Microbial degradation of organophosphorus pesticides using whole cells and enzyme extracts.

The use of microbial phosphotriesterases in the degradation of organophosphorus compounds employed as pesticides, plasticizers and petroleum additives is a sustainable alternative for bioremediation of water and soils, decontamination of particular foods and as poisoning antidote. Whole cells of six wild type microorganisms-Streptomyces phaeochromogenes, Streptomyces setonii, Nocardia corynebacterioides, Nocardia asteroides and two Arthrobacter oxydans-selected in our lab as phosphotriesterase sources, were further tested as biocatalysts in the hydrolysis of paraoxon, methyl paraoxon, methyl parathion, coroxon, coumaphos, dichlorvos and chlorpyrifos, highlighting 98% conversion of chlorpyrifos into its hydrolysis products using whole cells of S. phaeochromogenes at pH 8 and 40 °C. Immobilized whole cells and enzyme extracts were also assessed, observing as a general trend, that there is no significant variation in hydrolytic activity between them. These results suggest that according to the circumstances, immobilized whole cells (avoiding cellular disruption and centrifugation) or enzyme extracts (which can be handled more easily) could be used.

Screening of catalytically active microorganisms for the synthesis of 6-modified purine nucleosides.

Modified nucleosides can be prepared by microbial transglycosylation from cheaper nucleoside precursors using free or immobilised whole cells. An efficient screening method to find transglycosylation activity in microorganisms was developed for the synthesis of 6-modified purine nucleosides, such as 6-chloro-, 6-methoxy-, 6-iodo- and 6-mercaptopurine ribonucleoside. Out of 100 microorganisms screened, Bacillus stearothermophilus ATCC 12980 was the best for this purpose.

Design and applications of modified oligonucleotides.

Oligonucleotides have a wide range of applications in fields such as biotechnology, molecular biology, diagnosis and therapy. However, the spectrum of uses can be broadened by introducing chemical modifications into their structures. The most prolific field in the search for new oligonucleotide analogs is the antisense strategy, where chemical modifications confer appropriate characteristics such as hybridization, resistance to nucleases, cellular uptake, selectivity and, basically, good pharmacokinetic and pharmacodynamic properties. Combinatorial technology is another research area where oligonucleotides and their analogs are extensively employed. Aptamers, new catalytic ribozymes and deoxyribozymes are RNA or DNA molecules individualized from a randomly synthesized library on the basis of a particular property. They are identified by repeated cycles of selection and amplification, using PCR technologies. Modified nucleotides can be introduced either during the amplification procedure or after selection.

Design and applications of modified oligonucleotides

Oligonucleotides have a wide range of applications in fields such as biotechnology, molecular biology, diagnosis and therapy. However, the spectrum of uses can be broadened by introducing chemical modifications into their structures. The most prolific field in the search for new oligonucleotide analogs is the antisense strategy, where chemical modifications confer appropriate characteristics such as hybridization, resistance to nucleases, cellular uptake, selectivity and, basically, good pharmacokinetic and pharmacodynamic properties. Combinatorial technology is another research area where oligonucleotides and their analogs are extensively employed. Aptamers, new catalytic ribozymes and deoxyribozymes are RNA or DNA molecules individualized from a randomly synthesized library on the basis of a particular property. They are identified by repeated cycles of selection and amplification, using PCR technologies. Modified nucleotides can be introduced either during the amplification procedure or after selection

Trypanosoma cruzi arginine kinase characterization and cloning. A novel energetic pathway in protozoan parasites.

This work contains the first description of a guanidino kinase in a flagellar unicellular parasite. The enzyme phosphorylates L-arginine and was characterized in preparations from Trypanosoma cruzi, the ethiological agent of Chagas' disease. The activity requires ATP and a divalent cation. Under standard assay conditions (1 mM L-arginine), the presence of 5-fold higher concentrations of canavanine or histidine produced a greater than 50% enzyme inhibition. The base sequence of this enzyme revealed an open reading frame of 357 amino acids and a molecular weight of 40,201. The amino acid sequence shows all of the characteristic consensus blocks of the ATP:guanidino phosphotransferase family and a putative "actinin-type" actin-binding domain. The highest amino acid identities of the T. cruzi sequence, about 70%, were with arginine kinases from Arthropoda. Southern and chromosome blots revealed that the kinase is encoded by a single-copy gene. Moreover, Northern blot analysis showed an mRNA subpopulation of about 2.0 kilobases, and Western blotting of T. cruzi-soluble polypeptides revealed a 40-kDa band. The finding in the parasite of a phosphagen and its biosynthetic pathway, which are totally different from those in mammalian host tissues, points out this arginine kinase as a possible chemotherapy target for Chagas' disease.

Involvement of insulin-like growth factors-I and -II and their receptors in medroxyprogesterone acetate-induced growth of mouse mammary adenocarcinomas.

The role of the insulin-like growth factors (IGFs) system was investigated in hormone-dependent (HD) and -independent (HI) in vivo lines of the medroxyprogesterone acetate (MPA)-induced mammary tumor model in Balb/c mice. IGF-II protein and message showed a three- to four-fold increase in HD lines growing in MPA-treated mice, as compared with HD tumors growing in untreated mice. Progression to a hormone-independent phenotype in all these lines was accompanied by a high constitutive expression of IGF-II. Similar IGF-I mRNA levels were detected in HD and HI lines. Both IGF-I and -II messages arose from the malignant epithelial cells, as shown by in situ hybridization studies. A significant decrease in Man-6P/type II IGF-R content was detected in HD tumors growing in MPA-treated mice as compared with HD lines growing in untreated mice. On the other hand, in HI tumors, notwithstanding high IGF-II synthesis, the levels of Man-6P/type II IGF-R remain high. Competitive inhibition and affinity labeling studies showed an almost exclusive binding of IGF-II to Man-6P/type II IGF-R on tumor membranes. The involvement of IGFs in the growth of epithelial primary cultures of the C4-HD line was evaluated. Exogenous IGF-I potentiated MPA stimulatory effect at concentrations of 50-100 ng/ml. Treatment of C4-HD cells with antisense oligodeoxynucleotides (ASODNs) to type I IGF-R and to IGF-II RNA resulted in a dose-dependent inhibition of MPA-mediated cell proliferation. The inhibition caused by IGF-II ASODNs could not be overcome by the addition of IGF-II up to 150 ng/ml. ASODNs to type I IGF-R at 40 microg/ml reduced by 75% the number of type I IGF-R; ASODNs to IGF-II at 1 microM decreased by 83% the levels of IGF-II protein. Our results provide support for the involvement of IGF-I and -II in MPA-induced mammary tumor growth by autocrine pathways.

[Synthetic oligonucleotides and their application in gene therapy]. / Oligonucleótidos sintéticos y su aplicación en terapia génica.

Synthetic oligonucleotides are useful for research in molecular biology, for clinical diagnosis and for the development of new therapeutic agents. In fact, their application to gene therapy has given rise to a new field termed antisense, enabling the synthesis of a new generation of drugs. The procedure is based on coupling an oligonucleotide with mRNA to yield the protein whose production by the host is to be prevented, and thus through diverse pathways inhibiting gene expression. In medicine, a start has been made by applying antisense techniques in human drug trials, particularly as antiviral and antileukemic agents.

Oligonucleótidos sintéticos y su aplicación en terapia génica. / [Synthetic oligonucleotides and their application in gene therapy]

Synthetic oligonucleotides are useful for research in molecular biology, for clinical diagnosis and for the development of new therapeutic agents. In fact, their application to gene therapy has given rise to a new field termed antisense, enabling the synthesis of a new generation of drugs. The procedure is based on coupling an oligonucleotide with mRNA to yield the protein whose production by the host is to be prevented, and thus through diverse pathways inhibiting gene expression. In medicine, a start has been made by applying antisense techniques in human drug trials, particularly as antiviral and antileukemic agents.