10μM Concentration Research Articles

The Effect of Indole-3-Acetic Acid and Benzyladenine on Sequential Leaf Senescence on Helianthus Annuus L. Seedlings

ABSTRACTThe effect of IAA and BA on the mechanism of senescence which occurs in the cotyledons of Helianthus annuus L. seedlings showing epigenic germination has been investigated. In this study 0,01μM and 10μM IAA solutions were sprayed to the leaves and BA solutions with the same concentrations—to the cotyledons of 16 days-old seedlings. We have determined chlorophyll content, protein amount, peroxidase activity and the changes in the membrane permeability in the cotyledons of the treated and control seedlings. Compared to the control cotyledons IAA in the concentrations of 10μM and 0,01μM caused a breakdown of chlorophyll at the ratio of 64% and 54%, respectively. Soluble protein levels in the IAA at 10μM treatment decreased in the ratio of 12%. IAA treatments with 10μM and 0,01μM stimulated POD activity with about 11 and 8 times, respectively. On the other hand, it has been observated that 10μM and 0,01μM BA treatment retarted senescence, whereas 0,01μM BA and 0,01 μM IAA when were applied together enhanced senescence. It has been discussed that IAA which is known as an allosteric activators of cytokinin oxidase stimulates the cytokinin oxidations so it increases senescence that occurs in the cotyledons. In this research, according to the results it has been thought that the senescence signal may be a substance in the nature of IAA.

Cytotoxic activities of 4 natural products – carnosic acid, xanthohumol, caffeic acid phenethyl ester and (-) eburnamonine

In a cytotoxic or anti-proliferation assay, 20 compounds derived from natural products have been screened for cytotoxic effects against A549 cell line using MTT assay. Among them, carnosic acid, xanthohumol, caffeic acid phenethyl ester and (-) eburnamonine showed activies at concentrations of 50µM and 10µM. These four active compounds were subjected to cytotoxicity studies in two other cell lines (A431 and SK-BR3), using SRB assay. While carnosic acid, xanthohumol and (-) eburnamonine still showed cytotoxic activity, caffeic acid phenethyl ester showed no activity in A431 cell line and low activity against SK-BR3 cell line (IC50=258µM). Xanthohumol possessed the strongest cytotoxic effects against all 3 cell lines with IC50 values of 24.16µM, 16.14µM and 1.68µM in A549, A431 and SK-BR3 cells, respectively. Because A431 and SK-BR3 cells are known to over-express tyrosine kinase receptors, we have studied the four compounds for KDR and Her2 tyrosine kinase receptor inhibitory activity by using fluorescence polarization. The results showed that except caffeic acid phenethyl ester, which showed low inhibitory activity on KDR at a high concentration of 10µM, the other compounds were inactive. In conclusion, carnosic acid, xanthohumol and (-) eburnamonine showed cytotoxic activity in A549, A431 and SK-BR3 cell lines but their activities are not mediated though the inhibition of KDR or Her2 receptors. The cytotoxic activity of caffeic acid phenethyl ester is just limited to A549 cell line, not for the other two cell lines, which suggests a specific activity of this compound.

Treatment of normal and malignant cells with nucleoside analogues and etoposide enhances deoxycytidine kinase activity

Deoxycytidine kinase (dCK), one of the rate-limiting enzymes in the intracellular metabolism of many antileukaemic drugs, was shown to be stimulated after treatment of human tonsillar lymphocytes by 2-chloro-2′-deoxyadenosine (cladribine, CdA) (Sasvári-Székely, et al., Biochem Pharmacol 1998, 56, 1175–1179). Here we present a comparative study of different normal and malignant cells in respect to the activation of dCK by CdA. G-phase lymphocytes showed a higher sensitivity for dCK stimulation than S-phase cells. Normal and leukaemic peripheral blood mononuclear cells, as well as the promyelocytic cell line HL60 responded to CdA treatment by a 2–5-fold increase in activity of dCK. However, no significant stimulation was detected either in CCRF-CEM T-lymphoblastoid cells, or in K562 myeloid cells. Thymidine kinase (TK) activity was not stimulated in any cases. Treatment of these cells with several other analogues beside CdA, such as 2-chloro-2′-arabino-fluoro-2′-deoxyadenosine (CAFdA), 2-fluoro-1-β-d-arabinosyladenine (Fludarabine, FaraA) and 1-β-d-arabinosylcytosine (cytarabine, araC) gave similar results to CdA treatment. Enhancement of dCK activity could also be achieved with the topoisomerase II inhibitor, etoposide. In contrast, 2-chloro-riboadenosine (CrA) had no effect on the dCK at concentrations of 10μM or less, while dCyd and 5-aza-dCyd caused slight inhibition. These results indicate that treatment of cells with several inhibitors of DNA synthesis potentiates the dCK activity. The drugs widely differ in their stimulatory effect on dCK, and there are also ‘responsive’ and ‘non-responsive’ cells with respect to dCK activation. Thus, enhancement of the dCK activity by specific drugs in ‘responsive’ cells might give a rationale for combination chemotherapy.

Assessment of the embryotoxic potential of the total hydrolysis product of fumonisin B 1 using cultured organogenesis-staged rat embryos

Arninopentol (AP1) is the total hydrolysis product of fumonisin B 1 (FBI), the major and best characterized of the fumonisins, which are mycotoxins that are common contaminants of corn and corn meal. Some human populations expected to have significant exposure to AP1 have a high incidence of babies born with neural tube defects (NTD). The embryotoxicity of AP1 was evaluated in cultured rat embryos. Gestation day 9.5 embryos were exposed to 0, 3, 10, 30, 100 or 300 μM AP1 throughout the entire 45-hr culture period. At 100 μM AP1, growth and overall development were reduced significantly. There was also a significant increase in the incidence of abnormal embryos. 29% of the embryos had NTD, and 36% of the embryos had other abnormalities. At 300 μM API, the incidence of NTD was 15%, and 85% of the embryos had other abnormalities. These findings suggest that AP1, at concentrations of 100μM and above, can induce NTD in organogenesis-stage cultured rat embryos. However, these NTD are in conjunction with significant overall retardation of growth and development as well as significant increases in the incidence of other defects. These studies also showed, when compared with previous findings, that API is over 100-fold less toxic than FB1 to cultured rat embryos.

Sodium trimetaphosphate fights tooth decay

Arninopentol (AP1) is the total hydrolysis product of fumonisin B1 (FBI), the major and best characterized of the fumonisins, which are mycotoxins that are common contaminants of corn and corn meal. Some human populations expected to have significant exposure to AP1 have a high incidence of babies born with neural tube defects (NTD). The embryotoxicity of AP1 was evaluated in cultured rat embryos. Gestation day 9.5 embryos were exposed to 0, 3, 10, 30, 100 or 300 μM AP1 throughout the entire 45-hr culture period. At 100 μM AP1, growth and overall development were reduced significantly. There was also a significant increase in the incidence of abnormal embryos. 29% of the embryos had NTD, and 36% of the embryos had other abnormalities. At 300 μM API, the incidence of NTD was 15%, and 85% of the embryos had other abnormalities. These findings suggest that AP1, at concentrations of 100μM and above, can induce NTD in organogenesis-stage cultured rat embryos. However, these NTD are in conjunction with significant overall retardation of growth and development as well as significant increases in the incidence of other defects. These studies also showed, when compared with previous findings, that API is over 100-fold less toxic than FB1 to cultured rat embryos.

3″-Azido-3′-Deoxythymidine and 2′,3′-Dideoxycytidine Do Not Inhibit Gene-Specific DNA Repair in Hamster Cells

3″-Azido-3′-deoxythymidine (AZT) was the first approved drug for the treatment of the AIDS; however, despite its usefulness, AZT often produces side effects that require cessation of therapy. 2′,3′-Dideoxycytidine (ddC) is a related anti-retroviral agent in advanced stages of clinical testing. A previous report demonstrated that AZT decreased the repair of UV-induced DNA strand breaks in mammalian cells after ultraviolet (UV) irradiation. We studied the effect of AZT and ddC on DNA repair from the hamster DHFR gene of the major UV-induced DNA lesion, cyclobutane pyrimidine dimers (CPDs). We conclude that neither AZT nor ddC inhibited DNA replication or the gene-specific repair of CPDs in the hamster DHFR gene after 8 or 24 hrs of repair incubation at concentrations of 25μM and 10μM, respectively.

VLA-2 mediates the interaction of collagen with endothelium during in vitro vascular tube formation.

A confluent endothelial monolayer can be induced to form vascular tubes in response to collagen. We investigated possible mechanisms of collagen-induced tube formation by using antibodies to the VLA-2 integrin receptor and protein kinase C inhibitors. Pre-incubation of cells with anti-VLA-2 (which recognises both the α2 and β1 chains) and AK7 (which recognises only the α2 chain) showed a dose-dependent inhibition of tube formation. At 50 μg/ml, anti-VLA-2 completely inhibited collagen-induced tube formation, whereas AK7 caused only partial inhibition. Both chlorpromazine and trifluoperazine, at concentrations of 10μM, prevented tube formation (> 40% inhibition), In summary, the VLA-2 integrin receptor plays a role in the induction of tube formation by type I collagen. Protein kinase C may be activated during this process.

Inhibition of Thymidylate Synthetase Activity Induced in Varicella-Zoster Virus Infected Cells by (E)-5-(2-bromovinyl)-2′-deoxyuridine

The authors investigated the effect' of thymidylate synthetase (TS) on the antivariclllazoster virus (VZV) activity of ( E)5-(2-bromovinyl)-2′-deoxyuridine (BVDU). TS catalyses the conversion of deoxyuridylate (dUMP) to thymidylate (dTMP) and is a key enzyme in pyrimidine biosynthesis, providing the only source of dTMP synthesized de novo in mammalian cells. VZV encodes a specialized viral form of TS. TS activity in cells infected with VZV (TIO-VZV and TK−-VZV) increased proportionally with focus formation. From kinetic analysis using the Michaelis-Menten equation, the authors determined a Km value of 6.6μm for dUMP of TS induced in VZV-infected cells and a corresponding value from mock-infected cells of only 2.8μm. BVDU inhibited the induction of TS activity in TK+-VZV-infected cells at concentrations under ×10−3μm, but did not inhibit TS activity of TK−-VZV- or mock-infected cells at concentrations as high as 10μm. Inhibitory activity of BVDU against TS induced in TK+-VZV-infected cells appears to occur when BVDU is phosphorylated to BVDU monophosphate by viral pyrimidine kinase. These results suggest that the selective inhibitory action of BVDU on VZV replication depends on a specific interaction with both viral TK and TS. 5-Bromodeoxyuridine and 5-iododeoxyuridine inhibited TS activities induced in both VZV (TK+, TK−) and mock-infected cells. Other antiherpes compounds [i.e. 1-β-D-arabinofuranosyl- E-5-(2-bromovinyl)uracil, 9- 2-hydroxyethoxymethyl)-guanine, arabinosyladenine, and others] did not inhibit TS activity in VZV-infected cells at concentrations Of 10μm.

Excitatory effects of cholecystokinin in rat hippocampus: pharmacological response compatible with ‘central’- or B-type CCK receptors

The effects of the sulfated octapeptide of cholecystokinin (CCK 8-S), the non-sulfated homolog (CCK 8-NS) as well as the C-terminal di-, tri- and tetrapeptidic fragments (respectively CCK 2, CCK 3 and CCK 4) were studied in vitro in rat hippocampal slices by extracellular recording of the spontaneous action potential discharge frequency of neurons located in the CA1 stratum pyramidalis. Bath-applied CCK 8-S concentration-dependently increased the action potential discharge frequency of hippocampal CA1-neurons in concentrations ranging from 0.05 to above 1 μM. Both CCK 8-NS and CCK 4 exhibited reversible and concentration-dependent excitatory effects. They were 4 and 10 times less potent than CCK 8-S,respectively, as concentrations of 2μM CCK 8-NS and 5 μM CCK 4 were needed to evoke the same excitation as that induced for a given neuron by 0.5 μM CCK 8-S. In contrast, none of the shorter fragments (CCK 2 and CCK 3) were effective in altering spontaneous discharge of CCK 8-S-sensitive neurons even at concentrations of 100μM. The pharmacologic profile of the excitatory response observed in the rat hippocampus follows the same pattern as the binding profile observed on brain membrane preparations. It is therefore concluded that the CCK receptors involved in this response seem to be related more to the ‘central’- or B-type CCK receptors rather than to the ‘peripheral’- or A-type CCK receptors.

Inhibitory action of verapamil and chlorpromazine on the release of slow-reacting substance of anaphylaxis from cat lung

Verapamil, a calcium channel blocker, significantly inhibited the release of slow-reacting substance of anaphylaxis (SRS-A) from cat lung fragments challenged with either the calcium ionophore, A23187, or Ascaris extract. This effect was observed when verapamil was present at the concentrations of 10μM and 50μM. Chlorpromazine 10μM significantly inhibited antigen-induced SRS-A release. Histamine release was low and no significant effect by verapamil or chlorpromazine could be shown.

Influenza A virus enhances the human polymorphonuclear leukocyte chemoluminescence response without effecting inhibition by trifluoperazine

Influenza A virus has been demonstrated to enhance superoxide generation and chemoluminescence (CL) in human polymorphonuclear leucoytes (PMNs) under in vitro conditions. Although the mechanisms of virus-enhanced neutrophil activity is not established, calmodulin concentrations are known to increase in some virus-transformed cells. In the following experiments, we evaluated the PMN response to the calmodulin-inhibitor trifluoperazine (TFP) after an incubation with influenza A virus. Human PMNs were isolated from whole blood and were incubated with either influenza A virus (at 50% egg-infective dose per 1 leukocyte) or noninfected allantoic fluid. After incubation with influenza virus, the CL response of isolated PMNs to opsonized zymosan particles was measured. The influenza virus-treated PMNs had a mean (±SEM, n = 16) increase in light emission of 59.5 ± 7.7%. TFP, in concentrations of 6μm, 8μm, and 10μM, inhibition of CL was similar in influenza virus and allantoic fluid-treated neutrophils. These data suggest that, although the influenza A virus enhanced the PMN “respiratory burst” to opsonized zymosan particles, it did not alter the cell response to one calmodulin inhibitor, TFP.

Neuronal opiate receptors in rabbit ear artery.

Dipeptide SD-41, tripeptides SD-25 and 34, and tetrapeptide SD-6 (at above 0.1μM) inhibited the contractions of rabbit ear artery ring segments induced by transmural nerve stimulation. The extent of inhibition was at most 70%, and in contrast at concentrations of above 10μM, the peptides augmented the contractions. This augmentation was more remarkable in the presence of naloxone. The antagonism by naloxone of the actions of the peptides indicates that the peptides act on neuronal opiate receptors. It is unlikely that rabbit ear artery contains μ-receptor because morphine and normorphine, which show μ-receptor agonist characteristics on guinea pig ileum, had no effect on the artery. Thus the peptides are probably not μ-agonists. The response of the artery was suppressed by the -agonists Met-enkephalin and D-Ala, D-Leu enkephalin. This suggests that δ-receptor is present in the artery and also that the SD-peptides act as δ-agonists. The fact that both SD-peptides and μ-receptor agonists dynorphin and ethylketocyclazocine partially blocked the arterial contractions suggests that SD-peptides are k-receptor agonists. Further evidence supporting this idea is that Mr 2266, a k-antagonist, was more effective than naloxone in antagonizing the actions of the SD-peptides and ethylketocyclazocine. From these results it is suggested that δ-receptor and k-receptor are opiate receptors in the rabbit ear artery and that short chain peptides (SD-peptides) have δ- and k-agonist characteristics.

CHANGES IN ACTIVITIES OF THYMIDYLATE SYNTHASE AND DIHYDROFOLATE REDUCTASE IN SEA URCHIN EGGS AFTER FERTILIZATION.

Thymidylate synthase activity in sea urchin eggs increases just after fertilization and decreases 30 min later. Then, cyclic variation in the activity occurs in association with the cleavage cycle. Dihydrofolate reductase activity in fertilized eggs is almost the same as in unfertilized eggs and shows no marked change within 3 hr after fertilization. Aminopterin, an analogue of dihydrofolate, inhibits dihydrofolate reductase, and arrests cleavage. On incubation in sea water containing aminopterin (20-100μM) from the time of fertilization, the development of Clypeaster and Pseudocentrotus eggs was arrested at the 32-64 cell stage, and that of Anthocidaris eggs was arrested at the morula stage. Dihydrofolate (100μM) counteracts the inhibitory effect of aminopterin on egg cleavage. Thymidine at concentrations above 10μM also prevents inhibition by aminopterin. Other deoxyribonucleosides at concentrations of 10μM to 100μM do not affect inhibition of egg cleavage by aminopterin. Deoxyadenosine at concentrations above 5 mM inhibits egg cleavage, but other deoxyribonucleosides have no effect.