ATM requirement in gene expression responses to ionizing radiation in human lymphoblasts and fibroblasts

The heritable disorder ataxia telangiectasia (AT) is caused by mutations in the AT-mutated (ATM) gene with manifestations that include predisposition to lymphoproliferative cancers and hypersensitivity to ionizing radiation (IR). We investigated gene expression changes in response to IR in human lymphoblasts and fibroblasts from seven normal and seven AT-affected individuals. Both cell types displayed ATM-dependent gene expression changes after IR, with some responses shared and some responses varying with cell type and dose. Interestingly, after 5 Gy IR, lymphoblasts displayed ATM-independent responses not seen in the fibroblasts at this dose, which likely reflect signaling through ATM-related kinases, e.g., ATR, in the absence of ATM function.     

Diversity of an ectomycorrhizal fungal community studied by a root tip and total soil DNA approach

Molecular methods based on soil DNA extracts are increasingly being used to study the fungal diversity of ectomycorrhizal (EM) fungal communities in soil. Contrary to EM root tip identification, the use of molecular methods enables identification of extramatrical mycelia in soil. To compare fungal diversity as determined by root tip identification and mycelial identification, six soil samples were analysed. Root tips were extracted from the six samples and after amplification, the basidiomycete diversity on the root tips was analysed by denaturing gradient gel electrophoresis (DGGE). The soil from the six samples was sieved, total soil DNA was extracted and after amplification, the basidiomycete diversity in the soil fractions was analysed by DGGE. Fourteen different bands were excised from the DGGE gel and sequenced; fungal taxon names could be assigned to eight bands. Out of a total of 14 fungal taxa detected in soil, 11 fungal taxa were found on root tips, of which seven were EM fungal taxa. To examine whether the sieving treatment would affect EM species diversity, two different sieve mesh sizes were used and in addition, the organic soil fraction was analysed separately. DGGE analysis showed no differences in banding pattern for the different soil fractions. The organic fraction gave the highest DGGE band intensities. This work demonstrates that there is a high correspondence between basidiomycete diversity detected by molecular analysis of root tips and soil samples, irrespective of the soil fraction being analysed.     

Association of CAPN1 316, CAPN1 4751 and TG5 markers with bovine meat quality traits in Mexico

We examined allele and genotype frequencies for the molecular markers CAPN1 316, CAPN1 4751 and TG5, and determined whether they are associated with beef quality traits in Mexican cattle. One hundred and twenty-four longissimus dorsi muscle samples were collected from cattle from north, central and southern Mexico. CAPN1 316 and CAPN1 4751 frequencies were determined using the allelic discrimination assay and the TG5 marker was typed by PCR-RFLP. Meat quality traits included intramuscular fat content (IMF) and tenderness determined by Warner-Bratzler shear force (WBSF) at 24 h postmortem. The association test was made using a mixed model, including genotypes, genetic group, and sampling location as fixed effects. Least squares means and significant interactions were compared using least significant differences based on the mixed procedure. CAPN1 316 CC was found at a low frequency (0.03) and has been reported as a favorable genotype associated with tenderness meat. Genotype frequencies for CAPN1 4751 were similar in favorable (CC) and unfavorable (TT) genotypes (0.26 and 0.28, respectively). The TG5 CC genotype had a frequency of 0.73, while the TT genotype frequency was 0.01. The means for WBSF and IMF were 4.08 ± 1.35 kg and 5.23 ± 2.14%, respectively. Sampling site and the CAPN1 316 genotypes significantly affected WBSF (P < 0.05). Samples collected from Hermosillo, Sonora, had the lowest WBSF (P < 0.05), while those collected in Veracruz were toughest (WBSF = 5.267 kg). The effect of GG and TG5 genotypes on IMF was significant (P < 0.05). CAPN1 316 and TG5 markers were found to be significantly associated with beef quality traits and thus will be useful for Mexican beef characterization.     

Differential distribution and association of repeat DNA sequences in the lateral element of the synaptonemal complex in rat spermatocytes

The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats, satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences play a key role in anchoring the chromosome to the protein scaffold of the SC. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cell-free propagation of prion strains

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). Disease is transmitted by the autocatalytic propagation of PrP(Sc) misfolding at the expense of the normal prion protein. The biggest challenge of the prion hypothesis has been to explain the molecular mechanism by which prions can exist as different strains, producing diseases with distinguishable characteristics. Here, we show that PrP(Sc) generated in vitro by protein misfolding cyclic amplification from five different mouse prion strains maintains the strain-specific properties. Inoculation of wild-type mice with in vitro-generated PrP(Sc) caused a disease with indistinguishable incubation times as well as neuropathological and biochemical characteristics as the parental strains. Biochemical features were also maintained upon replication of four human prion strains. These results provide additional support for the prion hypothesis and indicate that strain characteristics can be faithfully propagated in the absence of living cells, suggesting that strain variation is dependent on PrP(Sc) properties.     

Ternary complexes of Cu(II) ion with cimetidine and L-alanine

(Cu(CM)A)B(C6H5)4 and Cu(CM)A(OH) X H2O (CM = cimetidine and HA = L-alanine) were prepared and characterized by elemental analysis, TG-DTA, IR, and electronic spectral data and magnetic susceptibility measurements. The EPR spectrum of (Cu(CM)A)B(C6H5)4 shows a distorted octahedral environment for the Cu(II) ion.     

Effect of corticotropin-releasing factor on adrenal DBH and PNMT activity

The continuous administration of CRF (corticotropin-releasing factor) by the intraventricular route, 100 ng/day, to rats over a period of 7 days, results in significant increases of DBH (dopamine beta-hydroxylase) and PNMT (phenethanol N-methyltransferase) activities in the adrenal glands. The pattern of increase in DBH response to various doses of CRF does not correspond to the effects observed on plasma corticosterone, a result that suggests that CRF is acting to increase the adrenal enzyme by means other than through the pituitary-adrenal axis. In contrast, PNMT responds to CRF in a manner indicating a correlation with glucocorticoid availability. Moreover, PNMT induction by CRF persists even after adrenal denervation; it also occurs when CRF is given subcutaneously for 3 days, in a dose of 60 ng/day. Injection of reserpine did not potentiate the effect of intraventricularly administered CRF.     

Effects of guanfacine on three forms of distraction in the aging macaque

alpha-2 adrenoceptor agonists, such as clonidine and guanfacine, enhance attention in aged animals. According to one theory, alpha-2 receptor agonists improve attention by decreasing distractibility to task-irrelevant stimuli. In two healthy aging bonnet macaques, we investigated the effects of low-(0.001 mg/kg) and high-dose (0.05 mg/kg) acute intramuscular guanfacine versus saline control on accuracy (number of trials correct) in three tasks requiring attention: delayed matching-to-sample, one-target visual tracking (test of focused attention) and two-target visual tracking (test of divided attention). Each task employed distracting stimuli in a different paradigmatic context. One monkey responded to guanfacine at both doses with significant rises in accuracy on all three tasks. The second monkey showed significant accuracy improvement for high-dose guanfacine only. No sedation was observed. These results suggest that guanfacine improves attention and reduces distractibility in multiple task contexts in healthy aging primates.