Objections were raised immediately. Many of the supposed nanobacteria were less than 100 nanometres across, smaller than many viruses, which cannot replicate independently. Maniloff's work suggests that to contain the DNA and proteins needed to function, a cell must be at least 140 nanometres across. Kajander and Ciftcioglu, however, insisted that they had observed the nanoparticles self-replicating in a culture medium and claimed to have identified a unique DNA sequence. How could this be explained if the cells were not alive, they asked. Cisar has an answer to this. After studying nanoparticles found in saliva, his team published a paper in 2000 claiming that the DNA detected by the Finnish team was a contaminant from a normal bacterium. "It wasn't until we couldn't get any unique nucleic acids that we suddenly realised we were being tricked," he says. The paper also said that what looked liked self-replication was just an unusual process of crystal growth. "This just stopped everything in its tracks," says Virginia Miller, a member of Lieske's team. "It is cited as the gospel to why all the papers by Kajander are rubbish... The debate is very polarised and that has shocked me a bit." Some say the claims of Cisar's team are also fantastic. "They talk about 'self-propagating apatite'," says Jorgen Christoffersen, who studies biomineralisation at the University of Copenhagen in Denmark. "This is scientific nonsense." But scepticism about the claims by the Finnish researchers is heightened by the fact that they have financial interests. The group has set up a company called Nanobac Life Sciences in Tampa, Florida, which sells diagnostic kits for spotting nanobacteria. It is even developing treatments for conditions supposedly caused by them, despite the lack of evidence. None of the Mayo researchers, by contrast, holds any patents related to nanobacteria, Lieske says, nor do they have any financial stake in Nanobac. "We are an independent laboratory and we have provided new evidence," Miller says.
The paper went through seven cycles of revision before it was accepted by the American Journal of Physiology: Heart and Circulatory Physiology last week. "The review process, as painful as it was, forced us to look at the counter-arguments inside out, upside down and back to front, then repeat our experiments," Miller says. So what do they have to show for this effort? The researchers collected samples of calcified aneurysms (bulging blood vessels), arterial plaques and heart valves. They pulped the tissue, then filtered it to remove anything bigger than 200 nanometres and added the filtrate to a sterile medium. After a few weeks, the optical density of the liquid doubled, suggesting particles were self-replicating. Samples from aneurysms caused by a genetic disorder did not do this, nor did the density of the medium change if no filtrate was added. Some of the particles were removed, cleaned of their mineral coating, and then imaged with an electron microscope. This revealed small cell-like structures. This much is just repeating the work of Kajander and Ciftcioglu that has been so widely criticised. But the Mayo team has also come up with further results that are much harder to explain away. When the particles were "grown" in a flask, they absorbed uridine, one of the building blocks of RNA.
This suggests that RNA is being produced in the particles, the team says. However, even apatite crystals alone seemed to absorb some uridine, though not as much as the self-replicating particles. And when the Mayo team doused their tissue samples with an antibody that Nanobac claims binds to a protein unique to nanobacteria, they found it bound to diseased tissue, even when the calcium was washed away, but not to healthy tissue. On one sample of the self-replicating particles, they also compared the sites where the antibody bound with those where a DNA stain bound. "The nanobacteria antibody is binding to the same features that stain for DNA," says Miller. This is not enough for the critics. "What you have is umpteen weak arguments at best," Maniloff says. "This is not proof." One crucial piece of evidence would be finding DNA unique to these particles. "Just because other groups have not been able to identify a unique DNA sequence does not mean it does not exist," Miller says. "It just means the tools weren't right at the time." She says that the Mayo team has managed to isolate RNA and DNA, but she is not yet ready to talk about the results. "We are a conservative group, and that has stood us in good stead." Other scientists are also going after the DNA. Yossef Av-Gay, a microbiologist at the University of British Columbia in Vancouver, Canada, has been asked by Nanobac to work out what makes nanobacteria tick. "These particles are self-replicating, that is without doubt," Av-Gay says. But finding out what is inside them is complicated because they are so small and because the apatite shells absorb contaminants. "The problem is to distinguish between material absorbed from the environment and unique sequences from these organisms." Av-Gay too will say nothing about what his studies have revealed. "The story seems to be gearing towards the idea that these are not bacteria, but maybe a new living form. It is a very interesting story, but you won't get the answer now."
Written by Jenny Hogan, Boston
New Scientist issue: 22 May 2004
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