Thomas Alva Edison, one of the most prolific inventors ever to live, thought that the habit of sleeping was acquired by our cave-dwelling ancestors who had no option but to fall asleep in the darkness of the night. One of the reasons behind his inventing electric lamp was that the habit of sleeping could be made redundant and human productivity could be increased. Not a very bright idea! Actually, sleep/wakefulness, activity/rest patterns are indelibly hardwired into our very framework and not a bad habit attained by our forefathers. Chronobiology (the biology of time), a new exciting branch of medical sciences, tells us that the body runs several exquisite, pre-adaptive, programmable, cycles that alternate between the states of high and low activities. When human volunteers are kept in total darkness for prolonged periods of time, their bodies still go through the same daily cycles of sleep/wakefulness, core body temperatures and urinary output. This sleep-wakefulness cycle is approximately 24 hours long and is called circadian rhythm (circa: about; dies: day). Recent research shows that this time keeping is not confined to human beings but is a common thread running across the bioworld.

Just how rigid is this clock? Ask any international air traveler or a shift worker. While zipping across a time zone, you can adjust your wristwatch in an instant, the circadian clock takes about a week to catch up with the new time zone. During this period not only sleep but performance and general health are also affected. Shift work can also disrupt mental acuity to such an extent that some of the worst disasters that involved human error have occurred at night--a time the body has reserved for sleep. Some examples will clear the point: Chernobyl fiasco, 1:23 am; Bhopal tragedy, 3:00 am; and Titanic hit the iceberg at 1: am, remember?

Chronobiology

Chronobiology and its sidekick chronotherapeutics are having a tough time getting a nod of approval from the medical community. Hardly surprising, keeping in view that medical curricula have been teaching quite the opposite: homeostasis, which stipulates that the body tends to stay in a steady state equilibrium day in and day, regardless of the environmental conditions. But now chronobiology has started to turn the tables on the conventional physiology. Lets take the example of blood pressure. Clinical evidence suggests that most strokes and other cardiovascular events occur early in the morning. Examining the blood pressure graphs of people in 24 hours can solve this enigma. (See graph). Here we see that most persons (whether hypertensives or not) experience a rapid surge in the blood pressure. All these exciting advances fall under the umbrella of chronobiology and chronotherapeutics.

Studies carried out on normal as well as hypertensive people show that the sympathetic catecholamines, especially norepinephrine, start to build up in the blood very early in the morning. This is followed by increased renin activity, culminating in the accumulation of angiotensin I, which is a powerful vasoconstrictor. Both the above-mentioned processes lead to systemic vasoconstriction escalating the blood pressure.

Do we need the Clock?

Circadian cycles try to prepare man to face the challenges that each new morning brings. It makes sure that when you get up, all the system of your body are pepped up and are in full throttle and ready to embrace any challenge. It also enables the body to adapt to daily and seasonal environmental changes and enhance efficiency by chronologically separating catabolic and anabolic processes.

Keeping tightly in synch with the day/night cycles of the earth has obvious evolutionary advantages. "This allows organism to maintain an internal temporal order and to anticipate change." Says Dr. William Schwartz, a professor of neurology at the University of Massachusetts Medical School and a prominent researcher of circadian rhythms. " If you are a mouse," he adds, "it is useful to be able to anticipate when an eagle will fly and already be in your burrow, rather than be caught scurrying into it". The circadian rhythms are not confined to higher animals. From a human being to a fruit fly to a mold, the clock is ticking everywhere.

Where is the Clock Located?

In man as well as in other mammals, the master clock or the "pacemaker" is located are two bunches of neurons just above the optic chiasma (SCN), about 3 cm behind the eyes. If these neurons are removed surgically, the circadian rhythms go haywire, proving the indispensability of SCN. Each of the 10,000 or so neurons that form SCN is itself a tiny, but extremely accurate, clock. When the neurons are taken from experimental animals and kept in vitro, each neuron maintains its firing rhythm for several weeks with only a slight deviation from the 24-hour cycle. Further studies have shown that the cells of SCN are not unique in keeping time; many more such centers have been identified. One of the more important centers has been located in each retina. Many scientists believe that every living cell is a little oscillator in its own right.

But now these oscillators work and how they are synchronized with the environment? We will have to step into the turbulent waters of molecular genetics to answer these questions.

MOLECULAR GENETICS OF CIRCADIAN RYTHMS

The small, two-winged fruit fly Drosophila melanogaster -- the guinea pig of geneticists--has proven an invaluable aid for the unraveling of the molecular clockwork of circadian rhythms as well. In 1971 Konopla & Benzer found that mutations of a gene on the X chromosome of Drosophila could alter the length of the clock. They named this gene period (per). After a long lull, another clock gene on chromosome 2 was discovered and christened timeless (tim). Subsequent studies revealed that periodic expression of these genes is required for the clock to work. These genes then produce proteins that interact and through auto-regulatory feedback loops, constitute a single intracellular, self-sustaining circadian oscillator.

A day in a fly's life

The cycle begins early in the night with the initiation of transcription of per and tim. per and tim mRNAs slowly starts to accumulate and ultimately leave the nucleus. In the cytoplasm, these mRNAs bind with ribosomes and start to churn out PER and TIM proteins. PER is unstable but forms a stable hetrodimer with TIM. This binding suppresses cytoplasmic localization domains (CLDs) on each protein, allowing translocation of the complex into the nucleus. And guess what this complex does in the nucleus? It suppresses the expression of per and tim! This halts the loop for a while because no new mRNA are made and hence no PER and TIM can be synthesized. At this juncture, various enzymes attack the PER/TIM complex and slowly degrade them. This releases the inhibitory grip of PER/TIM hetrodimer on per and tim genes and the cycle starts over. One such cycle takes 24 hours. See diagram.

As mentioned earlier, this cycle is not passive. It can be altered by light. Experiments have shown that exposure to bright light leads to the degradation of TIM and alteration of the cycle. This is important because light sensitivity of TIM is responsible for the entrainment (synchronization) of the phase of circadian cycles to the environmental time of the day. Thanks to TIM's sensitivity to light, the internal clock is readjusted to local time whenever different time zones are crossed.

The mold chips in

While the molecular cogs and springs of the circadian clock of Drosophila were being pried open, some researchers focused on a bread mold Neurospora crassa. The mold is particularly straightforward to study from a circadian scientist's point of view because when inoculated to one side of a glass tube, the fungus produces pigmented spores in a strictly circadian schedule. In 1978 a gene frq was discovered in Neurospora that encodes FRQ. This protein was shown to feedback and prevent its own transcription, just like PER/TIM complex in Drosophila. Later on, Macino and his colleagues discovered two additional clock genes in genes Neurospora: white collar-1 and white collar-2, (wc-1 and wc-2). Wondering that these genes were so named because of their social position and job hierarchy? Actually, mutations in these genes produce molds that lack pigmentation at the outskirts of the colonies. One of the proteins that these genes encode, WC-2, was found to be the first positive feed back component of the clock, found to be crucial for the expression of frq. This finding was in stark contrast to all the previously known circadian proteins--PER, TIM and FRQ--all of which are negative feed back factors.

A PASsage to Mammals

Because mammals are made of an entirely different cloth, the same techniques that proved so handy to study clocks in flies and molds failed in mammals. The ice was finally broken by Joseph Takahashi and his colleagues at Northwestern University. They applied a radically different approach, called "forward genetics" to mice and finally pinned down a gene, Clock, mutations to which lead to arhythmicity. The corresponding protein, CLOCK, contains a 250- to 300-amino-acid-long motif, called PAS domain. This domain is (gasp!) almost identical to ones found on PER of Drosophila and WC-1 and WC-2 proteins of Neurospora. This enticing discovery suggests an evolutionarily ancient and fundamentally common origin of circadian rhythms.

All proteins with a PAS domain work by binding with another protein, and CLOCK is no exception. Its conjugation partner, found by Weitz and colleagues after a painstaking sifting of hundreds of likely candidates, is BMAL1. Unlike PER/TIM (which work negatively), but like WC-2, CLOCK/BMAL1 complex has turned out to be a positive feedback constituent of the clock, kindling the expression of Clock.

In addition to Clock, several other genes have also been identified in mice and hamsters, including mper1, mper2 and mper3. These genes are homologues of Drosophila per and play similar parts in the clock cycle.

This is the general mechanistic of the bioclock. At present, not all the pieces of the jigsaw are in place and many questions still remain to be answered. There are many posttranslational processes, other loops and many more genes that are yet to be identified.

The Big Picture

All this brings us back to the suprachiasmatic nucleus. All the clock genes are expressed in each neuron of the SCN and form a basic cellular circadian oscillator there. These individual oscillators couple to form a master "pacemaker". Running on these micro-clocks, the SCN exhibits a distinct firing rhythm. This forms a fulcrum, around which all behavioral and physiological rhythms of the body rotate.

As mentioned earlier, SCN is nestled just above the hypothalamus and this is no coincidence. SCN triggers the hypothalamus into action, which in turn stimulates the pituitary gland. As you might recall from your physiology texts, this gland is the nerve center of the endocrine system, directly or indirectly responsible for the release of such hormones as ACTH, cortisol, aldosterone and others. Current investigations show that these hormones are not released in a random order but follow clear schedules, closely in tow with the circadian clock of the body.

ACTH, for example, is released by the pituitary gland under the influence of the clock in the small hours of the night. ACTH travels to the adrenal glands and stimulates the release of aldosterone and cortisol. Both these hormones show telltale peaks, strongly indicating that their concentrations in the blood are time-controlled. See graph.

There has been considerable media hype over another circadian hormone, melatonin. The Internet is virtually teeming with websites trumpeting melatonin as a panacea, curing jet lag to stall ageing to boosting immune function. The power of the media can be adjudged by the fact that the sales of melatonin surpassed that of vitamin C in the USA last year. However, the exact role of melatonin is yet to be fully described in humans and various centers around the world are working on the hormone.

Chronotherapeutics

In 1997, G. D. Searle & Co. became the first pharmaceutical company to launch, in their own words, "The first formulation designed to align with circadian rhythm". It was the calcium channel blocker verapamil HCl, in a controlled-onset, extended-release formulation. It is advertised to blunt the early morning surge in blood pressure produced by circadian rhythm.

Chronotherapeutics does not stop at verapamil. There are many other drugs that work better when used at a specific time of the day. Examples are asthma and ulcer medication given before going to bed (acid secretion kicks up in the night) and NSAIDs at noon for osteoarthritis patients (which worsens in the evenings). See diagram. Other drugs that might be given by the clock include corticosteroids, analgesics, cholesterol-lowering agents, antihistamines, theophyline and anticancer drugs.

In not a very distant future, we might routinely see prescriptions that also include a column for a specific time of the day as well--6 p.m. sharp, for example--when the medicine is to be taken, and not just the conventional "once daily, with or without meals". The time of the bioclock is just around the corner.