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Path-breaking discoveries

Print edition : Nov 08, 2002 T+T-

Three scientists win the Prize in Physiology for seminal discoveries in the understanding of cell death.

THE Nobel Prize in Physiology or Medicine for 2002 has been awarded jointly to Sydney Brenner of the Molecular Sciences Institute, Berkeley, United States, John Sulston of the Wellcome Trust Sanger Institute, Cambridge, United Kingdom, and Robert Horvitz of the Massachusetts Institute of Technology (MIT), U.S., for their discoveries in "genetic regulation of organ development and programmed cell death''.

The origin of all the several hundreds of cell types in the human body is the fertilised egg. During the embryonic and foetal stages, the number of cells increases dramatically. These become mature and specialised to form the various tissues and organs in the body. In parallel with the generation of new cells there is the normal process of cell death and also a delicate and controlled elimination of specific cells, both in the foetus and the adult, to maintain an appropriate number of cells in the tissues. In an adult human being, more than a thousand billion cells are created every day. At the same time, a similar number must undergo "suicide death'', called "programmed cell death'', for a fine-tuned balance.

This year's Nobel laureates made seminal discoveries in the understanding of cell death. They used the nematode or worm Caenorhabditis elegans as the experimental model to study the process of cell division and differentiation from the fertlised egg to the adult and identify key genes regulating organ development and programmed cell death. Their studies showed that corresponding genes also exist in higher species, including man. It is of considerable biological and medical importance to understand how these complicated processes are controlled.

In the early 1960s Brenner realised that mammals are too complex for the basic studies of organ development and interplay between different cells, given the huge number of cells in them. A genetically amenable and multi-cellular organism simpler than mammals was required. The nematode C. elegans proved to be the ideal solution. This 1 mm-long worm has a short generation time and is transparent, and these enabled their direct observation under a microscope and the characterisation of the processes. The studies showed that in C. elegans 131 of the total 1,090 cells die reproducibly during development and this natural death is controlled by a unique set of genes. Brenner broke new ground in 1974, which provided the foundation for future research, by demonstrating that specific gene mutations could be introduced in the genome of C. elegans.

Different mutations were linked to specific genes and to specific effects on organ development. Sulston extended Brenner's work and developed techniques to study all cell divisions in the worm, from the fertilised egg to the 959 (1090 minus 131) cells in the adult organism. He also determined the cell lineage for a part of the nervous system and showed that this was conserved in every nematode. These findings proved that specific cells in the cell lineage always die through a programmed process and this could be monitored in the living organism. Specifically, he showed that the protein encoded by the gene called nuc-1 is needed for the degradation of the DNA (deoxyribonucleic acid) of the dead cell.

Horvitz took the above work further. In a series of elegant experiments, Horvitz investigated whether there was a genetic programme controlling cell death. In 1986 he identified the first two "death genes'', ced-3 and ced-4, and showed that these two were a prerequisite for a cell to die. He also showed that another gene, ced-9, protected cells from death by interacting with ced-3 and ced-4. He also identified a number of genes involved in the elimination of a dead cell. That the human genome also contained a ced-3-like gene is Horvitz's discovery.

PHYSICS

This year's Nobel Prize for Physics goes for the discoveries and detection of two new windows to the universe that have led to new branches in astronomy, neutrino astronomy and X-ray astronomy. One half of the prize has been awarded jointly to Raymond Davis Jr. of the University of Pennsylvania, U.S., and Masatoshi Koshiba of the International Centre for Particle Physics, University of Tokyo, Japan, for "pioneering contributions to astrophysics, in particular for the detection of cosmic neutrinos''. The second half has gone to Riccardo Giacconi of the Associated Universities Inc., Washington, "for pioneering contributions to astrophysics, which have led to the discovery of cosmic X-ray sources''.

Neutrinos are ghost-like particles that were postulated by Wolfgang Pauli in 1930 purely on theoretical grounds. They are chargeless and, until recently, were believed to be massless as well. They belong to the lepton family of elementary particles and come in association with their charged siblings the electron, the muon and the tau and thus exist as three distinct species. The flux of neutrinos from the sun, which are electron-neutrinos, is estimated to be very large: thousands of billions of solar neutrinos pass through our bodies every second without our noticing them. This is because neutrinos interact very weakly with matter and only one in a billion neutrinos would be stopped on its way through the earth. Hence their detection was thought to be extremely difficult.

The award to Davis and Koshiba is in recognition of their path-breaking research into the detection of neutrinos produced by nuclear fusion reactions in the centre of the sun. The observations of these neutrinos demonstrated conclusively that the sun is powered by the fusion of hydrogen nuclei into helium nuclei. The key to Davis' technique lay in the fact that while most reactions in the sun would create low-energy neutrinos, one creates a high-energy neutrino. Italian physicist Bruno Pontecorvo pointed out that it should be possible to detect this neutrino by its absorption by chlorine nuclei and their conversion to radioactive Argon whose half-life is about 50 days.

Davis constructed a new detector, a gigantic tank filled with 615 tonnes of chlorine-rich common dry-cleaning fluid, perchloroethylene. He placed this detector about 1,600 metres underground in Homestake Gold Mine in South Dakota to protect it from background cosmic radiation that would be absorbed by the overlying earth. Starting in 1967, Davis succeeded in capturing a total of 2,000 neutrinos from the sun over a period of 30 years. In the 1980s, with another gigantic detector filled with water tuned to a lower threshold neutrino energy, called Kamiokande, the Japanese group led by Koshiba was able to verify Davis' results. Kamiokande was also able to detect on February 23, 1987, neutrinos from a supernova explosion 170,000 light years away (one light year is equal to 1016 m) The explosion, now designated SN1987A, is estimated to have emitted 1058 neutrinos. Koshiba and company captured 12 of the 1016 neutrinos that passed through their detector and established that other cosmic sources of neutrinos existed as well.

The work of Davis and Koshiba has led to unexpected discoveries and a new intensive field of research, neutrino astronomy. However, the number of neutrinos reaching the earth from the sun in Davis' experiment was only one-third of that predicted. One of the explanations for this discrepancy called the`Solar Neutrino Puzzle', was that some electron-neutrinos somehow "oscillate'' into other neutrino species during their eight-minute flight from the sun. In 1996 Koshiba constructed a bigger detector called SuperKamiokande, which indicated that neutrinos may indeed convert to other species.

Last year the Sudbury Neutrino Observatory (SNO) in Ontario, Canada, demonstrated the existence of the phenomenon of neutrino oscillations, thus explaining the deficit observed by Davis. Neutrinos can oscillate only if they have mass, however small, and not strictly zero, as had been previously thought. By combining the results of various observations, neutrinos have been found to have a tiny mass, about a billionth of the electron mass. However, because of the enormous number of neutrinos in the universe, the total mass of neutrinos invisible matter is comparable to the mass of all visible matter in the universe.

The sun and all other stars emit electromagnetic radiation at different wavelengths, both visible and invisible (like X-rays). In order to investigate cosmic X-radiation, which is absorbed in the atmosphere, it is necessary to place X-ray detectors in space. In 1959, Giacconi constructed the first such instrument and launched it aboard a rocket, which flew for six minutes at a high altitude. As the rocket was rotating and the detector swept the sky, it detected a strong source of cosmic X-rays. This was later determined to be a distant UV star in the Scorpio constellation. In addition, an X-ray background, evenly distributed in the sky, too was discovered. Giacconi also detected sources of X-rays that most astronomers now believe to be harbouring blackholes. His contributions have laid the foundations of X-ray astronomy, which today is largely satellite-based. The first of the satellites was called Uhuru, which was launched in 1974.

Giacconi constructed the first X-ray telescope that was flown aboard the Einstein X-ray satellite in 1978. It has provided completely new and sharp images of the X-ray universe. In 1976 Giacconi initiated the construction of an improved and an even larger orbiting X-ray observatory, AXAF, which, for technical and financial reasons, could be realised only in 1999. Rechristened Chandra, this orbiting X-ray counterpart of the Hubble telescope has been beaming unsurpassed images of X-ray universe, in recent times. Notwithstanding this, satellites continue to be used for exploring aspects of the X-ray universe which Chandra does not probe.

CHEMISTRY

Development of novel techniques "for identification and structure analyses of biological macromolecules'' has won the 2002 Nobel Prize in Chemistry. One half of the prize goes to John B. Fenn of the Virginia Commonwealth University, Richmond, U.S., and Koichi Tanaka of Shimadzu Corporation, Kyoto, Japan, "for their development of soft desorption ionization methods for mass spectrometric analyses of biological macromolecules'' and the other half to Kurt Wutrich of the Swiss Federal Institute of Technology (ETH), Zurich, Switzerland, and the Scripps Research Institute, La Jolla, U.S., "for his development of nuclear magnetic resonance (NMR) spectroscopy for determining the three-dimensional structure of biological macromolecules''. Mass spectrometry (M.S.) and NMR are well-established techniques to study molecules but until recently were useful only for relatively small molecules.

These breakthroughs have enabled these techniques to embrace large biological macromolecules like proteins. The possibility of analysing biomolecules in detail has led to a better understanding of the process of life. Using these revolutionary methods, chemists can now rapidly and reliably identify what proteins a sample contains and also know their three-dimensional structures. Small variations in a protein structure can alter its functions significantly. M.S. allows the identification of a substance in a sample quickly on the basis of its mass. The method is so sensitive that it is possible to trace very small admixtures of molecules in a sample. Food stuff control, environmental analyses, doping and drug tests are areas where M.S. is routinely used. The evolution of the desorption technology, transferring macromolecules to ions in gaseous phase has formed the basis of its application to larger biomolecules in the last 20 years. Macromolecules may be large in comparison with other molecules but one is still dealing with very small structures (hundredths of a micrometre) and masses (10-19 g) .

The trick in desorption technology is to make the individual protein molecules let go of each other and spread out as a cloud of freely hovering, electrically charged protein ions. These are then accelerated in vacuum and their `time of flight (TOF)' to travel a given distance, which depends on their charge and mass, is used to estimate their masses. The fastest ones are the lightest and have the highest charge. The scientists behind two methods currently in use, which cause proteins to transform into the gas phase without losing their form and structure, have been awarded one half of the Nobel Prize in Chemistry.

In the first, which was developed by Fenn, the sample (in liquid phase) is sprayed using a strong electric field to produce small, charged, freely moving ions. The technique is known as electrospray ionisation (ESI). These molecules take on strong positive charges, thereby making their mass-to-charge ratio small enough for ordinary mass spectrometers to analyse. The other, developed by Tanaka, uses an intense laser pulse to do the same. This method is called soft laser desorption (SLD). Here the laser pulse blasts the sample (which is in solid or viscous phase) into tiny bits. The technique thereafter is similar to ESI. Tanaka's method forms the basis for several powerful laser desorption methods in use today. Both ESI and SLD have many areas of application. Sophisticated biochemical analyses, which seemed impossible only a few years ago, are now routinely performed thanks to these techniques.

Interactions between proteins are very important to understand the signal systems of life. Such biomolecule complexes can be examined using ESI, which is superior to other methods in the rapidity, sensitivity and the identification of the interaction. M.S.-based analytical methods are relatively cheap and thus are widely used in laboratories around the world. Today ESI and SLD are standard techniques for structure analyses of peptides, proteins and carbohydrates which make it possible to analyse quickly the protein content of intact cells and tissues.

In the pharmaceutical industry drug development has undergone a paradigm shift because of new M.S. techniques. In combination with other methods, ESI enables analyses of several hundred molecules a day. ESI-and SLD-based methods are now used in the rapid diagnosis of diseases such as malaria and ovarian, breast and prostate cancers. The development in the NMR technique for large biomolecules was essentially owing to Wutrich.