Peptide nucleic acid–DNA duplexes: Long range hole migration from an internally linked anthraquinone

The discovery that peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson–Crick base pairing rules opens fields in biochemistry, diagnostics, and medicine for exploration. Progress requires the development of modified PNA duplexes having unique and well defined properties. We find that anthraquinone groups bound to internal positions of a PNA oligomer intercalate in the PNA–DNA hybrid. Their irradiation with near-UV light leads to electron transfer and oxidative damage at remote GG doublets on the complementary DNA strand. This behavior mimics that observed in related DNA duplexes and provides the first evidence for long range electron (hole) transport in PNA–DNA hybrid. Analysis of the mechanism for electron transport supports hole hopping.

Climate and infectious disease: Use of remote sensing for detection of Vibrio cholerae by indirect measurement

It has long been known that cholera outbreaks can be initiated when Vibrio cholerae, the bacterium that causes cholera, is present in drinking water in sufficient numbers to constitute an infective dose, if ingested by humans. Outbreaks associated with drinking or bathing in unpurified river or brackish water may directly or indirectly depend on such conditions as water temperature, nutrient concentration, and plankton production that may be favorable for growth and reproduction of the bacterium. Although these environmental parameters have routinely been measured by using water samples collected aboard research ships, the available data sets are sparse and infrequent. Furthermore, shipboard data acquisition is both expensive and time-consuming. Interpolation to regional scales can also be problematic. Although the bacterium, V. cholerae, cannot be sensed directly, remotely sensed data can be used to infer its presence. In the study reported here, satellite data were used to monitor the timing and spread of cholera. Public domain remote sensing data for the Bay of Bengal were compared directly with cholera case data collected in Bangladesh from 1992–1995. The remote sensing data included sea surface temperature and sea surface height. It was discovered that sea surface temperature shows an annual cycle similar to the cholera case data. Sea surface height may be an indicator of incursion of plankton-laden water inland, e.g., tidal rivers, because it was also found to be correlated with cholera outbreaks. The extensive studies accomplished during the past 25 years, confirming the hypothesis that V. cholerae is autochthonous to the aquatic environment and is a commensal of zooplankton, i.e., copepods, when combined with the findings of the satellite data analyses, provide strong evidence that cholera epidemics are climate-linked.

Fluorescence correlation spectroscopy: Diagnostics for sparse molecules

The robust glow of molecular fluorescence renders even sparse molecules detectable and susceptible to analysis for concentration, mobility, chemistry, and photophysics. Correlation spectroscopy, a statistical-physics-based tool, gleans quantitative information from the spontaneously fluctuating fluorescence signals obtained from small molecular ensembles. This analytical power is available for studying molecules present at minuscule concentrations in liquid solutions (less than one nanomolar), or even on the surfaces of living cells at less than one macromolecule per square micrometer. Indeed, routines are becoming common to detect, locate, and examine individual molecules under favorable conditions.

DNA analysis and diagnostics on oligonucleotide microchips.

We present a further development in the technology of sequencing by hybridization to oligonucleotide microchips (SHOM) and its application to diagnostics for genetic diseases. A robot has been constructed to manufacture sequencing "microchips." The microchip is an array of oligonucleotides immobilized into gel elements fixed on a glass plate. Hybridization of the microchip with fluorescently labeled DNA was monitored in real time simultaneously for all microchip elements with a two-wavelength fluorescent microscope equipped with a charge-coupled device camera. SHOM has been used to detect beta-thalassemia mutations in patients by hybridizing PCR-amplified DNA with the microchips. A contiguous stacking hybridization technique has been applied for the detection of mutations; it can simplify medical diagnostics and enhance its reliability. The use of multicolor monitoring of contiguous stacking hybridization is suggested for large-scale diagnostics and gene polymorphism studies. Other applications of the SHOM technology are discussed.

Swiveling-domain mechanism for enzymatic phosphotransfer between remote reaction sites.

The crystal structure of pyruvate phosphate dikinase, a histidyl multiphosphotransfer enzyme that synthesizes adenosine triphosphate, reveals a three-domain molecule in which the phosphohistidine domain is flanked by the nucleotide and the phosphoenolpyruvate/pyruvate domains, with the two substrate binding sites approximately 45 angstroms apart. The modes of substrate binding have been deduced by analogy to D-Ala-D-Ala ligase and to pyruvate kinase. Coupling between the two remote active sites is facilitated by two conformational states of the phosphohistidine domain. While the crystal structure represents the state of interaction with the nucleotide, the second state is achieved by swiveling around two flexible peptide linkers. This dramatic conformational transition brings the phosphocarrier residue in close proximity to phosphoenolpyruvate/pyruvate. The swiveling-domain paradigm provides an effective mechanism for communication in complex multidomain/multiactive site proteins.

Isolation and molecular characterization of a human T-cell lymphotropic virus type II (HTLV-II), subtype B, from a healthy Pygmy living in a remote area of Cameroon: an ancient origin for HTLV-II in Africa.

We report characterization of a human T-cell lymphotropic virus type II (HTLV-II) isolated from an interleukin 2-dependent CD8 T-cell line derived from peripheral blood mononuclear cells of a healthy, HTLV-II-seropositive female Bakola Pygmy, aged 59, living in a remote equatorial forest area in south Cameroon. This HTLLV-II isolate, designated PYGCAM-1, reacted in an indirect immunofluorescence assay with HTLV-II and HTLV-I polyclonal antibodies and with an HTLV-I/II gp46 monoclonal antibody but not with HTLV-I gag p19 or p24 monoclonal antibodies. The cell line produced HTLV-I/II p24 core antigen and retroviral particles. The entire env gene (1462 bp) and most of the long terminal repeat (715 bp) of the PYGCAM-1 provirus were amplified by the polymerase chain reaction using HTLV-II-specific primers. Comparison with the long terminal repeat and envelope sequences of prototype HTLV-II strains indicated that PYGCAM-1 belongs to the subtype B group, as it has only 0.5-2% nucleotide divergence from HTLV-II B strains. The finding of antibodies to HTLV-II in sera taken from the father of the woman in 1984 and from three unrelated members of the same population strongly suggests that PYGCAM-1 is a genuine HTLV-II that has been present in this isolated population for a long time. The low genetic divergence of this African isolate from American isolates raises questions about the genetic variability over time and the origin and dissemination of HTLV-II, hitherto considered to be predominantly a New World virus.