News from HPCE '99: Correlating enzymatic activity and cell cycle

Britt Erickson reports from Palm Springs, CA. Anal. Chemi. , 1999, 71 (7), pp 244A–245A. DOI: 10.1021/ac9902852. Publication Date (Web): June 7, 201...
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demonstrate that G2/M cells with uniform DNA content comprise three groups of cells with unique metabolic patterns. Correlations between the metabolic probe uptake and certain enzymatic activities within

the three groups are also shown. The approach can be used to study cell-to-cell variation in any metabolic pathway if an appropriate metabolic probe can be fluorescently modified and taken up by the cells.

GOVERNMENT AND SOCIETY

Copyright competition Sweeping new copyright laws from Brussels could harm the international competitiveness of European science, warns the European Science Foundation (ESF), an international body whose membership includes 65 major scientific funding agencies in 22 countries. New European Union (EU) laws that aim to harmonize aspects of copyright for EU members are now under consideration. The net effect could be, for instance, that scientists who make a copy of a research paper to read in their offices without paying a fee would be breaking the law. EU ministers will soon debate the draft directive, but ESF hopes to apply pressure to ensure that such details are changed to prevent a legal and financial headache for scientists. According to Sir Roger Elliott, professor of physics at Oxford University and a Fellow of the Royal Society—one of the bodies represented by ESF—the main problem is that the "exceptions" within the proposed copyright laws do not allow for "fair use" as ii is currently practiced. "The directive from the EC does not contain any blanket exceptions of the sort that allow scientists and students

to make copies of material for their own personal study," he explains. "Instead, it talks of possible exceptions for the 'illustration of teaching and research', but this statement presents a terminological problem because it is so ambiguous." ESF suggests rewording this section to read "sole purpose of scientific research or for illustration for teaching", which it says would clarify the exception. The lack of a blanket statement could, according to ESF, "result in research being treated differently in different countries across Europe". The way the directive is written member states would be free to interpret and implement the exceptions as they see fit, which could lead to some scientists finding their access to the scientific literature threatened. This is where the problems start in terms of competitiveness. "European scientists would be placed at a disadvantage to their U.S. colleagues because the concept of fair use is more firmly entrenched [in the United States]," explains Elliott. ESF recommends allowing current national exceptions to be sustained For instance in the U K as Elliott points out it is still fair use for scientists and students to copy a research paper More important, from a commercial perspective, ESF suggests mat terms included

NIST opens new research facility. A dedication ceremony was held on March 8, 1999, to honor the opening of NIST's Advanced Chemical Sciences Laboratory (ACSL), a $75 million, 200,000-square-toot research facility with state-of-the-art equipment for modern molecular biology, biochemistry, and bioprocessing. The new building features walk-in cold rooms and ultraclean, highly controlled environments that will enable NIST to develop advanced measurements and standards for DNA diagnostics. In addition the new facility will allow NIST to extend its chemical measurement capabilities in support of industry environmental quality and national health

BUSINESS

HP to split In early March, Hewlett-Packard Company (HP) announced plans to split the company into two independent companies—one focused on measurement and the other focused on computers and imaging. The measurement company will comprise HP's test-and-measurement, components, chemical analysis, and medical businesses, which contributed $7.6 billion (U.S.) to HP's total revenues of $47.1 billion infiscalyear 1998. Edward W. Barnholt, currently HP executive vice president and general manager of the measurement organization, has been named chief executive officer of the measurement company. HP is considering an initial public offering for approximately 15 percent of the measurement company's outstanding shares by year-end. A name has not yet been selected for the new entity. The computing and imaging company will continue to operate under the HP HP does not anticipate significant workforce reductions as a result of the realignment. in the exceptions, such as "noncommercial" research, are so ambiguous that scientists could inadvertently break the copyright law. It points out that it would be very diiffcult to differentiate between commercial and noncommercial research in most academic settings. Elliott suggests that universities and related institutions be dubbed "noncommercial" rather than the individual pieces of research being carried out within their walls. If the restrictions become too irksome, scientists willfinda way around the problems, according to Elliott. "Already we are finding a growing movement among scientists and their employers to retain their personal copyright." He believes the present balance is equitable and the publishers are making a fair profit. But, with the emergence of electronic media, publishers have lobbied for tighter laws, and the balance is swinging the other way. The European Commission's draft, "Directive on the Harmonisation of Certain Aspects of Copyright and Related Rights in the Information Society", is available online at http://europa.eu.int/comm/dgl5/en/ index.htm. David Bradley

Analytical Chemistry News & Features, April 1, 1999 245 A

News

the front-face configuration, the excitation source is in front of the sample. This setup allows another polarizer to be placed between the reference fluorophore and the sample. The additional polarizer selects the perpendicular component of the sample emission and extends the range of the compensation angle to 90°. At this stage, Lakowicz says there's no clear advantage for either method of polarizing the emission from the reference fluorophore, although the stretchedfilmis easier to construct. "But you might not have a perfectly matched color, because your fluorophore might not be the same color in the film," he says. "With the same self-reference, you have no color matching

problems, but it means there's probably another solution within the device." The sensor's reliance on the human eye to detect the intensity differences might seem to limit the accuracy. Not so, according to Lakowicz. "We knew it was quite accurate, but we didn't have a number for it. We went back and checked," he says. "The human eye is good to 10% relative accuracy. That 10% relative accuracy corresponds to one to two degrees of angular displacement. We further translated that into concentration ranges. In terms of pH, it is close to being good enough for clinical accuracy. But at this moment, it is not as accurate as highly standardized blood pH measurements."

Thus far, the sensors have been used to measure the concentration of Rhodamine B in intralipid, a highly scattering solution that mimics the scattering properties of skin, and to measure pH using 6-carboxyfluorescein. With compensation angles typically accurate to 1°, the pH values are accurate to ±0.1 pH unit. Lakowicz envisions the polarization sensors being used in a variety of settings. "The neat trick is that you're doing fluorescence sensing, which has numerous applications in biotechnology, DNA analysis, high-throughput screening, and drug discovery, with nothing more than a light source, your eyes, and a couple of polarizers." Celia Henry

NEWS FROM HPCE ’99 Britt Erickson reports from Palm Springs, CA.

Coordination ionspray MS Most nonpolar compounds are not amenable to electrospray ionization (ESI) because they lack a site for protonation or deprotonation. To allow such compounds to be characterized by MS, Ernst Bayer and co-workers at Universitat Tubingen (Germany) have developed a novel chemical ionization method, called coordination ionspray (CIS), which transforms the analytes to positively or negatively charged coordination compounds. In most cases the kinetics of complex formation are rapid, so postcolumn formation does not lead to band broadening when the technique is coupled with HPLC, CE, or capillary electrochromatography. Terpenes, neutral aromatic compounds, vitamins A and D, carotenoids, and unsaturated fatty acids are transformed on line via either pre- or postcolumn reaction and nebulized into the mass spectrometer as palladium (II) or silver© complexes, sugars and saccharides as boron complexes, and neutral polypeptides as lithium complexes. In tandem MS mode, coordinated daughter-ion fr3.|?ments are often obtained providing additional structural information (e.g. Ag+-coordinated fragments of unsaturated fatty acids allow the assignment of the position of the double bond) In contrast to ESI, CIS does not require strong electric fields or the presence of sites that can be protonated or deprotonated. In addition, CIS-MS offers sensitivity in the 244 A

Olefins analyzed by CIS-MS.

lower femtomole region. CIS-MS combined with a separation technique such as HPLC or CE opens up new possibilities for the characterization of a wide variety of neutral or weakly polar compounds, which cannot be subjected to ESI-MS.

Correlating enzymatic activity and cell cycle Single-cell analysis offers a means for detecting variations from one cell to the next within a population of cells. Such variations arise largely from cells being at different stages in the cell cycle. In contrast to normal cells, cancer cells proliferate rapidly. Because cancer cells progress through the cell cycle asynchronously, the enzymatic activity of tumor cells varies significantlyfromcell to cell. Classic cytometry techniques, such as flow cytometry and fluorescent image cytometry, have been used to study variations in enzymatic activities associated with different phases of the cell cycle; however, only a limited number of enzymes (rarely more than

Analytical Chemistry News & Features, April 1, 1999

three) can be assayed simultaneously. Sergey Krylov, Norman Dovichi, and coworkers at the University of Alberta (Canada) have developed an approach for assaying multiple enzymatic activities in individual cells by combining CE and fluorescent image microscopy. To monitor enzymatic activities, cells are incubated with afluorescentlylabeled metabolic probe. The probe is taken up by the cells and converted by several enzymes to metabolic products, which retain the fluorescent label. A single cell is injected into the capillary and lysed. The products are then separated by CE and detected by laser-inducedfluorescence.Knowing the concentration of the metabolic products allows for the indirect assessment of the enzymatic activities in individual cells. The cell's phase in the cell cycle is determined by measuring the amount of DNA in the cell before it is injected into the capillary. Cellular DNA content varies according to the phase in the cell cycle—in the Gl phase, cells contain one set of chromosomes; in the S phase, DNA content increases dramatically as a result of DNA replication; and in the G2 and M phases, cells contain two complete sets of chromosomes. Hoescht 33342—a cell-permeable, DNA-intercalating fluorescent dye—is used to stain DNA proportionally. Hoescht fluorescencefroma single cell is measured with a photomultiplier tube detector mounted on afluorescentmicroscope. By measuring the amount of DNA in individual cells they C3.n be ciscribed to one of three phases Gl S or G2/M The researchers use the technique to