Dimethylamino Purine Research Articles

Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence.

Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1Thr161, and p-CDK1Thr14-Tyr15, were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations (p < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments (p < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of POU5F1 were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of CDX2 did not show differences. In addition, the BCL2A1/BAX ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment (p < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.

Developmental potential of artificially produced chimeric embryos following transfer to surrogate Goats

An experiment was conducted to study the developmental potential of goat chimeric embryos. A total of 8393 mature oocytes were randomly divided into two groups. Gr 1: oocytes (n=5222) were activated with 5 μM calcium ionophore for 5 to 7 min followed by treatment with 2.0 mM DMAP (dimethylaminopurine) for 4 h in mCR2 aa (modified Charles Rosenkrans 2 amino acid) medium. Gr 2: oocytes (n=3171) were used for in vitro fertilization (IVF). Inner cell mass from hatched blastocysts of parthenogenetic activated embryos were used to produce embryonic stem cell-like cells while 2 cell embryos obtained from IVF were utilized to produce tetraploid embryos. Aggregation efficiency, compact morula and blastocyst formations were 84.93±8.13, 80.53±10.55 and 7.05±3.08%, respectively. A total of 47 chimeric goat embryos were transferred into 17 Sirohi female recipients, resulted in in vivo development of foetus in two recipients. In first recipient, foetus looked like a very thick and hyperechoic amniotic ring until 49 days; thereafter, it was completely resorbed. In second recipient, foetus and amniotic ring were visible as hypoechoic structure from 50 days onwards; thereafter it was completely resorbed at 58 days of embryo transfer. In conclusion, the study indicated that foetal resorption was observed at various stages of pregnancy after transfer of chimeric embryos into recipients. It appears that placental atrophy may be the principal cause of the loss of foetuses.

FTIR and FT-Raman spectra of 6-(dimethylamino)purine and its theoretical studies of anharmonic vibrational analysis using quantum chemical calculations

The present work explores the anharmonic vibrational and electronic spectral features of 6-(dimethylamino) purine (DMAP) compound with the help of the FTIR (4000−400 cm−1), FT-Raman (4000−50 cm−1) and UV–vis (600−180 nm) spectra as well as DFT calculations. Anharmonic frequencies on the structure of 6-dimethylaminopurine (DMAP) were computed using the generalized vibrational perturbation theory (GVPT2), vibrational self-consistence field (VSCF) theory and correlation-corrected vibrational self consistence field (PT2-VSCF) method in DFT/6−311 G(d,p) framework. The fully anharmonic frequencies calculated using different methods are in remarkable agreement with the experiments as reflected from RMSE and MAD values and show large improvement over the harmonic approximation. The magnitudes of coupling between pairs of normal modes were calculated using quartic force field (2MR-QFF) approximation. The interactions between pair of modes due to mode-mode coupling have significantly affected the vibrational frequencies. The effect of intermolecular interaction (N–H⋅⋅⋅⋅⋅N) on the geometrical parameters and vibrational frequencies were studied using comparison of results of the monomeric and dimeric structure of the titled molecule. The electronic spectra in the methanol, ethanol and water solvents have been analyzed using the TD-DFT approach and these are compared with the experiment. Furthermore, the associated nonlinear optical (NLO) properties, frontier molecular orbitals and molecular electrostatic potential (MEP) surface analysis have been studied.

Expression of apoptosis related genes in buffalo embryos produced through in vitro fertilization and parthenogenetic activation

In present study immature oocytes were obtained from buffalo slaughterhouse ovaries and were subjected to in vitro maturation (IVM) in TCM-199 + 10% FBS + 5 µg/ml pFSH + 0.81 mM sodium pyruvate + 5% buffalo follicular fluid for 24 h in a CO2 incubator (5% CO2, 90–95% relative humidity) at 38.5°C. The maturer oocytes were used for IVF and the cleaved embryos were cultured for 8 days, while the parthenotes were produced with exposure of 7% ethanol, 6- dimethyl aminopurine and cultured for 8 days in mCR2aa medium. The relative abundance of BCL-XL transcripts in immature, matured oocytes; and embryos produced through IVF i.e. 2-cell, 4-cell, 8-16-cell, morula and blastocyst stage were 25.33±0.90, 12.67±1.20, 37.67±0.90, 30.67±0.30, 23.67±0.90, 18.33±0.90 and 27.00±1.20 respectively, while in parthenogenesis these values were 23.67±0.88, 13.67±1.20, 23.67±1.20, 22.34±0.88, 24.34±0.88, 33.67±0.88, 45.34±1.20 respectively. Relative abundance of BAX transcripts by IVF were 23.0±0.60, 0.33±0.10, 4.00±0.60, 5.00±0.60, 0.37±0.06, 13.0±0.66 and 56.7±0.90; and by parthenonenesis were 22.3±0.90, 0.13±0.03, 13.67±0.90, 14.0±0.60, 15.33±0.90, 64.67±2.20 and 55.0±2.10 respectively. In conclusion, expression pattern of apoptosis related genes revealed that incidence of apoptosis was significantly higher in IVF and parthenogenetically produced buffalo embryos at stages like immature oocytes, morula and blastocyst stage than the early cleavage stage embryos.

Glycoxydation promotes vascular damage Via MAPK‐ERK/JNK pathways

Oxidation and glycation enhance foam cell formation via MAPK/JNK in euglycemic and diabetic subjects. Here, we investigated the effects of glycated and oxidized LDL (glc-oxLDL) on MAPK-ERK and JNK signaling pathways using human coronary smooth muscle cells. Glc-oxLDL induced a broad cascade of MAPK/JNK-dependent signaling transduction pathways and the AP-1 complex. In glc-oxLDL treated coronary arterioles, tumor necrosis factor (TNF) α increased JNK phosphorylation, whereas protein kinase inhibitor dimethylaminopurine (DMAP) prevented the TNF-induced increase in JNK phosphorylation. The role of MKK4 and JNK were then investigated in vivo, using apolipoprotein E knockout (ApoE(-/-)) mice. Peritoneal macrophages, isolated from spontaneously hyperlipidemic but euglycemic mice showed increases in both proteins and phosphorylated proteins. Compared to streptozotocin-treated diabetic C57BL6 and nondiabetic C57BL6 Wt mice, in streptozotocin-diabetic ApoE(-/-) mice, the increment of foam cell formation corresponded to an increment of phosphorylation of JNK1, JNK2, and MMK4. Thus, we provide a first line of evidence that MAPK-ERK/JNK pathways are involved in vascular damage induced by glycoxidation.

37 EFFECTS OF TRICHOSTATIN A TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Faulty epigenetic reprogramming is a likely major cause of the low success rate observed in all mammals produced through somatic cell nuclear transfer (SCNT). It has been reported that treatment of reconstructed mouse embryos with the potent histone deacetylase inhibitor, trichostatin A (TSA), results in significantly increased developmental capacity of SCNT preimplantation embryos and live offspring (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 240, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089; Kishigami et al. 2006 J. Reprod. Dev. 53, 165–170). Studies investigating similar reprogramming capabilities of TSA in bovine SCNT embryos report conflicting results (Akagi et al. 2007 Reprod. Fertil. Dev. 19, 24 abst; Iwamoto et al. 2007 Reprod. Fertil. Dev. 19, 48 abst). In this study, the effects of TSA treatment on in vitro development of bovine SCNT embryos were examined. Bovine fetal fibroblasts were cultured under contact inhibition for 2 to 5 days and used as donor cells for SCNT. Oocytes were aspirated from abattoir-derived ovaries, and matured in vitro for 18 h prior to enucleation. Reconstructed SCNT couplets were electrofused, and then activated 24 h post-maturation using 5 µm ionomycin followed by 2 mm dimethylaminopurine (DMAP) for 4 h. SCNT embryos were subjected to 0 (control; C-NT) or 50 nm TSA for 13 h post-ionomycin (hpi) TSAa-NT) or 13 hpi + 6 h starting from 40 hpi (TSAb-NT). IVF embryos were produced as an additional control. All embryos were cultured in KSOM supplemented with 3 mg mL–1 BSA for 7.5 days, with 5% FBS added on Day 3. Experiments were repeated 3 or 7 times, and data were analyzed a -way ANOVA procedure. Developmental rates to the blastocyst stage and total cell number of blastocysts were determined. Total cell numbers were determined by fixing blastocysts in 4% paraformaldehyde, and staining with bisbenzimide 33342, followed by microslide mounting and visualization using an epifluorescence microscope. No difference was observed in cleavage rates among the four treatment groups, C-NT, TSAa-NT, TSAb-NT, and IVF, with the rates being 66%, 75%, 73.1%, and 82.3%, respectively (P = 0.33); nor was any improvement seen in the rate of blastocyst development of TSAa-NT or TSAb-NT over C-NT embryos: 36%, 40.2%, and 30.2%, respectively (P = 0.22). Furthermore, there was no significant difference in mean total cell number of blastocysts among treatment groups: C-NT, 120.2; TSAa-NT, 124.2; TSAb-NT, 129.3; and IVF, 141.1 (P = 0.29). These results suggest that 50 nm TSA treatment immediately following activation does not affect the development of bovine SCNT preimplantation embryos.

Role of Anti-Endothelial Cell Antibody in the Development of Coronary Arterial Lesions in Kawasaki Disease

Background and Objectives：Anti-endothelial cell antibodies (AECA) are found in the sera of many patients with Kawasaki disease (KD). In this study, the pathogenic role of AECA in the development of coronary arterial lesions of KD was investigated. Subjects and Methods：Serum IgM-AECA concentrations were measured in 22 KD patients. Cultured human coronary artery endothelial cells (HCAEC) were incubated with either acute or convalescent phase sera, and their expressions of intercellular adhesion molecule-1 (ICAM-1) assessed. IgM fractions of the sera were purified, and their ability to induce ICAM-1 mRNA and protein expressions evaluated. To address the signal transduction pathways involved in IgM-AECA-induced ICAM-1 expression, the blocking effect of four protein kinase inhibitors, PD98059, SB203580, dimethylaminopurine (DMAP) and parthenolide were measured. Results：IgM-AECA was present in 14 out of 22 (64%) acute KD sera. ICAM-1 expression of HCAEC incubated with acute KD sera (117.1±46.7) and AECA-positive acute KD sera (143.3±37.5) were significantly higher than those of the convalescent KD sera (88.9±14.4, p<0.05) or AECA-negative acute KD sera (71.2±11.8, p<0.05), respectively. IgM-AECA from KD patients significantly induced ICAM-1 protein and mRNA expression. The upregulation of ICAM-1 expression was significantly inhibited by SB203580, DMAP and parthenolide, but not by PD98059. Conclusion：IgM-AECA was detected in the sera of about 2/3 of acute KD patients, which activated endothelial cells by upregulation of ICAM-1 expression, possibly via p38, JNK MAPK and NF-κB signal transduction pathways. Thus, IgM-AECA may play a pathogenic role in the development of coronary arterial lesions in KD patients. (Korean Circulation J 2006;36:723-731)

Dimethylaminopurine inhibits metabolic effects of insulin in primary adipocytes

Dimethylaminopurine (DMAP) has previously been used as an inhibitor of phosphorylation in studies of meiotic events, and more recently to investigate TNFα signaling, because of its potential to inhibit activation of c-jun N-terminal kinase (JNK). Here we have addressed the effects of DMAP on metabolic insulin responses in adipocytes and on intracellular insulin signaling molecules. At 100 μmol/L, DMAP completely inhibited the ability of insulin to counteract lipolysis in isolated adipocytes. Insulin-induced lipogenesis and glucose uptake was inhibited to a lesser degree in a concentration-dependent manner starting at 10 μmol/L DMAP. Insulin-induced tyrosine phosphorylation of the insulin receptor was not affected by DMAP. Insulin-induced activation of protein kinase B, a known mediator of insulin action, was not inhibited by 100 μmol/L, but to a low extent by 1 mmol/L DMAP in intact cells. This inhibition was not sufficient to affect activation of the downstream protein kinase B substrate phosphodiesterase 3B. The inhibition of activation of JNK as a possible mechanism whereby DMAP affects insulin-induced antilipolysis, lipogenesis, and glucose uptake, was investigated using the JNK inhibitor SP600125. At 100 μmol/L, SP600125 completely reversed the antilipolytic effect of insulin, as well as partially inhibited insulin-induced lipogenesis and glucose-uptake, indicating that JNK may be involved in mediating these actions of insulin. Inhibition of JNK by DMAP may therefore partly explain the negative impact of DMAP on insulin action in adipocytes.

Different activation treatments for successful development of bovine oocytes following intracytoplasmic sperm injection.

In this study, the developmental capacity and cytogenetic composition of different oocyte activation protocols was evaluated following intracytoplasmic sperm injection (ICSI) of in vitro matured bovine oocytes. Motile spermatozoa selected by Percoll density gradient were treated with 5 mM dithiothreitol (DTT) and analysed for ultrastructural changes of the head using transmission electron microscopy (TEM). The alterations in sperm morphology after DTT treatment for different times (15, 30 and 60 min) were 10%, 45-55% and 70-85%, respectively. Further, a partial decondensation of sperm heads was observed after DTT treatment for 30 min. Oocytes were injected with sperm treated with DTT for 30 min. In group 1, sperm injection was performed without any activation stimulus to the oocytes. In group 2, sham injection without sperm was performed without activating the oocytes. Oocytes injected with sperm exposed to 5 microM ionomycin for 5 min (group 3), 5 microM ionomycin + 1.9 mM dimethylaminopurine (DMAP) for 3 h (group 4) and 5 microM ionomycin + 3 h culture in M199 + 1.9 mM DMAP (group 5) were also evaluated for cleavage, development and chromosomal abnormality. Cleavage and development rates in groups 1, 2 and 3 were significantly (p < 0.05) lower than those in groups 4 and 5. The incidence of chromosomal abnormality in the embryos treated directly with DMAP after ionomycin (group 4) was higher than in group 5. We conclude that immediate DMAP treatment after ionomycin exposure of oocytes results in arrest of release of the second polar body, and thus leads to changes in chromosomal pattern. Therefore, the time interval between ionomycin and DMAP plays a crucial role in bovine ICSI.

Studies on the cytoplasmic protein tyrosine kinase activity of the Antarctic psychrotrophic bacteriumPseudomonas syringae

The Antarctic psychrotrophic bacterium Pseudomonas syringae contains a 66-kDa cytoplasmic protein which was found to by phosphorylated on a tyrosine residue [Ray, M.K. et al. (1994) FEMS Microbiol. Lett. 122, pp. 49-54]. To investigate the nature of the cytoplasmic protein tyrosine kinase and its role in the bacterial physiology, we carried out some biochemical studies of the enzyme in vitro in the presence of exogenous peptide substrates and expression studies in vivo at low and high temperature during various phases of growth. The results suggest that the protein tyrosine kinase associated with the cytoplasmic fraction of the bacterium has certain similarities and dissimilarities with the known eukaryotic tyrosine kinases. The protein tyrosine kinase could phosphorylate exogenous substrate corresponding to the N-terminal peptide of p34cdc2 kinase but could not do so on poly(Glu:Tyr). The enzyme could not be inhibited by genistein, staurosporine and dimethyl aminopurine, but could be inhibited by piceatannol which is a known competitive inhibitor of the peptide binding site of mammalian protein tyrosine kinases. The enzyme activity in the cytoplasm is uniquely inhibited by sodium orthovanadate (IC50 = 20 microM) which is a known protein tyrosine phosphatase inhibitor. The expression studies show that the enzyme is produced more at a higher temperature (22 degrees C) of growth than at lower temperature (4 degrees C) and during the stationary phase of growth of P. syringae.

Studies on the cytoplasmic protein tyrosine kinase activity of the Antarctic psychrotrophic bacterium Pseudomonas syringae

The Antarctic psychrotrophic bacterium Pseudomonas syringae contains a 66-kDa cytoplasmic protein which was found to be phosphorylated on a tyrosine residue [Ray, M.K. et al. (1994) FEMS Microbiol. Lett. 122, pp. 49–54]. To investigate the nature of the cytoplasmic protein tyrosine kinase and its role in the bacterial physiology, we carried out some biochemical studies of the enzyme in vitro in the presence of exogenous peptide substrates and expression studies in vivo at low and high temperature during various phases of growth. The results suggest that the protein tyrosine kinase associated with the cytoplasmic fraction of the bacterium has certain similarities and dissimilarities with the known eukaryotic tyrosine kinases. The protein tyrosine kinase could phosphorylate exogenous substrate corresponding to the N-terminal peptide of p34 cdc2 kinase but could not do so on poly(Glu:Tyr). The enzyme could not be inhibited by genistein, staurosporine and dimethyl aminopurine, but could be inhibited by piceatannol which is a known competitive inhibitor of the peptide binding site of mammalian protein tyrosine kinases. The enzyme activity in the cytoplasm is uniquely inhibited by sodium orthovanadate (IC 50=20 μM) which is a known protein tyrosine phosphatase inhibitor. The expression studies show that the enzyme is produced more at a higher temperature (22°C) of growth than at lower temperature (4°C) and during the stationary phase of growth of P. syringae.

Inhibition of Tumor Necrosis Factor Signal Transduction in Endothelial Cells by Dimethylaminopurine

Tumor necrosis factor (TNF) promotes diverse responses in endothelial cells that are important to the host response to infections and malignancies; however, less is known of the postreceptor events important to TNF action in endothelial cells than in many other cell types. Since phosphorylation cascades are implicated in cytokine signaling, the effects of the protein kinase inhibitor dimethylaminopurine (DMAP) on TNF action in bovine aortic endothelial cells (BAEC) were investigated. In BAEC, TNF promotes phosphorylation of eukaryotic initiation factor 4E (eIF-4E), c-Jun N-terminal kinase (JNK) and ceramide-activated protein kinase activities, Jun-b expression, prostacyclin production, and, when protein synthesis is inhibited, cytotoxicity. DMAP abrogated or significantly attenuated each of these responses to TNF, without affecting the specific binding of TNF to its receptors. Histamine, another agent active in the endothelium, promotes phosphorylation of elongation factor-2 (EF-2) and prostacyclin production, but not phosphorylation of eIF-4E in BAEC. Histamine-stimulated EF-2 phosphorylation was not inhibited and prostacyclin production was unaffected by DMAP. These observations demonstrate that a distinct signal transduction cascade, which can be selectively inhibited by DMAP, promotes the response of BAEC to TNF. Thus, we have identified a reagent, DMAP, that may be useful for characterizing the TNF signal transduction pathway.

Effect of isobutylmethylxanthine and related drugs on the receptor response (ERG; a-wave) of the frog retina at various extracellular calcium concentrations.

Various drugs known or expected to increase the levels of cyclic nucleotides in cells were applied to isolated superfused frog retinae, and their influence on the aspartate-isolated a-wave was studied. Isobutylmethylxanthine (IBMX), triacetylguanosine (TAG), and dimethylaminopurine (DAMP) strongly influenced the responses elicited from dark-adapted retinae by flashes of light: With all three drugs the response amplitude was increased, and latency and time to peak were prolonged. If, on the other hand, the retinae were light-adapted by background light of various intensities, the drugs showed different effects on the response amplitude: IBMX either did not influence the amplitude at all or even caused a decrease (4 of 6 experiments), DAMP decreased the amplitude and TAG caused an increase of the amplitude in 2 of 3 experiments. But latency and time to peak were still prolonged by all three drugs. When dark-adapted retinae were superfused with IBMX or TAG Ringer solution and simultaneously calcium concentration was raised, different effects of calcium on the three measured parameters of the a-wave were observed: By increasing the extracellular calcium concentration the increase of the amplitude caused by drugs was reversed, down or even below the control level, whereas latency and time to peak remained prolonged. Thus, both an increased calcium level and light adaptation had the same effect, namely to reverse only that part of the drug effect concerning the amplitude but not latency or time to peak of the response. The data suggest that calcium and cyclic nucleotides act through different ways in the rod cells.

Composition of the serum and lymph of rats with aminonucleoside-induced nephrosis.

Recently a nephrotic syndrome, characterized by proteinuria, hypoalbuminemia, hypercholesterolemia, hyperlipemia, and edema, has been produced in rats by the subcutaneous injection of an aminonucleoside (6 dimethylamino purine, 3-amino-d-ribose). The serum changes in nephrosis may be reflexed in lymph and tissue fluid. Information on the composition of lymph in nephrosis is, however, limited. The present studies were carried out to evaluate the role of lymph in carrying various components in renal or nephrotic edema to the general circulation.