Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission

The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90–110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details.

The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p

The yeast nonchromosomal gene [URE3] is due to a prion form of the nitrogen regulatory protein Ure2p. It is a negative regulator of nitrogen catabolism and acts by inhibiting the transcription factor Gln3p. Ure2p residues 1–80 are necessary for prion generation and propagation. The C-terminal fragment retains nitrogen regulatory activity, albeit somewhat less efficiently than the full-length protein, and it also lowers the frequency of prion generation. The crystal structure of this C-terminal fragment, Ure2p(97–354), at 2.3 Å resolution is described here. It adopts the same fold as the glutathione S-transferase superfamily, consistent with their sequence similarity. However, Ure2p(97–354) lacks a properly positioned catalytic residue that is required for S-transferase activity. Residues within this regulatory fragment that have been indicated by mutational studies to influence prion generation have been mapped onto the three-dimensional structure, and possible implications for prion activity are discussed.

The metabolic implications of intracellular circulation

Two views currently dominate research into cell function and regulation. Model I assumes that cell behavior is quite similar to that expected for a watery bag of enzymes and ligands. Model II assumes that three-dimensional order and structure constrain and determine metabolite behavior. A major problem in cell metabolism is determining why essentially all metabolite concentrations are remarkably stable (are homeostatic) over large changes in pathway fluxes—for convenience, this is termed the [s] stability paradox. For muscle cells, ATP and O2 are the most perfectly homeostatic, even though O2 delivery and metabolic rate correlate in a 1:1 fashion. In total, more than 60 metabolites are known to be remarkably homeostatic in differing metabolic states. Several explanations of [s] stability are usually given by traditional model I studies—none of which apply to all enzymes in a pathway, and all of which require diffusion as the means for changing enzyme–substrate encounter rates. In contrast, recent developments in our understanding of intracellular myosin, kinesin, and dyenin motors running on actin and tubulin tracks or cables supply a mechanistic basis for regulated intracellular circulation systems with cytoplasmic streaming rates varying over an approximately 80-fold range (from 1 to >80 μm × sec−1). These new studies raise a model II hypothesis of intracellular perfusion or convection as a primary means for bringing enzymes and substrates together under variable metabolic conditions. In this view, change in intracellular perfusion rates cause change in enzyme–substrate encounter rates and thus change in pathway fluxes with no requirement for large simultaneous changes in substrate concentrations. The ease with which this hypothesis explains the [s] stability paradox is one of its most compelling features.

Targeted gene inactivation in petunia by PCR-based selection of transposon insertion mutants.

Establishment of loss-of-function phenotypes is often a key step in determining the biological function of a gene. We describe a procedure to obtain mutant petunia plants in which a specific gene with known sequence is inactivated by the transposable element dTph1. Leaves are collected from batches of 1000 plants with highly active dTph1 elements, pooled according to a three-dimensional matrix, and screened by PCR using a transposon- and a gene-specific primer. In this way individual plants with a dTph1 insertion can be identified by analysis of about 30 PCRs. We found insertion alleles for various genes at a frequency of about 1 in 1000 plants. The plant population can be preserved by selfing all the plants, so that it can be screened for insertions in many genes over a prolonged period.

Re-expression of the alpha 2 beta 1 integrin abrogates the malignant phenotype of breast carcinoma cells.

To assess the role of altered alpha 2 beta 1 integrin expression in breast cancer, we expressed the alpha 2 beta 1 integrin de novo in a poorly differentiated mammary carcinoma that expressed no detectable alpha 2-integrin subunit. Expression of the alpha 2 beta 1 integrin resulted in a dramatic phenotypic alteration from a fibroblastoid, spindle-shaped, non-contact-inhibited, motile, and invasive cell to an epithelioid, polygonal-shaped, contact-inhibited, less motile, and less invasive cell. Although expression of the alpha 2 subunit did not alter adhesion to collagen, it profoundly altered cell spreading. Re-expression of the alpha 2 beta 1 integrin restored the ability to differentiate into gland-like structures in three-dimensional matrices and markedly reduced the in vivo tumorigenicity of the cells. These results indicate that the consequences of diminished alpha 2 beta 1-integrin expression in the development of breast cancer and, presumably, of other epithelial malignancies are increased tumorigenicity and loss of the differentiated epithelial phenotype.

Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases.

Most helicases studied to date have been characterized as oligomeric, but the relation between their structure and function has not been understood. The bacteriophage T7 gene 4 helicase/primase proteins act in T7 DNA replication. We have used electron microscopy, three-dimensional reconstruction, and protein crosslinking to demonstrate that both proteins form hexameric rings around single-stranded DNA. Each subunit has two lobes, so the hexamer appears to be two-tiered, with a small ring stacked on a large ring. The single-stranded DNA passes through the central hole of the hexamer, and the data exclude substantial wrapping of the DNA about or within the protein ring. Further, the hexamer binds DNA with a defined polarity as the smaller ring of the hexamer points toward the 5' end of the DNA. The similarity in three-dimensional structure of the T7 gene 4 proteins to that of the Escherichia coli RuvB helicase suggests that polar rings assembled around DNA may be a general feature of numerous hexameric helicases involved in DNA replication, transcription, recombination, and repair.

Barnstable, Massachusetts 15 Minute Digital Raster Graphic

This layer is a digital raster graphic of the historic 15-minute USGS topographic quadrangle map of Barnstable, Massachusetts. The edition date is 1893 and the map was reprinted in 1907. A digital raster graphic (DRG) is a scanned image of a U.S. Geological Survey (USGS) standard series topographic map, including all map collar information. The image inside the map neatline is geo-referenced to the surface of the earth and fit to the Universal Transverse Mercator projection. The horizontal positional accuracy and datum of the DRG matches the accuracy and datum of the source map. The names of quadrangles which border this one appear on the map collar in their respective positions (N,S,E,W) in relation to this map.

Barre, Massachusetts 15 Minute Digital Raster Graphic

This layer is a digital raster graphic of the historic 15-minute USGS topographic quadrangle map of Barre, Massachusetts. The suvery (ground condition) date is 1887, the edition date is March, 1894 and the map was reprinted in 1942. A digital raster graphic (DRG) is a scanned image of a U.S. Geological Survey (USGS) standard series topographic map, including all map collar information. The image inside the map neatline is geo-referenced to the surface of the earth and fit to the Universal Transverse Mercator projection. The horizontal positional accuracy and datum of the DRG matches the accuracy and datum of the source map. The names of quadrangles which border this one appear on the map collar in their respective positions (N,S,E,W) in relation to this map.

Becket, Massachusetts 15 Minute Digital Raster Graphic

This layer is a digital raster graphic of the historic 15-minute USGS topographic quadrangle map of Becket, Massachusetts. The survey (ground condition) date is 1886. A digital raster graphic (DRG) is a scanned image of a U.S. Geological Survey (USGS) standard series topographic map, including all map collar information. The image inside the map neatline is geo-referenced to the surface of the earth and fit to the Universal Transverse Mercator projection. The horizontal positional accuracy and datum of the DRG matches the accuracy and datum of the source map. The names of quadrangles which border this one appear on the map collar in their respective positions (N,S,E,W) in relation to this map.

Belchertown, Massachusetts 15 Minute Digital Raster Graphic

This layer is a digital raster graphic of the historic 15-minute USGS topographic map of the Belchertown, Massachusetts quadrangle. The suvey (ground condition) dates are 1885 and 1887; the edition date is November, 1893. A digital raster graphic (DRG) is a scanned image of a U.S. Geological Survey (USGS) standard series topographic map, including all map collar information. The image inside the map neatline is geo-referenced to the surface of the earth and fit to the Universal Transverse Mercator projection. The horizontal positional accuracy and datum of the DRG matches the accuracy and datum of the source map. The names of quadrangles which border this one appear on the map collar in their respective positions (N,S,E,W) in relation to this map.

Berlin (N.Y.) Massachusetts 15 Minute Digital Raster Graphic

This layer is a digital raster graphic of the historic 15-minute USGS topographic quadrangle map entitled Berlin, (N.Y.) which also shows towns and features in Massachusetts. The survey dates (ground condition) for this map are 1885-88, and the edition date is 1890. A digital raster graphic (DRG) is a scanned image of a U.S. Geological Survey (USGS) standard series topographic map, including all map collar information. The image inside the map neatline is geo-referenced to the surface of the earth and fit to the Universal Transverse Mercator projection. The horizontal positional accuracy and datum of the DRG matches the accuracy and datum of the source map. The names of quadrangles which border this one appear on the map collar in their respective positions (N,S,E,W) in relation to this map.

Blackstone, Massachusetts 15 Minute Digital Raster Graphic

This layer is a digital raster graphic of the historic 15-minute USGS topographic map of the Blackstone, Massachusetts quadrangle. The survey date (ground condition) of this map is 1886 and the edition date is October, 1893. A digital raster graphic (DRG) is a scanned image of a U.S. Geological Survey (USGS) standard series topographic map, including all map collar information. The image inside the map neatline is geo-referenced to the surface of the earth and fit to the Universal Transverse Mercator projection. The horizontal positional accuracy and datum of the DRG matches the accuracy and datum of the source map. The names of quadrangles which border this one appear on the map collar in their respective positions (N,S,E,W) in relation to this map.

Boston (1900), Massachusetts 15 Minute Digital Raster Graphic

This layer is a digital raster graphic (DRG) of the historic 15-minute USGS topographic map of the Boston, Massachusetts quadrangle. The survey date (ground condition) of this map ranges from 1898 to 1900, the edition date is July, 1903 and it was reprinted in 1918. A digital raster graphic (DRG) is a scanned image of a U.S. Geological Survey (USGS) standard series topographic map, including all map collar information. The image inside the map neatline is geo-referenced to the surface of the earth and fit to the Universal Transverse Mercator projection. The horizontal positional accuracy and datum of the DRG matches the accuracy and datum of the source map. The names of quadrangles which border this one appear on the map collar in their respective positions (N,S,E,W) in relation to this map.

Boston North (1943), Massachusetts 15 Minute Digital Raster Graphic

This layer is a digital raster graphic (DRG) of the historic 15-minute USGS topographic map of the Boston North, Massachusetts quadrangle. The survey date (ground condition) of this map is 1943, the edition date is 1946. A digital raster graphic (DRG) is a scanned image of a U.S. Geological Survey (USGS) standard series topographic map, including all map collar information. The image inside the map neatline is geo-referenced to the surface of the earth and fit to the Universal Transverse Mercator projection. The horizontal positional accuracy and datum of the DRG matches the accuracy and datum of the source map. The names of quadrangles which border this one appear on the map collar in their respective positions (N,S,E,W) in relation to this map.

Boston South (1950), Massachusetts 15 Minute Digital Raster Graphic

This layer is a digital raster graphic (DRG) of the historic 15-minute USGS topographic map of the Boston South, Massachusetts quadrangle. The survey date (ground condition) of this map is 1943, it was revised in 1949 and reprinted with corrections in 1950. A digital raster graphic (DRG) is a scanned image of a U.S. Geological Survey (USGS) standard series topographic map, including all map collar information. The image inside the map neatline is geo-referenced to the surface of the earth and fit to the Universal Transverse Mercator projection. The horizontal positional accuracy and datum of the DRG matches the accuracy and datum of the source map. The names of quadrangles which border this one appear on the map collar in their respective positions (N,S,E,W) in relation to this map.