A new procedure for the measurement of the protein-kinase-catalyzed incorporation of the gamma-32P-phosphoryl group of ATP into phospho-proteins and phosphopeptides.

Separate estimations of intact apurinic sites and single-strand breaks in DNA necessitates the use of neutral sucrose gradients for sedimentation analysis after denaturation with formamide or with NaOH followed by reneutralization. The number of breaks per strand in the denatured sample, relative to a control, can be determined with the computer program of Gillespie et al. ⁶; the particular equation for denatured DNA in neutral sucrose gradient that relates the molecular weight and the sedimentation rate is given. The reliability of the whole technique was proven in an experiment with T7 phage [³²P]DNA in which the ³²P transmutations into ³²S were the origin of the strand breaks.     

Patterned monolayers of nitronyl nitroxide radicals

We report here the results of the preliminary characterization of the monolayer obtained both by self-assembling and microcontact printing of a di-alkyl sulfide nitronyl nitroxide derivative, 11-decyl sulfanyl-undecanyl nitronyl nitroxide of which we describe the synthesis. The sulfide unit has been introduced in order to allow the grafting of the molecule to the gold surface as well as to improve the stability of the organic radical with respect to different grafting agents like thiols, whereas the two long alkyl chains have been introduced to enhance the packing order of the molecules in a self assembled monolayer structure.X-band ESR was used to demonstrate the persistence of the paramagnetic character of the radical in the self-assembled monolayers, and to study its relatively large mobility. The microcontact printed monolayer was characterized by AFM, suggesting a non-negligible mobility of the molecules on the surfaces and a strong tilting of the molecules on the surface.     

Comparison of the conventional cervical smear and liquid-based cytology: results of a controlled, prospective study in the Abruzzo Region of Italy

OBJECTIVE: To compare conventional cervical testing (CCT) and liquid-based cytology (LBC) within a randomized trial performed during 2001-2002 in the Abruzzo Region of Italy, including a cost-outcome comparative analysis. STUDY DESIGN: Study subjects were recruited in the framework of a controlled, randomized study organized in the Abruzzo Region. Women aged 2 6-64 years were randomized to an active arm (LBC) or control arm (CC1). The particip ating laboratories had no previous ex perience with LBC. RESULTS: The inadequacy rate was 4.3% in CCT and 1.3% in the LBC arm (D < 0.001). Atypical squamous cells of undetermined sign ifi cance and atypical glands of undetermined significance reports were more frequent at CCT vs. LBC. A small, insignificant excess of low grade squamous intraepithelial lesions or high grade squamous epithelial lesions+ reports was observed in the LBC arm. The cervical intraepithelial neoplasia 2+ (CIN2+) detection rate was not statistically different in the 2 arms (CCT=0.54%, LBC= 0.66%, p = 0.28). In the overall series positive predictive value was slightly but not significantly higher in the LBC arm. LBC increased costs by 4.2% per both screened women and CIN2+ detected. CONCLUSION: The study reflects the introductory phase of LBC in laboratories without prior LBC experience. In this setting LBC reduced the inadequacy rate and decreased reading and was at least as sensitive as and more specific than CCT. Utilization of LBC in organized screening programs will be based on local feasibility, considering that the high cost of LBC is only partially compensated for by other benefits, such as residual cellular material, available for molecular testing, including human papillomavirus testing.     

Glucocorticoid-attenuated response genes induced in the lung during endotoxemia

Cytokines and other mediators whose induction in inflammatory lung disease is attenuated by glucocorticoids are potential targets for development of selective anti-inflammatory treatments. We refer to genes with these regulatory characteristics as glucocorticoid-attenuated response genes, or GARGs. Systematic identification of GARGs has not been attempted previously in vivo. Using an endotoxemia model in adrenalectomized mice, we constructed a subtracted lung library enriched in endotoxemia-induced genes and identified candidate GARGs by differential hybridization screening. Northern analysis confirmed induction in the lung during endotoxemia and attenuation by glucocorticoids of 36 genes of diverse types. The majority were genes of unknown function not previously implicated in the pulmonary response to inflammation, including a new member of a 2'-5'-oligoadenylate synthetase-like family and a novel lung inducible Neuralized-related C3HC4 RING protein. Our results suggest that a full understanding of glucocorticoid effects on lung inflammation will require elucidation of the roles of an extensive network of glucocorticoid-modulated genes.     

Structural requirements for the enzymatic phosphorylation of phosvitin.

Some structural features required for the enzymatic phosphorylation of phosvitin by purified rat liver cytosol phosvitin kinase have been investigated by testing the activity of such an enzyme toward phosphopeptides differing in size and chemical composition, obtained by pronase or acid hydrolysis of phosvitin. The results obtained can be summarized as follows: (a) Phosvitin kinase phosphorylates even fairly simple phosphopeptides (mol.wt 1000-2000) at rates comparable with intact phosvitin. (b) Acetylation of both phosvitin and pronase phosphopeptides completely prevents their phosphorylation indicating that some lysine residues are strictly required for the phosvitin kinase reaction. (c) Accordingly polyphosphorylserine blocks Ser(P)n which are very actively phosphorylated in phosvitin and pronase phosphopeptides, do not undergo any more enzymatic phosphorylation once isolated as such in a form free of other amino acids. (d) The activity of phosvitin kinase toward substrates probably devoid of Ser(P)n blocks suggests that there are not required for the protein kinase reaction. However, they apparently enhance the phosphorylation rate of the peptide substrates, likely by making easier their binding to the enzyme. It is proposed therefore that the peptidic unit able to undergo phosphorylation by rat liver cytosol phosvitin kinase consists of one or more phosphorylserine residues having in their close proximity a lysine residue playing a critical role in the mechanism of transphosphorylation.     

Bone marrow cell chromosomal aberrations and styrene biotransformation in mice given styrene on a repeated oral schedule

Styrene's capacity to induce chromosomal aberrations was studied in bone marrow cells of CD1 male mice. No mutagenic effect could be detected after either a 4-day treatment course with daily oral doses of 500 mg/kg or a 70-day course with daily oral doses of 200 mg/kg. Urinary elimination of styrene metabolites related to styrene-7,8-oxide formation (i.e. phenylethylene glycol, mandelic acid, benzoic acid, phenylglyoxylic acid and total mercapturic acids) was quantitatively evaluated in the group of mice given the 200 mg/kg dose. In parallel, kinetic studies were made on styrene and styrene-7,8-oxide blood concentrations in the same group of animals. These determinations were carried out on days 1 and 70 of treatment by spectrophotometric, gas chromatographic and mass fragmentographic procedures.Not even nanograms of styrene-7,8-oxide were found in the blood of styrene-treated mice. This suggests that the metabolite does not migrate from the cellular compartment where it is formed being immediately metabolized or irreversibly bound to cellular structures.This observation could well explain the lack of mutagenic effects observed.     

From multidomain particles to organic radicals: The multifaceted magnetic properties of tobacco and cigarette ash

We present a detailed investigation by magnetic measurements and EPR spectroscopy of the magnetic fraction of cigarettes and its variation with the smoking process. Our results indicate that both before and after smoking magnetic particles with a broad size distribution occur, presumably mainly composed by iron oxides. The size distribution becomes broader with the smoking process producing particles which range from the nanoscale to the multidomain dimension. Both techniques evidence the presence of a paramagnetic fraction whose main components are Mn(II) and Fe(III), as well as an organic radical. The amount of the paramagnetic component is strongly reduced with the combustion. Moreover ICP analysis reveals that a part of other metal ions are volatilized. In conclusion this investigation points out the relevant and complex magnetic properties of both ashes and tobacco which should then be considered as a possible source of data contamination to be avoided in molecular magnetism laboratories.