Nitroimidazole conjugates of bis(thiosemicarbazonato)Cu(II) - Potential combination agents for the PET imaging of hypoxia

Combination agents comprising two different pharmacophores with the same biological target have the potential to show additive or synergistic activity. Bis(thiosemicarbazonato)copper(II) complexes (e.g. ⁶⁴Cu-ATSM) and nitroimidazoles (e.g. ¹⁸F-MISO) are classes of tracer used for the delineation of tumor hypoxia by positron emission tomography (PET). Three nitroimidazole-bis(thiosemicarbazonato)copper(II) conjugates were produced in order to investigate their potential as combination hypoxia imaging agents. Two were derived from the known bifunctional bis(thiosemicarbazone) H₂ATSM/A and the third from the new precursor diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3-(4-N-ethylamino-3-thiosemicarbazone) - H₂ATSM/en. Oxygen-dependent uptake studies were performed using the ⁶⁴Cu radiolabelled complexes in EMT6 carcinoma cells. All the complexes displayed appreciable hypoxia selectivity, with the nitroimidazole conjugates displaying greater selectivity than a simple propyl derivative used as a control. Participation of the nitroimidazole group in the trapping mechanism is indicated by the increased hypoxic uptake of the 2- vs. the 4-substituted ⁶⁴Cu-ATSM/A derivatives. The 2-nitroimidazole derivative of ⁶⁴Cu-ATSM/en demonstrated superior hypoxia selectivity to ⁶⁴Cu-ATSM over the range of oxygen concentrations tested. Biodistribution of the radiolabelled 2-nitroimidazole conjugates was carried out in EMT6 tumor-bearing mice. The complexes showed significantly different uptake trends in comparison to each other and previously studied Cu-ATSM derivatives. Uptake of the Cu-ATSM/en conjugate in non-target organs was considerably lower than for derivatives based on Cu-ATSM/A.     

Dimerization of 2,6-dimethoxyphenol by Aspergillus flavus: evidence for the reaction occurring close to mycelia

Aspergillus flavus grown on 2,6-dimethoxyphenol as sole carbon source produced tetramethoxy-p-dibenzoquinone by a free radical mechanism. The product was identified by H-nmr and ms. Scanning electron microscopy and light microscopy were used to follow the growth of mycelia and the attachment of crystals to the mycelial surfaces. Formation of dimer was inhibited by the presence of glucose in the medium.     

Stereospecificity of C4 nicotinamide hydrogen transfer of the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase

The stereospecificity of the reaction catalysed by the spinach chloroplast enzyme NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NADP+ oxidoreductase (phosphorylating), EC 1.2.1.13) with respect to the C4 nicotinamide hydrogen transfer was investigated. NADPH deuterated at the C4 HA position was synthesized using aldehyde dehydrogenase. 1H-NMR spectroscopy was used to examine the NADP+ product of the GPDH reaction for the presence or absence of the C4 deuterium atom. Chloroplast NADP-dependent glyceraldehyde-3-phosphate dehydrogenase retains the deuterium at the C4 HA position (removing the hydrogen atom), and is therefore a B (pro-S) specific dehydrogenase.     

Four viral genes independently contribute to attenuation of live influenza A/Ann Arbor/6/60 (H2N2) cold-adapted reassortant virus vaccines.

Clinical studies previously demonstrated that live influenza A virus vaccines derived by genetic reassortment from the mating of influenza A/Ann Arbor/6/60 (H2N2) cold-adapted (ca) donor virus with epidemic wild-type influenza A viruses are reproducibly safe, infectious, immunogenic, and efficacious in the prevention of illness caused by challenge with virulent wild-type virus. These influenza A reassortant virus vaccines also express the ca and temperature sensitivity (ts) phenotypes in vitro, but the genes of the ca virus parent which specify the ca, ts, and attenuation (att) phenotypes have not adequately been defined. To identify the genes associated with each of these phenotypes, we isolated six single-gene substitution reassortant viruses, each of which inherited only one RNA segment from the ca parent virus and the remaining seven RNA segments from the A/Korea/1/82 (H3N2) wild-type virus parent. These were evaluated in vitro for their ca and ts phenotypes and in ferrets, hamsters, and seronegative adult volunteers for the att phenotype. We found that the polymerase PA gene of the ca parent specifies the ca phenotype and that the PB2 and PB1 genes independently specify the ts phenotype. The PA, M, PB2, and PB1 genes of the ca donor virus each contribute to the att phenotype. The finding that four genes of the ca donor virus contribute to the att phenotype provides a partial explanation for the observed phenotypic stability of ca reassortant viruses following replication in humans.     

Regulation of protein kinase C by Zn(2+)-dependent interaction with actin.

Zn2+ influences diverse cellular processes by poorly understood mechanisms. Some of these effects may be mediated by the protein kinase C (PKC) family of enzymes, since an influx of Zn2+ greatly increases their binding of regulatory ligand phorbol ester and induces their translocation from cytosol to the cytoskeleton. Using a model with purified components, we now show that Zn2+ acts by forming a phospholipid-dependent complex of PKC with filamentous actin, which results in expression of new binding sites for phorbol ester and phosphorylation of actin. These results provide a basis for the observed localization of PKC at actin-membrane junctions, in-vivo.     

Activation of a cryptic gene by excision of a DNA fragment.

The cryptic bgl operon in Escherichia coli K-12 strain 1011A contains a 1.4-kilobase-pair fragment of foreign DNA within the bglF structural gene. The active allele found in its descendant strain, MK1, required the precise excision of that insertion for its activation. Molecular and genetic approaches have shown that strain 1011A possessed an active (bglR+) rather than a silent wild-type (bglR0) allele of the regulatory region and that this change was caused by a point mutation. Our model for the retention of cryptic genes (B. G. Hall, S. Yokoyama, and D. H. Calhoun, Mol. Biol. Evol. 1:109-124, 1983) suggested that the insertion might have been selected to silence a disadvantageous bglR+ allele. We examined the genealogy of strain MK1 and found that the insertion of foreign DNA was not selected for that reason, since it preceded the change to bglR+. This means that the change to bglR+ was also not selected, since the presence of the insertion would not allow expression of the operon. We have calculated the probability of isolating a bglR+ mutation by chance alone as less than 10(-8). We suggest that mutation rates estimated under the usual conditions of exponential growth may be irrelevant to the frequencies of these events under natural conditions.     

Cloprostenol induced luteal regression in the beef cow. II. Histological evaluation of corpora lutea and correlation with luteal progesterone

Forty-two cycling, multiparous beef cows (percentage-Brahman) were injected twice at 11-d intervals with 500mug Cloprostenol (a prostaglandin F(2alpha) analog) to induce luteolysis. Cows were randomly assigned for ovariectomy at 12 hr intervals from 0 to 72 hr post-injection. Corpora lutea were excised and one-half of the corpus luteum was stored in phosphate-buffered formalin until mounting and staining with hematoxylin and eosin. The other half of the CL was snap-frozen for determination of progesterone content and concentration. Luteal cell density increased following Cloprostenol injection and was significantly correlated with a shift from predominantly healthy Type I cells to predominantly degenerating Types III and V cells. Cell mitosis tended to decrease by 12 hr and was lower by 24 hr post-injection. Cell pyknosis increased by 24 hr post-injection and was correlated with the decrease in percentage of healthy luteal cells. No pattern was detectable in cell karyorrhexis. Histological regression of the CL was inversely correlated with CL progesterone content. Therefore, we conclude that a reduction in cell mitosis is the earliest morphological sign of degeneration of the CL and that the CL follows a well-defined sequence of regression which is accompanied by a decrease in progesterone content.