Purification and characterization of mouse hypoxanthine-guanine phosphoribosyltransferase.

Journal ArticleHypoxanthine-guanine phosphoribosyltransferase (HGPR transferase) (EC 2.4.2.8) has been purified approximately 4500-fold to apparent homogeneity from mouse liver. The procedure involves the use of affinity chromatography and was designed to be readily adaptable to small scale isolations. The enzyme appears to be composed of 3 subunits of identical molecular weight (27,000 per subunit). The subunit molecular weight has also been determined by the analysis of radioactively labeled HGPR transferase immunoprecipitated from wild type and mutant (HGPR transferase) mouse tissue culture cell lines

Yeast super-suppressors are altered tRNAs capable of translating a nonsense codon in vitro.

Journal ArticletRNA isolated from two different yeast super-suppressor strains translates a known nonsense mutation in vitro, whereas tRNA from a closely related nonsuppressing strain does not. Suppression was assayed by translation of RNA isolated from an amber coat mutant of bacteriophage Qbeta (GB11) in a protein-synthesizing system derived from mouse tissue culture cells (L cells). Suppressed forms of Qbeta coat protein synthesized in vitro were quantitatively detected by a specific immunoprecipitation assay. The L-cell protein-synthesizing system also responds to E. coli suppressor tRNA. This indicates that the biochemical mechanism for nonsense suppression is very similar in yeast and E. coli. These findings also provide additional evidence that the amber codon (UAG) functions as one of the mammalian chain-terminating codons. Since the suppression assay utilizes protein-synthesizing components isolated from mammalian cells, it should prove useful in the search for mammalian nonsense suppressor

Selective degradation of abnormal proteins in mammalian tissue culture cells.

Journal ArticleThe degradation rates of several missense mutants of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) in mouse L cells are compared to those of the wild-type enzyme. Although the rates of total protein breakdown in the mutant cell lines are identical to that of the parental L cell line, defective molecules of hypoxanthine-guanine phosphoribosyltransferase present in the mutant cell lines are degraded much faster than the wild-type enzyme. The level of defective phosphoribosyltransferase molecules present in the mutant cell lines is inversely proportional to the breakdown rate. This observation indicates that the major factor determining the concentrations of the defective phosphoribosyltransferases is their specific degradation rate. These results strongly support the hypothesis that abnormal proteins are selectively degraded in mammalian cells

Observations of X-rays and Thermal Dust Emission from the Supernova Remnant Kes 75

We present Spitzer Space Telescope and Chandra X-ray Observatory observations of the composite Galactic supernova remnant Kes 75 (G29.7-0.3). We use the detected flux at 24 microns and hot gas parameters from fitting spectra from new, deep X-ray observations to constrain models of dust emission, obtaining a dust-to-gas mass ratio M_dust/M_gas ~0.001. We find that a two-component thermal model, nominally representing shocked swept-up interstellar or circumstellar material and reverse-shocked ejecta, adequately fits the X-ray spectrum, albeit with somewhat high implied densities for both components. We surmise that this model implies a Wolf-Rayet progenitor for the remnant. We also present infrared flux upper limits for the central pulsar wind nebula.Comment: 7 pages, 2 tables, 4 figures, uses emulateapj. Accepted for publication in Ap

Educational Needs of Southern Forest Landowners

South-central United States forest landowners were surveyed to determine their forestry-related educational needs and appropriate methods for promoting effective programs covering desired topics. The majority of respondents had not participated in past educational programs because they were unaware of their existence. Therefore, forestry professionals and university Extension personnel should inform and encourage nonindustrial private forest (NIPF) landowners to take advantage of available opportunities. They should also use tax rolls to develop forest landowner databases. Once developed, newsletters, pamphlets, brochures, or letters should be mailed to increase forest landowner knowledge and awareness of forestry-related educational programs and activities

Mutations at position 184 of human immunodeficiency virus type-1 reverse transcriptase affect virus titer and viral DNA synthesis

AbstractMethionine at position 184 of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) was changed to valine, isoleucine, threonine, or alanine in an HIV-1-based vector. The vectors were analyzed for replication capacity and for resistance to the nucleoside analog 2′,3′-dideoxy-3′thiacytidine (3TC) using a single-cycle assay. Viruses containing the valine or isoleucine mutations were highly resistant to 3TC and replicated almost as well as the wild-type virus. The virus containing the threonine mutation was resistant to 3TC, but replicated about 30% as well as the wild-type. The alanine mutation conferred partial resistance to 3TC, but replicated poorly. The amounts of viral DNA synthesized decreased in 3TC-treated cells when the cells were infected with wild-type virus and the M184A mutant. The effect of these mutations on the generation of the ends of the linear viral DNA was determined using the sequence of the 2-LTR circle junctions. The M184T mutation increased the proportion of 2-LTR circle junctions containing a tRNA insertion, suggesting that the mutation affected the RNase H activity of RT

Mutations in human immunodeficiency virus type 1 reverse transcriptase that make it sensitive to degradation by the viral protease in virions are selected against in patients

AbstractMutations in the thumb subdomain of reverse transcriptase (RT) of HIV-1 can cause this enzyme to be degraded in virions by the viral protease (PR). Many of these mutations confer a temperature-sensitive phenotype on RT and viral replication. The degradation of RT by PR appears to take place after Gag-Pol has been processed. We show here that mutations in other parts of RT, including the RNase H domain, can make RT PR-sensitive and temperature-sensitive. These data explain why some mutations in the RNase H domain, which had little or no effect on the polymerase activity of purified recombinant RT, had a profound effect on viral titer. Because the PR-sensitive phenotype significantly reduced viral titer, we previously suggested that these mutations would be selected against in patients. We also show that RT mutations that are known to confer a temperature sensitive phenotype are rarely found in the Stanford database

Why Do HIV-1 and HIV-2 Use Different Pathways to Develop AZT Resistance?

The human immunodeficiency virus type 1 (HIV-1) develops resistance to all available drugs, including the nucleoside analog reverse transcriptase inhibitors (NRTIs) such as AZT. ATP-mediated excision underlies the most common form of HIV-1 resistance to AZT. However, clinical data suggest that when HIV-2 is challenged with AZT, it usually accumulates resistance mutations that cause AZT resistance by reduced incorporation of AZTTP rather than selective excision of AZTMP. We compared the properties of HIV-1 and HIV-2 reverse transcriptase (RT) in vitro. Although both RTs have similar levels of polymerase activity, HIV-1 RT more readily incorporates, and is more susceptible to, inhibition by AZTTP than is HIV-2 RT. Differences in the region around the polymerase active site could explain why HIV-2 RT incorporates AZTTP less efficiently than HIV-1 RT. HIV-1 RT is markedly more efficient at carrying out the excision reaction with ATP as the pyrophosphate donor than is HIV-2 RT. This suggests that HIV-1 RT has a better nascent ATP binding site than HIV-2 RT, making it easier for HIV-1 RT to develop a more effective ATP binding site by mutation. A comparison of HIV-1 and HIV-2 RT shows that there are numerous differences in the putative ATP binding sites that could explain why HIV-1 RT binds ATP more effectively. HIV-1 RT incorporates AZTTP more efficiently than does HIV-2 RT. However, HIV-1 RT is more efficient at ATP-mediated excision of AZTMP than is HIV-2 RT. Mutations in HIV-1 RT conferring AZT resistance tend to increase the efficiency of the ATP-mediated excision pathway, while mutations in HIV-2 RT conferring AZT resistance tend to increase the level of AZTTP exclusion from the polymerase active site. Thus, each RT usually chooses the pathway best suited to extend the properties of the respective wild-type enzymes

Touching the heart of HIV-1 drug resistance: the fingers close down on the dNTP at the polymerase active site

AbstractComparison of the recently solved structure of HIV-1reverse transcriptase (RT)-DNA-dNTP ternary complex with the previously solved structure of RT-DNA binary complex suggests mechanisms by which the HIV-1 RT becomes resistant to nucleoside-analog inhibitors, drugs currently used in the treatment of AIDS

Using Actiwatch to monitor circadian rhythm disturbance in Huntington' disease: A cautionary note

Huntington's disease (HD) is an inherited neurodegenerative disorder that is well recognised as producing progressive deterioration of motor function, including dyskinetic movements, as well as deterioration of cognition and ability to carry out activities of daily living. However, individuals with HD commonly suffer from a wide range of additional symptoms, including weight loss and sleep disturbance, possibly due to disruption of circadian rhythmicity. Disrupted circadian rhythms have been reported in mice models of HD and in humans with HD. One way of assessing an individual's circadian rhythmicity in a community setting is to monitor their sleep/wake cycles, and a convenient method for recording periods of wakefulness and sleep is to use accelerometers to discriminate between varied activity levels (including sleep) during daily life. Here we used Actiwatch® Activity monitors alongside ambulatory EEG and sleep diaries to record wake/sleep patterns in people with HD and normal volunteers. We report that periods of wakefulness during the night, as detected by activity monitors, agreed poorly with EEG recordings in HD subjects, and unsurprisingly sleep diary findings showed poor agreement with both EEG recordings and activity monitor derived sleep periods. One explanation for this is the occurrence of 'break through' involuntary movements during sleep in the HD patients, which are incorrectly assessed as wakeful periods by the activity monitor algorithms. Thus, care needs to be taken when using activity monitors to assess circadian activity in individuals with movement disorders