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Pretty colours and human imaging

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The goal: Much of Nobel laureate Roger Tsien’s lab’s current focus has been on developing probes that could be used in clinical practice.
The goal: Much of Nobel laureate Roger Tsien’s lab’s current focus has been on developing probes that could be used in clinical practice.

N. GOPAL RAJ

Colourful proteins vividly highlighted a process that led to Alzheimer’s disease in the brain of a genetically-engineered mouse

“A lot of this work is driven by my love for pretty colours,” exclaimed Roger Y. Tsien in the course of a talk at the Indian Science Congress in Thiruvananthapuram recently. The palette of glowing proteins he created won him a share of the Nobel Prize for Chemistry in 2008.

By modifying the structure of naturally occurring compounds found in jellyfish and corals, Dr. Tsien and his co-workers produced several fluorescent proteins that emitted a range of colours when light was shone on them. The genes for such proteins could be put into cells.

Protein activity

It was then possible to watch when and where host cell genes get switched on and off, when protein products are born, where they travel, with what other proteins they interact and how long they survive, he explained in his Nobel lecture delivered in December 2008.

In Thiruvananthapuram, Dr. Tsien showed some striking examples of the use of such fluorescent proteins. In a video, waves of red, marking the build-up of calcium, swept across as cells of a zebrafish embryo divided. Colourful proteins vividly highlighted a process that led to Alzheimer’s disease in the brain of a genetically-engineered mouse. With one protein that glowed red and another green, aggressively multiplying cancer cell lines could be distinguished from those that did not.

But such light did not penetrate well through mammalian flesh. The reason was that red blood absorbed most colours of visible light.

To get beyond that, fluorescent proteins that emitted light in the far red and infra-red regions of the spectrum were needed, he pointed out.

The paper

Last May, his research group at the University of California in San Diego in the United States published a paper in the journal Science announcing the development of a genetically-encoded fluorescent protein that produced infra-red light.

A light-sensing protein from the bacterium Deinococcus radiodurans was “chopped up and modified until it now glows in the infra-red,” he said. “This is now enabling many, many people, including us, to look at processes inside living mice.”

It may be possible to produce fluorescent proteins that emitted light farther into the infra-red spectrum, he told this correspondent. There were others in the same family of light-sensing proteins that absorbed light in the infra-red region. So if they ever fluoresced, they would give off light at even longer wavelengths.

Meanwhile, Xiaokun Shu, the post-doc in Dr. Tsien’s lab who worked on the infra-red fluorescent protein, has gone in a different direction. He has taken a protein from the plant Arabidopsis thaliana, cut it in half and mutated it. The result was a small bit of protein that generated a highly reactive form of oxygen known as ’singlet oxygen’. This mini singlet-oxygen-generator made it possible to visualise proteins at far below optical resolution. When a protein was tagged with it, “we can see it first by fluorescence but then later on we can see it at the electron microscope level,” said Dr. Tsien during his talk at the Science Congress, adding that this work had not yet been published.

Genetically-encoded fluorescent proteins are powerful tools. But for both ethical and practical reasons, these proteins cannot be put into humans.

Current focus

Much of his lab’s current focus has therefore been on developing probes that could be used in clinical practice.

Probes that selectively mark cancer cells so that they can be readily detected during a whole body scan would be very useful.

“We are not mice. We will need magnetic resonance imaging to do whole body scanning,” he pointed out.

The approach his group has taken is to use a small bit of protein with several positive charges on it.

Such a positively charged bit of protein would ordinarily stick to the negatively charged surface of cells.To prevent this from happening indiscriminately, the positively charged bit is connected by a linker to a negatively charged piece of protein so that the two charges neutralise each other.

But cancer cells have a protein-chewing enzyme on the outside that recognises and breaks the linker. The positively-charged bit can then stick to the cancer cells and be engulfed by them. A chemical, such as the element gadolinium, can be attached to the positively-charged portion to provide enhanced contrast during magnetic resonance imaging.

Such a probe had been shown to pick out tumours in mice, said Dr. Tsien.

The compound was administered as an injection and only the cancer cells were tagged. The rest of it was excreted in urine.

The same technique could be used to mark cancer cells with a fluorescent material that helped surgeons remove the entire tumour.

It could also be employed to target drugs at cancer cells with a high degree of specificity, thereby improving the efficacy of treatment.

Method modification

The method could be modified to identify portions of blood vessels that had been narrowed by the fatty deposits and where blood clots were forming. Such blood clots can lead to a heart attack or stroke.

In this case, the probe would have a linker that could be broken by thrombin, an enzyme involved in the formation of clots.


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