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1.
The externally controlled cleavage of covalently linked prodrugs, proteins, or solid-phase formulation vehicles offers potential advantages for controlled drug or gene delivery. A series of o-nitrobenzyl ester compounds (1-8) were synthesized to allow a systematic study of photolability. The o-nitrobenzyl ester was strictly required for photolability, while imido esters were not photolabile. The degradation kinetics of 1-o-phenylethyl ester was an order of magnitude faster than that of o-nitrobenzyl ester. Tosylate, phosphate, and benzoate derivatives of 1-o-nitrophenylethyl displayed similar photolability (>80% decomposition within 10 min at 3.5 mW/cm2 at 365 nm). O-o-Nitrobenzyl O',O'-diethyl phosphate displayed the fastest decomposition at photoirradiation condition (3.5 mW/cm2, 365 nm) suitable for biological systems. We report the synthesis and photo-decomposition of 1-o-nitrophenylethyl derivatives amenable for the creation of photolabile prodrugs or formulation particles for drug depots, DNA condensation, or tissue engineering applications. 相似文献
2.
Centrifugal elutriation was used to separate 9L rat brain tumour cells into fractions enriched in the G1, S, or G2/M phases of the cell cycle. Cells enriched in early G1, phase were recultured, grown in synchrony, and harvested periodically for analysis of their DNA distribution and polyamine content. Mathematical analysis of the DNA distributions indicated that excellent synchrony was obtained with low dissersion throughout the cell cycle. Polyamine accumulation began at the time of seeding, and intracellular levels of putrescine, spermidine, and spermine increased continuously during the cell cycle. In cells in the G2/M phase of the cell cycle, putrescine and spermidine levels were twice as high as in cells in the G1, phase. DNA distribution and polyamine levels were also analysed in cells taken directly from the various elutriation fractions enriched in G1, S, or G2/M. Because we did not obtain pure S or G2/M populations by elutriation or by harvesting synchronized cells, a mathematical procedure—which assumed that the measured polyamine levels for any population were linearly related to the fraction of cells in the G1, S, and G2/M phases times the polyamine levels in these phases and that polyamine levels did not vary within these phases—was used to estimate ‘true’ phase-specific polyamine levels (levels to be expected if perfect synchrony were achieved). Estimated ‘true’ phase-specific polyamine levels calculated from the data obtained from cells either sorted by elutriation or obtained from synchronously growing cultures were very similar. 相似文献
3.
HeLa cells were synchronized for S-phase DNA synthesis by the double thymidine-block procedure. A comparison was made of the polyamine content and S-phase DNA synthesis in cells from control cultures and cultures to which an inhibitor of polyamine biosynthesis, alpha-difluoromethylornithine, was added to the synchronization medium. Control cells showed a peak of synchronous DNA synthesis at 3 h and a maximum concentration of polyamines at 6-9 h after release of the second thymidine block. Cells from cultures containing the inhibitor were severely inhibited in the synthesis of DNA and contained no putrescine and only traces of spermidine while the spermine content was lowered by as much as 80%. Supplementation of cultures containing alpha-difluoromethylornithine with a polyamine, at the time of release of the second thymidine block, replenished the intracellular pool of the administered polyamine and partially restored S-phase DNA synthesis, with a lag of 3-6 h. Almost complete restoration of DNA synthesis in cells depleted of polyamines was achieved by the addition of a polyamine to cultures at least 10 h before release of the second thymidine block. The lag in initiation of synchronous S-phase DNA synthesis was eliminated in these cells. It is concluded that reversal by polyamines of the deficiency in S-phase DNA synthesis, in polyamine-depleted HeLa cells, is a time-dependent process indicative of the necessity for the replenishment of replication factors or their organization into an active replication complex. 相似文献
4.
A biotinylated photocleavable polyethylenimine (B-PC-PEI) was designed and synthesized for the capture and controlled release of nucleic acids from solid supports. B-PC-PEI was synthesized via a three-step reaction process and verified by 1H NMR and mass spectrometry. In aqueous solution, the o-nitrobenzyl group within B-PC-PEI was efficiently cleaved by 5 min of 365 nm light exposure from a distance of 20 cm (9 mW/cm2). When coupled to streptavidin-coated beads, the PEI domain of Cy5-labeled B-PC-PEI was released by 365 nm light exposure. In contrast, a Cy5-labeled biotinylated PEI (B-PEI) was used as a control and negligible fluorescence loss was observed. Cy5-labeled siRNA was electrostatically captured to streptavidin-coated beads preabsorbed with B-PC-PEI or B-PEI, and flow cytometry demonstrated significant loss of fluorescence from the bead surface after 5 min of light exposure only for B-PC-PEI, demonstrating controlled release of siRNA from the bead surface. Finally, the release of the Cy5-labeled siRNA into the supernatant was quantified. The release of Cy5-siRNA into the supernatant was significantly greater after 5 min of light exposure for B-PC-PEI/streptavidin beads compared to 0 min exposure and remained unchanged for B-PEI/streptavidin beads. B-PC-PEI facilitates capture and triggered release of surface-tethered nucleic acids with light exposure and is fully compatible with streptavidin-based applications. 相似文献
5.
It is well known that the positively charged polyamines have a DNA-stabilizing function and that polyamine depletion alters chromatin function. We have previously shown that polyamine depletion causes an S phase prolongation, and others have shown that there is an accumulation of Okazaki-like fragments in polyamine-depleted cells. In the present study, we have used the comet assay to investigate polyamine depletion-induced DNA strand breaks. Three breast cancer cell lines and one normal-like breast cell line were treated with the polyamine analogue N(1),N(11)-diethylnorspermine or with the polyamine biosynthesis inhibitor 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664). The comet assay showed that polyamine depletion resulted in DNA strand breaks. We also show that these DNA strand breaks occurred in cells where there was no expression of gamma-H2AX, which is a marker of DNA double-strand breaks. Thus, our conclusion is that polyamine depletion causes DNA single-strand breaks, which may be the cause for the observed delay in S phase progression. 相似文献
6.
Polyamine dependence of normal cell-cycle progression 总被引:8,自引:0,他引:8
Oredsson SM 《Biochemical Society transactions》2003,31(2):366-370
7.
A photoresponsive S-(o-nitrobenzyl)-l-cysteine N-carboxyanhydride (NBC-NCA) monomer was for the first time designed, and the related poly(S-(o-nitrobenzyl)-l-cysteine)-b-poly(ethylene glycol) (PNBC-b-PEO) block copolymers were synthesized from the ring-opening polymerization (ROP) of NBC-NCA in DMF solution at 25 °C. Their molecular structures, physical properties, photoresponsive self-assembly, and drug release of PNBC-b-PEO were thoroughly investigated. The β-sheet conformational PNBC block within copolymers presented a thermotropic liquid crystal phase behavior, and the crystallinity of PEO block was progressively suppressed over the PNBC composition. The characteristic absorption peaks of these copolymers at about 310 and 350 nm increased over UV irradiation time and then leveled off, indicating that the o-nitrobenzyl groups were gradually photocleaved from copolymers until the completion of photocleavage. The PNBC-b-PEO copolymers self-assembled into spherical nanoparticles in aqueous solution, presenting a photoresponsive self-assembly behavior, together with a size reduction of nanoparticles after irradiation. The anticancer drug doxorubicin can be released in a controlled manner by changing the light irradiation time, which was induced by gradually photocleaving the PNBC core of nanoparticles. This work provides a facile strategy not only for the synthesis of photoresponsive polypeptide-based block copolymers but also for the fabrication of photoresponsive nanomedicine potential for anticancer therapy. 相似文献
8.
Dissociation of tumour-promoter-induced effects on prostaglandin release, polyamine synthesis and cell proliferation of 3T3 cells.
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The phorbol ester 12-O-tetradecanoylphorbol 13-acetate induces tumour promotion, inflammation, cell proliferation and prostaglandin release. Recent reports suggest that the prostaglandins released by 12-O-tetradecanoylphorbol 13-acetate (TPA) initiate a cascade of events leading to polyamine synthesis and cell proliferation. In experiments designed to test this contention, it was found that addition of TPA (1 microM to 1 nM) to confluent mouse 3T3 fibroblasts successively caused the release of prostaglandins E2 and I2, induction of the enzyme ornithine decarboxylase (EC 4.1.1.17), stimulation of [3H]thymidine incorporation into DNA, and cell proliferation. Pretreatment of the cells with the anti-inflammatory steroid dexamethasone (1 microM) or the non-steroidal anti-inflammatory drug indomethacin (1 microM) inhibited TPA-induced prostaglandin release. However, dexamethasone enhanced the other effects of TPA, whereas indomethacin was ineffective. Addition of prostaglandin E2 to the cultures did not induce ornithine decarboxylase activity and cell proliferation. Pretreatment of the cells with 1,3-diaminopropane (1 mM) or alpha-methylornithine (5 mM), inhibitors of polyamine synthesis, decreased TPA-induced ornithine decarboxylase activity without affecting DNA synthesis. TPA stimulated [3H]thymidine incorporation into DNA, even when the ornithine decarboxylase activity was completely blocked. These data suggest that the proliferative effect of TPA on 3T3 cells is independent of prostaglandin release and polyamine synthesis. 相似文献
9.
Facchini A Borzí RM Marcu KB Stefanelli C Olivotto E Goldring MB Facchini A Flamigni F 《Journal of cellular physiology》2005,204(3):956-963
The activation of the NF-kappaB pathway by pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFalpha), can be an important contributor for the re-programming of chondrocyte gene expression, thereby making it a therapeutic target in articular diseases. To search for new approaches to limit cartilage damage, we investigated the requirement of polyamines for NF-kappaB activation by TNFalpha in human C-28/I2 chondrocytes, using alpha-difluoromethylornithine (DFMO), a specific polyamine biosynthesis inhibitor. The NF-kappaB pathway was dissected by using pharmacological inhibitors or by expressing a transdominant IkappaBalpha super repressor. Treatment of C-28/I2 chondrocytes with TNFalpha resulted in a rapid enhancement of nuclear localization and DNA binding activity of the p65 NF-kappaB subunit. TNFalpha also increased the level and extracellular release of interleukin-8 (IL-8), a CXC chemokine that can have a role in arthritis, in an NF-kappaB-dependent manner. Pre-treatment of chondrocytes with DFMO, while causing polyamine depletion, significantly reduced NF-kappaB DNA binding activity. Moreover, DFMO also decreased IL-8 production without affecting cellular viability. Restoration of polyamine levels by the co-addition of putrescine circumvented the inhibitory effects of DFMO. Our results show that the intracellular depletion of polyamines inhibits the response of chondrocytes to TNFalpha by interfering with the DNA binding activity of NF-kappaB. This suggests that a pharmacological and/or genetic approach to deplete the polyamine pool in chondrocytes may represent a useful way to reduce NF-kappaB activation by inflammatory cytokines in arthritis without provoking chondrocyte apoptosis. 相似文献
10.
Abstract. We have previously found that DNA replication was affected within one cell cycle after seeding Chinese hamster ovary (CHO) cells in the presence of the polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO). We could, however, not rule out if this was due to an effect on the G1 /S transition and/or on DNA synthesis elongation. In the present paper, we use a bromodeoxyuridine-flow cytometric method to more specifically study the G1 /S transition, the S phase length, and the progression of cells from S phase through G2 + M and into G1 , after seeding plateau phase CHO cells at low density in the absence or presence of 5 mM DFMO. We report here that DFMO-induced polyamine depletion increased the length of the S phase within one cell cycle after seeding of CHO cells in the presence of the inhibitor. No effect on the G1 /S transition was observed until 2 days after seeding, suggesting that a DFMO-induced lengthening of the G1 phase occurred later than the effect on S phase progression. These results imply that the G2 + M phase was not prolonged until 2 days after seeding CHO cells in the presence of DFMO. 相似文献
11.
Choi SK Verma M Silpe J Moody RE Tang K Hanson JJ Baker JR 《Bioorganic & medicinal chemistry》2012,20(3):1281-1290
Photochemistry provides a unique mechanism that enables the active control of drug release in cancer-targeting drug delivery. This study investigates the light-mediated release of methotrexate, an anticancer drug, using a photocleavable linker strategy based on o-nitrobenzyl protection. We evaluated two types of the o-nitrobenzyl-linked methotrexate for the drug release study and further extended the study to a fifth-generation poly(amidoamine) dendrimer carrier covalently conjugated with methotrexate via the o-nitrobenzyl linker. We performed the drug release studies by using a combination of three standard analytical methods that include UV/vis spectrometry, (1)H NMR spectroscopy, and anal. HPLC. This article reports that methotrexate is released by the photochemical mechanism in an actively controlled manner. The rate of the drug release varies in response to multiple control parameters, including linker design, light wavelength, exposure time, and the pH of the medium where the drug release occurs. 相似文献
12.
Chemical ligation approaches facilitate the chemoselective assembly of unprotected peptides in aqueous solution. Here, two photolabile auxiliaries are described that enlarge the applicability of native chemical ligation to non-cysteine targets. The auxiliaries, designed to allow reaction with thioester peptides, generate an amide bond between the two initial fragments. The o-nitrobenzyl tertiary benzylamide that is formed at the ligation junction can be transformed into a native amide group under mild photolysis conditions. The veratryl auxiliary was found to be excessively labile during peptide purification and ligation. However, the auxiliary based on the o-nitrobenzyl group shows all the necessary properties for a general application in routine peptide and protein synthesis. In addition, the auxiliary linked to the N-terminus can be efficiently photolyzed, suggesting a new approach for the generation of photocaged amines. Synthesis, solid phase introduction onto peptide chains, ligation properties and photolysis in water are described, and a careful study of compatibility of the method with potentially fragile peptide side chains is reported. 相似文献
13.
14.
Transglutaminase 2 (TG2) has been reported to be involved in cell growth through the formation of epsilon-(gamma-glutamyl) lysine (Gln-Lys) or N-(gamma-glutamyl) polyamine (Gln-polyamine). We have recently reported that the inhibition of Gln-Lys cross-linking by the formation of Gln-spermidine led to the increase of DNA synthesis in regenerating rat liver. TG2 may catalyze the replacement reaction between Lys residues in protein and polyamines. In the present study, we attempted to develop an experimental model for ascertaining this replacement reaction. We examined whether or not TG2 exhibited the association and dissociation reaction of Gln-polyamine bond in protein, using N,N-dimethylcasein (DC). The dissociated polyamines were identified by autoradiography. The dissociation of [(14)C] polyamines from DC bond [(14)C] polyamines complex by TG2 could occur in the presence of non-radioactive polyamines as second amine donor, whereas in the absence, could not almost occur. Moreover, it was indicated that this release of old [(14)C] polyamine bonded to DC was due to binding of added new [(14)C] polyamine to Gln residues in DC. These results demonstrate that TG2 catalyzes the replacement reaction between added [(14)C] polyamine and DC bond [(14)C] polyamine. The dissociation and association reaction may both occur together, the new DC-polyamine complex being formed at the same time as the dissociation of old DC-polyamine complex, since readying a second amine donor is necessary to dissociate DC-polyamine complex. These results indicate that this experimental model is successful in the study of TG2-catalyzed dissociation and association reaction of Gln-polyamine bond in protein. 相似文献
15.
Growth stimulation of either fetal rat liver cells or rat embryo fibroblasts in culture results in considerable increases in intracellular polyamine levels as cells proceed through the cell cycle. Treatment of such cell cultures with appropriate levels of two inhibitors of polyamine synthesis, namely α-hydrazino ornithine and methylglyoxal bis(guanylhydrazone), can essentially completely block these increases in cellular polyamine content. Under such conditions, where the elevation in intracellular polyamine content is prevented, cell cultures are nevertheless able to initiate DNA synthesis and subsequently synthesize DNA at rates comparable to untreated control cultures that have been growth-stimulated. These two cell types therefore contain sufficient polyamines when in a resting state (G1) to enable them to enter from G1 into S phase and traverse S phase at normal rates in the absence of further polyamine synthesis. The recruitment of cells into the first cell cycle, through serum stimulation of growth, therefore appears not to be mediated or regulated by the increases in intracellular levels of polyamines that occurs under these conditions. Conversely, the arrest of growth of these cell types resulting from serum deprivation is not mediated by a limitation of intracellular polyamine content. 相似文献
16.
Huth S Hoffmann F von Gersdorff K Laner A Reinhardt D Rosenecker J Rudolph C 《The journal of gene medicine》2006,8(12):1416-1424
BACKGROUND: Plasmid DNA (pDNA) dissociation from polyamine gene vectors after cellular uptake has not been well characterized. A more detailed understanding of this process could lead to more efficient gene transfer agents. Since RNA is present in the cytoplasm at high concentrations and due to its structural similarity to DNA, we were interested in its conceivable interaction with polyamine gene vectors. METHODS: In a first set of experiments gene vectors were incubated in cell lysate and pDNA release was investigated by Southern blot analysis with or without RNase A pretreatment and by confocal laser scanning microscopy. Further, interaction of polyamine gene vectors with RNA was investigated by fluorescence quenching assay. These methods were complemented by a functionality assay using isolated nuclei. RESULTS: The incubation of gene vectors with cell lysate resulted in the dissociation of pDNA from the complexes. This effect was abolished when the cell lysate was pretreated with RNase A. The addition of RNA in the absence of cell lysate led also to a dissociation of pDNA. This process commenced instantaneously after the addition of RNA as analyzed by fluorescence quenching. When gene vectors were incubated in cell lysate containing isolated nuclei, the dissociation of pDNA from the polyamine gene vectors occurred preferentially extranuclearally as confirmed by confocal laser scanning microscopy. These results were further corroborated in a functional assay. CONCLUSIONS: These data suggest that RNA induces pDNA dissociation from the polyamine gene vectors. Furthermore, this process apparently occurs in the cytoplasm before the gene vectors enter the nucleus. 相似文献
17.
Pedreño E López-Contreras AJ Cremades A Peñafiel R 《Journal of inorganic biochemistry》2005,99(10):2074-2080
Polyamines are ubiquitous polycations that participate in cellular processes such as growth, differentiation and cell death. Among the different functions ascribed to these organic cations, the polyamine spermine is known to protect DNA from the damage produced by reactive oxygen species (ROS) generated by different agents including copper ions. We have found that spermine exerts opposite effects on DNA strand breakage induced by Fenton reaction depending on metal concentration. Whereas at low concentration of the transition metals, 10 microM copper or 50 microM Fe(II), 1 mM spermine exerted a protective role, at metal concentrations higher than 25 microM copper or 100 microM Fe(II), spermine stimulated DNA strand breakage. The promotion of the damage induced by spermine was independent of DNA sequence but decreased by increasing the ionic concentration of the media or by the presence of metal-chelating agents. Moreover, spermine did not increase the oxidation of 2-deoxyribose by metal/H2O2 when DNA was substituted by 2-deoxyribose as a target for damage. Our results corroborate that spermine may protect DNA and 2-deoxyribose from the damage induced by ROS but also demonstrate that under certain conditions spermine may promote DNA strand breakage. The fact that this promoting effect of spermine on ROS-induced damage was observed only in the presence of DNA suggests that this polyamine under certain conditions may facilitate the interaction of copper and iron ions with DNA leading to the formation of ROS in close proximity to DNA. 相似文献
18.
19.
Polyamine Biosynthetic Enzymes in the Cell Cycle of Chlorella: Correlation between Ornithine Decarboxylase and DNA Synthesis at Different Light Intensities 总被引:4,自引:4,他引:0
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During the life cycle of Chlorella vulgaris Beijerinck var vulgaris fa. vulgaris growing synchronously, the specific activity of ornithine decarboxylase peaked at the 2nd hour of the cycle, whereas that of arginine decarboxylase changed only slightly, increasing towards the end of the cycle. The endogenous level of putrescine and spermidine on a per cell basis increased gradually up to the 8th hour of the cycle, and declined thereafter. Thus, the peak of ornithine decarboxylase activity and the polyamine increase preceded both DNA replication (which took place between the 6th and 8th hours of the cycle) and autospore release (which started at the 8th hour). A 2-fold increase in the light intensity caused doubling of the DNA content, resulting in doubling of the number of autospores per mother cell. It also brought about a 2-fold increase in the specific activity of ornithine decarboxylase and polyamine content, the peaks being at the same hour of the cycle under high and low light intensities. The increase in cell number and polyamine content in a Chlorella culture grown under high light intensity was inhibited by α-difluoromethyl ornithine, a specific inhibitor of ornithine decarboxylase, this inhibition being partially reversed by putrescine.
It is suggested that in C. vulgaris the sequence of events which relates polyamine biosynthesis to cell division is as follows: increased ornithine decarboxylase activity, accumulation of polyamines, DNA replication, and autospore release.
相似文献20.
Regulation of tRNA methyltransferase activities by spermidine and putrescine. Inhibition of polyamine synthesis and tRNA methylation by alpha-methylornithine or 1,3-diaminopropan-2-ol in Dictyostelium.
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Inhibitors of polyamine synthesis (alpha-methylornithine and 1,3-diaminopropan-2-ol) were used to study the relationship between polyamine synthesis and specific methylations of tRNA in Dictyostelium discoideum during vegetative growth. Polyamine concentrations were found to be 10 mM for putrescine, 1.6 mM for spermidine and 7 mM for 1,3-diaminopropane throughout the growth stage. On treatment of growing amoebae with alpha-methylornithine or with 1,3-diaminopropan-2-ol (each at 5 mM), the syntheses of putrescine, spermidine and 1,3-diaminopropane were arrested within 4h. After polyamine synthesis had ceased, the incorporation of methyl groups into tRNA was considerably decreased under conditions that had no effect on the incorporation of uridine into tRNA, or on net syntheses of protein and of DNA. The following nucleosides in tRNA were concerned: 1 methyladenosine, 5-methylcytidine, 7-methylguanosine, 2-methylguanosine, N2N2-dimethylguanosine and 5-methyluridine (ribosylthymine). The corresponding tRNA methyltransferases, determined in Mg2+-free enzyme extracts, proved to be inactive unless polyamines were added. Putrescine and/or spermidine at concentrations of 10 mM or 1-2 mM respectively stimulate the transmethylation reaction in vitro to a maximal rate and to an optimal extent at exactly the same concentrations as found in vegetative cells. In contrast, 1,3-diaminopropane, which is formed from spermidine, does not affect the methylation of tRNA in vitro at physiological concentrations. Putrescine and/or spermidine stabilize the tRNA methyltransferases in crude extracts in the presence but not in the absence of the substrate tRNA. The results support the view that S-adenosylmethionine-dependent transmethylation reactions can be regulated by alterations of polyamine concentrations in vivo. 相似文献