Effect of unusual polyamines on cleavage of DNA by restriction enzymes

The effect of unusual polyamines, such as thermine, caldopentamine, caldohexamine, tris(3-aminopropyl)amine, or tetrakis(3-aminopropyl)ammonium, on the activities of various restriction endonucleases was investigated by using an Escherichia coli plasmid as a substrate, which contains a high GC content fragment from an extreme thermophile. Restriction enzymes used were SmaI, BanII, NaeI, RsaI, and TaqI. Most of the polyamines tested were inhibitory to the enzyme activities. The larger and more branched a polyamine was, the more the activities of nucleases were inhibited. The inhibition was positively correlated with the polyamine concentration. The sites protected by a polyamine were identical to those protected by other polyamines, and also identical to those which were less sensitive to the restriction enzyme in the absence of polyamines. No sequence specificity was seen among these sites.     

Localization of aldosterone-producing adenoma: venous sampling in primary aldosteronism

Adrenal venous sampling of blood was performed for nine patients with aldosterone-producing adenoma (APA). Measurement of adrenal venous aldosterone is useful for localization of APA but difficult, because catheterization of the right adrenal vein is not easy, and the blood is diluted by nonadrenal flow. To solve these problems, levels of aldosterone (A; ng/dl) and cortisol (C; micrograms/dl) were measured in samples from the left adrenal vein (LAV) and the inferior vena cava (IVC), and the LAV A/C and (LAV A/C)/(IVC A/C) ratios were calculated. These ratios were also obtained for 16 patients with essential hypertension. The adenoma could be localized in three of the nine cases by the measurement of aldosterone alone, but the use of a LAV A/C ratio greater than 5 x 10(-3) and a (LAV A/C)/(IVA A/C) ratio less than 1.0 as criteria separated the patients into those with a left APA, right APA, or essential hypertension. Consequently, adrenal venous sampling and the calculation of these ratios enables preoperative localization of APA with more accuracy, especially when the tumor is small or the result of CT and adrenal scintigraphy is not consistent.     

Accumulation pattern of arachin and its subunits in maturation of groundnut seeds

The accumulation pattern of arachin and its subunits in growinggroundnuts was investigated. Soluble proteins were extractedfrom the kernels at twelve different stages of maturation (4–16weeks after pegging). Fractionation showed arachin, conarachinII, 5S and 2S protein components with sucrose gradient centrifugation.Ten weeks after pegging, only 35% of the maximum amount of arachinhad accumulated, whereas conarachin II was 85%, the 5S component89%, and the 2S component 76%. Arachin, however, increased rapidlyin the later stage of maturation. No change in the subunit ratioin arachin during seed growth was observed on the patterns ofsodium dodecyl sulfate-gel electrophoresis and gel isoelectricfocusing in the presence of urea. The ratio of the arachin subunitscontained in urea-extractable fraction of the kernels was constantthroughout seed development and was consistent with the subunitratio in arachin. On the other hand, the arachin subunits inthe free forms, if any, accounted for less than 1% of the associatedarachin subunits. Probably, the arachin subunits synthesizedin equimoles are associated into arachin without individualdeposition and are accumulated as arachin associates in growingseeds. (Received July 17, 1980; )     

Molecular motion and order in oriented lipid multibilayer membranes evaluated by simulations of spin label ESR spectra. Effects of temperature,cholesterol and magnetic field

A simulation method to interpret electron spin resonance (ESR) of spin labelled amphiphilic molecules in oriented phosphatidylcholine multibilayers in terms of a restricted motional model is presented. Order and motion of the cholestane spin label (3-spiro-doxyl-5α-cholestane) incorporated into egg yolk phosphatidylcholine, dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine, pure and in mixture with cholesterol, were studied at various termperatures. With egg yolk phosphatidylcholine identical sets of motional parameters were obtained from simulations of ESR spectra obtained at three microwave frequencies (X-, K- and Q-band). With dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine analyses of the spectra show that phase transitions occur in samples containing up to 30 mol % cholesterol. The activation energy for the motion of the spin label is about three times larger above than below the phase transition, indicating a more collective motion in the liquid crystalline state than in the gel state. In the liquid crystalline state the activation energy is larger in the pure phosphatidylcholines than with cholesterol added. Additions of cholesterol to egg phosphatidylcholine induces a higher molecular order but does not appreciably affect correlation times. This is in contrast to dipalmitoylphosphatidylcholine where both order and correlation times are affected by the presence of cholesterol. The activation energies follow the same order as the transition temperatures: dipalmitoylphosphatidylcholine > dimyristoylphosphatidylcholine > egg yolk phosphatidylcholine, suggesting a similar order of the cooperativity of the motion of the lipid molecules. Magnetic field-induced effects on egg phosphatidylcholine multibilayers.     

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T₀ plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T₁ generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T₀ plants, we could remove foreign DNA easily by genetic segregation in the T₁ generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

     

Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish

A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.