O6-methylguanine in the SV40 origin of replication inhibits binding but increases unwinding by viral large T antigen.

To study the effect of the potentially cytotoxic base O6-methylguanine (O6-meG) on the initiation of DNA replication, double-stranded oligonucleotides corresponding to the SV40 origin of replication were constructed in which O6-meG replaced guanine in one strand. Out of 14 methylated residues, 10 were present in the Binding sites for T antigen (3 in Binding Site 1 and 7 in Binding Site 2). Binding of purified T antigen to the substituted oligonucleotide was considerably reduced in comparison to the unsubstituted one, as measured by nitrocellulose filter binding. Both the ATP-dependent and ATP-independent binding of T antigen were affected by the presence of the methylated base. Band shift analysis revealed an altered pattern of delayed-migrating complexes of T antigen with the O6-meG-containing oligonucleotide. Competition experiments, in which unmodified oligonucleotides containing Binding Site 1 or 2 were included in the binding assays, indicated that the affinity of T antigen for the O6-meG modified sites was reduced. When partially duplex oligonucleotides containing either Binding Site 1 or Site 2 of the origin of replication were used as substrates for the helicase activity of T antigen, the presence of O6-meG increased the extent of T antigen catalysed displacement of single-stranded DNA fragments.     

Mutagenicity of halogenated aliphatic hydrocarbons in Salmonella typhimurium,Streptomyces coelicolor and Aspergillus nidulans

Eight structurally related halogenated aliphatic hydrocarbons mono-, di- and trichloroacetaldehyde (the last in the anhydrous and hydrate form), mono-, di- and trichloroethanol and allyl chloride, were tested for their ability to induce gene mutations in prokaryotic and eukaryotic microorganisms. The genetic systems employed were the Salmonella reversion test with strain TA1535 and TA100, with and without metabolic activation, a forward and a back-mutation system in S. coelicolor and two forward mutation systems in A. nidulans. Each compound was tested with the spot and plate incorporation assay techniques.Mono-, di- and trichloroacetaldehyde were mutagenic in all the microorganisms employed; all the halogenated ethanols were positive in A. nidulans, while in S. typhimurium and in S. coelicolor the only active forms were respectively the mono- and dichloroderivatives. Allyl chloride was active in S. typhimurium and S. coelicolor and negative in A. nidulans.The technical approach as well as the complex influence of different factors (toxicity, volatility and stability) on the genetic response of each of the compounds under test did not allow to obtain more than a qualitative relationship between mutagenic potency and chemical structure.     

Isolation and partial characterization of a glial hyaluronate-binding protein

A glial hyaluronate-binding protein (GHAP) with an isoelectric point of 4.3-4.4 was isolated from human brain white matter. The 60-kDa glycoprotein appeared to be quite resistant to proteolysis, and comparison with GHAP from a viable glioma removed at surgery showed that the protein isolated from autopsy material was not a degradation product resulting from postmortem autolysis. The protein was localized immunohistochemically with mouse monoclonal and rabbit polyclonal antibodies in cerebral white matter. Only small amounts could be found in the gray matter. After enzymatic deglycosylation, an immunoreactive 47-kDa polypeptide was obtained. Two amino acid sequences of GHAP showed a striking similarity (up to 89%) with a highly conserved region of cartilage proteins (bovine nasal cartilage proteoglycan and rat and chicken link protein). However, the amino acid composition and other amino acid sequences suggested that there are also differences between brain-specific GHAP and cartilage proteins.     

Masking of epitopes in tissue sections

Summary Antisera to chicken brain antigen (CBA) isolated by hydroxyapatite chromatography from 8 M urea extracts following repeated extractions with phosphate buffer selectively decorate neurofilaments (NF) in neuronal perikarya, dendrites and axons. The antisera also reacted with GFA protein, the astrocyte-specific intermediate filament protein, as indicated by the adsorption of NF immunoreactivity following passage of the antisera through columns prepared with purified GFA protein. Moreover, the antisera stained the polypeptides of the NF triplet (70 kd, 150 kd, 200 kd) and GFA protein by the immunoblotting procedure. Monoclonal antibodies selectively decorating NF in tissue sections were isolated from a fusion of mouse myeloma cells with spleen cells of mice immunized with CBA. By the immunoblotting procedure the antibodies decorated the 150 kd NF polypeptide and GFA protein. No staining of glial filaments or any other structure on tissue sections was also observed with antibodies derived from another fusion strongly reacting with GFA protein on immunoblots. All antibodies (monoclonal and polyclonal) appeared to react with the same region of the GFA polypeptide as indicated by immunoblots of cleavage products.     

Isolation of a large aggregating proteoglycan from human brain.

A large proteoglycan (365 kDa), identified with monoclonal antibodies raised against chondroitin sulfate, was isolated from human brain. The isolation required anion-exchange chromatography followed by gel filtration through a Sephacryl S-500 column. The proteoglycan bound specifically to [3H]hyaluronate (HA). The binding was not reduced by high salt concentrations (up to 4 M) and was inhibited at low pH (< 4.0). The binding was inhibited by the octamer and decamer (but not the hexamer) oligosaccharides of HA. Limited proteolysis of the proteoglycan gave rise to a relatively stable polypeptide (80 kDa). The amino-terminal sequence of the 80-kDa polypeptide was identical to the cDNA-derived amino-terminal sequence of versican, a large human fibroblast proteoglycan. A monoclonal antibody raised against bovine proteoglycans and recognizing the versican core protein reacted by immunoblotting with the proteoglycan isolated from human brain. The antibody was used to localize the proteoglycan in acetone-fixed cryostat sections of bovine spinal cord. The localization of the proteoglycan in the central nervous system was identical to that previously reported for glial hyaluronate-binding protein (GHAP), a 60-kDa glycoprotein of the brain extracellular matrix (ECM). However, a major difference was observed with respect to the sensitivity of the two antigens to hyaluronidase. As previously reported, GHAP was released from the tissue by hyaluronidase digestion, whereas the proteoglycan persisted under these conditions. We conclude that the protein-hyaluronate aggregates in brain ECM contain both GHAP and versican, that GHAP is only retained in the ECM by its interaction with hyaluronate, and that the proteoglycan is anchored in some other manner and probably connects cell surfaces with the ECM since it was not released by hyaluronidase digestion.     

Time course of induction of oxytocin messenger ribonucleic acid levels in the hypothalamic paraventricular nucleus of ovariectomized rats following gonadal steroid administration

The nonapeptide oxytocin (OT) is important for uterine contractility at parturition, milk ejection during lactation, and the induction of maternal behavior. OT messenger ribonucleic acid (mRNA) levels increase in the paraventricular and supraoptic nuclei (PVN and SON) of late pregnant and lactating rats and are modulated by the steroid milieu that accompanies these states. Specifically, sequential exposure to estradiol (E₂) and progesterone (P) followed by P withdrawal 48 hrs prior to sacrifice increases PVN, and to a lesser but significant degree, SON OT mRNA. To better define the time course of induction of OT mRNA levels following P withdrawal, ovariectomized Sprague-Dawley rats were treated with empty or steroid-filled capsules. On day 1, animals received an E₂-filled or empty capsule, followed by P-filled or empty capsules on day 3. On day 14, P-filled or empty capsules were removed and animals were sacrificed 24, 36, or 48 hrs later. The hypothalamic PVN were analyzed for OT mRNA by in situ hybridization histochemistry. Significant differences in PVN OT mRNA were found among the groups (P < 0.0001, Kruskal-Wallis). Animals in the 48 hr (P = 0.007) and 36 hr (P = 0.005), but not the 24 hr, steroid-treated groups had significantly increased OT mRNA relative to their respective sham-treated cohorts (Mann-Whitney U test). The relative abundance of PVN OT mRNA differed among the steroid-treated groups (Kruskal-Wallis, P < 0.0003), with highest levels at 48 hr. We conclude that increases in PVN OT mRNA occur by 36 hrs, and are highest at 48 hrs, after P withdrawal in the E₂-primed rat. Future studies will determine if OT-mediated changes in behavior or physiology that surround parturition are related to these changes in OT mRNA.     

Photocontrol of betaxanthin synthesis in Celosia plumosa seedlings

Light stimulates the betaxanthin accumulation in Celosia plumosa. The induction is partially reversed by far-red and inhibited by actinomycin D, puromycin, salicylaldoxime and 2,4-dinitrophenol, while 3-(3,4-dichlorophenyl)-1,1-dimethylurea has an inhibitory effect only when photosynthesis is operative. In darkness betaxanthins synthesis is promoted by kinetin.     

Edaphic influences of ophiolitic substrates on vegetation in the Western Italian Alps

Background and aims

Soils derived from serpentinite (serpentine soils) often have low macronutrient concentrations, exceedingly low Ca:Mg molar ratios and high heavy metal concentrations, typically resulting in sparse vegetative cover. This combined suite of edaphic stresses is referred to as the “serpentine syndrome.” Although several plant community-level studies have been conducted to identify the most important edaphic factor limiting plant growth on serpentine, the primary factor identified has often varied by plant community and local climate. Few studies to date have been conducted in serpentine plant communities of alpine or boreal climates. The goal of our study was to determine the primary limiting edaphic factors on plant community species composition and productivity (cover) in the alpine and boreal climate of the Western Alps, Italy.

Methods

Soil properties and vegetation composition were analyzed for several sites underlain by serpentinite, gabbro, and calc-schist substrates and correlated using direct and indirect statistical methods.

Results

Boreal forest soils were well-developed and tended to have low pH throughout the soil profile resulting in high Ni availability. Alpine soils, in comparison, were less developed. The distinct serpentine plant communities of the Western Alps are most strongly correlated with high levels of bioavailable Ni associated with low soil pH. Other factors such as macronutrient deficiency, low Ca:Mg molar ratio and drought appear to be less important.

Conclusions

The strong ecological influence of Ni is caused by environmental conditions which increase metal mobilization.     

ATR and ATM differently regulate WRN to prevent DSBs at stalled replication forks and promote replication fork recovery

Accurate response to replication arrest is crucial to preserve genome stability and requires both the ATR and ATM functions. The Werner syndrome protein (WRN) is implicated in the recovery of stalled replication forks, and although an ATR/ATM‐dependent phosphorylation of WRN was observed after replication arrest, the function of such modifications during the response to perturbed replication is not yet appreciated. Here, we report that WRN is directly phosphorylated by ATR at multiple C‐terminal S/TQ residues. Suppression of ATR‐mediated phosphorylation of WRN prevents proper accumulation of WRN in nuclear foci, co‐localisation with RPA and causes breakage of stalled forks. On the other hand, inhibition of ATM kinase activity or expression of an ATM‐unphosphorylable WRN allele leads to retention of WRN in nuclear foci and impaired recruitment of RAD51 recombinase resulting in reduced viability after fork collapse. Altogether, our findings indicate that ATR and ATM promote recovery from perturbed replication by differently regulating WRN at defined moments of the response to replication fork arrest.     

A rare case of Plasmodium (Haemamoeba) relictum infection in a free-living Red Knot (Calidris canutus rufa, Scolopacidae)

The Red Knot (Calidris canutus rufa) is a Nearctic migrant shorebird that breeds in the Canadian Arctic and spends the winter season in coastal sites in South America. A rare case of a blood protozoan was found by molecular analyses from an adult bird captured during spring migration at the last refuelling stopover in Delaware Bay USA in 2006. The parasite was identified as Plasmodium relictum belonging to subgenus Haemamoeba based on the shape of meronts, roundish gametocytes, and its position in the erythrocytes from the blood smears examination. A partial cytochrome b sequence was a 100% match to a sequence of Plasmodium relictum, sequence Genbank accession number: id DQ659543.1 (lineage code haplotype P5). This is the first report of avian malaria in a wild individual of C. c. rufa.