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Replication of the parvovirus MVM. II. Isolation and characterization of intermediates in the replication of the viral deoxyribonucleic acid.

The time course of the appearance of intracellular viral DNA has been studied in mouse L cells infected with the single-stranded DNA virus MVM (minute virus of mice) by using a selective extraction procedure. Approximately half of this DNA elutes from hydroxyapatite as single-stranded DNA. It is sensitive to Escherichia coli exonuclease I and shows a sedimentation profile similar to DNA from the virus, suggesting that it is progeny viral DNA. The remainder of the selectively extracted DNA elutes from hydroxyapatite in the position of double-stranded DNA and is resistant to exonuclease I. Most of this DNA has a sedimentation coefficient of 14 to 16S, indicating that its molecular weight is twice that of the viral DNA. Denaturation renders the majority of the double-stranded DNA sensitive to exonuclease I, but a significant fraction renatures spontaneously in a monomolecular fashion, indicating that it has a cross-linked or hairpin structure. Chromatography of the double-stranded DNA on benzoylated diethylaminoethyl cellulose resolves two components, one with duplex structure and one which contains single-stranded regions. A short pulse label late in infection predominantly labels the latter class of DNA, suggesting that it contains replicating intermediates. The possible roles of these various forms of DNA in the replication of the viral genome are discussed.

Animals↗

Wine, biodiversity, technology, and antioxidants.

Two chemical classes of flavonoids, the flavan-3-ols (catechins and proanthocyanidins) and the anthocyanins, are the natural antioxidants present at the highest concentration in red grape and wine. In the berry, the anthocyanins are localized in the skins, similarly to other highly bioactive phenolics of grape such as the resveratrols and the flavonols, while the flavan-3-ols are contained both in the skins and seeds. During winemaking, only a fraction of the grape flavonoids are selectively extracted into the wine, with a time course and a final yield strongly depending on the grape variety. The knowledge of the diverse and cultivar-specific characteristics of the grape is therefore critical to the appropriate design of the winemaking process. By means of a selective extraction method specifically designed to mimic the winemaking process, it was possible to analyze the "phenolic potential of red grape," thus obtaining quantitative information about amount and localization of the extractable flavonoids in the grape. Twenty-five high-quality red grape cultivars (V. vinifera) were studied, including 4 of the worldwide leading cultivars and 21 Italian cultivars with the highest reputation for the production of both young and aged premium red wines. The results clearly indicate that the grape variety plays a central role in determining both the absolute amount of the flavonoids, and the distribution between the berry skin and seeds of the flavan-3-ols. The very high biodiversity of the red grape cultivars in terms of flavonoids indicate a largely under-exploited opportunity to produce a range of diverse premium wines with optimized levels of natural antioxidants.

Anthocyanins↗

Phosphorylation of molluscan paramyosin.

1. In Mytilus edulis two proteins of the contractile apparatus can be phosphorylated by cyclic AMP dependent protein kinases: a 295,000 d protein of unknown function, and paramyosin. 2. Paramyosin isolated from thick filaments by the selective extraction method contains the 106,000 d monomer only, whereas paramyosin extracted from ethanol ether dried powder contains equal amounts of the 108,000 d, and the 106,000 d monomers, and traces of the 104,000 d monomer. 3. Paramyosin isolated from ethanol ether dried powder incorporates up to four times the amount of 32P than paramyosin isolated by the selective extraction method. 4. Cytoplasmatic protein kinases show a higher affinity towards paramyosin as a phosphoryl acceptor than protein kinases associated with paramyosin. 5. Paramyosin of 5-HT treated catch muscles is phosphorylated 2 to 4 times better than paramyosin of ACh treated or untreated catch muscles.

Animals↗

Chelating sorbents based on silica gel and their application in atomic spectrometry.

This mini-review covers chelating sorbents anchored to silica gel and their analytical applications for the preconcentration, separation and determination of trace metal ions, focussing mainly on the last 20 years. The article summarizes also the experience gathered by our research group in the synthesis and characterization of new modified silica gels "via silanization", and their affinity toward selective extraction and separation of trace elements. The introduction of 1,5-bis(di-2-pyridyl)methylene thiocarbohydrazide silica gel (DPTH-gel) and methylthiosalicylate silica gel (TS-gel) chelating sorbents in trace and ultratrace analysis provide vital breakthroughs in preconcentration methods. These home-made materials allow certain analytes to be selectively extracted from complex matrices without matrix interference and good detection limits. The advantages of these new chelating sorbents in comparison with 8-hydroxyquinoline chelating sorbent immobilized on silica gel are discussed.

Chelating Agents↗

Staining of DNA-phosphate groups and DNA-aldehyde molecules with dyes of the azine group.

The investigation reports on the use of safranine-SO2 and phenosafranine-SO2, prepared with N HCl or oxalic acid plus potassium metabisulphite, for staining rat liver sections following Feulgen procedure. It has been found that optimum staining of DNA-aldehyde molecules is possible with safranine-SO2 and phenosafranine-SO2, prepared with N HCl and potassium metabisulphite, upto a duration of one week after the preparation of the dye-reagents. Thereafter, staining intensity of the nuclei produced by the dye-reagents is gradually diminished. Staining of acid-hydrolysed sections is also possible with aqueous solutions of these dyes. Moreover, DNA-phosphate groups can also be stained with aqueous solutions of these dyes after selective extraction of RNA with cold phosphoric acid. The in situ absorption spectra of nuclei, stained for DNA-aldehyde molecules with safranine-SO2, phenosafranine-SO2 and aqueous solutions of these dyes, have been presented in this paper. Also presented herein are absorption data of nuclei stained with these dyes after selective extraction of RNA. It has been found that absorption-peaks of nuclei stained differently are different from one another. The implications of these findings have been discussed.

Aldehydes↗

Reaction of the leukocytes of melanoma patients and control donors, including pregnant women, with melanoma- and fetus-derived materials.

Using a one-stage capillary leukocyte migration inhibition assay, we examined peripheral blood leukocytes from 97 patients with malignant melanoma, 194 pregnant women, and 123 non-pregnant donors for their reactivity with materials from fetal and melanomatous tissues. As in previous studies, we found melanoma extracts selectively to inhibit melanoma patients' leukocytes. First-trimester fetal extracts also selectively inhibited melanoma patients' leukocytes, suggesting their cross-reactivity with melanoma-derived antigens. We could not localize the source of the inhibitory materials within the fetuses. We found no evidence that pregnant women were selectively immunized to fetal or melanoma extracts, regardless of their parity or the stage of their pregnancy.

Cell Migration Inhibition↗

[Hair: a powerful biological marker for exposure to xenobiotics].

Human hair analysis is now recognized for evaluating someone exposure to xenobiotics: drugs of abuse, pharmaceuticals and polluants. This paper describes analytical methods than can be used by biologists. For drugs of abuse after decontamination, hydrolysis of hair, selective extraction and derivatization, determinations are performed by gas chromatography-mass spectrometry (GC/MS). Calibration uses deuterated standards. The cut-off value is 0.5 ng/mg for 6-monoacetylmorphine (heroin) and amphetamines and a benzoylecgonine/cocaine ratio > 0.05 for cocaine. Measurement of metabolites from endogenous metabolism sign the exposure (6-monoacetylmorphine and morphine for heroin, benzoylecgonine for cocaine, delta 9-tetrahydrocannabinol carboxylic by-product for cannabis). For drugs, after selective extraction, determinations are performed by GC/MS or liquid chromatography coupled to a diode array detector. Main applications concern drug monitoring to complement blood determinations or when blood collection is missing, as evidence of hidden, illicit or criminal drug exposure. Finally it is a powerfull tool for clinical diagnosis especially when late biological investigations are performed.

Biomarkers↗

Pb scavenging from a freshwater lake by Mn oxides in heterogeneous surface coating materials.

Selective extraction techniques were used to assay the importance of specific solid phases in Pb binding by heterogeneous surface coating materials (biofilms) in Cayuga Lake, NY. Hydroxylamine hydrochloride (NH(2)OH.HC1) was used to extract easily reducible Mn oxides, and sodium dithionite (Na(2)S(2)O(4)) was used to extract Mn and Fe oxides in two sets of biofilm samples retrieved from the lake. Pb remaining after extraction was removed by extraction with 10% HNO(3), determined by analysis of Pb(208) using a sector field mass spectrometer with an inductively coupled plasma ion source (ICP-MS), and compared to the total extractable Pb. The results indicate that the greatest contribution to total Pb binding to the heterogeneous surface coating materials was from Mn oxides. Pb adsorption capacity of Mn oxides exceeded that of Fe oxides on a molar basis by approximately an order of magnitude. The high reactivity observed for natural Mn oxides indicates that they are biogenic in origin, consistent with expectations based on the relative biotic and abiotic rates of Mn(II) oxidation under circumneutral conditions. Collectively, these results confirm expectations based on prior observations of adsorption of added Pb by Cayuga Lake biofilms before and after selective extraction, and also confirm predictions for Pb phase association in the lake based on the behavior of laboratory surrogates for adsorptive surfaces.

Adsorption↗

Identification of an activity in B-cell extracts that selectively impairs the formation of an immunoglobulin mu s poly(A) site processing complex.

The immunoglobulin mu heavy-chain transcription unit is differentially expressed during B-cell development, producing mRNAs that encode secreted (mu s) and membrane-bound (mu m) forms of the heavy-chain polypeptide. Whereas the mu s mRNA and the mu m mRNA are produced in approximately equal abundance in B cells, an increase in the utilization of the mu s poly(A) site contributes to the production of the mu s mRNA as the predominant form in a plasma cell. Previous experiments have demonstrated a correlation between the formation of a stable complex on a poly(A) site and the relative function of the poly(A) site. We have thus investigated the parameters determining the interaction of these factors with the immunoglobulin poly(A) sites. Assays of complex formation involving the two immunoglobulin poly(A) sites by using HeLa cell activities revealed the formation of stable complexes with no apparent difference between the mu s site and the mu m site. In contrast, the mu s-specific complex was markedly less stable when a B-cell extract was used. Fractionation of B-cell extracts has revealed an activity that specifically destabilizes the mu s polyadenylation complex, suggesting that the function of this poly(A) site may be regulated by both positive- and negative-acting factors.

Animals↗

Residual waste from Hanford tanks 241-C-203 and 241-C-204. 1. Solids characterization.

Bulk X-ray diffraction (XRD), synchrotron X-ray microdiffraction (microXRD), and scanning electron microscopy/ energy-dispersive X-ray spectroscopy (SEM/EDS) were used to characterize solids in residual sludge from single-shell underground waste tanks C-203 and C-204 at the U.S. Department of Energy's Hanford Site in southeastern Washington state. Cejkaite [Na4(UO2)(CO3)3] was the dominant crystalline phase in the C-203 and C-204 sludges. This is one of the few occurrences of cejkaite reported in the literature and may be the first documented occurrence of this phase in radioactive wastes from DOE sites. Characterization of residual solids from water leach and selective extraction tests indicates that cejkaite has a high solubility and a rapid rate of dissolution in water at ambient temperature and that these sludges may also contain poorly crystalline Na2U207 [or clarkeite Na[(UO2)O(OH)](H2O)0-1] as well as nitratine (soda niter, NaNO3), goethite [alpha-FeO(OH)], and maghemite (gamma-Fe2O3). Results of the SEM/EDS analyses indicate that the C-204 sludge also contains a solid that lacks crystalline form and is composed of Na, Al, P, O, and possibly C. Other identified solids include Fe oxides that often also contain Cr and Ni and occur as individual particles, coatings on particles, and botryoidal aggregates; a porous-looking material (or an aggregate of submicrometer particles) that typically contain Al, Cr, Fe, Na, Ni, Si, U, P, O, and C; Si oxide (probably quartz); and Na-Al silicate(s). The latter two solids probably represent minerals from the Hanford sediment, which were introduced into the tank during prior sampling campaigns or other tank operation activities. The surfaces of some Fe-oxide particles in residual solids from the water leach and selective extraction tests appear to have preferential dissolution cavities. If these Fe oxides contain contaminants of concern, then the release of these contaminants into infiltrating water would be limited by the dissolution rates of these Fe oxides, which in general have lowto very low solubilities and slow dissolution rates at near neutral to basic pH values under oxic conditions.

Microscopy, Electron, Scanning↗

Antipsychotic medication for challenging behaviour in people with learning disability.

BACKGROUND: The term 'challenging behaviour', in the absence of psychiatric disorder, encompasses a wide range of behaviours that may be harmful to people or property, may be difficult to manage and may limit access to community facilities. Antipsychotic medications have been used to modify such behaviours in people with learning disability, but there is little evidence to suggest that the benefits outweigh the risks. OBJECTIVES: To determine the effectiveness of antipsychotic medication for people with learning disability and challenging behaviour. SEARCH STRATEGY: Biological abstracts, the Cochrane Library, the Cochrane Schizophrenia Group's Register, EMBASE, MEDLINE, PsycLIT were searched. Further references were sought from published trials and pharmaceutical companies. Trials were reliably identified and data extracted. SELECTION CRITERIA: All randomised controlled trials of antipsychotic medication versus placebo. DATA COLLECTION AND ANALYSIS: Reviewers independently evaluated and analysed data on an intention to treat basis. Data were evaluated at 4 and 8 weeks as longer follow-up data were not available. Reviewers assumed that those subjects lost to follow-up had a bad outcome. MAIN RESULTS: Only three randomised controlled trials could be included in the analyses. These provided no evidence of whether antipsychotic medication helps or harms adults with learning disability and challenging behaviour. REVIEWER'S CONCLUSIONS: There is limited data on this important issue and more research is urgently needed.

Adult↗

Residual waste from Hanford tanks 241-C-203 and 241-C-204. 2. Contaminant release model.

Release of U and 99Tc from residual sludge in Hanford waste tanks 241-C-203 and 241-C-204 atthe U.S. Department of Energy's (DOE) Hanford Site in southeastern Washington state was quantified by water-leaching, selective extractions, empirical solubility measurements, and thermodynamic modeling. A contaminant release model was developed based on these experimental results and solid-phase characterization results presented elsewhere. Uranium release was determined to be controlled by two phases and occurred in three stages. In the first stage, U release is controlled by the solubility of tejkaite, which is suppressed by high concentrations of sodium released from the dissolution of NaNO3 in the residual sludges. Equilibrium solubility calculations indicate the U released during this stage will have a maximum concentration of 0.021 M. When all the NaNO3 has dissolved from the sludge, the solubility of the remaining cejkaite will increase to 0.28 M. After cejkaite has completely dissolved, the majority of the remaining U is in the form of poorly crystalline Na2U2O7 [or clarkeite Na[(UO2)O(OH)](H20)0-1]. In contact with Hanford groundwater this phase is not stable, and becquerelite becomes the U solubility controlling phase, with a calculated equilibrium concentration of 1.2 x 10(-4) M. For Tc, a significant fraction of its concentration in the residual sludge was determined to be relatively insoluble (20 wt % for C-203 and 80 wt % for C-204). Because of the low concentrations of Tc in these sludge materials, the characterization studies did not identify any discrete Tc solids phases. Release of the soluble fraction of Tc was found to occur concomitantly with NO3-. It was postulated that a NaNO3-NaTcO4 solid solution could be responsible for this behavior. The Tc release concentrations for the soluble fraction were estimated to be 2.4 x 10-6 M for C-203 and 2.7 x 10(-5) M for C-204. Selective extraction results indicated that the recalcitrant fraction of Tc was associated with Fe oxides. Release of the recalcitrant fraction of Tc was assumed to be controlled by dissolution of Fe oxide in the form of ferrihydrite. Based on this assumption and measured values for the ratio of recalcitrant Tc to total Fe in each bulk sludge, the release concentration of the recalcitrant fraction of Tc was calculated to be 3.9 x 10(-12) M for C-203 and 10.0 x 10(-12) M for C-204.

Radioactive Waste↗

An evaluation of selected DNA extraction strategies for short tandem repeat typing.

Organic and Chelex extraction methods are compared to five commercially available DNA extraction kits. For forensic purposes, comparison of different extraction strategies is based on the ability of the methods to economically recover high yields of amplifiable DNA from small blood samples in a quick, simple and safe manner. The findings of this study indicate that resin-based methods, such as Chelex, are best able to meet these criteria.

DNA↗

Liquid-phase microextraction of basic drugs--selection of extraction mode based on computer calculated solubility data.

The extractability of 58 different basic drugs by 3-phase liquid-phase microextraction (LPME) was studied. Extraction recoveries were correlated to solubility data and log D data calculated with a commercial computer program. The basic drugs were extracted from 1.5 mL water samples (pH 13) through approximately 15 microL of dodecyl acetate immobilized within the pores of a porous polypropylene hollow fibre (organic phase), and into 15 microL of 10 mM HCl (acceptor solution) present inside the lumen of the hollow fibre. Compounds with a calculated solubility below 1 mg/mL at pH 2 were poorly recovered and remained principally in the organic phase. For these drugs, 2-phase LPME may be used as an alternative technique, where the aqueous acceptor phase is replaced by an organic solvent. In the solubility range 1-5 mg/mL, most drugs were effectively extracted (recovery >30%), whereas drugs belonging to the solubility range 5-150 mg/mL were all extracted with recoveries above 30% by 3-phase LPME. The hydrophilic nature of most drugs with solubilities above 150 mg/mL prevented them from entering the organic phase, and only those with log D >1.8 were effectively recovered by 3-phase LPME. For drugs with log D < 1.8 (and solubility >150 mg/mL), carrier-mediated LPME was found to be the preferred technique, where an ion-pair reagent (octanoic acid) was added to the sample. In the case of carrier-mediated LPME, the volume of sample was decreased to 100 microL to facilitate rapid extractions. Based on the present work, the extractability of new compounds may easily be predicted to speed up method development. Extractions were also accomplished from plasma samples, where interactions between proteins and the drugs may reduce the extraction recovery. However, dilution of the plasma samples with water and adjustment of pH into the alkaline region effectively suppressed drug-protein interactions for most of the drugs studied.

Humans↗

Behavioural response of Colorado potato beetle (Leptinotarsa decemlineata) larvae to selected plant extracts.

Potato leaves were treated with 2, 20 or 200 g kg(-1) solutions of extracts of five plant species collected in Turkey, and then exposed to larvae of Colorado potato beetle, Leptinotarsa decemlineata (Say). During the first 24 h of exposure, leaf consumption was not affected by 2 g kg(-1) extracts, whereas significantly more leaf tissue remained on leaves treated with 20 g kg(-1) extracts of Arctium lappa L., Bifora radians M Bieb, Humulus lupulus L. or Xanthium strumarium L. than on untreated control leaves. Feeding was not affected by the 20 g kg(-1) extract of Verbascum songaricum Schrenk ex Fisch & Mey. Extracts of all species at 200 g kg(-1) reduced larval feeding, with H. lupulus and X. strumarium providing the greatest protection. Observations of larval behaviour over the first 15 min of exposure to these extracts revealed that the interaction of beetles with leaf tissue was significantly affected by plant extracts. Feeding frequency was not affected by 2 g kg(-1) extracts but was reduced by all higher concentrations. Feeding was inhibited completely by 20 g kg(-1) of H. lupulus extract and reduced significantly compared with the controls by all other extracts. Suppression of feeding was caused by all extracts at 200 g kg(-1), with V. songaricum providing 91% reduction in feeding duration. Rejection behaviour, in which larvae did not return to the leaf after their interaction with it, was rare on 2 g kg(-1) extracts but seen in over 60% of larvae on 20 g kg(-1) extracts and over 80% on 200 g kg(-1) extracts. The present results demonstrate that these extracts have significant ability to protect potato leaves for up to 24 h by prevention of feeding behaviour by L. decemlineata. Further studies are needed to determine the potential of these plant extracts, or their active components, for use in biologically based pest management strategies.

Animals↗

Screening of selected plant extracts for in vitro inhibitory activity on human immunodeficiency virus.

As part of our screening of anti-AIDS agents from natural sources, extracts of 15 medicinal plants widely used in the folk medicines of North America and Europe were evaluated in vitro. Most of the extracts tested were relatively nontoxic to human lymphocytic MT-2 cells, but only the extracts of Hysopp officinalis and Dittrichia viscosa exhibited anti-HIV activity in an in vitro MTT assay. The 50% hydroalcohol extract of Hysopp officinalis and the aqueous extract of Dittrichia viscosa showed inhibitory effects against HIV-1 induced infections in MT-2 cells at concentrations ranging from 50 to 100 microg/mL and 25 to 400 microg/mL, respectively. Both extracts showed no appreciable cytotoxicity at these concentrations.

Anti-HIV Agents↗