DNA restriction--modification enzymes of phage P1 and plasmid p15B. Subunit functions and structural homologies

We have purified the type III restriction enzymes EcoP1 and EcoP15 to homogeneity from bacteria that contain the structural genes for the enzymes cloned on small, multicopy plasmids and which overproduce the enzymes. Both of the enzymes contain two different subunits. The molecular weights of the subunits are the same for both enzymes and antibodies prepared against one enzyme cross-react with both subunits of the other. Bacteria containing a plasmid derivative in which a large part of one of the structural genes has been deleted have a restriction- modification+ phenotype and contain only the smaller of the two subunits. This subunit therefore must be the one that both recognizes the specific DNA sequence and methylates it in the modification reaction (the restriction enzyme itself also acts as a modification methylase). We have purified the P1 and P15 modification subunits from these deletion derivatives and have shown that in vitro they have the expected properties: they are sequence-specific modification methylases. In addition, we have demonstrated that strains carrying the full restriction/modification system also contain a pool of free modification subunits that might be responsible for in vivo modification.     

Effect of alkylation with streptozotocin on the secondary structure of DNA

S₁ nuclease hydrolysis and hydroxyapatite chromatography were used to study the effect of the alkylating antibiotic, streptozotocin, on the secondary structure of DNA. Native calf thymus DNA was alkylatedin vitro with increasing concentrations of streptozotocin and subjected to S 1 nuclease hydrolysis. An increasing degree of DNA degradation was seen, suggesting a destabilization of the secondary structure. Indirect evidence, deduced from alkaline hydrolysis, effect of NaCl on S₁ nuclease hydrolysis, and hydroxyapatite chromatographic analysis of alkylated DNA, suggested a significant alkylation of DNA phosphates in addition to DNA bases. Nictotinamide has been reported to alter the cytotoxic and carcinogenic effects of streptozotocin. Our experiments indicate that in the presence of nicotinamide, streptozotocin causes the formation of a greater proportion of alkylated bases in relation to alkyl phosphotriesters. This may have significance in relation to the differential cytotoxicity of streptozotocin in the absence and presence of nicotinamide.     

H19/Igf2 Expression and Methylation of Histone 3 in Mice Chimeric Blastocysts

Background:Currently, the efficient production of chimeric mice and their survival are still challenging. Recent researches have indicated that preimplantation embryo culture media and manipulation lead to abnormal methylation of histone in the H19/Igf2 promotor region and consequently alter their gene expression pattern. This investigation was designed to evaluate the relationship between the methylation state of histone H3 and H19/Igf2 expression in mice chimeric blastocysts.Methods:Mouse 129/Sv embryonic stem cells (mESCs) expressing the green fluorescent protein (mESCs-GFP) were injected into the perivitelline space of 2.5 days post-coitis (dpc) embryos (C57BL/6) using a micromanipulator. H3K4 and H3K9 methylation, and H19 and Igf2 expression was measured by immunocytochemistry and q-PCR, respectively, in blastocysts. Results:Histone H3 trimethylation in H3K4 and H3K9 in chimeric blastocysts was significantly less and greater, respectively (p< 0.05), than in controls. H19 expression was significantly less (p< 0.05), while Igf2 expression was less, but not significantly so, in chimeric than in control blastocysts.Conclusion:Our results showed, that the alteration ofH3K4me3 and H3K9me3 methylation, change H19/Igf2 expression in chimeric blastocysts.Key Words: Chimeric blastocysts, H19/Igf2, Histone 3 (H3) methylation     

Single-walled carbon nanotube interactions with HeLa cells

This work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma – mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOX™ Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.     

Role of ATP in the cleavage mechanism of the EcoP15 restriction endonuclease

The EcoP15 restriction endonuclease forms complexes at specific sites on unmodified DNA both in the presence and in the absence of S-adenosyl-l-methionine. ATP acts as an allosteric effector of EcoP15 and induces DNA cleavage followed by release of the enzyme from the DNA. The efficiency of endonucleolytic scission varies from site to site. The nucleotide sequences at sites that are cleaved at a high frequency were compared.     

Sciatic nerve transection in neonatal rats induces apoptotic neuronal death in L5 dorsal root ganglion

Transection of a peripheral nerve in neonatal rats induces death of the axotomized neurons which may be due to either necrosis or apoptosis. In the present investigation, neuronal cell death in L5 dorsal root ganglion was evaluated after unilateral sciatic nerve transection in rats at 1, 3, 5, 7 and 10 days age. After 5 days, right (experimental) and left (control) dorsal root ganglia in all groups were removed, fixed, processed and embedded for either light or electron microscopy. Normal nucleoli were counted in paraffin embedded serial sections, and correction factors for split and multiple nucleoli were applied as well as the physical disector. The number of neurons in the right dorsal root ganglia, as compared with the controls, was significantly lower in all groups, and the percentage of the reduction at 1, 3, 5, 7 and 10 days was 32.4, 27.2, 23.8, 22.8 and 21.8% respectively. On the other hand, the results of neuronal counts using the disector method showed 34.0, 25.7, 20.2, 20.0 and 14.2% reduction in the number of neurons at 1, 3, 5, 7 and 10 days, respectively. The microscopic and ultrastructural results indicated that there were typical morphological changes similar to those of apoptosis, including condensed basophilic nuclei, formation of nuclear caps, cell shrinkage and apoptotic body formation. We concluded that there is an increase in apoptosis in dorsal root ganglia following sciatic nerve axotomy with the greatest neuronal loss on postnatal day 1.     

Effect of alkylation with N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea on the secondary structure of DNA

We have earlier reported that alkylation of DNA by the chemical carcinogen dimethyl sulphate, which mainly alkylates N-7 of guanine and N-3 of adenine, causes the formation of partially denatured regions in double-stranded DNA (Rizvi RY, Alvi NK & Hadi SM, Biosci. Rep. 2, 315-322, 1982). It is known that the major site of alkylation in DNA by N-ethyl-N-nitrosourea (EtNu) are the phosphate groups. N-methyl-N-nitrosourea (MeNu), on the other hand, causes the alkylation of mainly guanine residues. We have therefore studied the effect of these two alkylating carcinogens on the secondary structure of DNA. DNA alkylated with increasing concentrations of EtNu and MeNu was subjected to alkaline and S1 nuclease hydrolysis. Thermal melting profiles of alkylated DNA were also determined using S1 nuclease. The results indicated that alkylation by the two alkylating agents had a differential effect on the secondary structure of DNA. EtNu-alkylated DNA was found to be more thermostable than native DNA at neutral pH. It was however more alkali-labile than MeNu-alkylated DNA. The greater stability of EtNu-alkylated DNA was considered to be due to abolition of negative charges on phosphate alkylation.     

Effect of aspirin on prostaglandin synthesis by human platelets

When platelet rich plasma is exposed to N-ethylmaleimide, a ten fold increase in measurable prostaglandin E synthesis occurs. This effect is almost completely abolished within 2 hours of ingestion of 600 mg of aspirin by human volunteers. Recovery of this platelet function is slow for the first two days, returning sharply to normal over the next six days and plateauing approximately 8 days following initial removal from aspirin. It is suggested from these studies that platelet prostaglandin E production following NEM may be a useful test of platelet function.