Modulating Effects of Core Oligoadenylate on the Voltage-Operated Potassium Channels in Rat Pheochromocytoma Cells

We studied modulating influences of a core oligoadenylate, 2,5-ApApA, on the voltage-operated potassium channels; the agent was injected into cloned cells of the rat pheochromocytoma PC-12. Diffusion of 2,5-ApApA from a micropipette into the cell evoked clear changes in the current-voltage relationships of the integral potassium current; when positive shifts of the membrane potential reached about +20 mV, a saturation phenomenon was observed. The dependence of the probability for open state of the voltage-operated potassium channels on the membrane potential was calculated using normalization of the potassium conductance graphs; it satisfactorily fit Boltzmann's equation. Under the influence of 2,5-ApApA, activation of the potassium channels became more strongly dependent on the voltage. Within the first minutes of the action of core oligoadenylate, the potassium conductance changed by e times at a shift of the membrane potential by 12 mV, while after a stationary level of the 2,5-ApApA effect had been attained (approximately from the 25th min), the same change in the potassium conductance needed only an 8-mV shift. We conclude that 2,5-ApApA-evoked conformation modifications in the structure of the potassium channels in the cells of rat PC-12 pheochromocytoma can result from an increase in the sensitivity of voltage sensors in the above-mentioned channels to changes in the membrane potential.     

Isolation of trace amounts of 3′,4′-dehydro-4′-deoxydothistromin from peanut plants naturally infected with Cercospora personata

Trace amounts of the anthraquinonoid toxic metabolite, 3,4-dehydro-4-deoxydothistromin have been identified for the first time, from peanut tissues naturally infected by Cercospora personata. Characteristic uv spectral and Chromatographic properties of the metabolite and its tetraacetate as well as the mass spectrum of the latter have established its identity.     

Degradation products of myelin proteins in a light CNS subcellular fraction

The fraction floating on 0.32 M sucrose when normal mammalian spinal cord homogenate is submitted to discontinuous density gradient centrifugation is highly enriched in Marchi-positive material. In situ this material is located along paranodal myelin sheath segments. We here show by immunoblotting that degradation products of the myelin-associated glycoprotein (MAG) and of the enzyme 2,3-cyclic nucleotide 3-phosphodiesterase (CNP) is present in the Marchi-positive floating fraction but is not found in the myelin fraction. Since previous biochemical analyses of the floating fraction show a gross composition closely resembling myelin and since metabolic studies show the specific activity of incorporated amino acids to proceed with time from beavier to lighter myelin subfractions the results strongly suggest that normally occurring Marchi-positive bodies represents an intermediate stage in myelin catabolism.     

An efficient synthesis of 3′-amino-3′-deoxythymidine derivatives

An efficient method of reduction of 3-azido-3-deoxythymidine and its 5-protected derivatives to 3-aminothymidine derivatives on a palladium catalyst using ammonium formate as a source of hydrogen was suggested.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 147–150.Original Russian Text Copyright © 2005 by Seregin, Chudinov, Yurkevich, Shvets.     

Which 3-ribofuranosyl-substituted purine 5′-phosphates undergo template-directed oligomerization?

Summary We have studied the oligomerization reactions of the 2-methylimidazolide derivatives of 3-isoisoguanosine 5-phosphate (2) and 3-isoxanthosine 5-phosphate (5) in the presence of a variety of homopolynucleotide templates. In no case did we observe a substantial template-facilitated production of long oligomers. Polyuridylic acid directed the synthesis of low molecular-weight products from both monomers. Polycytidylic acid, polyadenylic acid, polyinosinic acid, and polyguanylic acid were ineffective as templates in the systems that we investigated.     

Membranotropic effects of peptides from the V3 loop of HIV-1

The V3 loop from HIV-1 envelope glycoprotein gp120 is involved in viral entry and determines the cellular tropism and HIV-1-induced cell–cell fusion. Earlier we have shown that V3 loop peptides representing the sequences of syncytia-inducing HIV strains have high membranotropic activity. These peptides caused the lysis of liposomes of various lipid compositions, could fuse negatively charged liposomes and induced hemolysis of erythrocytes. In contrast, peptides mimicking the sequences of non-syncytia-inducing viruses showed no lytic or fusion activities at the same concentrations. Now we have found that the V3 loop synthetic peptides containing the conserved GPGR region, derived from T-lymphotropic strains (BRU and MN), as opposed to peptides containing the GPGQ region, are able to cause a pronounced membrane permeabilization (dissipation of the pH and the of human peripheral blood lymphocytes, erythrocytes and plasma membrane vesicles at micromolar concentrations with a dose-dependent kinetics. Analysis of the secondary structures of the peptides by circular dichroism revealed conformational changes in V3 loop peptides depending on solvent hydrophobicity: from random coil in water to an -helix/-sheet conformation in trifluoroethanol. Such structural changes of the V3 loop together with the membrane insertion of the gp41 N-terminal fusion peptide may promote the formation of the fusion pore during virus–cell fusion.     

The Solution Structure of [d(CGC)r(amamam)d(TTTGCG)]2

The solution structure and hydration of a DNARNA hybrid chimeric duplex [d(CGC)r(a_ma_ma_m)d(TTTGCG)]₂ in which the RNA adenines were substituted by 2-O-methylated riboadenines was determined using two-dimensional NMR, simulated annealing, and restrained molecular dynamics. Only DNA residue 7T in the 2-OMe-RNA DNA junction adopted an O4-endo sugar conformation, while the other DNA residues including 3C in the DNA 2-OMe-RNA junction, adopted C1-exo or C2-endo conformations. The observed NOE intensity of 2-O-methyl group to H1 proton of 4a_m at the DNA 2-OMe-RNA junction is much weaker than those of 5a_m and 6a_m. The 2-O-methyl group of 4a_m was found to orient towards the minor groove in the trans domain while the 2-O- methyl groups of 5a_m and 6a_m were found to be in the gauche (+) domain. In contrast to the long-lived water molecules found close to the RNA adenine H2 and H1 protons and the methyl group of 7T in the RNA-DNA junction of [d(CGC)r(aaa)d(TTTGCG)]₂, there were no long-lived water molecules found in [d(CGC)r(a_ma_ma_m)d(TTTGCG)]₂. This is probably due to the hydrophobic enviroment created by the 2-O-methylated riboadenines in the minor groove or due to the wider minor groove width in the middle of the structure. In addition, the 2-O-methylation of riboadenines in pure chimeric duplex increses its melting temperature from 48.5°C to 51.9°C. The characteristic structural features and hydration patterns of this chimeric duplex provide a molecular basis for further therapeutic applications of DNARNA hybrid and chimeric duplexes with 2-modified RNA residues.     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

Interactions between esterified whey proteins (alpha-lactalbumin and beta-lactoglobulin) and DNA studied by differential spectroscopy

Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated -lactoglobulin and of methylated -lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated -casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210–340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210–340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.     

New Phosphonoformic Acid Derivatives of 3′-Azido-3′-Deoxythymidine

New 5-alkyl ethoxy- and aminocarbonylphosphonates of 3-azido-3-deoxythymidine (AZT) were synthesized, and their antiviral properties in HIV-1-infected cell cultures and stability to chemical hydrolysis were studied. The AZT 5-aminocarbonylphosphonates were shown to be significantly more stable in phosphate buffer (pH 7.2) than the corresponding ethoxycarbonylphosphonates. The therapeutic (selectivity) index of some of the compounds exceeded that of the parent AZT due to their higher antiviral activity.     

New syntheses of α-methyl- and α,α′-dimethylspermine

-Methylspermine and ,-dimethylspermine were synthesized in high overall yields starting from N-(benzyloxycarbonyl)-3-aminobutanol in order to study polyamine biochemistry in vitro and in vivo.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 200–205.Original Russian Text Copyright © 2005 by Grigorenko, Vepsalainen, Jarvinen, Keinanen, Alhonen, Janne, Khomutov.     

Calmodulin genes in zebrafish (revisited)

Calmodulin (CaM), a ubiquitous protein, ancestral in early eukaryotes, regulates a large number of physiologically important functions by activating other proteins, some of them enzymes, usually in response to changes in the local concentration of calcium ions. Invertebrates possess one gene that codes for CaM. Among vertebrates, mammals display three genes that code for a 100% identical CaM molecule, while for zebra fishes etc., a non-mammalian vertebrate, we reported earlier the existence of four such genes. The number of multiple genes coding for a 100% identical CaM molecule present in the zebra fish genome, however, is corrected here, from the four, as previously suggested, to six (alpha, alpha2, beta, beta2, gamma and gamma 2). Identification of each of these genes is readily achieved upon examination of the characteristic 5 and 3 UTRs within their respective mRNAs even though we do not know at present what role these UTRs might play. A scanning of the 3 UTRs for short homology elements among the six genes (and a comparison with the human type I, II, and III CaM 3 UTRs) also suggests that duplication processes for three genes resulted in the formation of six such genes. As they become available, the promoter regions for these six genes should be scanned for possible identification of putative regulatory elements if we are to understand the need for the uniquely rigid evolutionary maintenance of these six genes. A comparison of the promoter regions for the beta and beta 2 genes is presented in this paper. A few common short homologous elements appear to be retained in these generally highly variant two regions, but conclusions about differential expression controls must be delayed until the promoter regions for all the other CaM genes have been examined.     

Apical and lateral shoot apex-specific expression is conferred by promoter of the seed storage protein β-phaseolin gene

A previous analysis with deletion mutants of the native -phaseolin gene demonstrated that removal of a negative element 5 upstream of–107 permitted phaseolin expression in stem cortex and secondary root (Burowet al., 1992). Here we employed the -glucuronidase (GUS) reporter gene to visualize, by histochemical staining, the cell type-specificity of phaseolin expression in stem and root, and to understand further the spatial control of the -phaseolin gene. The 782 bp 5 upstream promoter and its deletion mutants were fused to the GUS gene, and these chimaeric genes were used to transform tobacco. Histochemical staining for GUS activity demonstrated that phaseolin promoters truncated downstream of –227 conferred cell-type specific expression in internal/external phloem and protoxylem of mature stem. Surprisingly, GUS staining was prominent in both apical and lateral shoot apices of plants that contain the full-length –782 promoter and mutant promoters deleted up to –64. GUS expression was extended to all cell types of shoot tips, including epidermis, cortex, vasculature, procambium and pith. Expression in vasculature of petioles was limited to plants with promoters truncated to –106 and –64. The current results are in agreement with our previous findings with the native phaseolin gene: that the major positive element (–295/–228) is sufficient for seed-specific late-temporal expression of the phaseolin gene. We conclude that the 5 upstream sequence of the -phaseolin gene directs spatially- and temporally-controlled gene expression in developing seeds during the reproductive phase, but also confers expression in shoot apices during the vegetative phase of plant development.     

Generation of fertile transplastomic soybean

We describe here the development of a plastid transformation method for soybean, a leguminous plant of major agronomic interest. Chloroplasts from embryogenic tissue of Glycine max have been successfully transformed by bombardment. The transforming DNA carries a spectinomycin resistance gene (aadA) under the control of tobacco plastid regulatory expression elements, flanked by two adjacent soybean plastome sequences allowing its targeted insertion between the trnV gene and the rps12/7 operon. All generated spectinomycin resistant plants were transplastomic and no remaining wild type plastome copies were detected. No spontaneous mutants were obtained. The transformation efficiency is similar to that of tobacco plastids. All transplastomic T0 plants were fertile and T1 progeny was uniformly spectinomycin resistant, showing the stability of the plastid transgene. This is the first report on the generation of fertile transplastomic soybean.     

cDNA cloning and characterization of a 3′/5′-O-methyltransferase for partially methylated flavonols from Chrysosplenium americanum

Enzymatic O-methylation of plant secondary metabolites is an important mechanism for the inactivation of reactive hydroxyl groups and for the modification of their solubility. A cDNA clone (pFOMT3) encoding the gene for the 3/5-O-methylation of partially methylated flavonols was isolated from Chrysosplenium americanum (Saxifragaceae). We used a PCR fragment obtained with degenerate oligonucleotides designed from conserved regions of various O-methyltransferases (OMTs). The pFOMT3 cDNA sequence shows about 67–85% similarity to other plant OMT sequences. The recombinant protein expresses strict specificity for positions 3/5 (meta) of partially methylated flavonols, but does not accept quercetin or caffeic acid for further methylation. Southern blot analysis of the genomic DNA probed with an OMT sequence suggests the presence of a number of related genes in this species, consistent with the multiple enzymatic methylations involved in the biosynthesis of polymethylated flavonols in this plant.     

Continuous production of 5′-ribonucleotides from yeast RNA by hydrolysis with immobilized 5′-phosphodiesterase and 5′-adenylate deaminase

The production of 5-IMP and 5-GMP by enzymatic conversion from RNA using a continuous two packed-bed reactor was investigated. 5-Phosphodiesterase (5PD) and 5-adenylate deaminase (5AD) were immobilized in an acrylic resin to produce derivatives with about 15 U/g of support. The kinetic properties of the enzymes were described by Michaelis-Menten models: no significant differences were found in the K _m value of the free and immobilized 5AD (60 and 20 m, respectively), whereas for 5PD the K _m value was one order of magnitude higher for the immobilized enzyme (4.85 mg RNA/ml), probably due to diffusional limitations. Both enzymes remained stable after 8 h of use in a continuous packed-bed reactor whereas the half lives of the free enzymes were 193 min and 240 min at 40°C and 70°C for 5AD and 5PD, respectively. A procedure is proposed for the design of a continuous two packed-bed column process.F. Olmedo and F. Iturbe are with the Depto. de Alimentos y Biotecnologia, Facultad de Química, UNAM, México 04510, D.F., Mexico. J. Gomez-Hernández is with the Depto. de Biotecnología, UAM-1, Apdo. Postal 55-535, México 09340, D.F., Mexico. A. López-Munguía is with the Instituto de Biotecnología, Apartado Postal 510-3, Cuernavaca, Mor. 62271, Mexico     

Serotoninergic innervation of the alimentary canal of the Colorado potato beetle,Leptinotarsa decemlineata: structural and functional aspects

Phenol (aryl) sulfotransferases (PSTs) provide a conjugative pathway that detoxifies hydroxylated aromatic xenobiotics by esterification with sulfate. Both human and bovine airways have been reported to use this pathway, and in this investigation the bovine system is examined. PST activity in tracheal through fourth generation bronchial mucosal cytosols was 0.1–0.35 nmol/mg protein/min. Activity was generally greater in more distal bronchi and in parenchymal extracts, which contained 0.6–3 nmol/mg/min PST activity. Comparison of the PST activities of bronchial and parenchymal cytosols indicated similar pH activity profiles, although steady state kinetic measurements revealed different K_m values for the acceptor substrate 2-naphthol (13.7 M for bronchial, 31.3 M for parenchymal). Anion exchange chromatography indicated two PST isoforms being expressed in different ratios. Immunoblot analysis with mouse antibovine PST revealed a closely spaced doublet at 32 kDa in both bronchial mucosal and parenchymal cytosolic extracts; however, this doublet was unequally stained in parenchymal extracts. Immunohistochemical analyses revealed faint positive staining of the tracheobronchial epithelium. Greatest immunostaining was observed in the nonciliated secretory epithelial cells of the bronchioles, whereas surrounding smooth muscle, endothelial cells, and alveoli were immunonegative. These results are consistent with the known locations of other detoxification enzymes within the airways.