Structural studies on an inhibitory antibody against Thermus aquaticus DNA polymerase suggest mode of inhibition

TP7, an antibody against Thermus aquaticus DNA polymerase I (TaqP), is usedas a thermolabile switch in 'hot start' variations of PCR to minimizenon-specific amplification events. Earlier studies have established thatTP7 binds to the polymerase domain of TaqP, competes with primer templatecomplex for binding and is a potent inhibitor of the polymerase activity ofTaqP. We report crystallographic determination of the structure of an Fabfragment of TP7 and computational docking of the structure with the knownthree-dimensional structure of the enzyme. Our observations stronglysuggest that the origin of inhibitory ability of TP7 is its binding toenzyme residues involved in DNA binding and polymerization mechanism.Although criteria unbiased by extant biochemical data have been used inidentification of a putative solution, the resulting complex offers aneminently plausible structural explanation of biochemical observations. Theresults presented are of general significance for interpretation of dockingexperiments and in design of small molecular inhibitors of TaqP, that arenot structurally similar to substrates, for use in PCR. Structural andfunctional similarities noted among DNA polymerases, and the fact thatseveral DNA polymerases are pharmacological targets, make discovery ofnon-substrate based inhibitors of additional importance.     

Phase Relationships and Physical Properties of Homologous Compounds in the Zinc Oxide-Indium Oxide System

Equilibrium phase relationships in the ZnO-In₂O₃ system were determined between 1100° and 1400°C using solid-state reaction techniques and X-ray diffractometry. In addition to ZnO and In₂O₃, nine homologous compounds, Zn _k In₂O _{k +3} (where k = 3, 4, 5, 6, 7, 9, 11, 13, and 15), were observed. Electrical conductivity and diffuse reflectance of the k = 3, 4, 5, 7 and 11 members were measured before and after annealing at 400°C for 1 h under forming gas (4%₂-96% N₂). Room-temperature conductivity increased as k decreased, because of increased carrier concentration as well as increased mobility. In general, transparency in the wavelength range of 450-900 nm increased as k increased. Reduction in forming gas resulted in increased conductivity and reduced transparency for all compounds measured. The highest room-temperature conductivity measured, 270 S/cm, was that of reduced Zn₃In₂O₆.     

Doses to fetal and maternal organs from intravenous 59FeCl3 or 57CoCl2

Doses from intravenous intakes of 59Fe or 57Co chloride during pregnancy were estimated. Near term fetal organ doses were derived via the MIRDOSE3 newborn phantom, with mean dose/cumulated activity (S) values rescaled for compatibility with near term fetus whole body S. A detailed in vivo biodistribution database provided indications of residence times in important maternal and fetal organs. 59Fe doses to the fetus whole body from early to late pregnancy were 7-11 mGy MBq(-1) (8.5-14.3 mSv MBq(-1)), similar to that to the mother. Doses to near term fetal spleen (59 mGy MBq(-1)), liver (36), and red marrow (9) were similar or higher than to the mother. 57Co doses to fetus whole body from early to late pregnancy were 0.7-3.3 mGy MBq(-1) (2.5-8.2 mSv MBq(-1)), similar or higher than to the mother. Doses to near term fetal small intestine (34 mGy MBq(-1)). liver (4.7) and red marrow (2.7) were similar or higher than to the mother.     

Method for repetitive measurement of the electrostatic chargingtendency of liquid dielectrics

The construction of an apparatus for the measurement of the electrostatic charging tendency (ECT) of a dielectric liquid is described. A sealed reciprocating piston arrangement permits a liquid to be repetitively passed through a charge separating element. The use of a gold element is shown to minimize the effects of moisture and control temporal effects. The system is evaluated by testing samples of transformer oil drawn from the field. In general, the new system ranks the oils in the same way as a conventional assessment against a paper filter, although anomalies have been found which are an impediment to adoption     

Oxidative modification of high-density lipoprotein 3 induced by human polymorphonuclear neutrophils. Protective effect of pentoxifylline

The function of high-density lipoproteins (HDLs) in reverse cholesterol transport is impaired if HDLs are subjected to oxidative stress. Polymorphonuclear neutrophils (PMNs), which have been detected in the earliest stages of atherosclerotic lesions, are one of the most likely sources of the reactive oxygen species that cause such stress. In this study, we investigated the effect of a PMN oxidative burst on HDL3. We also studied the impact on these events of pentoxifylline, a drug that regulates granulocyte function. HDL3 (370 nmol.mL-1 cholesterol-HDL) was incubated with PMNs (2 x 106. mL-1) in NaCl/Pi in the presence or absence of an iron chelate complex (10 microm Fe-nitrilotriacetic acid) at 37 degreesC for 60 min or 24 h. Phorbol myristate acetate (PMA) or formyl-methionylleucyphenylalanine (fMetLeuPhe) was used to stimulate PMNs. In iron-free NaCl/Pi medium, PMA-stimulated PMNs had a 40% lower HDL3 alpha-tocopherol content, whatever the incubation time. In NaCl/Pi medium containing iron, there was 80% less HDL3 alpha-tocopherol at 60 min, and HDL3 alpha-tocopherol had almost disappeared after 24 h. In this latter condition, the amount of thiobarbituric acid-reactive substances was significantly higher than the respective control HDL3 (P < 0.05) and oxidation of HDL3 by PMA-stimulated PMNs was associated with cross-linking of apoprotein AI, which was detected by SDS/PAGE. Similar results were obtained with fMetLeuPhe-stimulated PMN except that HDL3 alpha-tocopherol was consumed much more slowly during the first 60 min. Pretreatment of PMNs with various concentrations of pentoxifylline (0.001-20 mm) led to the concentration-dependent inhibition of oxidative modification of HDL3 induced by stimulated PMNs. The addition of 20 mm pentoxifylline in the most extreme oxidative stress conditions resulted in 70% of HDL3 alpha-tocopherol being maintained, with no formation of thiobarbituric acid-reactive substances and a lower level of apoprotein AI cross-linking. Thus HDL3 is susceptible to oxidative modifications induced by stimulated PMNs, in the presence of an exogenous source of iron. Pentoxifylline inhibited the oxidative modification of HDL3 by PMNs.     

Requirement for phosphatidylinositol-3'-kinase in cytokine-mediated germ cell survival during fetal oogenesis in the mouse

Apoptosis is responsible for primordial germ cell (PGC) attrition in the developing fetal ovary. In monolayer cultures of murine PGC, stem cell factor (SCF) and leukemia inhibitory factor (LIF) independently promote survival in vitro; however, the relevance of these data to fetal ovarian oogonium and oocyte survival, as well as the intracellular events involved in transducing the antiapoptotic actions of these cytokines in germ cells, remain to be elucidated. In this report, we investigated the effects of SCF and LIF, alone and in combination, on the survival of oogonia and oocytes, and elaborated on components of the signal transduction pathway used by these molecules, after validating a method of culturing fetal mouse ovaries. We further employed this system to also test the hypothesis that insulin-like growth factor-I (IGF-I), a classic antiapoptotic molecule, and transforming growth factor-beta (TGF-beta), a classic pro-apoptotic molecule, interact with the SCF/LIF pathway and function in a reciprocal fashion to precisely regulate germ cell numbers during fetal oogenesis. Freshly isolated embryonic day 13.5 ovaries contained nonapoptotic germ cells, as determined by histologic analysis of cellular morphology and in situ 3'-end-labeling of DNA integrity. In vitro culture of fetal ovaries without tropic support for 24, 48, and 72 h resulted in a time-dependent induction of germ cell apoptosis, such that most oogonia and oocytes present after 72 h were apoptotic. Morphometric analysis of serially sectioned ovaries indicated that the numbers of nonapoptotic germ cells remaining after 24, 48, and 72 h of culture were 78%, 38%, and 10%, respectively, of the number present before culture (P < 0.05 for all time points vs. 0 h). Inclusion of SCF (100 ng/ml) together with LIF (100 ng/ml) in the culture medium significantly attenuated germ cell apoptosis, with the SCF/LIF-treated ovaries retaining 5.5-fold more oogonia and oocytes after 72 h of culture as compared with control ovaries deprived of tropic support (P < 0.05). However, SCF or LIF, when added separately, had no (SCF) or little (LIF) inhibitory effect on germ cell apoptosis. Provision of 50 ng/ml IGF-I maintained survival of approximately two-thirds of the germ cells in cultured ovaries (P < 0.05), whereas a combination of all three growth factors (SCF, LIF, IGF-I) completely preserved the fetal ovary in culture to that resembling a freshly-isolated gonad. Cotreatment with 25 ng/ml TGF-beta partially reversed the survival actions of IGF-I or SCF/LIF, such that only one-third of the starting number of oogonia/oocytes remained after 72 h of culture (P < 0.05). Lastly, the antiapoptotic effects of SCF/LIF or IGF-I were almost entirely eliminated by cotreatment of fetal ovaries with either one of two inhibitors of phosphatidylinositol-3'-kinase (PI3K), LY294002 (5 microM) or wortmannin (50 nM), whereas cotreatment with an inhibitor of p70 S6 kinase (rapamycin, 25 ng/ml) was without effect. These data indicate that the combined actions of SCF, LIF, and IGF-I are required for maximal inhibition of apoptosis in germ cells of fetal mouse ovaries, and that the PI3K signaling pathway is an essential component of cytokine-mediated female germ cell survival. Moreover, TGF-beta can partially override the antiapoptotic actions of SCF/LIF or IGF-I in oogonia and oocytes, suggesting the existence of a complex signaling network that ultimately determines fetal ovarian germ cell fate.     

Effects of viral respiratory disease education and surveillance on antibiotic prescribing

BACKGROUND AND OBJECTIVES: We describe a partnership between family practice residency clinics and a state public health virology laboratory that has produced comprehensive viral respiratory disease education and surveillance. Family practice residents have been provided with education on respiratory viruses and the results of ongoing viral surveillance. The preliminary effects of this program on antibiotic prescribing by senior residents are evaluated in this paper. METHODS: We used a questionnaire to assess the acceptance by family practice residents of the educational component and the utility of ongoing viral surveillance. We used chart review to evaluate rates of antibiotic prescribing and the number of patients diagnosed per year with acute upper respiratory infection and acute bronchitis by senior residents in 1992 (preexposure) and 1996 (postexposure). RESULTS: By the third year of training, most residents (79%) reported receiving adequate training regarding common viral respiratory diseases. Moreover, residents reported that they were less likely to prescribe antibiotics to patients presenting with respiratory infections when provided with specific information on circulating viral pathogens. Antibiotic prescribing in the postexposure group was 68% lower for upper respiratory infection (URI) and 45% lower for a composite of URI and bronchitis. CONCLUSIONS: Education and monitoring of circulating respiratory viruses can result in familiarity with common disorders in primary care and reduce unnecessary antibiotic use.     

Three-channel solar radiometer for the determination of atmospheric columnar water vapor

The design of a three-channel solar radiometer used to determine total columnar atmospheric water-vapor amounts is presented. The main channel is located in the 0.94-μm water-vapor band, and two other channels are located in adjacent nonabsorption regions of the solar spectrum and are used to remove scattering effects from the main channel. Water-vapor transmittance is determined by means of a modified Langley approach, and these transmittances are converted to columnar water vapor by means of a band model developed at the University of Arizona. Several cases are presented in which columnar water-vapor amounts are determined through the use of the instrument and method described here. These results are compared with sounding-balloon results. Tests of the method indicate that columnar water vapor may be retrieved with an uncertainty of less than 10%.     

Low-density properties of gaseous,spin-aligned atomic hydrogen

The ground-state properties of bulk atomic hydrogen are calculated for the low-density gas, assuming that the atoms interact via the ³ _u ⁺ pair potential. The Monte Carlo technique is applied to a system consisting of 32 hydrogen atoms in a box, with periodic boundary conditions. These results are compared with those derived from the hard-sphere Bose-Einstein gas model.Work supported by NASA Grant No. NGR 06-002-159.