The effect of x-ray radiation and hypoxia on the proteolytic activity of the normal rat spleen and during tumor growth

The influence of X-radiation on activity of lysosomal enzymes (D, L, H cathepsins) in rat spleen tissue and in inoculated rat sarcoma 45 has been investigated. Intact rats and rats with tumors were subjected to whole-body and sarcoma 45 to local irradiation with doses of 0.155 C/kg and 0.31 C/kg in conditions of breathing gas hypoxic mixture containing 90% of nitrogen and 10% of oxygen (GHM-10). The combined exposure to radiation and GHM-10 was shown to produce a certain protective action (e.g. normalized cathepsin activity) in the spleen. In the tumor tissue the protective effect of GHM-10 was absent.     

Changes induced by sarcolysine in vivo in the composition of DNA-bound lipids in sarcoma 37

The two DNA fractions were isolated from sarcoma 37 by the use of the phenol method: supramolecular complex of DNA (SC DNA, 60%) and "phenol" nuclear matrix DNA (PNM DNA, 40%). The lipids in SC DNA represented of light and tightly bound components, the latter was similar to the lipid composition of PNM DNA. SC DNA contains 20 micrograms of neutral lipids (NL) and 6.5 micrograms of phospholipids (PL), while PNM DNA contains 9.8 micrograms of NL and 3.5 micrograms of PL per mg DNA. SC DNA-bound lipids of sarcoma 37 are deficient in free cholesterol (FC, 13%), but rich in cholesterol esters (CE, 39%) and free fatty acids (FFA, 23%); very rich in cardiolipin (CL, 43%) and phosphatidylethanolamine (PE, 28%), but deficient in phosphatidylcholine (PC, 12%). The tumor contains triglycerides (TG) that is absent in DNA of the normal cells. The injection of sarcolysine (10 micrograms/kg) markedly increased (1.5-3 times) the content of all LN and PL fractions in SC DNA, which was accompanied by both the accumulation of FC, TG, PC and the reduction of the remaining lipid fractions in PNM DNA. It is supposed, that DNA-bound lipids may be the target for the action of sarcolysine.     

Hydrolytic cleavage of DNA by quercetin zinc(II) complex

Quercetin zinc(II) complex was investigated focusing on its hydrolytic activity toward DNA. The complex successfully promotes the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complex with DNA. The rate of conversion of SC to NC is 1.68x10(-4) s(-1) at pH 7.2 in the presence of 100 microM of the complex. The hydrolytic cleavage of DNA by the complex is supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay, and T4 ligase ligation.     

Changes in the composition of DNA-bound nonhistone protein in the thymus and liver of gamma-irradiated rats

A supramolecular DNA complex (SC DNA) and DNA of a phenol nuclear matrix (PNM DNA) were extracted, by the phenol method, from rat thymus and liver 15 min following 10 Gy gamma-irradiation. The method of electrophoresis in polyacrylamide gel was used to study a composition of nonhistone proteins firmly bound to these DNA fractions. Irradiation was shown to induce the occurrence of new proteins and redistribution of proteins between SC DNA and PNN DNA of rat organs.     

Radioprotective action of GHM-10 when administered with dextran sulfate and heparin

A single administration of dextransulfate (40 mg/kg, 1-3 days before irradiation), or a double injection of heparin (250 units/kg, 24 hr and 15 min before irradiation) potentiated a weak radioprotective effect of gas hypoxic mixture (GHM-10) on animals exposed to absolutely lethal doses.     

DNA/HSA interaction and nuclease activity of an iron(III) amphiphilic sulfonated corrole

The DNA binding of amphiphilic iron(III) 2,17‐bis(sulfonato)‐5,10,15‐tris(pentafluorophenyl)corrole complex (Fe–SC) was studied using spectroscopic methods and viscosity measurements. Its nuclease‐like activity was examined by using pBR322 DNA as a target. The interaction of Fe–SC with human serum albumin (HSA) in vitro was also examined using multispectroscopic techniques. Experimental results revealed that Fe–SC binds to ct‐DNA via an outside binding mode with a binding constant of 1.25 × 10⁴ M^–1. This iron corrole also displays good activity during oxidative DNA cleavage by hydrogen peroxide or tert‐butyl hydroperoxide oxidants, and high‐valent (oxo)iron(V,VI) corrole intermediates may play an important role in DNA cleavage. Fe–SC exhibits much stronger binding affinity to site II than site I of HSA, indicating a selective binding tendency to HSA site II. The HSA conformational change induced by Fe–SC was confirmed by UV/Vis and CD spectroscopy. Copyright © 2015 John Wiley & Sons, Ltd.     

Inhibition of human immunodeficiency virus type 1 concerted integration by strand transfer inhibitors which recognize a transient structural intermediate

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) inserts the viral DNA genome into host chromosomes. Here, by native agarose gel electrophoresis, using recombinant IN with a blunt-ended viral DNA substrate, we identified the synaptic complex (SC), a transient early intermediate in the integration pathway. The SC consists of two donor ends juxtaposed by IN noncovalently. The DNA ends within the SC were minimally processed (~15%). In a time-dependent manner, the SC associated with target DNA and progressed to the strand transfer complex (STC), the nucleoprotein product of concerted integration. In the STC, the two viral DNA ends are covalently attached to target and remain associated with IN. The diketo acid inhibitors and their analogs effectively inhibit HIV-1 replication by preventing integration in vivo. Strand transfer inhibitors L-870,810, L-870,812, and L-841,411, at low nM concentrations, effectively inhibited the concerted integration of viral DNA donor in vitro. The inhibitors, in a concentration-dependent manner, bound to IN within the SC and thereby blocked the docking onto target DNA, which thus prevented the formation of the STC. Although 3'-OH recessed donor efficiently formed the STC, reactions proceeding with this substrate exhibited marked resistance to the presence of inhibitor, requiring significantly higher concentrations for effective inhibition of all strand transfer products. These results suggest that binding of inhibitor to the SC occurs prior to, during, or immediately after 3'-OH processing. It follows that the IN-viral DNA complex is "trapped" by the strand transfer inhibitors via a transient intermediate within the cytoplasmic preintegration complex.     

Characterization of MMS-sensitive mutants of Neurospora crassa

Several MMS-sensitive mutants of Neurospora crassa were compared with the wild-type strain for their relative sensitivities to UV, X-ray, and histidine. They were also compared for the frequency of spontaneous mutation at the loci which confer resistance to p-fluorophenylalanine. The mutants were also examined for possible defects in meiotic behavior in homozygous crosses and for any change in the inducible DNA salvage pathways (as indicated by their ability to utilize DNA as the sole phosphate source in the growth medium). On the basis of these characterizations, the present MMS-sensitive mutants of Neurospora can be placed into three groups. The first group includes three mutants, mus-(SC3), mus-(SC13), and mus-(SC28). These are slow growers, insensitive to histidine with no apparent meiotic defects and may have reduced frequency of spontaneous mutation. In addition, their mycelial growth is sensitive to MMS but the conidial viability following MMS, UV or X-ray treatment appears normal or only slightly more sensitive than the wild-type. The second group includes only one mutant, mus-(SC15); its mycelial growth is very sensitive to MMS but the conidial survival following treatment with MMS or UV appears normal; however, the conidial survival following exposure to X-ray is significantly reduced. This mutant shows an increase (more than 10-fold) frequency of spontaneous mutation, but behaves normal like the wild-type with respect to fertility, growth rate and insensitivity to histidine. The third group includes mutants mus-(SC10), mus-(SC25), and mus-(SC29). These mutants are very sensitive to UV, X-rays and MMS and to histadine but have normal growth rates on minimal medium. Mutant mus-(SC10), but not mus-(SC25) and mus-(SC29), has an increased (11 ×) frequency of spontaneous mutation. On the basis of data presented, the MMS sensitivity of the first group of mutants cannot be ascertained to arise from a defect in the DNA repair pathways; instead, it may stem from altered cell permeability or other pleotropic effects of the mus mutations. However, it can be suggested that the second and third group of mus mutants may indeed result from a defect in the DNA repair pathways controlled by the mus genes; this conclusion is based on their cross-sensitivity to a number of DNA-damaging agents such as MMS, UV and/or X-ray, high frequencies of spontaneous mutation (mutator effects) and defects in meiotic behavior.     

Thiol-induced fragmentation of chromosomal DNA]

Supramolecular complexes of DNA (SC DNA) were isolated from loach sperm, loach erythrocytes and hen erythrocytes by the phenol method. By the use of UV-sedimentation on neutral 5-20% sucrose gradient, we studied the effect of 2-mercaptoethanol (ME), dithiothreitol (DTT) and NaBH4 on SC DNA at different pH and long-time incubation (5 and 10 days). It appeared that ME treatment at pH 4.4 fragmented SC DNA of three objects into subunits of size 5 x 10(5)D. Incubation with DTT at pH 8 in the presence of EDTA resulted in subunits of size 1-2 x 10(7)D. However, NaBH4 at pH 8 failed to induce fragmentation of SC DNA. It is shown that ME-induced at pH 4.4 fragmentation is accompanied by a decrease in hyperchromatic effect of subunits, indicating the presence of "sticky" ends. Thus, ME-induced fragmentation of SC DNA results from a "clayting" double-strand break, involving, on an average, 180 bp.     

The length of chromatin loops in meiotic prophase I of warm-blooded vertebrates depends on the DNA compositional organization

In meiotic prophase I, chromatin fibrils attached to the lateral elements of the synaptonemal complexes (SC) form loops. SCAR DNA (synaptonemal complex associated regions of DNA) is a family of genomic DNA tightly associated with the SC and located at the chromatin loop basements. Using the hybridization technique, it was demonstrated that localization of SCAR DNA was evolutionarily conserved in the isochore compositional fractions of the three examined genomes of warm-blooded vertebrates—human, chicken, and golden hamster. The introduction of the concept of the comparative loops (CL) of DNA that form of chromatin attach to SC in the isochore compositional fractions provided the calculation of their length. An inverse proportional relationship between the length of CL DNA and the GC level in the isochore compartments of the studied warm-blooded vertebrate genomes was revealed. An exception was the GCpoorest L1 isochore family. For different compositional isochores of the human and chicken genomes, the number of genes in the CL DNA was evaluated. A model of the formation of GC-rich isochores in vertebrate genomes, according to which there was not only an increase in the GC level but also the elimination of functionally insignificant noncoding DNA regions, as well as joining of isochores decreasing in size, was suggested.     

Isolation and characterization of a cDNA encoding a synaptonemal complex protein.

A gene encoding a 65-kilodalton antigen of the rat synaptonemal complex, SC65, has been cloned by screening rat testis lambda gt11 and lambda ZAPII cDNA expression libraries using polyclonal antibodies against rat synaptonemal complex proteins. The longest open reading frame, initiating at an ATG codon in the cDNA, encodes a protein of 431 amino acids, with a relative molecular mass of 50,000. Immunological analysis locates the SC65 gene product on the synaptonemal complex between the pairing faces of the parallel aligned cores of homologous chromosomes in spermatocytes. Of the rat tissues examined, the SC65 gene is transcribed in testis, brain, and heart at similar levels, and in the liver at a much lower level. The DNA sequence extending about 80 base pairs downstream of the translation termination codon has 93% similarity to the identifier sequence present in the rat genome in 1 x 10(5)-1.5 x 10(5) copies and in cDNA clones of precursors of brain-specific mRNAs. The amino acid sequence encoded by the SC65 gene contains an acidic region in the C-terminal domain of the protein, potential glycosylation sites, and at least one possible phosphorylation site. The protein shows no overall similarity to proteins of known function, nor is there similarity to protein sequences present in GenBank or EMBL data bases.     

Synaptonemal complex analysis in spermatocytes and oocytes of turbot, Scophthalmus maximus (Pisces, Scophthalmidae).

A surface-spreading synaptonemal complex (SC) technique was used to analyze spermatocytes and oocytes of turbot (Scophthalmus maximus) to visualize the process of chromosome synapsis. The total SC length was 205 +/- 12 microm in males and 172 +/- 29 microm in the only female analyzed. A representative SC karyotype of turbot was obtained. Each SC showed lateral elements of equal length. No bivalent exhibiting atypical synaptic behaviour that could be associated with heteromorphic sex chromosomes was observed, either in males or in the female. The DNA content of turbot was evaluated in eight individuals of both sexes by flow cytometry analysis. The 2C mean DNA content of turbot (1.308 +/- 0.009 pg/cell) was among the lowest observed within fishes. No statistical differences in DNA content were revealed between the sexes [Wilcoxon/Mann-Whitney test; P(W(x) = 0.243)]. The SC/DNA content ratio observed in turbot was the highest reported to date in bony fishes (Osteichthyes).     

Molecular anatomy of the Streptococcus pyogenes pSM19035 partition and segrosome complexes

Vancomycin or erythromycin resistance and the stability determinants, δω and ωεζ, of Enterococci and Streptococci plasmids are genetically linked. To unravel the mechanisms that promoted the stable persistence of resistance determinants, the early stages of Streptococcus pyogenes pSM19035 partitioning were biochemically dissected. First, the homodimeric centromere-binding protein, ω2, bound parS DNA to form a short-lived partition complex 1 (PC1). The interaction of PC1 with homodimeric δ [δ2 even in the apo form (Apo-δ2)], significantly stimulated the formation of a long-lived ω2·parS complex (PC2) without spreading into neighbouring DNA sequences. In the ATP·Mg2+ bound form, δ2 bound DNA, without sequence specificity, to form a transient dynamic complex (DC). Second, parS bound ω2 interacted with and promoted δ2 redistribution to co-localize with the PC2, leading to transient segrosome complex (SC, parS·ω2·δ2) formation. Third, δ2, in the SC, interacted with a second SC and promoted formation of a bridging complex (BC). Finally, increasing ω2 concentrations stimulated the ATPase activity of δ2 and the BC was disassembled. We propose that PC, DC, SC and BC formation were dynamic processes and that the molar ω2:δ2 ratio and parS DNA control their temporal and spatial assembly during partition of pSM19035 before cell division.     

酵母双杂交筛选与果蝇C(2)M相互作用的蛋白

同源染色体联会时形成的联会复合体(Synaptonemal complex, SC)是由减数分裂前期Ⅰ多种蛋白质聚集而成的超级复合结构。生殖细胞特异性的核蛋白C(2)M(Crossover suppressor on 2 of Manheim)在染色体上高度聚集可以诱导SC的形成。本文采用酵母双杂交方法,利用C(2)M的诱饵表达载体筛选果蝇cDNA文库,共发现40个可能与C(2)M相互作用的蛋白,包括多种DNA及组蛋白结合蛋白、ATPase、转录调节因子。从筛选的结果中,选取wech和Psf1基因构建了转基因果蝇,并在生殖细胞中进行了基因沉默,结果显示联会复合体的消失受到延迟。上述结果表明Wech和Psf1蛋白可能与C(2)M形成复合物,共同参与联会复合体的形成或其稳定性的维持。     

Immunocytogenetics. II. Human autoantibodies to synaptonemal complexes

Synaptonemal complex (SC) autoantibodies are spontaneously produced by patients with various autoimmune diseases. Immunofluorescence staining of pachytene cells localized the antigen to the central element or transverse filaments of the SC but not to the lateral elements. Specific antibody labeling was confined to the SC at synapsis. Cytochemical tests showed that the SC autoantigen is a basic protein possibly bound to DNA. An unusual characteristic of the SC autoantigen is its species specificity. Patients were found whose sera selectively labeled the SCs of other humans, mice, or newts. The combining of anti SC and anti-kinetochore antibodies provides a new immunocytochemical method for the analysis of SC karyotypes. The optimum conditions for preparation of pachytene cells for visualization by indirect immunofluorescence were determined. The nature and functions of the SC antigen, as well as possible applications of SC-specific autoantibodies in cytogenetics and cell biology, are discussed.     

Meiotic cohesin complexes are essential for the formation of the axial element in mice

Cohesin is a conserved multisubunit protein complex that participates in chromosome segregation, DNA damage repair, chromatin regulation, and synaptonemal complex (SC) formation. Yeast, but not mice, depleted of the cohesin subunit Rec8 are defective in the formation of the axial elements (AEs) of the SC, suggesting that, in mammals, this function is not conserved. In this paper, we show that spermatocytes from mice lacking the two meiosis-specific cohesin subunits RAD21L and REC8 were unable to initiate RAD51- but not DMC1-mediated double-strand break repair, were not able to assemble their AEs, and arrested as early as the leptotene stage of prophase I, demonstrating that cohesin plays an essential role in AE assembly that is conserved from yeast to mammals.     

Isolation and characterization of three monoclonal antibodies to human serum low density lipoprotein apoprotein B

Human serum low density lipoprotein (LDL) is a large (Mr = 2-3 X 10(6), complex particle composed of lipid, protein and carbohydrate. We obtained about 40 mouse spleen-myeloma hybrid cell lines which produce antibodies against LDL. Three of them, SC2, SC3 and SC10, have been cloned and subcloned and their antibody products characterized. They recognize three non-overlapping epitopes in native LDL. Two of them, SC3 and SC10, also are capable of recognizing very low density lipoprotein, (VLDL), whereas SC2 reacts only weakly with VLDL. All three antigenic determinants remain intact, and accessible to antibodies on the LDL protein apo B, prepared by delipidation in a 'non-denaturing' detergent, sodium deoxycholate. However, apo B prepared by organic solvent, ether-ethanol, or sodium dodecyl sulfate (SDS) delipidation, while reacting strongly with SC10, is only poorly recognized by SC2 or SC3. Proteolysis of LDL with trypsin, chymotrypsin, Staphylococcus aureus protease, papain or thermolysin gives, in each case, several non-identical protein fragments which are separable by SDS-polyacrylamide gel electrophoresis. Upon immunoblotting, some of these fragments are now recognized by either SC3 or SC10 but not SC2, some are recognized by both SC3 and SC10, and others are immunologically unreactive. The protein bands that are separated by SDS gel electrophoresis are composed of several non-identical fragments and contain the antigenic sites to differing degrees. Some of the immunologically reactive fragments do not appear to contain carbohydrate. Reduction and carboxymethylation do not destroy the immunoreactivity of LDL toward any of the antibodies; however, modification of lysine residues by citraconic anhydride markedly diminishes the reactivity of LDL toward SC3. It is likely that the two antibodies SC3 and SC10 are directed against different linear amino acid sequences or very stable domains, whereas the third, SC2, is directed against a more fragile conformational domain of apo B.     

Spread of herpes simplex virus to the spinal cord is independent of spread to dorsal root ganglia

Levels of herpes simplex virus 1 (HSV-1) and HSV-2 DNA in dorsal root ganglia (DRG) and spinal cord (SC) were quantified after inoculation of guinea pig genitals and footpads. In genital infection, viral DNA reached SC and DRG simultaneously (at 2 to 3 days after inoculation) but was more abundant in SC than in DRG. After inoculation of footpads, which lack parasympathetic innervation, the viruses spread more efficiently to DRG than to SC. These results show important differences between genital and footpad infections, including independence of spread to DRG and SC, and imply that autonomic neurons may play an important role in the pathogenesis of viral latency after genital inoculation.     

Ultrastructure and composition of the synaptonemal complex in spread and negatively stained spermatocytes of the golden hamster and the albino rat

The ultrastructure of the synaptonemal complex (SC) has been studied in spermatocytes of the golden hamster and the albino rat, spread on liquid surfaces and negatively stained with uranyl acetate. The conditions for a reproducible procedure for spreading the SC have been specified. Spreading on water causes large losses of material from the complex. Spreading on 0.45–0.9% NaCl in water results in good preservation of the SC. Ethanol dehydration introduces irreversible changes in the shape of the chromatin fibers and the components of the complex. Digestions with DNase and proteases, extraction with 2M NaCl and fixation in an aqueous solution of formaldehyde permit analysis of the components of the SC. The lateral elements of the SC are formed by three components: 1) the bulk material which is protease sensitive, DNase resistant, insoluble in 2M NaCl and partially soluble in water; 2) the axial attachment regions of the chromatin fiber; and 3) an axial and linear filament, 65 Å wide, which is DNase sensitive. It is suggested that this linear 65 Å filament contains a single linear DNA molecule to which the chromatin fibers are attached. The central element of the SC is made of fibrillar material, most of which is DNase resistant and protease sensitive. Fibrils 25 Å wide cross the central space and merge with the central element. The cross fibrils and the central element are labile in solutions containing less than 0.45% NaCl. — From the present results and previous data on diplotene axes (Solari, 1970), it is concluded that the lateral elements of the SC of hamster and rat spermatocytes are undivided during pachytene. It is suggested that the singleness of the axes in the lateral elements is based on the presence of a single DNA molecule axially located in the lateral elements, and that the chromatin fibers are symmetrically attached to this DNA molecule.     

Meiosis in Mesostoma Ehrenbergii Ehrenbergii. IV. Recombination Nodules in Spermatocytes and a Test of the Correspondence of Late Recombination Nodules and Chiasmata

Male meiosis in Mesostoma ehrenbergii ehrenbergii (2x = 10) is characterized by extreme restriction of chiasma formation; 3 pairs of chromosomes form bivalents at metaphase I which are associated by single very distally localized chiasma, while two pairs of chromosomes remain as unpaired univalents. Electron microscopical three-dimensional reconstruction analysis of serial sections has been applied to 20 pachytene spermatocyte nuclei. In each nucleus three short stretches of synaptonemal complex (SC) were found, confined to a localized branched lobe of the nucleus, confirming the findings of an earlier study. The majority of reconstructed nuclei show that each of the three SC segments has a single prominent recombination nodule ("late" RN) associated with it. Late RNs in this system therefore show an excellent correspondence with metaphase I chiasmata, in contrast to a previous report. M.e. ehrenbergii is therefore not an exception to the hypothesis that meiotic exchange requires a functional late RN. A few nuclei had two, one or no RNs; these presumably represent nuclei that are not at the stage of maximum RN presence. Although M. e. ehrenbergii shows pronounced chiasma localization at the light microscope level, at the ultrastructural level RNs are widely distributed along the 5-10 microns of SC formed in each bivalent, indicating that genetic exchange are not restricted to particular localized sites but occur at a large number of DNA sequence.