Fast method for detecting, measuring endotoxin developed - C&EN

Nov 6, 2010 - Fast method for detecting, measuring endotoxin developed ... develops, displaying itself as shock and fever frequently as a prelude to d...
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are not dependent, as other leukemia cells are, on acquiring this amino acid from extracellular fluids. The effect of L-asparaginase treatment is to destroy such extracellular L-asparagine by hydrolyzing it to L-aspartic acid. This hydrolysis terminates the availability of L-asparagine to leukemia cells that depend on a supply of this amino acid. To establish the activity of the synthetase enzyme, the workers first prepared a mixture of ingredients needed in the synthesis of L-asparagine. These compounds include L-asparagine synthetase, aspartic acid, adenosine triphosphate, glutamine, and magnesium ions. The reaction of these compounds yields L-asparagine, adenosine monophosphate, pyrophosphate, and glutamic acid, the New York City group notes. After preparing this assay system for measuring the enzyme activity of L-asparagine synthetase, Dr. Meister continues, a tumor preparation is treated with the solution. Following a 10- to 60-minute reaction with the tumor at 37.6° C. (body temperature), the workers remove the synthetase enzyme from the solution with a protein precipitant. They then analyze the supernatant solution by paper electrophoresis to show that Lasparagine is indeed formed as a result of the synthetase activity. They find, too, a reduction in the concentration of aspartic acid which is proportional to the amounts that are aminated to L-asparagine. "We now understand at the enzyme level an important biochemical difference between normal cells and cancer cells, Dr. Meister tells C&EN. "We're really encouraged with these findings inasmuch as they allow us to therapeutically exploit the metabolic defects of cancer cells."

Fast method for detecting, measuring endotoxin developed Using cells taken from the blood of horseshoe crabs, a Johns Hopkins University scientist has developed a quick test-tube method for detecting and measuring the disease-producing micromolecule endotoxin. Dr. Jack Levin's work—the most sensitive in vitro technique yet developed for endotoxin—promises to assist studies of the mechanism by which endotoxin causes a variety of pathological effects in humans and other mammals. He reported his findings in Atlantic City at the 52nd meeting of the Federation of American Societies for Experimental Biology. Endotoxin is a high-molecularweight lipopolysaccharide and a con14 C&EN APRIL 22, 1968

stituent of cell wall of most gram-negative bacteria. In severe gram-negative infections a condition called endotoxemia often develops, displaying itself as shock and fever frequently as a prelude to death. One of its more pathologically relevant effects is to promote clotting within the blood vessels so that the host's normal clotting mechanism becomes exhausted with resulting uncontrolled bleeding after a wound. Competition among molecular pathologists is intense over determining how endotoxin interacts biochemically with the host's metabolic processes. Does it work through a protein or a chemical mediator? Or does it act directly on the various systems it affects? Dr. Levin's technique, a result of a bit of seashore serendipity by Dr. Frederick Bang of Johns Hopkins, gives biochemists, medical research scientists, and pharmacologists a fast assay with a potential sensitivity for the detection of endotoxin in human blood serum of 0.005 microgram per ml. Other in vitro methods are 100 times less sensitive. The method is based on the ability of endotoxin to form a gel when mixed with an extract of amoebacytes, the circulating blood cells of Limulus, the ancient horseshoe crab. The process consists simply of adding endotoxin or solutions containing it to the extract. The extent and rate of gelation are measured by light scattering and can be used to measure endotoxin concentration. Overall, the technique takes about an hour. "So it seems to me that we have a very sensitive and quantitative technique with which to study the physicochemical reactions of endotoxin with protein," Dr. Levin says. "By studying the nature of this reaction with the protein in the Limulus extract, we can obtain insight into the kinetics of the process, and perhaps how endotoxin acts to initiate or accelerate coagulation, and how it affects blood vessels." Dr. Bang's role in the study began about 10 years ago. His collaboration on the problem with Dr. Levin started five years later.

Are pulsars from intelligent sources or from neutron stars? When pulsars were described by Dr. Frank Drake to the American division of the International Scientific Radio Union earlier this month, the possibility that they are being emitted by an intelligent source far out in space could not be entirely dismissed. But Dr. Drake, head of Cornell University's Arecibo Ionospheric Ob-

servatory in Puerto Rico, is an astronomer. On a scientific basis he considers the arguments against pulsars coming from intelligent beings to be persuasive, although not conclusive. He reported to ISRU that three of four pulsars are generating pulses of 38 to 40 thousandths of a second. The intervals between them are from 1.0 second to 1.3 seconds. These observations support a "neutron star" theory put forward by British astronomers to explain the pulsars. The British, using the Mullard Radio Astronomy Observatory at Cambridge University in November 1967, detected a series of remarkably regular signals or pulses. The radiotélescope was operating at a frequency of 81.5 MHz. Each pulse lasted 0.3 second, repeated every 1.337 seconds. The regularity, constant to better than 1 in 10 7 , suggested to the British a man-made origin of the signals. The absence of parallax showed that the source was lying far outside the solar system but still in our galaxy. The Cambridge team leader Anthony Hewish dubbed the source a pulsar. Further work detected three other pulsars with properties similar to the first. The Cambridge team found that duration of emission of the pulses at any frequency never exceeded 0.016 second. The source size thus could not be bigger than 4.8 Χ 10 3 kilo­ meter. Furthermore, sharpness of the signal, and a lack of parallax greater than two minutes placed the pulsar at a distance of 100 to 300 light years. In astronomical measures, this is a short distance. The British astronomers threw out the idea of pulsars being associated with intelligent life because the fan­ tastic amount of energy needed to gen­ erate the signals suggested a natural phenomenon. They worked up two theories, one based on plasma oscilla­ tion, the other a binary theory of star pairs spinning around each other. Their final idea was that the signals are natural oscillations of dying stars that have shrunk by gravitational contrac­ tion into neutron stars. A neutron star is an incredibly dense body of atoms which have collapsed to form neutrons tightly packed and spinning at extra­ ordinary speeds. Existence of such a star had already been postulated but never discovered. While arguments against the pul­ sars being artificial in origin are not conclusive, the neutron star theory fits some of the observations. Such a star would spin at the rate required to pro­ duce energy to generate the signals. But theory is theory. Arecibo-based Americans and Cambridge-based Brit­ ish have a good deal more listening to do. Perhaps the pulsars are listening now to us listening to them.