Influence of human B-casomorphin-7 on specific binding of 3H-spiperone to the 5-HT2-receptors of rat brain frontal cortex

Using radio-receptor analysis, it has been demonstrated that human beta-casomorphin-7 (Tyr-Pro-Phe-Val-Glu-Pro-Ile) displaces 3H-spiperone from 5-HT2-receptors of rat brain frontal cortex. IC50 of human beta-casomorphin-7 was 8 microM. These data suggest that one of the mechanisms of neurotropic action of beta-casomorphin-7 is might be associated with its influence on the serotoninergic system.     

Brain-derived heat shock protein 70-peptide complexes induce NK cell-dependent tolerance to experimental autoimmune encephalomyelitis

Heat shock proteins (Hsp) are markedly up-regulated at sites of inflammation during autoimmune diseases like experimental autoimmune encephalomyelitis (EAE). In this study, we show that Hsp70-peptide complexes (pc) isolated from brains of mice with EAE prevented the development of EAE clinically and pathologically when administered before proteolipid protein 139-151 (PLP139-151) immunization. In contrast, pure Hsp70 or Hsp70-pc derived from brains of healthy mice or other inflamed tissue did not modulate the expression of EAE. In animals in which EAE had been suppressed by Hsp70-pc, lymphocytes showed increased cell death in response to PLP139-151 that correlated with elevated IFN-gamma and NO production. Coculture of spleen cells from Hsp70-pc immunized mice with spleen cells from untreated EAE mice, in addition to depletion experiments, showed that NK cells reduced reactivity to PLP139-151. Transfer of NK cells from Hsp70-pc-immunized mice to recipients sensitized for EAE abolished disease development. Thus, we propose that Hsp70 demonstrate the ability to bind to peptides generated during brain inflammation and to induce a regulatory NK cell population that is capable of preventing subsequent autoimmunization for EAE.     

Evenly tritium-labeled peptides and their in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50-150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium-labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a tg scale of biological samples both in vitro and in vivo.     

Gene mapping of 28S and 5S rDNA sites in the spined loach Cobitis taenia (Pisces, Cobitidae) from a diploid population and a diploid–tetraploid population

We compare the chromosomal 28S and 5S rDNA patterns of the spined loach C. taenia (2n = 48) from an exclusively diploid population and from a diploid–polyploid population using 28S and 5S rDNA probe preparation and labelling, and fluorescence in situ hybridization (FISH). The 5S rDNA was located in two to three chromosome pairs, and separated from the 28S loci for the males and one female (F1) from the diploid population. Loaches from a diploid–polyploid population, and one female (F2) from the diploid population were characterized by at least one chromosome pair with 5S and 28S overlapping signals. The fishes differed mainly in their number of 28S rDNA loci, located on 3–6 chromosomes. All individuals from both populations were characterized by one acrocentric chromosome bearing a 28S rDNA signal on the telomeres of its long arm. The number of major ribosomal DNA in the karyotype of C. taenia by FISH was always higher than the number of Ag-NORs. Our data confirm the extensive polymorphism of NORs in both populations, as already has been observed in closely related Cobitis species, and less polymorphic 5S rDNA pattern. However, this preliminary result highlights the need for a wider scale study.     

Inhibitory antibodies to human angiotensin-converting enzyme: fine epitope mapping and mechanism of action

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the 3D-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E.mAb and E.S.mAb complexes, respectively; however, only complex E.S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.     

Evenly tritium labeled peptides in study of peptide in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50–150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a μg scale of biological samples both in vitro and in vivo.     

Replication-associated activities of purified human papillomavirus type 11 E1 helicase

Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.     

Fluoxetine results in misleading conclusions on fish behavior

Fluoxetine is an antidepressant medicine causing relaxation and mood improvement in people, with silencing certain personality traits in some cases. The question arise if such phenomena can be observed in nontarget organisms such as fish. Fluoxetine affects fishes behavior; however, it is not known if the medicine affects its “personality.” This study aimed to evaluate the reaction of the invasive Neogobius fluviatilis and native Gobio gobio individuals to fluoxetine at environmental concentration of 360 ng/L. We prepared three variants of the experiments: (a) behavioral trials with unexposed fishes, (b) behavioral trials with the same fishes after 21 days of fluoxetine exposure, and (c) behavioral trials with the same fishes after 21‐day depuration period, that is, without fluoxetine. The fishes reaction time (RT), that is, difference in time spent on reaching food with and without the necessity of overcoming the obstacle, was analyzed. Additionally, the personality, bold or shy, traits of each fish individual, was assigned. The results indicated that environmental concentrations of the antidepressant influenced RT. The average RT of the fishes cultured with fluoxetine was by 7‐min shorter in comparison with the nonexposed control. Share of individuals exposed to fluoxetine assigned as bold raised to 71.4% in comparison with 46.4% in nonexposed control. This sheds new light on wild fishes behavior caught from freshwater. Environmental concentrations of the antidepressant influenced the time of fishes reaction and share individuals assigned as bold. Moreover, 21‐day recovery lasting might be not enough to get fluoxetine effect on fishes.     

Macrozoobenthos of two Antarctic glacial coves: a comparison with non-disturbed bottom areas

There are only few studies on shallow Antarctic benthic communities associated with habitats affected by intense mineral sedimentation inflow. The analysis of macrofaunal communities associated with two shallow, isolated glacial coves was performed in Admiralty Bay (King George Island) and compared with non-disturbed sites. Multivariate analyses (hierarchical classification, nMDS) clearly separated glacial cove communities (two assemblages) from the sites situated outside both basins (two assemblages). The community influenced by the streamflow of glacial discharge of meltwater situated in the area with sandy–clay–silt sediments had a very low species richness, diversity and abundance. It was dominated by eurytopic, motile deposit feeding polychaetes such as Mesospio moorei, Tharyx cincinnatus and Leitoscoloplos kerguelensis as well as the bivalve Yoldia eightsi. The second glacial community of the area located at a grater distance from the outlet of the stream was characterized by sandy–clay–silt and clay–silt deposits and showed also a low diversity and species richness. The most abundant here were peracarid crustaceans, with the dominant opportunistic feeder Cheirimedon femoratus. Community from the non-disturbed area with silty–clay–sand, and silty–sand sediments had higher species richness and diversity. The assemblage of fauna from the sandy bottom has values of those two indexes similar to those found in the disturbed areas.     

Analysis of mercury levels in historical bone material from syphilitic subjects--pilot studies (short report)

The aim of the present work is to study the remains of seven individuals with typical symptoms of tertiary syphilis in terms of mercury content in bones, thereby verifying whether they were subjected to treatment and, if they were, how long their organisms were exposed to toxic mercury fumes. Mercury was used, mainly in the Middle Ages and in the early modern period, as a preventive measure in case of individuals suffering from syphilis, a venereal disease, and also leprosy. Syphilitic patients treated this way should demonstrate increased concentration of mercury in their bones. The skeletons studied in the present work originate from various archaeological sites in southern and north-central Poland. The analyses concerned individuals with diagnosed syphilis as well as healthy individuals who constituted the control group. The analyses were performed by the LA-ICP-MS technique, with the use of laser Nd: YAG, Macro, 266 nm, New Wave, USA, coupled with Spectrometer Elan DRC-e Perkin Elmer, USA. The content analysis of the studied bone material revealed with high probability that the contact method of mercurial treatment was used only in the case of two women from north-central Poland, deceased at the turn of the 15th century at the earliest.