Preparation of long sticky ends for universal ligation-independent cloning: Sequential T4 DNA polymerase treatments

Ligation-independent cloning (LIC) is a useful method for efficient directional cloning of a PCR product. LIC requires a specially designed vector containing a long stretch of sequence that is missing any one of the four nucleotides. When the linearized vector is treated with T4 DNA polymerase, in the presence of the absent base, long single-stranded overhangs are generated that are suitable for cloning. In this study, long and efficient sticky ends for LIC were produced by sequential T4 DNA polymerase treatments at non-specific sequences on a commercially available vector. All restriction enzyme sites become available in the current LIC.     

The nature of adolescent competencies predicted by preschool delay of gratification.

Delay of gratification, assessed in a series of experiments when the subjects were in preschool, was related to parental personality ratings obtained a decade later for 95 of these children in adolescence. Clear and consistent patterns of correlations between self-imposed delay time in preschool and later ratings were found for both sexes over this time span. Delay behavior predicted a set of cognitive and social competencies and stress tolerance consistent with experimental analyses of the process underlying effective delay in the preschool delay situation. Specifically, children who were able to wait longer at age 4 or 5 became adolescents whose parents rated them as more academically and socially competent, verbally fluent, rational, attentive, planful, and able to deal well with frustration and stress. Comparisons with related longitudinal research using other delay situations help to clarify the important features of the situations and person variables involved in different aspects of delay of gratification. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effects of various amino acids on gastric lesions induced by acetylsalicylic acid (ASA) and gastric secretion in pylorus-ligated rats

The simultaneous oral administration of various amino acids such as L-lysine, L-arginine, L-histidine, L-serine and others at 750, 250 or 83.3 mg/kg in pylorus-ligated rats produced a marked prevention of the gastric mucosal damages caused by oral acetylsalicylic acid (ASA) at 100 mg/kg. In regard with L-lysine and L-arginine, it was assumed that these amino acids might inhibit the ASA-induced gastric lesions through neutralization of acid because of the high alkalinity of these amino acids. In addition, the lesser effect of the hydrochoride salts of these amino acids as compared with the free form on ASA-induced gastric lesions was observed. The other effective amino acids markedly prevented the back diffusion of acid in response to ASA, suggesting as one of the possible mechanisms of lesion formation. However, L-cysteine, which exerted insignificant effect on ASA-induced gastric lesions, also prevented the back diffusion of acid even though the Na+ concentration had not returned to the control level.     

Visualization of leukocyte dynamics in the choroid with indocyanine green

PURPOSE: To report a method to evaluate leukocyte dynamics in the choroidal circulation with indocyanine green (ICG) angiography. METHODS: Nonpigmented and pigmented rats were administered ICG solution intravenously. The fundus image was obtained with a scanning laser ophthalmoscope and recorded on magnetic tapes at a video rate (30 frames/second). The images were analyzed with a personal computer-based image analysis system. RESULTS: On ICG angiography, hyperfluorescent dots were seen moving along the choroidal vessels and in the retinal vessels several minutes after injection. These fluorescent dots were thought to be circulating leukocytes stained with ICG. The micrographs of blood smears after ICG injection showed intense fluorescence of leukocytes. Computer-assisted image analysis allowed tracing of these fluorescent dots using a frame-by frame method. CONCLUSIONS: Results of this study indicated that ICG can be used for vital staining of leukocytes and that it is possible to evaluate leukocyte movement in the choroidal circulation in vivo in rats.     

Novel DNA detection system of flow injection analysis (2). The distinctive properties of a novel system employing PNA (peptide nucleic acid) as a probe for specific DNA detection

In order to realize immediate detection of a double stranded DNA amplified by Polymerase Chain Reaction (PCR), we applied Peptide Nucleic Acid (PNA) to the probe of DNA detection system using Surface Plasmon Resonance (SPR). We report our success in immediate detection of PCR products solution with high sequence-specificity.     

High concentrations of cholecystokinin octapeptide suppress protein kinase C activity in guinea pig pancreatic acini

In pancreatic acini, calcium-mobilizing agents increase intracellular calcium and stimulate the production of diacylglycerol, and then activate protein kinase C (PKC). However, there are few studies which have examined the activation of PKC in intact acini. To examine the activation of PKC in intact acini by calcium-mobilizing agents, we measured the binding of [3H]phorbol-12,13-dibutyrate (PDBu) to intact acini. Acini were incubated with 10 nM [3H]PDBu at 25 degrees C with or without agents. The binding reactions were terminated by filtration. The filters were counted by a scintillation counter after washing. Acini possessed a single class of binding sites to PDBu, with Kd = 70 nM. CCK-8 and carbachol upregulated the binding affinity of PKC to PDBu in the acini. The ability of calcium-mobilizing agents to increase binding of [3H]PDBu to the acini had a close correlation to their ability to stimulate the amylase secretion from the acini, and higher concentrations of CCK-8 for amylase secretion suppressed binding of [3H]PDBu to the acini. 8Br-cAMP, 8Br-cGMP, and calcium ionophore did not inhibit the maximal activation of PKC induced by CCK-8. The calmodulin inhibitor W7 did not reverse the inhibitory effect of higher concentrations of CCK-8 on PKC activation. These results indicate that calcium-mobilizing agents upregulate the binding affinity of PKC to PDBu in intact acini, and that higher concentrations of CCK-8 for amylase secretion may activate the intracellular mechanism that inhibits PKC activity in acini. This inhibitory mechanism was mediated by some other mechanism other than cAMP-, cGMP-, calcium- and calmodulin-dependent mechanisms.     

Decreased GA1 content caused by the overexpression of OSH1 is accompanied by suppression of GA 20-oxidase gene expression

We previously reported that overexpression of the rice homeobox gene OSH1 led to altered morphology and hormone levels in transgenic tobacco (Nicotiana tabacum L.) plants. Among the hormones whose levels were changed, GA1 was dramatically reduced. Here we report the results of our analysis on the regulatory mechanism(s) of OSH1 on GA metabolism. GA53 and GA20, precursors of GA1, were applied separately to transgenic tobacco plants exhibiting severely changed morphology due to overexpression of OSH1. Only treatment with the end product of GA 20-oxidase, GA20, resulted in a striking promotion of stem elongation in transgenic tobacco plants. The internal GA1 and GA20 contents in OSH1-transformed tobacco were dramatically reduced compared with those of wild-type plants, whereas the level of GA19, a mid-product of GA 20-oxidase, was 25% of the wild-type level. We have isolated a cDNA encoding a putative tobacco GA 20-oxidase, which is mainly expressed in vegetative stem tissue. RNA-blot analysis revealed that GA 20-oxidase gene expression was suppressed in stem tissue of OSH1-transformed tobacco plants. Based on these results, we conclude that overexpression of OSH1 causes a reduction of the level of GA1 by suppressing GA 20-oxidase expression.     

Doxorubicin cardiotoxicity: diastolic cardiac myocyte dysfunction as a result of impaired calcium handling in isolated cardiac myocytes

We examined intracellular calcium transients of isolated single cardiac myocytes from rats with doxorubicin (DOX)-induced cardiomyopathy with simultaneous measurement of cell motion. DOX was administered i.p. to Sprague-Dawley rats at 2.5 mg/kg once a week for 10 weeks. Field-stimulated calcium transients and simultaneous cell motion in single myocytes were measured in the presence or absence of isoproterenol using fura-2/AM. Histopathologic examination revealed slight changes. The time courses of both calcium transients and cell motion were significantly prolonged by DOX. There was a slight but not significant reduction in parameters of contractility in both calcium transients and cell motion. The beta-adrenoceptor responsiveness of both calcium transients and cell motion was not significantly impaired compared with the controls. Our data indicated that, despite the slight histologic changes in the heart in DOX-induced cardiomyopathy, impaired sequestration of intracellular free calcium ions in individual myocytes may be one factor leading to diastolic dysfunction. Monitoring of diastolic function is important to detect early cardiotoxicity caused by DOX.