Thyroliberin and its analog lacking hormonal properties stimulate respiratory center activity in the frog Rana temporaria

It has been shown that thyroliberin and its synthetic analogue PR-546 injected into the lymphatic sacs (4.10(-7)-4.10(-9) g/kg) or applied to the medulla oblongata in the bulbar frogs (4.10(-10)-4.10(-12) g/kg) significantly increase the rate of motor respiratory volleys in the hypoglossal nerve. In preparations with the irregular level of the activity of the respiratory center, these drugs enhanced stabilization of the initial level of the rhythm of motor respiratory discharges.     

Interaction of a cysteine-containing peptide with DNA

Cystine peptide dimer (Lys-Gly-Val-Cys-Val-N2H2Dns)2 with S-S bridge was synthesized and its interactions with DNA and synthetic polynucleotides have been studied by optical spectroscopy methods. By recording fluorescent titration curves we have shown that the affinity of the peptide to different synthetic polynucleotides decreases in the order: poly(dG).poly(dC) greater than poly(dA).poly(dT) greater than poly(dGC).poly(dGC). The stability of complexes to increasing concentrations of NaCl diminishes in the same order. The association constant is about 20-fold greater for peptide binding to poly(dG).poly(dC) than to poly(dA).poly(dT). By using circular dichroism and fluorescence measurements we have shown that the peptide competes for the binding sites on DNA with two minor-groove binding antibiotics--distamycin A and sybiromycin. These results have suggested that the peptide also binds in the DNA minor groove. Investigation of the interactions between such peptides and DNA may be useful for constructing ligands with combined specificity to DNA.     

Interaction of trivaline with single-stranded polyribonucleotides]

Binding of tripeptide H-Val3-(NH)2-Dns (TVP) to polyribonucleotides was studied by fluorescence methods, circular and flow linear dichroism, equilibrium dialysis and electron microscopy. It was found that TVP binds to poly(U) in monomer, dimer and tetramer forms with binding constants of about 10(3), 40, 18.10(4) M, respectively. The cooperativity parameter for peptide dimer binding is 2000. The peptide forms tetramer complexes with poly(A), poly(C), poly(G) also. The formation of a complex between the peptide tetramer and nucleic acid is accompanied by a significant increase in the fluorescence intensity. The cooperative binding of TVP dimers to poly(U), poly(A), poly(C) is accompanied by a dramatic decrease in the flexibility of polynucleotide chains. However, it has a small effect (if any) on the flexibility of the poly(G) chain. The observed similarity of thermodynamic, optical and hydrodynamic++ properties of TVP complexes with single-stranded and double-stranded nucleic acids may reflect a similarity in the geometries of peptide complexes with nucleic acids. Electron microscopy studies show that peptide binding to poly(U) and dsDNA leads to compactization of the nucleic acids caused by interaction between the peptide tetramers bound to a nucleic acid. At the first stage of the compactization process the well-organized rod-like particles are formed, each consisting of one or more single-stranded polynucleotide fibers. Increasing the peptide concentration stimulates a side-by-side association and folding of the rods with the formation of macromolecular "leech-like" structures with the thickness of 20-50 nm.     

Stereochemistry and kinetics of interaction with DNA of the antineoplastic antibiotic olivomycin

The kinetics of interaction of antitumor glycoside antibiotic olivomycin with DNA has been investigated. The existence of two relaxation times in the experimental kinetics curves indicates that two types of antibiotic--DNA complex are formed. We have measured the rate constants of association and dissociation processes and determined their temperature dependences. It is suggested, that one of the complex form results from nonspecific interaction between glycoside residues of the antibiotic molecule and sugar-phosphate backbone of DNA whereas the other type of complex exhibits a pronounced specificity for GC-rich regions on DNA. The binding specificity probably results from formation of a H-bond between the antibiotic chromophore ring and guanine 2-amino group. A stereochemical model for olivomycin-DNA complex is proposed. According to this model the antibiotic chromophore and glycoside residues are located in the narrow groove of DNA.     

Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

Deep-level diagnostic value of the rDNA-ITS region

The similarity of certain reported angiosperm rDNA internal transcribed spacer (ITS) region sequences to those of green algae prompted our analysis of the deep-level phylogenetic signal in the highly conserved but short 5.8S and hypervariable ITS2 sequences. We found that 5.8S sequences yield phylogenetic trees similar to but less well supported than those generated by a ca. 10-fold longer alignment from rDNA-18S sequences, as well as independent evidence. We attribute this result to our finding that, compared to 18S, the 5.8S has a higher proportion of sites subject to vary and greater among-site substitution rate homogeneity. We also determined that our phylogenetic results are not likely affected by intramolecular compensatory mutation to maintain RNA secondary structure nor by evident systematic biases in base composition. Despite historical homology, there appears to be no ITS2 primary sequence similarity shared sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that groups, however, share sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that ITS region sequences can diagnose organismal origins and phylogenetic relationships at many phylogenetic levels and provide a useful paradigm for molecular evolutionary study.      

Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.