Acrylamide binding to the DNA and protamine of spermiogenic stages in the mouse and its relationship to genetic damage

Mice received an intraperitoneal injection of 14C-labeled acrylamide (AA) at an exposure of 125 mg/kg to equal that used in genetic studies carried out by Shelby et al. (1986). Subsequently, spermatozoa were recovered from the reproductive tracts of the animals over a 3-week period and assayed for the amount of bound AA. A strong increase in the level of binding occurred in late-spermatid to early-spermatozoa stages; these same stages are also genetically most sensitive to the action of AA. At all time points, alkylation of DNA within the sperm accounted for a very small fraction (generally less than 0.5%) of the total sperm-head alkylation. However, alkylation of protamine, a protein unique to sperm cells, was found to be correlated with total sperm-head alkylation and accounted for essentially all of the AA binding. Two radioactive adducts were found in hydrolysed protamine samples, one of which co-eluted with a standard of S-carboxyethylcysteine. Protamine alkylation appears to be a significant cause of acrylamide-induced genetic damage in spermiogenic cells of the mouse.     

Inhalation exposure-rate of ethylene oxide affects the level of DNA breakage and unscheduled DNA synthesis in spermiogenic stages of the mouse

The effect of ethylene oxide (EtO) inhalation-exposure rate on the induction of DNA breakage in late spermatids and on unscheduled DNA synthesis (UDS) in early spermatids was studied. The exposures were 450 parts per million (ppm) for 4 h, 900 ppm for 2 h, and 1800 ppm for 1 h. Thus, the total exposure was always 1800 ppm-h. Both DNA breakage and UDS were found to increase by a factor of approximately 3 in going from the low to high EtO concentration, suggesting that the molecular dose of EtO to the testis had increased by a similar factor. Our results are consistent with the EtO exposure-rate effect found by Generoso et al. (1986) for induction of dominant-lethal mutations in late spermatids and early spermatozoa.     

A review of the genetic effects of ethyl methanesulfonate

Ethyl methanesulfonate (EMS) is a monofunctional ethylating agent that has been found to be mutagenic in a wide variety of genetic test systems from viruses to mammals. It has also been shown to be carcinogenic in mammals. Alkylation of cellular, nucleophilic sites by EMS occurs via a mixed SN1/SN2 reaction mechanism. While ethylation of DNA occurs principally at nitrogen positions in the bases, because of the partial SN1 character of the reaction, EMS is also able to produce significant levels of alkylation at oxygens such as the O6 of guanine and in the DNA phosphate groups. Genetic data obtained using microorganisms suggest that EMS may produce both GC to AT and AT to GC transition mutations. There is also some evidence that EMS can cause base-pair insertions or deletions as well as more extensive intragenic deletions. In higher organisms, there is clear-cut evidence that EMS is able to break chromosomes, although the mechanisms involved are not well understood. An often cited hypothesis is that DNA bases ethylated by EMS (mostly the N-7 position of guanine) gradually hydrolyze from the deoxyribose on the DNA backbone leaving behind an apurinic (or possibly an apyrimidinic) site that is unstable and can lead to single-strand breakage of the DNA. Data also exist that suggest that ethylation of some chromosomal proteins in mouse spermatids by EMS may be an important factor in causing chromosome breakage.     

Identification of transcription factors that bind to the 5′-UTR of the barley PHO2 gene

Plant Molecular Biology - In barley and other higher plants, phosphate homeostasis is maintained by a regulatory network involving the PHO2 (PHOSPHATE2) encoding ubiquitin-conjugating...     

Polynuclear complexes of copper(II) with the tetra-aza ligand 3,6-bis(2′-pyridyl)pyridazine(L). Crystal structure of the bromine derivative [Cu2L(OH)Br3]n

The tetra-aza ligand 3,6-bis(2′-pyridyl)pyridazine (L), when reacting in appropriate conditions with Cu(II) halides, gives rise to polynuclear complexes of general formula [Cu₂L(OH)X₃]_n (X = Cl or Br). The bromine derivative has been studied by X-ray analysis. The crystals are twins by merohedry of class I, space group Pn (P2₁/n apparent space group), with the following cell constants: a = 13.691(5), b = 6.245(3), c = 10.298(4) Å, β = 103.92(5)°. The structure was refined by least-squares techniques to a final R factor of 0.066. The structure consists of binuclear units joined to each other through bridging bromine atoms to form a polymeric array. The two independent copper atoms of the dinuclear moiety are five-coordinated with a geometry which is intermediate between a square-pyramid and a trigonal-bipyramid.     

Properties of sesame oil by detailed 1H and 13C NMR assignments before and after ozonation and their correlation with iodine value,peroxide value,and viscosity measurements

Gaseous ozone chemically reacts with unsaturated triglyceride substrates leading to ozonated derivatives with a wide potential applications, ranging from the petrochemical to the pharmaceutical industry. To date, an ultimate understanding of the ozone reactivity during sesame oil ozonation process as well as detailed ¹H and ¹³C NMR assignments are lacking. A practical advantage of NMR is that a single NMR sample measurement can explain many issues, while similar analysis by traditional methods may require several independent and time-consuming measurements. Moreover, significant relationships among NMR spectra and both conventional chemical analysis and viscosity measurements have been found. Eventually, NMR could play an important role for quality attributes of ozonated oil derivatives.     

Interaction of deoxyhemoglobin with the cytoplasmic domain of murine erythrocyte band 3

The partial pressure of oxygen constitutes an important factor in the regulation of human erythrocyte physiology, including control of cell volume, membrane structure, and glucose metabolism. Because band 3 is thought to be involved in all three processes and because binding of hemoglobin (Hb) to the cytoplasmic domain of band 3 (cdb3) is strongly oxygen-dependent, the possibility that the reversible association of deoxyhemoglobin (deoxyHb) with cdb3 might constitute an O(2)-dependent sensor that mediates O(2)-regulated changes in erythrocyte properties arises. While several lines of evidence support this hypothesis, a major opposing argument lies in the fact that the deoxyHb binding sequence on human cdb3 is not conserved. Moreover, no effect of O(2) pressure on Hb-band 3 interactions has ever been demonstrated in another species. To explore whether band 3-Hb interactions might be widely involved in O(2)-dependent regulation of erythrocyte physiology, we undertook characterization of the effect of O(2) on band 3-Hb interactions in the mouse. We report here that murine band 3 binds deoxyHb with significantly greater affinity than oxyHb, despite the lack of significant homology within the deoxyHb binding sequence. We further map the deoxyHb binding site on murine band 3 and show that deletion of the site eliminates deoxyHb binding. Finally, we identify mutations in murine cdb3 that either enhance or eliminate its affinity for murine deoxyHb. These data demonstrate that despite a lack of homology in the sequences of both murine band 3 and murine Hb, a strong oxygen-dependent association of the two proteins has been conserved.     

Dosimetry studies on the ethylation of mouse sperm DNA after in vivo exposure to (3H)ethyl metanesulfonate

Methods for determining the chemical dose of ethyl methanesulfonate (EMS) to the DNA of mouse spermatozoa in the vasa deferentia and epididymides have been developed. These include procedures for the removal of contaminating protamine, which, like DNA, possesses nucleophilic sites that can be ethylated by EMS. At least 99% of all sperm protamine (at a 95% confidence level), as well as any other cellular contaminants, is removed during purification of the DNA. The purified DNA recovered from spermatozoa gives no indication of a preferential recovery of either (G+C)-rich or (A+T)-rich regions of the mouse genome: the [¹⁴C]dT/[³H]dC ratios for whole sperm and sperm DNA were the same for each animal tested.The spermatozoa of males used in the dosimetry studies were labeled with [¹⁴C]thymidine, and then the animals were given various [³H]EMS doses intraperitoneally. A constant exposure time of 4 h was used. The ratios of ³H and ¹⁴C activities in whole sperm and purified sperm DNA were used to measure the percentage of the total sperm ethylation occurring in the DNA. The maximum percentage found was about 18% in the dose range of 100–400 mg/kg. Values for the ethylations per nucleotide (E/N) ranged from ～ 10^?7 at 3.3 mg/kg up to ～ 10^?4 at 400 mg/kg, and the data indicated that E/N increased with the 1.5 power of the dose. E/N was also measured in testicular DNA, and the values obtained were close to those found for spermatozoan DNA.The results of such chemical dosimetry studies will be far-reaching in the interpretation of molecular events responsible for genetic alterations. As an example, dominant lethal studies by others, using EMS in the dose range considered in the present paper, have shown little or no effect until two or more days after injection of the mutagen into male mice. Since many sperm DNA ethylations are found after a 4-h exposure to EMS it appears that most of these DNA ethylations are not genetically important, at least in the production of dominant lethals, and that perhaps genetic damage occurs only at rarely ethylated DNA sites.     

Receptor-mediated delivery of siRNAs by tethered nucleic acid base-paired interactions

We report a new strategy for cell-type-specific delivery of functional siRNAs into cells. The method involves the noncovalent attachment of siRNAs to ligand-conjugated oligodeoxynucleotides via nucleic acid base-paired interactions. The resulting complexes can be directly applied to cells, leading to specific cellular uptake and gene silencing. The method is simple, economical, and can be easily adapted for other cell surface receptors. Here we show the application of this method for the delivery of siRNAs to folate receptor-expressing cells.