away the metabolites for disposal. For this, the heart has to contract in order to generate enough force for the ventricles (atria too) to have a standard ejection fraction. Not only does it contract, it also generates the impulses necessary for its contraction, a property called automaticity, by which it generates its own rhythm.

The cells in our bodies are bathed in a sea of fluid. This fluid, called the extracellular fluid or ECF, as it is located outside the cells. The ECF is rich in sodium ions (Na+), while their concentration inside the cells are much less. Conversely, the concentration of potassium ions (K+) is more inside than outside. It is due to this difference in concentration of ions on either side of the cell membrane, a transmembrane voltage is produced. These ions being polar in nature and water soluble, can not penetrate the lipid cell membrane. But they can gain entry, through specialized pores called ion channels.

The sodium ion channels, channels through which sodium ions pass, has 2 gates: an activation gate and an inactivation gate. What controls these gates is not known for sure, but it could either be an energy barrier controlling its entry or a flexible peptide chain-like stuff that alters the conformation of the gates to restrict the ion's access. Extracellular calcium ions also restrict sodium ions to go through and thus has a stabilizing effect. These gates are called voltage controlled gates, since the opening or closure of the gates are controlled by transmembrane voltage. These gates are inactivated at voltages above -55mV (i.e. say -40 mV) and no sodium ion can pass. Below -70 mV , the gates are open, allowing free flow of Na+ along the electrochemical gradient, from the exterior to the interior of the cell.

There are areas of heart other than the sinus node which have intrinsic automaticity i.e. they are capable of generating their own rhythms, such as the Purkinje fibers. But the SA Node (sinoatrial node) is the normal pace-maker, since it fires at the highest rate. In the nodal tissue, where the cell voltage hovers around -60 to -55 mV (millivolt), the sodium ion channels are inactivated at this voltage. Since, the resting membrane potential (RMP) of the pacemaker lies in a region where the sodium channels are inactivated, the firing of the SA Node depends on other ions, particularly calcium ions (Ca++). These nodal pacemaker cells (possibly P cells, containing little organelles) are inherently 'leaky' to calcium ions. So, Ca++ enters these cells making the cells' interiors less negative, (Ca++ is a cation bearing 2 positive charges. A cation is a positively charged ion, so called because it is attracted towards the cathode). Once the cell gains enough positive charge, to become a little more positive, about -40 mV, as shown in the figure (prepotential), there is a sudden spurt of calcium influx (impulse). T type (transient) calcium channels are responsible for the prepotential while L (long lasting) type calcium channels are responsible for the impulse. In addition to the influx of calcium ions from the ECF, calcium is also liberated locally from the sarcoplasmic reticulum of the nodal cells, known as the 'calcium spark'. So, the cell is now fully depolarized, as shown. But soon calcium channels close and influx stops. Potassium channels open. Since the concentration of K+ is more inside, K+ leaves the cell making the interior more negative. The efflux of K+ and stopping of further influx of Ca++ repolarizes the cell, making it ready for another cycle. Ca++ leaks again and the cycle repeats.

The colored animation on the top left shows the spread of cardiac impulse, from the SA Node. Click on the animation if it doesn't animate on its own.

Last modified: Mar20 2009
Reference: Basic and Clinical Pharmacology, Bertram G Katzung, 9th ed, page 220

Dendrites and Memories

In my previous post 'Spiny Dendrites', I discussed about dendrites' role in learning and memory as well as its role in evoking consciousness. Exactly how these dendrites learn and more importantly memorize, has not been clearly understood, but they are being discovered at a rapid pace.

On the dendritic cell membrane, nerve endings from various other neurons converge at the synapse, the gap between those neuronal processes and the neurone on which it is converging upon. The synapses are generally chemically coupled (by neurotransmitters), but they may also be electrically coupled (as in the lateral vestibular nucleus) or both electrical and chemical coupling may be found (conjoint). Chemical coupling is the most common. In this mode of signal transmission, whenever a presynaptic nerve gets an electrical signal (in the form of an action potential), it releases a neurotransmitter (the chemical coupler) into the synaptic cleft. The post-synaptic nerve (the nerve it verges upon, downstream) senses these chemicals (acetylcholine, adrenaline, glutamic acid, GABA etc) by virtue of 'receptors', located mostly in the 'post synaptic densities' (thickened portions of post-synaptic dendritic/neuronal cell membranes, adjacent to that part of the presynaptic membranes releasing the neurotransmitters, i.e. the synaptic vesicles).

Recently, it has been discovered that PSD-95 (post synaptic density-95), a key protein constituent (of postsynaptic densities), on which other proteins are assembled, needed to be phosphorylated, for it to act correctly (as a scaffold, for other proteins to act). Researchers suggested that this (phosphorylation) could help improve cognitive function in schizophrenia, autism and depression. I, personally, think that they will be of use as nootropics too.

In another development in neurobiology, it was shown that memories were burned into the neurons (as you burn 'memories' into a CD using Nero or other programs), and these basic units of memories had to be constantly recycled so that the memories remained there. These memory units, in the form of 'receptors' (of the neurotransmitters; which were located in the postsynaptic neuronal membranes), were in constant motion within the 'fluid' cell membranes. This 'recycling' might explain how the dendritic spines maintain their characteristic identity despite constant molecular turnover. This research might help us fight neurological disorders, such as Alzheimer's disease, schizophrenia, or learning disorders like autism.

Molecular Motors

There are miniature machines in the cells of our bodies, which carry molecules on their 'legs' and walk on their 'heads', as they move from one part of the cell to the other. This way, they transport synaptic vesicles, small bags filled with neurotransmitters, from the nerve cell-body to the end of the neuron, the synaptic knobs. They also pull the chromosomes apart during cell division: mitosis and meiosis. In can also transport molecules from the synaptic knobs towards its cell body (retrograde transport). In this mode of transport, the nerve terminals pick up molecules (by endocytosis) such as nerve growth factors and send them to the cell-body. By this mechanism, they also pick up numerous viruses (polio virus that cause poliomyelitis etc.), toxins (toxins of Clostridium tetani; of tetanus) and send them to the interior as well, to its own detriment.

Broadly speaking, the molecular motors are of two types: those which move over actin (a filamentous molecule) and those which walk on microtubules. Kinesin is one such molecule which walks on microtubules. It has two heads. One head binds to ATP molecules (the energy currency of the cells) and hydrolyzes it to derive energy; while the other head bends and 'swings' forward. This way they produce a seemingly continuous motion. The cargo is bound to the other end (so lets we call them legs). Kinesins, with some exceptions, typically transport molecules towards the + end (polymerizing end) of microtubules. Dyneins, are quite like kinesins, but they carry molecules towards the -ve end of microtubules. It is of two types: cytoplasmic dynein and axonemal dynein. Axonemal dyneins are found in the cilia or flagella of cells, allowing the cell to move about by 'beating'.

Myosin on the other hand, walks along actin microfilaments. When we move our muscles, one of its heads hydrolyzes ATP molecules, and derives energy from it. It harnesses this energy to 'bend its other head', in what we call a power stroke. Thus, in doing so, it brings actin microfilaments closer (via this power stroke). Your muscle contracts, as a result. This is known as the 'ratchet theory' or walk along theory (of muscle contraction).

This video clip beautifully illustrates how kinesin does a perfect 10:

Microtubules: The Cytoskeletal Rail-Road

The cells have a rich cytoskeletal network, consisting of microfilaments, microtubules, intermediate filaments, and proteins that hold them to the interior of the cell. These cytoskeletal structures subserve various functions such as giving the cells its shape and allows small organelles inside the cells to go to other places in the cells. For example, in a nerve cell, the synaptic vesicles, which contain neurotransmitters, are synthesized in the cell bodies. These neurotransmitter filled vesicles are then transported by 'molecular motors' form the cell body to the synaptic knobs, situated at the other end of the neuron.

Not only do they (microtubules) serve as a 'rail line' on which molecular motors move (walk), they also act as 'traction wires' which pull chromosomes apart during cell division (mitosis). Thus in cancer, when cell division is excessive, drugs which interfere with microtubule formation (polymerization) can be employed effectively to curb this cell replication gone haywire. Vinca alkaloids (obtained from the Vinca rosea plant) such as vincristine (used in Hodgkin's Lymphoma, small cell lung cancer etc.), vinblastine (used in ALL or acute lymphoblastic leukemia in children), vinorelbine (used in NSCLC or non small cell carcinoma of the lung); and taxanes such as taxol (paclitaxel, used for the treatment of cancers of the breast and ovary) extracted from the western yew tree, are molecules which stabilize microtubules and thus arrest cell division.

Microtubules are composed of smaller subunits made of proteins, alpha tubulin and beta tubulin. Alpha and beta tubulins together form a heterodimer (hetero since alpha and beta tubulins are different molecules). 13 such hetero dimers join to form a ring and many such rings are joined in a stacked manner to form this hollow structure called microtubules. Microtubules are dynamic structures with one end forming by polymerization (+end) and the other end destroying by depolymerization (-end), as this 10 second video clip illustrates.

The Unsung Hero (ine) of Genetics

We all credit Watson and Crick for their discovery that the DNA molecule was arranged in a double helix pattern. But how many of us know about this charming and intelligent lady, Rosalind Franklin, who made their discoveries much easier, by 'providing' them with the distinctive diffraction X-ray photograph, termed 'Photograph 51'?

Working in scientific arena was traditionally a man's domain then, and women were frowned upon. Naturally, as expected, she also had been subject to much harassment by her male colleagues. At the same time, she also used to make fun of her male colleagues.

While working on Signer DNA (DNA molecules, extracted from the thymus gland of calf; used for their distinctive X-ray diffraction pattern ), at King's College, London, she found out that there were two forms of DNA; a 'wet' form (B-DNA), that was longer and a 'dry' form, that was shorter. They continued with working on the wet forms.

Watson and Crick were also behind the same trail of determining the nature of DNA, but they were far behind any possible breakthrough. They did not even know about Chargaff's Rule, that stated that for every Adenine molecule, there was an equal number of Thymine molecule, and the number of Cytosine molecules were equal to that of the Guanine molecule (A=T, G=C). By sheer luck, Watson chanced upon Photograph 51 (picture shown here), 9 months after it was kept in a vault by Rosalind. He was quick enough to deduce the 'double helical' structure by intuition and reasoning. The 'x' like speckled banding had enough tell-tale signs.

Watson, Crick and Wilkins were awarded the Nobel Prize in Physiology or Medicine, in 1962, for their discovery about nucleic acids (not exclusively for DNA). Rosalind was long dead by then. She died of ovarian carcinoma in 1958, possibly due to extreme radiation exposure. It is also true that some of her own family members also died of cancer and that cancer incidences were particularly high in Ashkenazi Jews, which she was. Whatever the cause of her death were, the contribution she made toward the understanding of DNA structure, have certainly paved the way for modern genetics. In our minds, she will continue to dwell forever.