Preparation of tritiated oostatic peptides for study of radioactivity incorporation in flesh fly Neobellieria bullata.

A series of insect oostatic peptides containing 3,4-dehydroproline in the C-terminal part or inside of the peptide chain was synthesized and tritiated by addition of (3)H2 to double bond of 3,4-dehydroproline residue. (3)H-label was introduced also into tyrosine residue of oostatic tetra- and pentapeptides by isotopic exchange of benzyl beta-hydrogens. In this way, three types of tritiated peptides were prepared, different in the radiolabeled amino acid position: [(3)H] Tyr-Asp-Pro-Ala-OH, H-Tyr-Asp-[(3)H] Pro-Ala-OH, [(3)H] Tyr-Asp-Pro-Ala-Pro-OH, H-Tyr-Asp-[(3)H] Pro-Ala-Pro-OH, H-Tyr-Asp-Pro-Ala-[(3)H] Pro-OH, H-Tyr-Asp-Pro-Ala-Pro(5)-[(3)H] Pro-OH and H-Asp-[(3)H] Pro-OH. These peptides made possible a highly sensitive comparative study on radioactivity incorporation into head and ovaries of the flesh fly Neobellieria bullata, which revealed this process to proceed differently. The reasons of the found differences are discussed.

Heterotrophic fixation of CO(2) in soil.

The occurrence of heterotrophic CO(2) fixation by soil microorganisms was tested in several mineral soils differing in pH and two artificial soils (a mixture of silica sand, alfalfa powder, and nutrient medium inoculated with a soil suspension). Soils were incubated at ambient ( approximately 0.05 vol%) and elevated ( approximately 5 vol%) CO(2) concentrations under aerobic conditions for up to 21 days. CO(2) fixation was detected using either a technique for determining the natural abundance of (13)C or by measuring the distribution of labeled (14)C-CO(2) in soil and bacteria. The effects of elevated CO(2) on microbial biomass (direct counts, chloroform fumigation extraction method), composition of microbial community (phospholipid fatty acids), microbial activity (respiration, dehydrogenase activity), and turnover rate were also measured. Heterotrophic CO(2) fixation was proven in all soils under study, being higher in neutral soils. The main portion of the fixed CO(2) (98-99%) was found in extracellular metabolites while only approximately 1% CO(2) was incorporated into microbial cells. High CO(2) concentration always induced an increase in microbial activity, changes in the composition of the microbial community, and a decrease in microbial turnover. The results suggest that heterotrophic CO(2) fixation could be a widespread process in soils.

Synthesis and application of methyleneoxy pseudodipeptide building blocks in biologically active peptides.

Pseudodipeptides H-Phepsi[CH2O]Phe-OH, H-Tyrpsi[CH2O]Asp-OH and H-Propsi[CH2O]-D-Thr-OH were synthesized using the intramolecular Williamson reaction via substituted morpholin-3-one ring with the nitrogen atom protected with bulky Boc group. This protection and the substituent at C5 position induced the stereospecific alkylation at the C2 position introducing the side chain of the C-terminal amino acid mimetic. In the first pseudodipeptide a quenching of the enolate with benzaldehyde was followed by dehydration and corresponding double bond was hydrogenated with high stereospecific purity. In the other pseudodipeptides, this alkylation was carried out directly by tert-butyl 2-bromoacetate or acetaldehyde. However, in the latter reaction an R configuration of C3 substituent in conjugated lactame ring was determined using a NOE NMR. Consequently, after opening this ring by acidic hydrolysis, the C-terminal part of corresponding pseudodipeptide possessed the side-chain of D-Thr mimetic, contrary to former one. Synthesized pseudodipeptides were introduced into HIV protease inhibitors and into peptides with oostatic activity.

Synthesis and effect of shortened oostatic decapeptide (TMOF) analogs with isosteric structures on reproduction of Neobellieria bullata.

Oligopeptides 2a-2d derived from the oostatic decapeptide (TMOF) sequence, H-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-OH (1a) and containing isosteric structures were synthesized and assayed to determine their effect during reproduction in the flesh fly Neobellieria bullata. The N-terminal linear tetra- and pentapeptides 2a, 2b containing the Pro-psi[CH2O]Ala isosteric linkage affect egg development in 80-90% of ovarioles resulting in some resorbed egg chambers, abnormal yolk deposition, the formation of large eggs with irregular yolk granules and proliferation of follicular epithelium. In comparison with their nonisosteric precursors 1b, 1c they exhibit even more accelerated oostatic activity. However, peptides 2c, 2d containing a Pro-psi[CH2S]Ala isosteric linkage are less active.

Cyclic analogs of insect oostatic peptides: synthesis, biological activity, and NMR study.

Cyclic peptides 2a-2c, derived from the sequence of the C-terminal shortened analogs of the oostatic decapeptide H-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-OH (1a), were synthesized and assayed on their effect in a reproduction of the flesh fly Neobellieria bullata. The cyclization of the N-terminal linear tetra- and pentapeptides 1b and 1c to the cyclotetra- and cyclopentapeptides 2b and 2c decreased the oostatic activity by one order of magnitude. The cyclodecapeptide 2a, which emerged spontaneously during the pentapeptide cyclization, was quite inactive. Comparative 1H and 13C NMR study on a conformation of the cyclopeptides 2a-2c, and their linear precursors 1b and 1c revealed that a space structure of the cyclic analogues 2b and 2c is too restricted to adopt a biological conformation necessary for receptor binding and therefore only minor oostatic activity is observed after their application. The lack of the oostatic activity in the case of the more flexible dimeric analogue 2a is ascribed to the size of its molecule and its overall shape that is not compatible with a receptor binding.

Selection of a pesticide with low environmental impact.

The conditions were analyzed for evaluation of a mean soil biodegradation of a pesticide within a certain region. Different soils from two Czech and two German regions were used for a highly sensitive analysis based on a laboratory incubation of each isolated active soil strain with a radiolabeled pesticide. It was proved for all analyzed soils that the biodegradation activity of the total biomass was caused by only a small part of the present microbial strains. Not only bacteria, but also fungi and yeast have to be taken into consideration in a pesticide biodegradation. The biodegradation products were quantified by radio high-performance liquid chromatography.

Synthesis, radiolabeling and biological activity of peptide oostatic hormone and its analogues.

A series of Pro peptides containing the sequence of the oostatic hormone 3d and its shorter analogues 3a-3c differing in a number of the C-terminal Pro residues was prepared for a study of its effect on oogenesis in Sarcophaga bullata Parker (Diptera). Peptides 3a-3d were synthesized in solution by the fragment condensation of Boc-Tyr-Asp(OtBu)-Pro-Ala-Pro-OH (2f) with Pro oligopeptides H-(Pro)2-5-OtBu. The amino-terminal protected pentapeptide acid 2f was prepared by a stepwise procedure from TFA.H-Ala-Pro-OMe using Boc-Pro-OH, Z-Asp(OtBu)-OSu and Boc-Tyr-OSu. The H(Z)-(Pro)2-5-OtBu oligopeptides 1a-1h were synthesized from Z-Pro-OH and H-Pro-OtBu by a combination of stepwise procedure and fragment condensation. The 125I-labeled molecules of the octapeptide 3b and decapeptide 3d were used for radiotracer distribution studies. Evidence of content of the labeled peptide material in various parts of the insect body (ovaries, head, intestine) is presented. The time distribution of the labeled material in the insect organs was correlated with results of histological analysis of ovaries treated by nonlabeled peptides. The peptides assayed affected processes of egg development in 20-60% of ovarioles. The decapeptide 3d caused changes consisting in some resorbed egg chambers and normal appearance of vitellogenic eggs, whereas the octapeptide 3b caused abnormal yolk deposition and formation of big eggs with irregular yolk granules, proliferation of follicular epithelium in some egg chambers and about the same amount of resorbed egg chambers as decapeptide. These structural differences are complementary to the different values of organ radioactivities.

Putative regulatory sequence in human papillomavirus type 16 E2 open reading frame.

A 114 bp fragment of the human papillomavirus type 16 (HPV16) E2 open reading frame (nt. 3142-3255) containing a putative estrogen responsive element (ERE) was amplified and cloned into pBLCAT2 plasmid in both sense (p159-4) and anti-sense (p164) orientation. The plasmids were transfected into human breast-cancer cell line MCF-7 containing estrogen receptor and the cultures were kept in the presence or absence of beta-estradiol. The chloramphenicol acetyltransferase (CAT) activity was not influenced by estrogen. However, a silencer effect was observed both in cultures transfected with p159-4 and p164 plasmids. We prepared and cloned synthetic fragments containing the putative ERE and failed to prove that the palindrome in the putative ERE was responsible for the silencer activity.

Degradation of a radiolabeled juvenile hormone analog using two insect species.

A synthetic insect juvenile hormone analog (a juvenoid), ethyl N-[2-[4-[[2,2-(ethylenedioxy)cyclohexyl]methyl]phenox]ethyl]carbam ate, which has displayed high biological activity against different insect species and high stability under field conditions, was selected as a biologically active model compound for a study of a juvenile hormone analog degradation. The biologically active compound itself and its three diversely radiolabeled derivatives were applied to the flesh fly (Sarcophaga bullata) or the tsetse fly (Glossina palpalis), respectively. Monitoring of a fate of the applied juvenile hormone analog was carried out using a detection method of the radioactivity microdistribution within the whole insect body in combination with a radio high performance liquid chromatography (radio-HPLC), both of whole-body extracts made in different, but in advance scheduled, time intervals, and of extracts of insect excreta accumulated over an eight-day experiment.

"Mir" radiation dosimetry results during the solar proton events in September-October 1989.

Using data from dosimetry-radiometry system "Liulin" on board of "Mir"-space station the particle flux and doserate during September-October, 1989 has been studied. The orbit of the station was 379 km perigee, 410 km apogee and 51.6 degrees inclination. Special attention has been paid to the flux and doserate changes inside the station after intensive solar proton events (SPE) on 29 of September, 1989. The comparison between the doses before and after the solar flares shows increase of the calculated mean dose per day by factor of 10 to 200. During the SPE on the 29 of September the additional dose was 310 mrad. The results of the experiment are compared with the data for the solar proton fluxes obtained on the GOES-7 satellite.

Space radiation dosimetry with active detections for the scientific program of the second Bulgarian cosmonaut on board the Mir space station.

A dosimetry-radiometry system has been developed at the Space Research Institute of the Bulgarian Academy of Science to measure the fluxes and dose rates on the flight of the second Bulgarian cosmonaut. The dosimetry system is designed for monitoring the different space radiations, such as solar cosmic rays, galactic cosmic rays and trapped particles in the earth radiation belts. The system consists of a battery operated small size detector unit and a "read-write" and telemetry microcomputer unit. The sensitivity of the instrument (3.67 x 10(-8) rad/pulse) permits high resolution measurements of the flux and dose rate along the track of the Mir space station. We report our initial results for the period of the flight between the 7th and 17th June 1988.

Complexity of the genetic control of a structural marker in the CH1 homology region of pig IgG.

The occurrence of two variant peptides P1 and P2 originating in the vicinity of the heavy-light chain disulphide bridge in the CH1 homology region was examined in IgG samples prepared from 36 randomly selected pigs. The peptides labelled with 35S at the half-cystine residues were separated on two-dimensional peptide maps and their ratio was quantitatively evaluated by a non-destructive automated method using semiconductor detectors. Fourteen individuals were found to yield only the peptide P1, the remaining 22 individuals yielded both P1 and P2 with a mean ratio of 69.4: 30.6. A model of genetic determination of the amino acid interchange, serine-leucine, distinguishing the peptides P1 and P2 was suggested. It has been assumed that the gene for one gamma-chain subclass exists only in the form coding for the peptide P1 whereas the gene for another gamma-chain subclass exists in two allelic forms coding for either P1 or P2.

Distribution of cesium in the organism and its effect on the nucleotide metabolism enzymes.

After the intraperitoneal administration of 0.5 mEq 134 CsCI . kg -1 to mice, the maximum cesium level in the kidney's, heart, lungs and liver was found in the first hour (T 1/2 13 h), in the muscles after 8 h (T 1/2 180 h), in the brain after 24 h (T 1/2 140 h) and in the blood after 24 h. Maximum cesium levels were found in the muscles. Rats excreted about 17% of the administered dose in 24 h and 38% in 144 h. Most of the cesium (about 90%) is excreted in the urine. In rats, equalization of the plasma and RBC cesium levels takes longer than 6h. Cesium transport is not entirely dependent on the ATPase system, as shown by the results given by the crude mitochondrial fraction with a reduced potassium content. Among the various univalent ions studied, the effect of cesium on creatine kinase, 5'-nucleotidase, phosphodiesterase and deaminase activity was the smallest.