The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [³H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.     

Assignment of the sorbitol dehydrogenase locus to human chromosome 15 pter→q21

The locus for sorbitol dehydrogenase (SORD, E.C. 1.1.1.14) has been shown to segregate with hexosaminidase A and mannose phosphate isomerase in a series of human-Chinese hamster somatic cell hybrids. Cytogenetic analysis supports the assignment to chromosome 15 and further defines the gene locus to the region 15pterq21.This research was supported by the Medical Research Council of Canada (MT 4061), the Children's Hospital of Winnipeg Research Foundation, Inc., and the Department of Health, Province of Manitoba (H.S.W.).     

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.     

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.     

Chromosome-level genome assembly and characterization of Sophora Japonica

Sophora japonica is a medium-size deciduous tree belonging to Leguminosae family and famous for its high ecological, economic and medicinal value. Here, we reveal a draft genome of S. japonica, which was ∼511.49 Mb long (contig N50 size of 17.34 Mb) based on Illumina, Nanopore and Hi-C data. We reliably assembled 110 contigs into 14 chromosomes, representing 91.62% of the total genome, with an improved N50 size of 31.32 Mb based on Hi-C data. Further investigation identified 271.76 Mb (53.13%) of repetitive sequences and 31,000 protein-coding genes, of which 30,721 (99.1%) were functionally annotated. Phylogenetic analysis indicates that S. japonica separated from Arabidopsis thaliana and Glycine max ∼107.53 and 61.24 million years ago, respectively. We detected evidence of species-specific and common-legume whole-genome duplication events in S. japonica. We further found that multiple TF families (e.g. BBX and PAL) have expanded in S. japonica, which might have led to its enhanced tolerance to abiotic stress. In addition, S. japonica harbours more genes involved in the lignin and cellulose biosynthesis pathways than the other two species. Finally, population genomic analyses revealed no obvious differentiation among geographical groups and the effective population size continuously declined since 2 Ma. Our genomic data provide a powerful comparative framework to study the adaptation, evolution and active ingredients biosynthesis in S. japonica. More importantly, our high-quality S. japonica genome is important for elucidating the biosynthesis of its main bioactive components, and improving its production and/or processing.     

Effects of Luteolin on Human Breast Cancer Using Gene Expression Array: Inferring Novel Genes

Taraxacum officinale (dandelion) is often used in traditional Chinese medicine for the treatment of cancer; however, the downstream regulatory genes and signaling pathways mediating its effects on breast cancer remain unclear. The present study aimed to explore the effects of luteolin, the main biologically active compound of T. officinale, on gene expression profiles in MDA-MB-231 and MCF-7 breast cancer cells. The results revealed that luteolin effectively inhibited the proliferation and motility of the MDA-MB-231 and MCF-7 cells. The mRNA expression profiles were determined using gene expression array analysis and analyzed using a bioinformatics approach. A total of 41 differentially expressed genes (DEGs) were found in the luteolin-treated MDA-MB-231 and MCF-7 cells. A Gene Ontology analysis revealed that the DEGs, including AP2B1, APP, GPNMB and DLST, mainly functioned as oncogenes. The human protein atlas database also found that AP2B1, APP, GPNMB and DLST were highly expressed in breast cancer and that AP2B1 (cut-off value, 75%) was significantly associated with survival rate (p = 0.044). In addition, a Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the DEGs were involved in T-cell leukemia virus 1 infection and differentiation. On the whole, the findings of the present study provide a scientific basis that may be used to evaluate the potential benefits of luteolin in human breast cancer. Further studies are required, however, to fully elucidate the role of the related molecular pathways.