Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is... more Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is rarely administered. It was therefore of interest to determine whether bleomycin could be directed to cleave RNA or DNA at a specific site by conjugation to a complementary oligonucleotide. A 15 nt MYC complementary oligodeoxynucleotide (HMYC55) bearing a 5' bleomycin A5 (Blm) residue was designed to base-pair with nt 7047-7061 of human MYC mRNA. Reactivity of the Blm-HMYC55 conjugate (and mismatch controls) with a MYC mRNA 30-mer, a MYC DNA 30-mer, and a MYC 2'-O-methyl RNA 30-mer, nt 7041-7070, was analyzed in 100 microM FeNH(4)SO(4), 50 mM beta-mercaptoethanol, 200 mM LiCl, 10 mM Tris-HCl, pH 7.5, at 37 degrees C. Cleavage of the substrate RNA or DNA occurred primarily at the junction of the complementary DNA-target RNA duplex, 18-22 nt from the 5' end of the RNA. Reaction products with lower mobility than the target RNA or DNA also formed. Little or no reaction was observed with more than three mismatches in a Blm-oligodeoxynucleotide conjugate. Neither the short RNA or DNA cleavage fragments nor the low mobility products were observed in the absence of Fe(II), or the presence of excess EDTA. The target RNA was also cleaved efficiently by bleomycin within a hybrid duplex with a preformed single-nucleotide bulge in the RNA strand. New Blm-oligodeoxynucleotide conjugates containing long hexaethylene glycol phosphate based linkers between oligodeoxynucleotide and bleomycin were designed to target this bulge region. These conjugates achieved 8-18% cleavage of the target RNA, depending on the length of the linker. Blm-oligodeoxynucleotide conjugates thus demonstrated sequence specificity and site specificity against RNA and DNA targets.
Promoters of the genes for guanyl-specific ribonucleases, secreted by B. intermedius (binase) and... more Promoters of the genes for guanyl-specific ribonucleases, secreted by B. intermedius (binase) and B. pumilus (Rnase Bp) in phosphate deficient conditions, contain regions similar to appropriate consensus sequences in promoters of the PHO regulated genes of B. subtilis. A number of genes expressed in response to phosphate starvation in B. subtilis are regulated by the two component signal transduction system PhoP-PhoR. Expression of recombinant genes for binase and RNase Bp in B. subtilis strains with mutations in the regulatory protein genes of the PHO regulon was studied. Their expression is strongly regulated by the regulatory proteins of the B. subtilis PHO regulon. z
The properties of new chimeric oligodeoxynucleotides made of short sequences (tetramers, pentamer... more The properties of new chimeric oligodeoxynucleotides made of short sequences (tetramers, pentamers, octamers, and decamers) bridged by hexamethylenediol and hexaethylene glycol linkers have been investigated. These chimeric oligonucleotides showed an improved resistance toward snake venom 3'-phosphodiesterase, with an increased stability when a terminal 3'-3'-internucleotide phosphodiester bond is present. It also has been demonstrated that the hybrid complexes formed by bridged oligonucleotides and a complementary 20-mer RNA are able to elicit the activity of ribonuclease H (RNase H) from Escherichia coli. The substrate properties of chimeric oligonucleotides depend on the length of the oligonucleotide fragments bridged by linkers. Introduction of a nonnucleotide spacer into the native oligonucleotide only slightly hampers the extent of the RNA hydrolysis in the hybrid complexes, whereas a modification of the site of reaction is observed as a possible consequence of the steric disturbance due to the aliphatic linkers. Hence, these new chimeric oligonucleotides, namely, short oligonucleotide fragments bridged by nonnucleotide linkers, demonstrate a favorable combination of exonuclease resistance and high substrate activity toward RNase H. As a consequence, these chimeric oligonucleotides could be proposed as new, promising analogs to be used in the antisense strategy.
A new class of compounds--acridone derivatives--was tested using the direct fluorometric helicase... more A new class of compounds--acridone derivatives--was tested using the direct fluorometric helicase activity assay to determine the inhibitory properties of the derivatives towards the NS3 helicase of Hepatitis C virus (HCV). The compounds were also tested as putative transcription inhibitors of in vitro transcription based on the DNA-dependent T7 RNA polymerase. Most of the acridone derivatives tested were transcription inhibitors; however, only four of them inhibited the NS3 helicase at low concentrations (IC(50) from 3 microM to 20 microM) and were therefore selected for further studies on the mechanism of inhibition. The acridone derivatives probably act via intercalation into double-stranded nucleic acids but they may also interact directly with viral enzymes. Selected carboxamides were tested in the subgenomic HCV replicon system. Two of the compounds: N-(pyridin-4-yl)-amide and N-(pyridin-2-yl)-amide of acridone-4-carboxylic acid are efficient RNA replication inhibitors with selectivity indexes of 19.4 and 40.5, respectively, proving that the acridone derivatives may be regarded as potential antiviral agents.
An ultradian oscillation of protein synthesis was detected by synchronization of metabolic activi... more An ultradian oscillation of protein synthesis was detected by synchronization of metabolic activity in rat hepatocyte cultures. This oscillation occurs in dense cultures in fresh medium, but not in sparse ones. Metabolic synchronization of sparse cultures, however, was initiated by conditioned medium or addition of 0.3-0.5 microm of a mixture of bovine brain gangliosides to fresh culture medium along with either 0.06-0.2 microm GM1 or 0.1-0.2 microm GDIa. GTIb and GDIb did not produce oscillations, nor did human liver ganglioside GM3. High expression of GM1 ganglioside determinants in hepatocytes maintained in the conditioned medium purified polyclonal antibodies to GM1 was coupled with protein synthetic oscillatory activity, i.e. metabolic synchronization. Incubation of dense cultures with GM1-antibodies for 24 h decreased the amplitude of these oscillations. In sparse cultures maintained in fresh medium where protein synthesis showed no oscillatory pattern, GM1 expression was low.
The effect of different non-nucleotide inserts incorporated into oligonucleotide chains on their ... more The effect of different non-nucleotide inserts incorporated into oligonucleotide chains on their hybridization properties was studied by the method of thermal denaturation. Various types of alkyldiols and oligoethylene glycols were used as inserts modifying oligonucleotide backbone. Such modification of oligonucleotides caused the destabilization of their complementary complexes. It was shown that the hybridization properties of the modified oligonucleotides depend on several features of inserts: the type, number, length of insertions, and positions of interrupted dinucleotide steps in oligonucleotide chain.
Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 1998
It was confirmed unambiguously that the anomalously high plateau in the tRNA aminoacylation react... more It was confirmed unambiguously that the anomalously high plateau in the tRNA aminoacylation reaction catalyzed by Thermus thermophilus phenylalanyl-tRNA synthetase is a result of enzymatic synthesis of tRNA bearing two bound phenylalanyl residues (bisphenylalanyl-tRNA). The efficiency of bisphenylalanyl-tRNA formation was shown to be quite low : the second phenylalanyl residue is attached to tRNA approximately 50 times more slowly than the first one. The thermophilic synthetase can aminoacylate twice not only T. thermophilus tRNA Phe but also Escherichia coli tRNA Phe and E. coli tRNA Phe transcript, indicating that the presence of modified nucleotides is not necessary for tRNA Phe overcharging. Bisphenylalanyl-tRNA is stable in acidic solution, but it decomposes in alkaline medium yielding finally tRNA and free phenylalanine. Under these conditions phenylalanine is released from bisphenylalanyl-tRNA with almost the same rate as from monophenylalanyl-tRNA. In the presence of the enzyme the rate of bisphenylalanyl-tRNA deacylation increases. Aminoacylated tRNA Phe isolated from T. thermophilus living cells was observed to contain no detectable bisphenylalanyl-tRNA under normal growth of culture. A possible mechanism of bisphenylalanyl-tRNA synthesis is discussed. ß 1998 Elsevier Science B.V. All rights reserved.
A new strategy of selective DNA target modification was proposed. The using of reactive derivativ... more A new strategy of selective DNA target modification was proposed. The using of reactive derivatives of short oligonucleotides in the presence of flanking effector pair allows one to modify DNA target only when the perfect complementary complex of DNA target and oligonucleotide tandem is formed.
The osmotic water permeability (P os ) of cell membranes isolated from leaves of 40-, 50-and 60-d... more The osmotic water permeability (P os ) of cell membranes isolated from leaves of 40-, 50-and 60-day-old Mesembryanthemum crystallinum plants was estimated by measuring light-scattering kinetics using stopped-flow spectrophotometry. The measurements were performed on the plasma membrane (PM), purified tonoplast (TP), and TP-enriched vesicles. The PM and TP-enriched vesicles were obtained by partitioning the microsomal fraction in an aqueous polymer two-phase system, whereas the purified TP vesicles were prepared by microsomal vesicle flotation on a sucrose cushion. The P os of isolated membranes declined with plant age. The kinetic experiments showed that there was no difference between the P os of the PM and TP isolated from plants of all ages. A 24-h exposure of plants to 400 mM NaCl caused a decline in the P os as well. These findings suggest that, during M. crystallinum transition to CAM, which was induced by plant ageing or salinity, plant osmoregulatory responses included changes in the P os of the leaf-cell membranes. These variations in the P os are discussed in the context of adaptive mechanisms responsible for the maintenance of the water balance in the common ice plant.
Mutations in whirlin, a putative PDZ scaffold protein, have recently been shown to cause deafness... more Mutations in whirlin, a putative PDZ scaffold protein, have recently been shown to cause deafness and short cochlear hair cell stereocilia in whirler mice and recessive deafness (DFNB31) in humans. Through its PDZ domains, whirlin might organize a group of proteins into a functional complex required for stereocilia elongation. Identifying these protein partners will advance our understanding of the development of stereocilia and their function as mechanosensory organelles indispensable for normal hearing.
Această broşură este doar un ghid orientativ şi nicidecum o interpretare exactă a viselor . Sunt ... more Această broşură este doar un ghid orientativ şi nicidecum o interpretare exactă a viselor . Sunt multe cărți oculte de interpretare a viselor, cărți şi interpretări pe care noi nu le agreem ca şi copii ai lui Dumnezeu. Însă deoarece foarte, foarte mulți oameni visează diferite vise, vise care-i marchează, vise care îi răscolesc şi nu ştiu cum să se raporteze la mesajul lor, am considerat ca pe baza Bibliei să oferim o orientare generală asupra posibilității interpretării acestora.
Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is... more Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is rarely administered. It was therefore of interest to determine whether bleomycin could be directed to cleave RNA or DNA at a specific site by conjugation to a complementary oligonucleotide. A 15 nt MYC complementary oligodeoxynucleotide (HMYC55) bearing a 5' bleomycin A5 (Blm) residue was designed to base-pair with nt 7047-7061 of human MYC mRNA. Reactivity of the Blm-HMYC55 conjugate (and mismatch controls) with a MYC mRNA 30-mer, a MYC DNA 30-mer, and a MYC 2'-O-methyl RNA 30-mer, nt 7041-7070, was analyzed in 100 microM FeNH(4)SO(4), 50 mM beta-mercaptoethanol, 200 mM LiCl, 10 mM Tris-HCl, pH 7.5, at 37 degrees C. Cleavage of the substrate RNA or DNA occurred primarily at the junction of the complementary DNA-target RNA duplex, 18-22 nt from the 5' end of the RNA. Reaction products with lower mobility than the target RNA or DNA also formed. Little or no reaction was observed with more than three mismatches in a Blm-oligodeoxynucleotide conjugate. Neither the short RNA or DNA cleavage fragments nor the low mobility products were observed in the absence of Fe(II), or the presence of excess EDTA. The target RNA was also cleaved efficiently by bleomycin within a hybrid duplex with a preformed single-nucleotide bulge in the RNA strand. New Blm-oligodeoxynucleotide conjugates containing long hexaethylene glycol phosphate based linkers between oligodeoxynucleotide and bleomycin were designed to target this bulge region. These conjugates achieved 8-18% cleavage of the target RNA, depending on the length of the linker. Blm-oligodeoxynucleotide conjugates thus demonstrated sequence specificity and site specificity against RNA and DNA targets.
Promoters of the genes for guanyl-specific ribonucleases, secreted by B. intermedius (binase) and... more Promoters of the genes for guanyl-specific ribonucleases, secreted by B. intermedius (binase) and B. pumilus (Rnase Bp) in phosphate deficient conditions, contain regions similar to appropriate consensus sequences in promoters of the PHO regulated genes of B. subtilis. A number of genes expressed in response to phosphate starvation in B. subtilis are regulated by the two component signal transduction system PhoP-PhoR. Expression of recombinant genes for binase and RNase Bp in B. subtilis strains with mutations in the regulatory protein genes of the PHO regulon was studied. Their expression is strongly regulated by the regulatory proteins of the B. subtilis PHO regulon. z
The properties of new chimeric oligodeoxynucleotides made of short sequences (tetramers, pentamer... more The properties of new chimeric oligodeoxynucleotides made of short sequences (tetramers, pentamers, octamers, and decamers) bridged by hexamethylenediol and hexaethylene glycol linkers have been investigated. These chimeric oligonucleotides showed an improved resistance toward snake venom 3'-phosphodiesterase, with an increased stability when a terminal 3'-3'-internucleotide phosphodiester bond is present. It also has been demonstrated that the hybrid complexes formed by bridged oligonucleotides and a complementary 20-mer RNA are able to elicit the activity of ribonuclease H (RNase H) from Escherichia coli. The substrate properties of chimeric oligonucleotides depend on the length of the oligonucleotide fragments bridged by linkers. Introduction of a nonnucleotide spacer into the native oligonucleotide only slightly hampers the extent of the RNA hydrolysis in the hybrid complexes, whereas a modification of the site of reaction is observed as a possible consequence of the steric disturbance due to the aliphatic linkers. Hence, these new chimeric oligonucleotides, namely, short oligonucleotide fragments bridged by nonnucleotide linkers, demonstrate a favorable combination of exonuclease resistance and high substrate activity toward RNase H. As a consequence, these chimeric oligonucleotides could be proposed as new, promising analogs to be used in the antisense strategy.
A new class of compounds--acridone derivatives--was tested using the direct fluorometric helicase... more A new class of compounds--acridone derivatives--was tested using the direct fluorometric helicase activity assay to determine the inhibitory properties of the derivatives towards the NS3 helicase of Hepatitis C virus (HCV). The compounds were also tested as putative transcription inhibitors of in vitro transcription based on the DNA-dependent T7 RNA polymerase. Most of the acridone derivatives tested were transcription inhibitors; however, only four of them inhibited the NS3 helicase at low concentrations (IC(50) from 3 microM to 20 microM) and were therefore selected for further studies on the mechanism of inhibition. The acridone derivatives probably act via intercalation into double-stranded nucleic acids but they may also interact directly with viral enzymes. Selected carboxamides were tested in the subgenomic HCV replicon system. Two of the compounds: N-(pyridin-4-yl)-amide and N-(pyridin-2-yl)-amide of acridone-4-carboxylic acid are efficient RNA replication inhibitors with selectivity indexes of 19.4 and 40.5, respectively, proving that the acridone derivatives may be regarded as potential antiviral agents.
An ultradian oscillation of protein synthesis was detected by synchronization of metabolic activi... more An ultradian oscillation of protein synthesis was detected by synchronization of metabolic activity in rat hepatocyte cultures. This oscillation occurs in dense cultures in fresh medium, but not in sparse ones. Metabolic synchronization of sparse cultures, however, was initiated by conditioned medium or addition of 0.3-0.5 microm of a mixture of bovine brain gangliosides to fresh culture medium along with either 0.06-0.2 microm GM1 or 0.1-0.2 microm GDIa. GTIb and GDIb did not produce oscillations, nor did human liver ganglioside GM3. High expression of GM1 ganglioside determinants in hepatocytes maintained in the conditioned medium purified polyclonal antibodies to GM1 was coupled with protein synthetic oscillatory activity, i.e. metabolic synchronization. Incubation of dense cultures with GM1-antibodies for 24 h decreased the amplitude of these oscillations. In sparse cultures maintained in fresh medium where protein synthesis showed no oscillatory pattern, GM1 expression was low.
The effect of different non-nucleotide inserts incorporated into oligonucleotide chains on their ... more The effect of different non-nucleotide inserts incorporated into oligonucleotide chains on their hybridization properties was studied by the method of thermal denaturation. Various types of alkyldiols and oligoethylene glycols were used as inserts modifying oligonucleotide backbone. Such modification of oligonucleotides caused the destabilization of their complementary complexes. It was shown that the hybridization properties of the modified oligonucleotides depend on several features of inserts: the type, number, length of insertions, and positions of interrupted dinucleotide steps in oligonucleotide chain.
Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 1998
It was confirmed unambiguously that the anomalously high plateau in the tRNA aminoacylation react... more It was confirmed unambiguously that the anomalously high plateau in the tRNA aminoacylation reaction catalyzed by Thermus thermophilus phenylalanyl-tRNA synthetase is a result of enzymatic synthesis of tRNA bearing two bound phenylalanyl residues (bisphenylalanyl-tRNA). The efficiency of bisphenylalanyl-tRNA formation was shown to be quite low : the second phenylalanyl residue is attached to tRNA approximately 50 times more slowly than the first one. The thermophilic synthetase can aminoacylate twice not only T. thermophilus tRNA Phe but also Escherichia coli tRNA Phe and E. coli tRNA Phe transcript, indicating that the presence of modified nucleotides is not necessary for tRNA Phe overcharging. Bisphenylalanyl-tRNA is stable in acidic solution, but it decomposes in alkaline medium yielding finally tRNA and free phenylalanine. Under these conditions phenylalanine is released from bisphenylalanyl-tRNA with almost the same rate as from monophenylalanyl-tRNA. In the presence of the enzyme the rate of bisphenylalanyl-tRNA deacylation increases. Aminoacylated tRNA Phe isolated from T. thermophilus living cells was observed to contain no detectable bisphenylalanyl-tRNA under normal growth of culture. A possible mechanism of bisphenylalanyl-tRNA synthesis is discussed. ß 1998 Elsevier Science B.V. All rights reserved.
A new strategy of selective DNA target modification was proposed. The using of reactive derivativ... more A new strategy of selective DNA target modification was proposed. The using of reactive derivatives of short oligonucleotides in the presence of flanking effector pair allows one to modify DNA target only when the perfect complementary complex of DNA target and oligonucleotide tandem is formed.
The osmotic water permeability (P os ) of cell membranes isolated from leaves of 40-, 50-and 60-d... more The osmotic water permeability (P os ) of cell membranes isolated from leaves of 40-, 50-and 60-day-old Mesembryanthemum crystallinum plants was estimated by measuring light-scattering kinetics using stopped-flow spectrophotometry. The measurements were performed on the plasma membrane (PM), purified tonoplast (TP), and TP-enriched vesicles. The PM and TP-enriched vesicles were obtained by partitioning the microsomal fraction in an aqueous polymer two-phase system, whereas the purified TP vesicles were prepared by microsomal vesicle flotation on a sucrose cushion. The P os of isolated membranes declined with plant age. The kinetic experiments showed that there was no difference between the P os of the PM and TP isolated from plants of all ages. A 24-h exposure of plants to 400 mM NaCl caused a decline in the P os as well. These findings suggest that, during M. crystallinum transition to CAM, which was induced by plant ageing or salinity, plant osmoregulatory responses included changes in the P os of the leaf-cell membranes. These variations in the P os are discussed in the context of adaptive mechanisms responsible for the maintenance of the water balance in the common ice plant.
Mutations in whirlin, a putative PDZ scaffold protein, have recently been shown to cause deafness... more Mutations in whirlin, a putative PDZ scaffold protein, have recently been shown to cause deafness and short cochlear hair cell stereocilia in whirler mice and recessive deafness (DFNB31) in humans. Through its PDZ domains, whirlin might organize a group of proteins into a functional complex required for stereocilia elongation. Identifying these protein partners will advance our understanding of the development of stereocilia and their function as mechanosensory organelles indispensable for normal hearing.
Această broşură este doar un ghid orientativ şi nicidecum o interpretare exactă a viselor . Sunt ... more Această broşură este doar un ghid orientativ şi nicidecum o interpretare exactă a viselor . Sunt multe cărți oculte de interpretare a viselor, cărți şi interpretări pe care noi nu le agreem ca şi copii ai lui Dumnezeu. Însă deoarece foarte, foarte mulți oameni visează diferite vise, vise care-i marchează, vise care îi răscolesc şi nu ştiu cum să se raporteze la mesajul lor, am considerat ca pe baza Bibliei să oferim o orientare generală asupra posibilității interpretării acestora.
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