Crystals of wild-type, mutated, derivatized and complexed 50 S ribosomal subunits from Bacillus stearothermophilus suitable for X-ray analysis

Three-dimensional single crystals of wild-type and mutated 50 S ribosomal subunits from Bacillus stearothermophilus, as well as crystals of reconstituted subunits containing heavy-atom clusters and complexes of these subunits with tRNA and a short nascent polypeptide chain, were grown from polyethylene glycol in the presence of salts at low concentrations. Within experimental error, all these crystals are isomorphous, packed with monoclinic symmetry (C2) in unit cells of a = 300 A, b = 546 A, c = 377 (+/- 1%) A and beta = 112 degrees. Using synchrotron radiation at 85 to 100 K they diffract to 11 A resolution and can be irradiated for hours without disintegrating, so that a complete data set could be collected from a single crystal.     

Application of non-radioactive methods of DNA detection in analysis of human genetic disorders

We have applied two non-radioactive methods for detection of unique sequences in human genome: 1 polymerase chain reaction, 2 hybridization with digoxigenin-deoxyuridine 5-triphosphate labeled probes. With the polymerase chain reaction technique we were able to amplify short segments of genes coding for coagulation factors VIII and IX. Electrophoretical analysis of products of polymerase chain reaction enabled us to detect deletions causing hemophilia A or B. To analyse deletions in dystrophin gene, the most frequent cause of Duchenne muscular dystrophy, we have amplified several different fragments of this gene simultaneously. We have studied restriction fragment length polymorphism closely linked to the cystic fibrosis locus with digoxigenin-deoxyuridine 5 -triphosphate labeled probe p3.11 with sensitivity comparable to methods involving the use of radioisotopes.     

Alpha-smooth muscle actin is expressed in a subset of bone marrow stromal cells in normal and pathological conditions

A series of 217 trephine bone marrow biopsies from adult patients and specimens from 16 fetuses and 5 infants were examined for the presence of stromal myoid cells (MCs) using a monoclonal antibody recognizing alpha-smooth muscle actin. In the normal adult bone marrow, stromal cells did not contain alpha-smooth muscle actin, whereas during fetal life, many alpha-smooth muscle actin-containing MCs were connected with vascular sinusoids in the primitive bone marrow. This cell type reappeared in various characteristic distribution patterns in adult bone marrow during different neoplastic and non-neoplastic conditions including metastatic carcinoma, Hodgkin's disease, multiple myeloma, hairy cell leukemia, acute myeloid leukemia (FAB M4, 5, 7) and chronic myelo-proliferative diseases. In general, the appearance of MCs was associated with a slight to pronounced increase in the deposition of reticulin and collagen fibers. We propose that bone marrow MCs represent a distinct subpopulation of fiber-associated or adventitial reticular cells undergoing cytoskeletal remodeling in response to various stimuli.     

Latent autoantibodies to cardiolipin in the blood serum of healthy subjects

Pooled health blood donors' sera were fractionated by gel filtration and/or ion exchange chromatography on strong anion-exchanger. Measurement of anticardiolipin antibodies levels by ELISA method shows that gel filtration at pH 4.05 and 9.2 (complex-degradation conditions) leads to increase in summary levels while after elution at neutral pH such an effect did not appear. Light increasing of anticardiolipin levels was also noted after fractionation on QAE-Sephadex. Data obtained state that anticardiolipin autoantibodies amount in sera is greater than it can be detected by direct measurement in untreated serum samples. Existence of "hidden" anticardiolipin autoantibodies supposes the hypothesis about alternative way of humoral immunity regulation by blocking anti-self antigens activities with serum biopolymers.     

Electron microscopic study of the cytoskeleton of human podocytes

The ultrastructural study of man's cytoskeleton of podocytes is carried out. Populations of podocytes with two different types of structure of the cytoskeleton in dependence on age (2, 4, 6, 37 and 65 years) is revealed in kidneys. The first type of cytoskeleton of the podocyte is peculiar for children's age and is characterized by branched, high density microfilament network, expressed by system of microtubules and single myofilaments. The intermediate filaments here are either utterly absent or present so feebly they find themselves "disguised" by other strongly developed components of cytoskeleton and revealing them with the help of technique of electron microscope is impossible. In kidneys of adults, and especially of old aged persons podocytes with other type of organization of the cytoskeleton are mainly identified. The distinctive signs of the last are bundle arrangement of microfilaments, plural bundles of intermediate filaments and individual microtubules. This study permits to make a conclusion that during individual development and growing old in kidneys of high animals and man, probably, physiological changes causing morphological reconstruction of cytoskeleton which is accompanied by intensive development of intermediate filaments' system with simultaneous "involution" of microtubules and microfilaments' systems take place.     

Importance of associated microorganisms in the development of acute pneumonia

Sputum samples obtained from 106 patients with acute pneumonia have been studied by the quantitative microbiological method. Different microbial associations have been shown to play an important role in the development of acute pneumonia (67.9%). Microbiological studies have revealed the prevalence of pneumococci in the etiological structure of the disease. Staphylococci, hemophilic bacteria and Neisseria have been found to take part in the development of acute pneumonia in a lesser number of cases and more often occur in combination with pneumococci or form different associations themselves. The so-called "etiological" type of microbial associations has been determined, two or three microbial species being isolated at high concentrations (10(6) and over) and the percentage of patients with such combinations being 59.7 +/- 5.8%. The analysis of the clinical course of the disease has revealed that the presence of different microbial species in patients linked, to a great extent, with the clinical picture of the disease. These data may be used for prescribing adequate etiotropic therapy and for prognostication. Quantitative bacteriological studies carried out in the dynamics of the disease permit timely detection of changes in the microflora of the respiratory tracts, the evaluation of the effectiveness of antibacterial therapy in the process of its implementation and the rational correction of therapeutic measures.     

The action of membranotropic compounds with various structures on the parameters of the phase transition of dimyristoylphosphatidylcholine

The effect of substances of different nature on the thermodynamic characteristics of dimyristoylphosphatidylcholine (DMPC) phase transition by the differential scanning microcalorimetry has been studied. The substances disposed in hydrophobic part of membrane--alpha-tocopherol, ubiquinone Q10, ionol and vitamin K3 cause the decrease of enthalpy and cooperativity of phase transition. The substances which have the side hydrocarbon chain (tocopherol and ubiquinone Q10) compared with ones without it (ionol and vitamin K3) and reduced quinones (Q10 and vitamin K3) compared with the oxidized ones have stronger influence on the enthalpy and cooperativity of transition. The inclusion of the local anesthetic dicaine disposed mainly in the zone of polar heads of phospholipids into DMPC membranes decreases the temperature of phase transition considerably and practically does not change the cooperativity. A possibility to use the method of differential scanning microcalorimetry to estimate the localization of membrane tropic substances within lipid bilayer is under discussion.     

Molecular organization of human plasma alpha 2-macroglobulin and its trypsin complexes. A small-angle x-ray scattering investigation

The molecular organization of human plasma alpha 2-macroglobulin (alpha 2M), and its 1:1 and 1:2 trypsin complexes, have been investigated using the small-angle x-ray scattering method. All the experimental data can be explained by the same basic model, consisting of three oblate-shaped domains arranged in a sandwich-like structure. Each of the larger peripheral domains consists of two parallel elliptic cylinders associated side-by-side, whereas the smaller central domain consists of just one elliptic cylinder. In the native molecule the three domains are separated by regions of low protein density. Upon trypsin binding the dimensions of the four peripheral cylinders remain unchanged, but their positioning in space is reorganized so that the whole molecule becomes more compact. The model thus offers a plausible explanation for the mechanism of inactivating of the protease by entrapping it between the two larger domains. By comparing the shape and dimensions of the total molecule with those determined for the half-molecular fragment, obtained after reducing the intersubunit disulfide bonds, we propose that the fragment consists of just one of the peripheral domains plus half of the central domain. Different projections of the model are consistent with the electron micrographs of alpha 2M given in the literature. The model can also explain many of the physical and chemical properties recorded for alpha 2M and its protease complexes.     

Subunit composition of photosystem I and identification of center X as a [4Fe-4S] iron-sulfur cluster

A photosystem I (PS-I) preparation from barley (Hordeum vulgare L.) containing the reaction center protein P700-chlorophyll a-protein 1 (CP1) and smaller polypeptides with apparent molecular masses of 18, 16, 14, 9.5, 9, 4, and 1.5 kDa has been analyzed with respect to subunit stoichiometry. CP1 contains two homologous subunits with approximate masses of 82 kDa. CP1 and the smaller polypeptides were isolated, and the amino acid composition of each component and of the PS-I preparation was determined. Based on the amino acid composition data and the determined ability of each isolated polypeptide to bind Coomassie Brilliant Blue, the PS-I complex is shown to contain 1 mol of each of the homologous 82-kDa polypeptides as well as 1 mol of the 18-, 16-, 9.5-, and 9-kDa polypeptides for each mol of P700. The total polypeptide mass of the PS-I complex is 209 kDa excluding tryptophan and approximately 220 kDa including tryptophan. The two 82-kDa subunits present/P700 provide cysteine residues for binding only one Fe-S center. In conjunction with the earlier reported binding of four iron and four acid-labile sulfides to CP1/P700 (H?j, P. B., Svendsen, I., Scheller, H. V., and M?ller, B. L. (1987) J. Biol. Chem. 262, 12676-12684), this demonstrates the center X is a [4Fe-4S] cluster and eliminates the possibility of center X being composed of two [2Fe-2S] clusters.