Oostatic peptides containing D-amino acids: synthesis, oostatic activity, degradation, accumulation in ovaries and NMR study.

Analogs of the H-Tyr-Asp-Pro-Ala-Pro-OH pentapeptide with D-amino acid residues either in differing or in all of the positions of the sequences were prepared and their oostatic potency was compared with that of the parent pentapeptide. The D-amino acid residue containing analogs exhibited an equal or even higher oostatic effect in the flesh fly Neobellieria bullata than the parent peptide. Contrary to the rapid incorporation of radioactivity from the labeled H-Tyr-Asp-[3H]Pro-Ala-Pro-OH pentapeptide into the ovaries of N. bullata in vitro, the radioactivity incorporation from the labeled pentapeptides with either D-aspartic acid or D-alanine was significantly delayed. As compared to the parent pentapeptide, also the degradation of both the D-amino acid-containing analogs mentioned above proceeded at a significantly lower rate. The decreased intake of radioactivity, the lower degradation and finally also the high oostatic effect may be ascribed to the decreased enzymatic degradation of the peptide bonds neighboring the D-amino acid residues in the corresponding peptides. The introduction of the non-coded D: -amino acids thus enhances the oostatic effect in N. bullata owing to the prolonged half-life of the corresponding pentapeptides, which can thus affect more ovarian cells.

Study of oostatic peptide uptake and metabolism in developing ovaries of the flesh fly, Neobellieria bullata.

The uptake and metabolism of the oostatic pentapeptide analogue of trypsin modulating oostatic factor (TMOF), H-Tyr-Asp-Pro-Ala-Pro-OH (5P), in ovaries of Neobellieria bullata (Parker) (Diptera: Sarcophagidae) were analyzed during their developmental stages. During selected stages of yolk deposition, the fate of [3HPro(3)]5P after its in vivo injection was compared to its uptake after in vitro incubation of dissected ovaries. The ovaries were analyzed from 30 s to 180 min after incubation. A detection sensitivity of 60-100 fmol of the labeled 5P was achieved using radio-high performance liquid chromatography. While the uptake of the applied radioactivity strongly depended on the stage of vitellogenesis, especially for the in vitro experiment, degradation of 5P was very quick and independent of whether the label was injected or incubated with the ovaries, regardless of the developmental stage of ovaries. No tracers of 5P were detected at 30 s after applying the labeled 5P in all tests.

Radiochromatographic assay of metabolites of the oostatic peptide labeled in different positions of the peptide chain.

Reversed-phase high-performance liquid radio-chromatography (radio-HPLC) was set up to detect the time course of labeled degradation product formation of the pentapeptide H-Tyr-Asp-Pro-Ala-Pro-OH (5P), which has oostatic effects in different insect species. The detection limit of the system was in the range of 80-150 Bq. To follow formation of the degradation products, three amino acid residues in 5P were independently tritiated: Tyr1, Pro3 and Pro5. Each of the three tritiated peptides was analyzed after incubation with fresh hemolymph or ovaries of Neobellieria bullata. In the incubation mixture, free terminal amino acids and shortened sequences of 5P were identified. A metabolite of tyrosine represented the only exception; it was finally identified as water using degradation of [3H]Tyr by tyrosinase. Metabolic degradation of [3H]Tyr-5P was found to be considerably quicker than that of H-[3H]Tyr-Asp-Pro-Ala-OH (4P). The degradation of 5P was considerably slower in ovaries in comparison to hemolymph.

Biodegradation of juvenoid diastereoisomers: radio-HPLC and MS analysis.

Microbial degradation of two diastereoisomeric forms 2 and 3 of a selected juvenoid (insect juvenile hormone bioanalog), ethyl N-{2-{4-[(2-hydroxycyclohexyl)methyl]phenoxy}ethyl}carbamate was studied and the degradation products analyzed. Degradation experiments were performed using simple modeling under laboratory conditions. A Candida sp. strain T1, isolated from soil, was chosen as a biodegradation species. Radiolabeling of the studied compounds 2 and 3 was used in combination with radio-HPLC and MS analysis to increase the limits of detection, monitoring and isolation of trace quantities of the products of degradation and/or transformation. Resulting from the microbial processes using 2 or 3 as source compounds, three identical products (4-6) of their biodegradation were produced. Compound 2 also afforded two additional products (7, 8). Radio-HPLC analysis and separation, and subsequent MS analysis of the degradation mixtures resulted in identification of the degradation products. The degree and the rate of biodegradation of 2 and 3 were analyzed after 1, 3 and 7 days from the beginning of the experiment.

Radio-high-performance liquid chromatography for ecotoxicity assessment of insect growth regulators.

The described radiochromatographic method permits fast and high-sensitivity monitoring of soil biodegradation products of an insect growth regulator for its environmental risk assessment. We analyzed and compared two diastereoisomers of ethyl N-(2-(4-[(2-hydroxycyclohexyl) methyl]phenoxy)ethyl)carbamate, namely its cis-(1S,2S) isomer JN-W330 and a trans-(1R,2S) isomer JN-W331. Microbial conversion of the cis-isomer to the trans-isomer was proved by mass spectrometry analyzer. Among the chromatographic columns tested, the best separation was found with a 125 mm x 4 mm i.d. column packed with Supersphere 100 RP-C18, 5 microm and an acetonitrile-water gradient. The detection limit for the both isomers was in the range of 120-250 Bq (0.3-0.8 ng) at a concentration of 2 ng/ml with radiometric detection. The calibration curves for standard solutions were linear in the range of 150Bq-150kBq (r = 0.996). The method enabled us to compare the analyzed juvenoids with biologically active oostatic peptides in terms of their environmental safety.

Degradation of juvenile hormone analog by soil microbial isolates.

Juvenoids are efficient pesticides with relatively low toxicity to humans. However, few studies have evaluated the effect of degradation by soil microorganisms on their toxicity. The effects of bacterial, fungal and yeast isolates on aerobic decomposition of ethyl N-[2-[4-(2,2-ethylenedioxy-1-cyclohexylmethyl)phenoxy]ethyl] carbamate during eight weeks were determined. The effect of different concentration of glucose on their degradation activity is also analyzed.

The efficacy and safety of diacerein in the treatment of painful osteoarthritis of the knee: a randomized, multicenter, double-blind, placebo-controlled study with primary end points at two months after the end of a three-month treatment period.

OBJECTIVE: To determine whether the efficacy of diacerein persists at 2 months after the end of a 3-month treatment period, compared with placebo, in patients with painful knee osteoarthritis (OA). METHODS: After a 1-week nonsteroidal antiinflammatory drug washout period, patients received either diacerein or placebo for 3 months, followed by an off-treatment period of 3 months to determine the carryover effects of the drug. Although patients were followed up through month 6, the primary efficacy end point was the percent change from baseline in pain (Western Ontario and McMaster Universities Osteoarthritis Index [WOMAC] A) at month 5 (i.e., 2 months after the end of treatment) compared with placebo. The co-primary efficacy end point was the percent change from baseline in the total WOMAC score, also at month 5 versus placebo. RESULTS: Two hundred three patients were screened, and 168 patients with painful knee OA were randomized. One hundred sixty-five patients were analyzed in an intent-to-treat analysis. At month 5, diacerein showed statistically significant superiority versus placebo as assessed with both the WOMAC A (P < 0.0001) and the total WOMAC (P < 0.0001), demonstrating the carryover effect of the drug. This superiority was already evident from month 2 for pain (P = 0.001) and month 1 for total WOMAC (P = 0.0021). Diacerein was safe and well tolerated. No serious or previously undocumented adverse events were observed during the study. CONCLUSION: This is the first published study of a symptomatic slow-acting OA drug in which the time of assessment of the primary outcome end points was 2 months after the end of a 3-month treatment period. The results show that diacerein is safe and effective for the treatment of knee OA and has a long carryover effect.

Activity and mechanism of action of insect oostatic peptides in flesh fly.

The relationship between structure and activity of insect oostatic decapeptide (Aed-TMOF) analogues in flesh fly was analyzed. The highest oostatic activity was exhibited by the pentapetide and tetrapeptide analogues, H-Tyr-Asp-Pro-Ala-Pro-OH and H-Tyr-Asp-Pro-Ala-OH, respectively. The tetrapeptide, either native or tritiated, was used to study its metabolism in the ovaries and hemolymph and to detect putative binding sites in the flesh fly ovaries and head. A high metabolism of the tetrapeptide with a half-life in the hemolymph and ovaries less than 1h was determined. The initial limiting step in the degradation is tyrosine(1) cleavage. Other degradation products were detected only transiently in low quantities. Using tritiated tetrapeptide, we found that only very low specific binding was detected in the homogenates of ovaries and in the rough membrane preparation in the presence and absence of protease inhibitors.