Hybrid nanoreservoirs based on dextran-capped dendritic mesoporous silica nanoparticles for CD133-targeted drug delivery

In this study, the chemical features of dendritic mesoporous silica nanoparticles (DMSNs) provided the opportunity to design a nanostructure with the capability to intelligently transport the payload to the tumor cells. In this regard, doxorubicin (DOX)-encapsulated DMSNs was electrostatically surface-coated with polycarboxylic acid dextran (PCAD) to provide biocompatible dextran-capped DMSNs (PCAD-DMSN@DOX) with controlled pH-dependent drug release. Moreover, a RNA aptamer against a cancer stem cell (CSC) marker, CD133 was covalently attached to the carboxyl groups of DEX to produce a CD133-PCAD-DMSN@DOX. Then, the fabricated nanosystem was utilized to efficiently deliver DOX to CD133+ colorectal cancer cells (HT29). The in vitro evaluation in terms of cellular uptake and cytotoxicity demonstrated that the CD133-PCAD-DMSN@DOX specifically targets HT29 as a CD133 overexpressed cancer cells confirmed by flow cytometry and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. The potentially promising intelligent-targeted platform suggests that targeted dextran-capped DMSNs may find impressive application in cancer therapy.     

Effectiveness of chemical mutagenesis in multiple damage to one or both strands of plasmid DNA

Methods for site-directed multiple modification of DNA have been developed and used for modification of either one or two strands of plasmid DNA. Plasmid DNAs modified in the region of the tet gene were transformed into Escherichia coli cells and Tet colonies were screened. It was shown that multiple lesions in one DNA strand performed using either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or sodium bisulfite were effectively repaired in the cell by error-free mechanism. In contrast, modification of two DNA strands led to induction of mutations. The efficiency of mutagenesis in the case of modification of a local region of one DNA strand with sodium bisulfite and modification of the other strand with MNNG was 1.1-7.9%. Mutations were analysed by restriction mapping and sequencing. All of them were G----A transitions.     

Production of ethanol by alginate-entrapped Saccharomyces cerevisiae strain "14-12"

Cells of S. cerevisiae strain "14-12" of different ages were immobilized in sodium alginate and used for conversion of glucose to ethanol. Immobilized cells of 48 hr old were the most potential. Employment of high counts of alginate-entrapped cells shortened the period required for production of the maximal alcohol yield. However, the percentage surviving cells decreased with increasing initial cell counts. Maximal accumulation of ethanol (4.18 g/100 ml) was obtained after 4 days of static fermentation with 1.8 X 10(8) immobilized yeast cells. The residual viable cell count was found to represent 3-fold the surviving percentage in a control experiment using an inoculum of the free yeast cells. Immobilized yeast cells could convert about 85% of the available sugars to ethanol over 28 days of the repeated-batch fermentation. The immobilized cells retained 50% of their viability for 16 days. After 48 days of repeated fermentation only 6% of the yeast cells were viable, and on the 52nd day no viable cells could be detected.     

Digitalis receptor sugar binding site characteristics: A model based upon studies of Na+, K+-ATPase preparations with differing digitalis sensitivities

The structure-activity relationships of the genin moieties of digitalis glycosides are commonly elucidated by determining the inhibitory potency of a variety of genins toward the plasma membrane Na⁺, K⁺-ATPase; qualitatively these relationships appear to be fairly independent of the specific Na⁺, K⁺-ATPase preparation utilized for the analysis. To determine whether this is the case with regard to the sugar moieties of glycosides, the inhibitory effects of 12 monoglycosides of digitoxigenin toward four Na⁺, K⁺-ATPase preparations of different origin were measured. It was found that while recognition of the major structural determinants of sugar activity appeared to be independent of enzyme source, recognition of the minor structural determinants of activity showed some source dependence. It was also observed that the intrinsic sensitivity to sugar potentiation may be source dependent and unrelated to intrinsic sensitivity to inhibition by digitoxigenin. These observations are compatible with a model of the Na⁺, K⁺-ATPase sugar binding site(s) in which intrinsic sensitivity to sugar attachment as well as recognition characteristics (for sugar structural features) both determine the extent to which a sugar moiety may contribute to the activity of monoglycosides. Further, in these studies one of the Na⁺, K⁺-ATPase preparations employed was obtained from rat brain, a tissue known to contain a mixture of ouabain sensitive and insensitive isoforms. We have observed that the rigorous purification techniques employed appear to have selectively removed from or denatured the less ouabain sensitive al isoform found in this enzyme preparation.     

Loss of CpG dinucleotides from DNA. VI. Methylation of mitochondrial and chloroplast genes

From nucleotide sequences of mitochondrial and chloroplast genes the probable frequency of the CpG----TpG + CpA substitutions was determined. These substitutions may indicate the level of prior DNA methylation. It was found that the level of this methylation is significantly lower in mitochondrial DNA (mtDNA) and chloroplast DNA (chDNA) than in nuclear DNA (nDNA) of the same species. The species (taxon) specificity of mtDNA and chDNA methylation was revealed. A correlation was found between the level of CpG methylation in nDNA, and mtDNA and chDNA in different organisms. It is shown that cytosine residues in CpG were not subjected to significant methylation in the fungi and invertebrate mtDNA and also in the algae chDNA. In contrast, the vertebrate mtDNA bears the impress of CpG-supression, which is confirmed by direct data on methylation of these DNA. Here the first data on the possible enzymatic methylation of the plant mtDNA and chDNA were obtained. It was shown that the degree of CpG-suppression in the 5S rRNA nuclear genes of lower and higher plants is significantly higher in the chloroplast genes of 4,5S and 5S rRNA. From data on pea chDNA hydrolysis with MspI and HpaII it was established that in CCGG sequences this DNA is not methylated. The role of DNA methylation in increasing the mutation rate and in accelerating the evolutionary rates of vertebrate mtDNA is discussed.     

The chemical mutagen dimethyl sulphate induces homologous recombination of plasmid DNA by increasing the binding of RecA protein to duplex DNA.

The role of different DNA damages in the stimulation of homologous recombination was studied by using an in vivo plasmid recombination assay. Dimethyl sulphate (DMS) treatment of plasmid DNA induced a 20-50-fold increase in the frequency of recombinational events. DMS treatment also stimulated RecA protein binding to double-stranded DNA. In contrast, plasmid DNA containing uracil, which, like DMS, is also subject to repair, was less effective in stimulation of recombination. The ability of purified RecA protein to bind DMS-treated or uracil-containing DNA was tested by measuring its ATPase activity. The result indicates that DMS treatment, but not uracil incorporation, stimulates RecA protein binding to DNA. We conclude, that the main reason (or the first step) for stimulation of recombination by mutagens is activation of RecA binding to damaged DNA.     

Ethanol tolerance ofSaccharomyces cerevisiae and its relationship to lipid content and composition

Saccharomyces cerevisiae strain 14-12 is a highly ethanol-tolerant organism. It can grow in the presence of 13% ethanol but growth is completely prevented at 14% ethanol. A relationship was detected between yeast lipids and ethanol tolerance. A gradual decrease of lipid content was recorded as the concentration of supplemented ethanol increased. Moreover, free fatty acids were comparatively decreased in these lipid extracts. When separately added to media with 14% ethanol different lipids produced varied stimulatory effects on yeast growth. Maximum yield of yeast growth was obtained at 14% ethanol in the presence of lecithin, palmitic acid and cholesterol. Yeast lipids produced in the presence of these fractions are characterized by a relatively high percentage of free fatty acids. The change in the percentage of free fatty acids was shown to be the controlling factor in ethanol tolerance.     

Antigenic relatedness between human lecithin-cholesterol acyltransferase and phospholipases of the A2 family

A monoclonal antibody, B10, generated against pure human lecithin-cholesterol acyltransferase (EC 2.3.1.43) caused the inhibition of the esterolytic and cholesterol esterifying activities of the enzyme. This antibody also reacted with a number of pancreatic and snake venom phospholipases A2 species but not phospholipase A1. A concentration-dependent inhibition of phospholipase A2 was also seen in the presence of B10. Treatment of lecithin-cholesterol acyltransferase or B10-reacting phospholipases with phenacyl bromide, a reagent known to interact with the active site of phospholipase A2, inhibited both their esterolytic activity and their capacity to bind to B10. A dimeric phospholipase A2 species with a known occluded active site did not cross-react with B10. Thus, lecithin-cholesterol acyltransferase and some enzymes of the phospholipase A2 family share a common antigenic determinant which is probably located near or at their esterolytic active site.     

Neurotensin stimulates pancreatic exocrine secretion in rats

Neurotensin (NT) stimulates pancreatic exocrine secretion in dogs and humans. The purpose of this study was to examine the effect of exogenous neurotensin on pancreatic exocrine secretion in rats. Five Sprague-Dawley male rats were prepared with pancreatic, gastric and duodenal fistulas. Bile was shunted into the duodenum in order to collect pure pancreatic juice. 24 h later, neurotensin (0.05, 0.1, 0.2, 0.3, 1.0 nmol/kg) was infused intravenously in a random fashion. Pancreatic juice was collected every 10 min, and the volume was recorded and protein and bicarbonate were measured. Neurotensin stimulated, in a dose-related manner, the pancreatic secretion of water, protein and bicarbonate. Neurotensin may be involved in the physiologic control of pancreatic secretion in rats.     

Spontaneous binding ofYersinia enterocolitica to murine leukocytes

Twelve strains ofYersinia enterocolitica were examined for their ability to bind spontaneously to murine leukocytes. Each of eight HeLa cell invasive strains exhibited nonselective binding to peritoneal leukocytes, lymph node leukocytes, and thymocytes, whereas four noninvasive strains lacked binding properties. Like the HeLa cell invasion, the binding ofY. enterocolitica to leukocytes was much less efficient for bacteria grown at 37°C than for bacteria grown at 22°C. The binding properties were not influenced by the virulence plasmid that codes for Vwa⁺ phenotype. This leukocyte binding test is proposed as a simple assay for invasive properties ofY. enterocolitica.