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.     

The in vivo construction of 4-chloro-2-nitrophenol assimilatory bacteria

Pseudomonas sp. N31 was isolated from soil using 3-nitrophenol and succinate as sole source of nitrogen and carbon respectively. The strain expresses a nitrophenol oxygenase and can use either 2-nitrophenol or 4-chloro-2-nitrophenol as a source of nitrogen, eliminating nitrite, and accumulating catechol and 4-chlorocatechol, respectively. The catechols were not degraded further. Strains which are able to utilize 4-chloro-2-nitrophenol as a sole source of carbon and nitrogen were constructed by transfer of the haloaromatic degrading sequences from either Pseudomonas sp. B13 or Alcaligenes eutrophus JMP134 (pJP4) to strain N31. Transconjugant strains constructed using JMP134 as the donor strain grew on 3-chlorobenzoate but not on 2,4-dichlorophenoxyacetate. This was due to the non-induction of 2,4-dichlorophenoxyacetate monooxygenase and 2,4-dichlorophenol hydroxylase. Transfer of the plasmid from the 2,4-dichlorophenoxyacetate negative transconjugant strains to a cured strain of JMP134 resulted in strains which also had the same phenotype. This indicates that a mutation has occurred in pJP4 to prevent the expression of 2,4-dichlorophenoxyacetate monooxygenase and 2,4-dichlorophenol hydroxylase.     

Comparison of fiber types in skeletal muscles from ten animal species based on sensitivity of the myofibrillar actomyosin ATPase to acid or copper

Summary Comparisons were made of the histochemical characteristics of skeletal muscle from 10 animal species. The basic comparison was made from the staining patterns for the myofibrillar actomyosin ATPase produced by preincubation of fresh frozen cross-sections of muscle at alkaline pH (10.30) or acid pH (4.60) with those produced by preincubation in media containing Cu²⁺ at alkaline pH (10.30), near neutral pH (7.40), or acid pH (4.60). Muscle sections were also stained for reduced nicotinamide adenine dinucleotide tetrazolium reductase and alpha-glycerophosphate dehydrogenase to provide an indication of the relative oxidative and glycolytic capacity of the different fiber types. Type II fibers in mixed fibered muscles were either very sensitive, moderately sensitive, or relatively insensitive to inactivation of the myofibrillar actomyosin ATPase after acid preincubation. These fibers were identified as type IIA1, IIA2, and IIA3, respectively. The myofibrillar actomyosin ATPase of the type I fibers of these muscles, with the exception of those in mouse muscle, was activated by pretreatment with acid. A separation of animal species was possible based on the stability of the IIA1 fibers to inclusion of Cu²⁺ in the preincubation medium. For one group of animals (rat, mouse, monkey, man, dog, rabbit, and cow), a reciprocal relationship existed between lability to acid and stability to Cu²⁺ for type IIA1 and IIA3 fibers, respectively. For the second group of animals (horse, ass, and cat) there was a parallel relationship between lability or stability of the type IIA1 and IIA3 fibers to pretreatment with either acid or Cu²⁺ Visiting scholar from the Laboratory of Biomechanics and Physiology, College of General Education, Yamaguchi University, Yamaguchi 753, JapanSupported in part by Washington State Equine Research Program grant #105 3925 0042     

Evidence for isofunctional enzymes in the degradation of phenol, m- and p-toluate, and p-cresol via catechol meta-cleavage pathways in Alcaligenes eutrophus.

A study of the degradation of phenol, p-cresol, and m- and p-toluate by Alcaligenes eutrophus 345 has provided evidence that these compounds are metabolized via separate catechol meta-cleavage pathways. Analysis of the enzymes synthesized by wild-type and mutant strains and by strains cured of the plasmid pRA1000, which encodes m- and p-toluate degradation, indicated that two or more isofunctional enzymes mediated several steps in the pathway. The formation of three catechol 2,3-oxygenases and two 2-hydroxymuconic semialdehyde hydrolases was indicated from an examination of the ratio of the specific activities of these enzymes against various substrates. Evidence for two 2-hydroxymuconic semialdehyde dehydrogenases, two 4-oxalocrotonate isomerases and decarboxylases, and three 2-ketopent-4-enoate hydratases was derived from the induction of these enzymes under different growth conditions. Each activity was detected when the wild type was grown in the presence of m-toluate, but not when grown with phenol (except for a hydratase) or p-cresol, whereas in strains cured of pRA1000, growth with phenol or p-cresol, but not with m-toluate, induced these enzymes. Hydroxylation of phenol and p-cresol appears to be mediated by the same enzyme.     

Pseudomonas putida Mutants Defective in the Metabolism of the Products of meta Fission of Catechol and Its Methyl Analogues

A selection procedure is described which was used to isolate mutants of Pseudomonas putida strain U in the following enzymes of the meta-fission pathway of phenol and cresols: hydrolase, tautomerase, and decarboxylase. Strains deficient in the hydrolase are unable to use either o- or m-cresol as a sole carbon source and were shown to accumulate 2-hydroxy-6-keto-2,4-heptadienoate when incubated in the presence of o- or m-cresol. When 2-hydroxymuconic semialdehyde (plus nicotinamide adenine dinucleotide, oxidized form) was metabolized by phenol-induced extracts of tautomerase-deficient strains, the enol tautomer of 4-oxalocrotonate accumulated and was then converted slowly to the keto tautomer by a nonenzymatic reaction. Phenol-induced extracts of decarboxylase-deficient strains accumulated the keto tautomer of 4-oxalocrotonate from 2-hydroxymuconic semialdehyde (plus nicotinamide adenine dinucleotide, oxidized form). Strains with an inactive decarboxylase are unable to completely metabolize either phenol or p-cresol. Tautomerase-defective strains are unable to grow with p-cresol as the sole carbon source and grow only very slowly on phenol.     

The rodent non-genotoxic hepatocarcinogen Nafenopin and EGF alter the mitosis/apoptosis balance promoting hepatoma cell clonal growth

The responses of a series of rat hepatoma cell lines (FaO, HTC, RH1) to the rodent non-genotoxic hepatocarcinogen and per-oxisome proliferator (PP) Nafenopin were studied to determine if this PP acts with EGF, a naturally occurring liver growth regulator, to perturb the balance between mitosis and apoptosis. EGF enhanced the growth of FaO cells (well differentiated) and HTC cells (intermediate differentiation) but not of the poorly differentiated RH1 cell line. Nafenopin also increased FaO cell growth but, surprisingly, retarded the growth of both HTC and RH1 cells. Since population expansion kinetics result from mitosis and death, replicative DNA synthesis (RDS) and apoptotic cell death were measured in HTC cells. As expected, EGF elevated RDS and suppressed cell death. However, Nafenopin depressed HTC net population expansion via a suppression of cell death coupled to a more marked inhibition of RDS. This apparent paradox was investigated using soft agar cloning. This revealed sub-populations with differing growth kinetics suggesting selective clonal expansion via an alteration in the balance between mitosis and apoptotic cell death. At later stages, cells are refractory to EGF and Nafenopin, suggesting that genetic changes may have superseded such factor-dependence.     

Cloning and analysis of the polyhydroxyalkanoic acid synthase gene from an Acinetobacter sp.: Evidence that the gene is both plasmid and chromosomally located

Abstract The polyhydroxyalkanoic acid (PHA) synthase gene ( phaC_Ac ) of a species of Acinetobacter isolated from an activated sludge treatment plant was cloned by heterologous complementation in a poly-β-hydroxybutyrate (PHB) negative mutant of Alcaligenes eutrophus . Nucleotide sequence analysis of phaC_Ac revelaed an open reading frame of 1770 bp with potential to encode a 67.7 kDa protein. The deduced amino acid sequence displays high similarity to other PHA synthase proteins. Probing with an internal region of phaC_Ac revealed that the PHA sythase gene may be present in more than one copy and may occur at both plasmid and chromosomal locations in Acinetobacter spp. This is the first organisms for which evidence has been presented to suggest that a gene involved in PHA metabolism is plasmid-encoded. Purification of PHB granules from sucrose gradients identified proteins of 38 kDa, 41 kDa and 64 kDa which may have a role in PHB metabolism.     

Production of poly-\-hydroxybutyrate in Acinetobacter spp. isolated from activated sludge

Two strains of Acinetobacter sp. isolated from activated sludge actively removing phosphate were examined for their abilities to produce poly-\-hydroxybutyrate (PHB). When yield-limited by phosphate, strain RA3117 contained material that stained with Sudan Black, but contained only 0.9% PHB on a dry weight basis. This strain contained no sudanophilic material or PHB when limited by ammonia or sulphate. When strain RA3757 was limited by phosphate, ammonia or sulphate it produced 2.0, 7.8 and 11.5% PHB, respectively, on a dry weight basis. \-Ketothiolase and acetoacetyl-coenzyme A (CoA) reductase were only observed in RA3757 cell-free extracts. \-Ketothiolase was produced both in cells with and without PHB whereas acetoacetyl-CoA reductase was found only in cells accumulating PHB. When RA3757 was grown in ammonia-limiting medium with acetate, butyrate, caproate or ethanol as carbon source, similar levels of PHB were produced. When cells were grown on valerate, RA3757 produced 5.6 poly-\-hydroxyvalerate and 0.9% PHB on a dry weight basis. Correspondence to: J. W. May