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.     

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.     

Sequence of the ebgA gene of Escherichia coli: comparison with the lacZ gene

We have sequenced the ebgA (evolved beta-galactosidase) gene of Escherichia coli K12. The sequence shows 50% nucleotide identity with the E. coli lacZ gene, demonstrating that the two genes are related by descent from a common ancestral gene. Comparison of the two sequences suggests that the ebgA gene has recently been under selection. A significant excess of identical, rather than synonymous, codons used to encode identical amino acids at the same positions in the aligned sequences implies that some form of selection is operating directly at the DNA level. This selection is independent of, and in addition to, selection based on codon usage or on function of the gene products.      

A cryptic miniplasmid from the hyperthermophilic bacterium Thermotoga sp. strain RQ7.

An 846-bp cryptic plasmid has been discovered in the hyperthermophilic bacterium Thermotoga sp. strain RQ7. This is the first plasmid described for an organism from this ancient bacterial lineage and the smallest plasmid described to date for any organism. Nucleotide sequencing revealed a single open reading frame possibly encoding a 25,460-Da basic protein (212 amino acids). Upstream of the putative promoter lie five 11-bp direct repeats, each separated by 1 to 4 bp, while between the promoter and the open reading frame lies an 11-bp palindromic sequence. Its mode of replication is unknown, but its sequence bears similarities to those of plasmids which replicate by a rolling-circle mechanism.     

The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.