Hsc70‐induced Changes in Clathrin‐Auxilin Cage Structure Suggest a Role for Clathrin Light Chains in Cage Disassembly

The molecular chaperone, Hsc70, together with its co‐factor, auxilin, facilitates the ATP‐dependent removal of clathrin during clathrin‐mediated endocytosis in cells. We have used cryo‐electron microscopy to determine the 3D structure of a complex of clathrin, auxilin^401‐910 and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map, we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly .     

Kinetics of degradation of dipalmitoylphosphatidylcholine (DPPC) bilayers as a result of vipoxin phospholipase A2 activity: an atomic force microscopy (AFM) approach

In this paper we used AFM as an analytical tool to visualize the degradation of a phospholipid bilayer undergoing hydrolysis of the vipoxin's PLA(2). We obtained time series images during the degradation process of supported 1, 2-dipalmitoylphosphatidylcholine (DPPC) bilayers and evaluated the occurrence and the growth rate of the bilayer defects. The special resolution of the AFM images allowed us to measure the area and the perimeter length of these defects and to draw conclusions about the kinetics of the enzyme reaction. Moreover, we also report for some unique characteristics discovered during the vipoxin's PLA(2) action. Experimentally for the first time, we observed the appearance and the growth of three-dimensional (3D), crystal-like structures within the formed defects of the degraded bilayer. In an effort to explain their nature, we applied bearing image analysis to estimate the volume of these crystals and we found that their growth rate follows a similar kinetic pattern as the degradation rate of the supported bilayer.     

Modified natural clinoptilolite detoxifies small mammal's organism loaded with lead II: genetic, cell, and physiological effects

The detoxification capacity of the clinoptilolite modification KLS-10-MA used as food additive in small mammals, chronically lead-exposed, was proven for the first time. The modified clinoptilolite was prepared based on natural Bulgarian clinoptilolite deposits. As a powder, it was mechanically mixed at 12.5% concentration with the conventional forage for small rodents. Lead in the form of aqueous solution of Pb(NO(3))(2) was diluted in the drinking water. In the ecotoxicological experiment covering 90 days, imprinting control region laboratory mice were used. They were allocated into four groups: group 1, (control): animals fed with conventional food for small rodents and water; group 2: animals fed with conventional food + clinosorbent KLS-10-MA and water; group 3: animals fed with conventional food and water + Pb(NO(3))(2); and group 4: animals fed with conventional food + KLS-10-MA and water + Pb(NO(3))(2). A group of non-exposed healthy animals was fed with conventional forage mixed with KLS-10-MA to prove eventual toxicity of the sorbent and influence on growth performance. The changes in the chromosome structure, mitotic index, erythrocyte form, erythropoiesis, and body weight gain were recorded. On day 90, the following relations were established: Pb-exposed and clinoptilolite-supplemented mice exhibited 2.3-fold lower chromosome aberrations frequency, 2.5-fold higher mitotic index, and 1.5-fold higher percentage normal erythrocytes 1.3-fold higher body weight compared to Pb-exposed and unsupplemented animals. The obtained data showed that the sorbent is practically non-toxic. The results of the present study encourage a further elaboration of a reliable drug based on the tested substance in the cases of chronic lead intoxication.     

Cryo-electron microscopy of coagulation Factor VIII bound to lipid nanotubes

Factor VIII (FVIII) is a key protein in blood coagulation, deficiency or malfunction of which causes Haemophilia A. The sole cure for this condition is intravenous administration of FVIII, whose membrane-bound structure we have studied by Cryo-electron microscopy and image analysis. Self-assembled lipid nanotubes were optimised to bind FVIII at close to native conditions. The tubes diameter was constant at 30 nm and the lipid bilayer resolved. The FVIII molecules were well defined, forming an 8.5 nm thick outer layer, and appeared to reach the hydrophobic core of the bilayer. The two known FVIII atomic models were superimposed with the averaged 2D protein densities. The insertion of the FVIII within the membrane was evaluated, reaffirming that the membrane-binding C2 or C1-C2 domain(s) fully penetrate the outer leaflet of the lipid layer. The presented results lay the basis for new models of the FVIII overall orientation and membrane-binding mechanism.     

Binding of recombinant human coagulation factor VIII to lipid nanotubes

Cryo-electron microscopy has the power to visualise lipid membranes at the closest to in vivo conditions. The structure of the lipid bilayer can be well resolved and the interactions between lipid-protein and protein-protein molecules followed at the molecular level. We undertook an extended Cryo-electron microscopy study to follow the factor VIII binding to phosphatidylserine containing lipid nanotubes at different lipid composition. Obtaining well ordered tubes is required to define the factor VIII membrane-bound structure. The observed alterations in the arrangement of the protein molecules are indicative for the flexibility of the membrane-bound factor VIII. Understanding the significance of these conformational changes is essential to comprehend the function of factor VIII in coagulation and as a drug for Hemophilia A.     

Effect of UV Radiation and Other Abiotic Stress Factors on DNA of Different Wild Plant Species Grown in Three Successive Seasons in Alpine and Subalpine Regions

Plants in natural ecosystems are exposed to a combination of UV radiation, ionizing radiation (IR) and other abiotic factors. These factors change with the altitude. We investigated DNA alterations of some wild plants of different plant families in natural ecosystems at three altitudes in Rila Mountain, Bulgaria (1500, 1782, and 2925 m above sea level (a.s.l.) exposed to UV radiation, IR and other abiotic stresses, to assess the tolerance of plant species to the changing environmental conditions in three successive growth seasons. For this purpose, physicochemical, cytogenetic, and molecular methods were applied. DNA damage was assessed by micronucleus test and molecular method comet assay adapted and applied by us to wild plant species from Onagraceae, Rosaceae, Boraginaceae, Saxifragaceae, Orobanchaceae, Asteraceae and Poaceae families, growing at three different altitudes. Variability in the DNA sensitivity and the level of tolerance was observed among the plant species in response to combined abiotic factors assessed by induced DNA damage and gross beta activity. The studied representatives of Poaceae were less susceptible than the other studied species at all three altitudes and showed close level of DNA injuries to that of unaffected control plant grown in laboratory conditions. The lower levels of DNA damage of these wild plant species corresponded to their lower ability to accumulate radionuclides. There was a particularly pronounced low level of DNA injuries in the plant species at the highest altitude. The level of DNA damage showed correlation with the values of some abiotic environmental factors. The results would contribute to the elucidation of the extent of adaptation of plant species to the continuously changing environment and would be useful in selecting sensitive herbaceous monitor species for environmental impact assessment at mountain and alpine sites.     

Antimicrobial activity and protective properties of vaginal lactobacilli from healthy Bulgarian women

The role of vaginal Lactobacillus as an efficient barrier against invading pathogens is of considerable interest. The purpose of the present study was to assess in vitro the ability of 20 recently identified vaginal lactobacilli to protect the vagina. In order to evaluate their significance, the antimicrobial, hemagglutination (HA) and aggregation (Agg) activities, as well as acid and hydrogen peroxide (H(2)O(2)) production, were estimated. The cell-free cultures of eight strains showed a stable antimicrobial activity after elimination of the putative effects of lactic acid and H(2)O(2). Three of the isolated vaginal lactobacilli expressed a broad spectrum of anti-bacterial activity including Gram-negative pathogens. Strains with anti-Gardnerella and anti-herpes simplex virus type 2 activities were found. All tested isolates were H(2)O(2) producers, actively acidifying the growth media to pH 3.92+/-0.04, which is presumed to neutralize sexually transmitted infection pathogens. The major part (75%) expressed an HA activity and different Agg phenotypes, estimated as important properties in the competition with invading pathogens and in host defense. These results are encouraging and prompt further research of the characterized active strains and their possible application in prophylaxis of vaginal disorders.     

Sex chromosome pre-reduction in male meiosis of Lethocerus patruelis (St?l, 1854) (Heteroptera,Belostomatidae) with some notes on the distribution?of?the species

The karyotype and meiosis in males of giant water bug Lethocerus patruelis (Heteroptera: Belostomatidae: Lethocerinae) were studied using standard and fluorochrome (CMA₃ and DAPI) staining of chromosomes. The species was shown to have 2n = 22A + 2m + XY where 2m are a pair of microchromosomes. NORs are located in X and Y chromosomes. Within Belostomatidae, Lethocerus patruelis is unique in showing sex chromosome pre-reduction in male meiosis, with the sex chromosomes undergoing reductional division at anaphase I and equational division at anaphase II. Cytogenetic data on the family Belostomatidae are summarized and compared. In addition, the structure of the male internal reproductive organs of Lethocerus patruelis is presented, the contemporary distribution of Lethocerus patruelis in Bulgaria and in the northern Aegean Islands is discussed, and the first information about the breeding and nymphal development of this species in Bulgaria is provided.     

Combinations of CD45 isoforms are crucial for immune function and disease

Expression of the CD45 Ag in hemopoietic cells is essential for normal development and function of lymphocytes, and both mice and humans lacking expression exhibit SCID. Human genetic variants of CD45, the exon 4 C77G and exon 6 A138G alleles, which alter the pattern of CD45 isoform expression, are associated with autoimmune and infectious diseases. We constructed transgenic mice expressing either an altered level or combination of CD45 isoforms. We show that the total level of CD45 expressed is crucial for normal TCR signaling, lymphocyte proliferation, and cytokine production. Most importantly, transgenic lines with a normal level, but altered combinations of CD45 isoforms, CD45(RABC/+) and CD45(RO/+) mice, which mimic variant CD45 expression in C77G and A138G humans, show more rapid onset and increased severity of experimental autoimmune encephalomyelitis. CD45(RO/+) cells produce more TNF-alpha and IFN-gamma. Thus, for the first time, we have shown experimentally that it is the combination of CD45 isoforms that affects immune function and disease.