Rapid mapping of restriction sites of a DNA: restriction of agarose gel and two-dimensional analysis of end-labeled DNA.

A new two-dimensional technique for the mapping of restriction sites is presented. Linear DNA labeled at both ends is first partially digested with the restriction endonuclease for which a map is desired. Following electrophoresis of the partial digest in an agarose gel, complete digestion of the fragments in the gel matrix with a second restriction enzyme is carried out. Electrophoresis in the second dimension resolves two sets of labeled spots: one set from the left and the other from the right end. For a given band of the autoradiogram of the first dimension gel, the mobility of the band gives the size of the DNA fragment, and therefore the distance of a particular restriction site from one of the ends of the original linear DNA. The mobility of the labeled spot derived from this band in the second dimension gel allows one to distinguish whether the distance deduced above is from one end or the other. Additional information about the location of one set of restriction sites for one enzyme relative to those for a second enzyme can also be obtained using the two-dimensional method. The advantages of the technique are the small amount of DNA required and the rapidity with which many maps can be constructed from one labeled DNA. As a test of the method, maps for the HindIII and HaeIII cleavage sites of circular phage PM2 DNA have been obtained, after first converting the DNA to the linear form by digestion with HpaII.     

Suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and of incorporation of acetate into cholesterol in homozygous hypercholesterolemic fibroblasts by ferritin-low density lipoprotein conjugates.

Conjugates of ferritin with low density lipoproteins (LDL) were prepared and separated by sucrose gradient centrifugation. These conjugates, at cholesterol concentration of 100--132 microgram/ml, caused a greater than 90% suppression of hydroxymethylglutaryl coenzyme A reductase activity and of acetate incorporation into cholesterol in cultured skin fibroblasts from a normal subject as well as from a subject with homozygous familial hypercholesterolemia. The half maximal inhibition concentration was approx. 10 microgram/ml cholesterol for LDL and ferritin . (LDL)2 and 5 microgram/ml for (ferritin)2 . LDL in both cell lines. In contrast, native low density lipoproteins have only a minimal inhibitory effect in homozygous cells. The ability of the conjugates to stimulate the incorporation of oleate into cholesteryl esters was also equal in the two cell lines, although the conjugates were only 10% as active as low density lipoproteins in the normal cells. LDL reduced the ferritin . (LDL)2-mediated suppression of hydroxymethylglutaryl-CoA reductase activity in homozygous cells while ferritin . (LDL)2 reduced the LDL-mediated stimulation of cholesteryl ester formation in normal cells.     

Growth of Enterobacter aerogenes in a chemostat with double nutrient limitations.

The behavior of Enterobacter aerogenes during growth in chemostats limited by single and double nutrient restrictions was examined. On the assumption that different essential nutrients act to limit growth in different ways, we selected pairs of nutrients likely to affect different aspects of metabolism. Results show that macromolecular cell composition can be controlled by using more than one nutrient restriction. The polysaccharide content of the cells is readily manipulated by the ratio of carbon to nitrogen in the inlet nutrients. Also, at low dilution rates, ratios of protein to ribonucleic acid are dependent on the ratio of phosphate to nitrogen in the input nutrients. An examination of both acetic acid and metabolite production (as measured by ultraviolet absorbance of culture filtrates) showed that accumulation of these products was dependent on both dilution rate and type of nutrient limitation(s). These results were examined in terms of the problems of translation of batch to continuous culture processes and the use of selected nutrient limitations to control noncellular product formation.     

Generation of ATP by chloroplasts through solvent perturbation.

A two-stage method was discovered for generating ATP by chloroplasts in the dark at constant pH through solvent perturbation. With cold acetone as the perturbing solvent, the yield of ATP was found to increase with the volume percent of acetone in the first stage medium. The results are difficult to explain in term of the proton gradient model, but is consistent with the conventional model of prior water formation and subsequent ATP generation.     

A simple, sensitive, and specific assay for free choline in plasma.

This paper describes a sensitive and specific enzymatic-radioisotopic method for determining plasma choline. Assays may be performed without prior extraction of the tissue. Plasma is first heated to destroy enzymes that would otherwise produce free choline from that which is normally bound. The free choline in plasma is then converted to phosphorylcholine [³²P], in the presence of ATP-γ-³²P, in a reaction catalyzed by choline kinase. Phosphorylcholine [³²P], isolated by ion-exchange chromatography, is measured as an index of the concentration of free choline. The concentration of plasma choline in man and in several species of laboratory animals was determined, and found to range from 5.5 nmoles/ml in dogs to 15.4 nmoles/ml in guinea pigs. The concentration of free choline in plasma of adult rats raised on a choline-deficient diet was half that of littermate controls raised on a control diet supplemented with free choline.     

JQ-1 ameliorates schistosomiasis liver granuloma in mice by suppressing male and female reproductive systems and egg development of Schistosoma japonicum

Schistosomiasis is a serious and widespread parasitic disease caused by infection with Schistosoma. Because the parasite’s eggs are primarily responsible for schistosomiasis dissemination and pathogenesis, inhibiting egg production is a potential approach to control the spread and severity of the disease. The bromodomain and extra-terminal (BET) proteins represent promising targets for the development of epigenetic drugs against Schistosoma. JQ-1 is a selective inhibitor of the BET protein family. In the present study, JQ-1 was applied to S. japonicum in vitro. By using laser confocal scanning microscopy and EdU incorporation assays, we showed that application of JQ-1 to worms in vitro affected egg laying and the development of both the male and female reproductive systems. JQ-1 also inhibited the expression of the reproductive-related genes SjPlk1 and SjNanos1 in S. japonicum. Mice infected with S. japonicum were treated with JQ-1 during egg granuloma formation. JQ-1 treatment significantly reduced the size of the liver granulomas and levels of serum alanine aminotransferase and aspartate aminotransferase in mice and suppressed both egg laying and the development of male and female S. japonicum reproductive systems in vivo. Moreover, the mRNA expression levels of some proinflammatory cytokines were decreased in the parasites. Our findings suggest that JQ-1 treatment attenuates S. japonicum egg–induced hepatic granuloma due at least in part to suppressing the development of the reproductive system and egg production of S. japonicum. These findings further suggest that JQ-1 or other BET inhibitors warrant additional study as a new approach for the treatment or prevention of schistosomiasis.     

Electrospun Scaffold of Collagen and Polycaprolactone Containing ZnO Quantum Dots for Skin Wound Regeneration

Nanofibers(NFs)have been widely used in tissue engineering such as wound healing.In this work,the antibacterial ZnO quantum dots(ZnO QDs)have been incorporated into the biocompatible poly(ε-caprolactone)/collagen(PCL/Col)fibrous scaffolds for wound healing.The as-fabricated PCL-Col/ZnO fibrous scaffolds exhibited good swelling,antibacterial activity,and biodegradation behaviors,which were beneficial for the applications as a wound dressing.Moreover,the PCL-Col/ZnO fibrous scaffolds showed excellent cytocompatibility for promoting cell proliferation.The resultant PCL-Col/ZnO fibrous scaffolds containing vascular endothelial growth factor(VEGF)also exhibited promoted wound-healing effect through promoting expression of transforming growth factor-β(TGF-β)and the vascular factor(CD31)in tissues in the early stages of wound healing.This new electrospun fibrous scaffolds with wound-healing promotion and antibacterial property should be convenient for treating wound healing.