Genetic polymorphisms of superoxide dismutase in Parkinson's disease.

Oxidative stress reactions may contribute to the pathogenesis of Parkinson's disease (PD). The superoxide dismutases potentially play significant roles in PD by detoxifying superoxide radical. We developed genomic DNA and cDNA-based sequencing assays to identify genetic variants in the copper/zinc superoxide dismutase (SOD1) and manganese superoxide dismutase (SOD2) genes. No genetic variants were detected in the gene encoding SOD1 in DNA from 45 idiopathic PD cases and 49 controls from a population-based case-control study. However, we identified a previously described polymorphism of the mitochondrial targeting sequence consisting of a C47T in exon 2 of SOD2, which results in an alanine to valine substitution. We analyzed this SOD2 variant in DNA from 155 cases and 231 controls from the same study, using an allele-specific fluorogenic 5' nuclease assay, and found no differences in the distributions of allelic frequencies. These results indicate that SOD gene variants do not contribute to PD pathogenesis.

Capillary zone electrophoresis of pharmaceutical peptides.

In peptide analysis, capillary zone electrophoresis (CZE) gives complementary information to that obtained using high-performance liquid chromatography and capillary isotachophoresis. However, a prerequisite for the implementation of CZE for routine quality control purposes is a simple strategy quickly leading to an adequate separation system. A general approach towards the set-up of such systems is presented. A broad range of peptides were used as representative models, viz., adrenocorticotropic hormone (ACTH), the modified ACTH fragment Org 2766, endorphins, cholecystokinin and fragments thereof. In general, the pH and the concentration of the applied electrophoresis buffers are the most important parameters to be considered in the CZE of pharmaceutical peptides.

Capillary electrophoresis of peptides. Analysis of adrenocorticotropic hormone-related fragments.

Capillary electrophoresis can be used successfully to analyse small peptides to give additional information to that obtained using high-performance liquid chromatography (HPLC). The separation of a modified adrenocorticotropic hormone (4-9) fragment (Org 2766) and several of its fragments was investigated using capillary zone electrophoresis. Prediction of migration in aqueous systems using pKa-related data and the migration behaviour using sodium dodecyl sulphate in the buffer are discussed, as is the choice of buffer systems. The electrophoretic patterns are compared with the HPLC separation.

Synthesis and 11C-labelling of the ACTH fragment analogue H-Met(O2)-Glu-His-Phe-D-Lys-Phe-OH (Org 2766) via its homocystine-containing precursor.

The hexapeptide dimer (H-Hcy-Glu-His-Phe-D-Lys-Phe-OH)2 was synthesized using solution methods and characterized. Its conversion into H-Met(O2)-Glu-His-Phe-D-Lys-Phe-OH, Org 2766, was studied on a small scale in as short a time as possible; reduction of the disulfide bond using Na/NH3, reaction with CH3I, oxidation with H2O2 and catalyst and purification by HPLC were carried out starting with 2 mg of the dimer in a total preparation time of approximately 22 min, starting with the addition of CH3I. The preparation of the 11C-labelled analogue was carried out by methylation with 11CH3I. Restrictions imposed by working with carbon-11 will be discussed.

Complementary information from isotachophoresis and high-performance liquid chromatography in peptide analysis.

Reversed-phase high-performance liquid chromatography is a valuable analytical technique to support the synthesis, isolation and purification of peptides, as is illustrated by some critical separations. In addition to this technique, capillary isotachophoresis can give useful information on the purity determination of peptides and on the presence of ionic compounds of a non-peptidic nature. With regard to the latter aspect, isotachophoresis proved to be a suitable technique as a check on the effective removal of salts after preparative high-performance liquid chromatography.

The influence of gamma-irradiation upon the chemical and biological properties of insulin.

Partially purified insulin preparations of bovine and porcine origin, were subjected to gamma-irradiation with doses ranging from 1.0 up to 25 kGy (0.1-2.5 Mrad) at 0 degrees C or ambient temperature. The susceptibility of insulin to the irradiation was determined by chromatography, electrophoresis and assay of the biological activity. The sterilizing effect of the gamma-irradiation was investigated for Bacillus pumilus as well as for artificial mixtures of lactose and several bacilli. It is concluded that the sterilizing dose for the investigated insulins was greater than or equal to 2.2 kGy. At doses up to 25 kGy at 0 degree C no specific radiolytic products were detectable, whereas the biological activity was fully retained. The content of dimers and the content of related peptides appeared to increase gradually with the irradiation dose absorbed. No effects of long-term storage could be demonstrated on biological and chemical properties of insulin after 2.2, 4.5 and 7.5 kGy.

Comparison of the receptor binding properties of nandrolone and testosterone under in vitro and in vivo conditions.

Previous in vitro binding studies with androgen receptors in rat seminal vesicles (Toth M. and Zakar T., J. steroid Biochem. 17 (1982) 653-660) have shown that the difference in the effects of nandrolone (N) and testosterone (T) is caused by the fact that 5 alpha-reduction increases the affinity of T and decreases the affinity of N. We confirmed this result using androgen receptors in rat prostate and intact human MCF-7 cells. We also analysed the receptor binding properties of N, T, dihydronandrolone (DHN) and dihydrotestosterone (DHT) in vivo following a combined 2-h infusion of a physiological dose of [3H]T and the same dose of [3H]N in castrated male rats, which permitted a direct comparison of the accumulation of [3H]N, [3H]T, [3H]DHN and [3H]DHT in different sub-cellular fractions of various tissues. There was a considerable accumulation of radioactivity in the liver, but no retention of active compounds. In the prostate there was a preferential retention of [3H]DHT over [3H] DHN in the receptor fractions whereas in thymus, spleen and muscular tissues [3H]N and [3H]T were retained in equal amounts. The kidney showed a preferential retention of [3H]N over [3H]T. The present results explain the relatively strong effect of nandrolone compared to that of testosterone on target tissues devoid of 5 alpha-reductase activity (e.g. muscular tissue) compared to its relatively weak effect on tissues with a relatively high 5 alpha-reductase content (e.g. prostate).

Application of reversed-phase HPLC in some critical peptide separations.

The application of reversed-phase high performance liquid chromatography in peptide chemistry is illustrated. Its use is described for separation of closely related endorphins, as an optical purity criterion, for rapid determination of the species of origin of insulin preparations, and finally for analysis of small, basic (hydrophilic) peptides.

Analytical isotachophoresis in peptide chemistry. I. Analysis of anions in peptides.

The application of analytical capillary, carrier-free isotachophoresis as a rapid method (approx. 10-15 min per run) for the simultaneous determination of anions present in peptides (as counterion, contaminating ion or peptide anion) is described. Only small amounts of material, approx. 0.1-1.0 mg, are necessary to obtain information on the nature of the anion (qualitative) and of the amount of anion present (quantitative aspect).