Confirmation of quantitative trait loci affecting fatness in chickens

In this report we describe the analysis of an advanced intercross line (AIL) to confirm the quantitative trait locus (QTL) regions found for fatness traits in a previous study. QTL analysis was performed on chromosomes 1, 3, 4, 15, 18, and 27. The AIL was created by random intercrossing in each generation from generation 2 (G₂) onwards until generation 9 (G₉) was reached. QTL for abdominal fat weight (AFW) and/or percentage abdominal fat (AF%) on chromosomes 1, 3 and 27 were confirmed in the G₉ population. In addition, evidence for QTL for body weight at the age of 5 (BW5) and 7 (BW7) weeks and for the percentage of intramuscular fat (IF%) were found on chromosomes 1, 3, 15, and 27. Significant evidence for QTL was detected on chromosome 1 for BW5 and BW7. Suggestive evidence was found on chromosome 1 for AFW, AF% and IF%, on chromosome 15 for BW5, and on chromosome 27 for AF% and IF%. Furthermore, evidence on the chromosome-wise level was found on chromosome 3 for AFW, AF%, and BW7 and on chromosome 27 for BW5. For chromosomes 4 and 18, test statistics did not exceed the significance threshold.     

Methods for removing endogenous factors from CNS membrane preparations: Differences in [3H]GABA binding parameters and developmental-related effects

The present report describes a systematic study comparing and combining methods currently used for the removal of endogenous factors known to affect the interaction of GABA with its receptor. The effects of these methods were analyzed by performing [³H]GABA binding studies, and by measuring the amount of residual GABA left in the different membrane preparations. The effectiveness of these methods were also applied to different developmental stages. The results show that: 1) an exhaustive buffer washing procedure is necessary to accurately measure the maximal binding capacity (Bmax) of the low-affinity GABA binding site, and 2) the use of more drastic methods, including freeze-thawing and Triton treatment allows a clear demonstration of receptor heterogeneity and a precise measurement of the B_max of the high-affinity GABA binding site as well as increases the affinity of the low-affinity site. The analysis of the B_max values obtained with these different procedure in relation to the values of GABA removal, strongly indicates that the exhaustive washing procedure removes some unknown endogenous substances required for Triton treatment to exhibit its maximal effectiveness. Finally, a detailed analysis of K_d and B_max values obtained with these three methods in the developing nervous tissue shows the existence of significant differences with regard to their effectiveness in removing endogenous substances when applied in different developmental stages.Special issue dedicated to Dr. Claude Baxter.     

Cell-cycle phases and genetic profile of bone marrow-derived mesenchymal stromal cells expanded in vitro from healthy donors

Human mesenchymal stromal cells (MSCs) expanded in vitro for cell therapy approaches need to be carefully investigated for genetic stability, by employing both molecular and conventional karyotyping. Reliability of cytogenetic analysis may be hampered in some MSC samples by the difficulty of obtaining an adequate number of metaphases. In an attempt to overcome this problem, a methodology apt to evaluate the cell‐cycle structure on synchronous MSCs was optimised. Results obtained in five independent experiments by comparing cell‐cycle analysis of synchronous and asynchronous MSC populations evaluated at early and late culture passages documented that in synchronous MSCs, 30% of cells entered G2/M phase after about 27–28 h of culture, while in asynchronous MSCs only 8% of cells in G2/M phase could be observed at the same time point. Cytogenetic analysis on synchronous MSCs allowed us to obtain 20–25 valuable metaphases/slide, whereas only 0–4 metaphases/slide were detectable in asynchronous preparations. J. Cell. Biochem. 112: 1817–1821, 2011. © 2011 Wiley‐Liss, Inc.     

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.     

Synaptic Membrane Freezing Affects Modulatory Sites in Avian Central Nervous System GABAA Receptor

Studies were carried out to determine whether barbiturates and neurosteroids share common recognition sites at the GABA_A receptor complex in avian CNS. To achieve this, differentially prepared fresh and frozen synaptic membranes were used. Both the barbiturate, pentobarbital, and the neurosteroid, 3-hydroxy-5-pregnan-20-one, were able to stimulate GABA binding in both types of membranes. Stimulation differed markedly when both drugs were added jointly to different treated tissue. In frozen membranes drugs acted synergistically and were differentially displaced by picrotoxinin, while in fresh ones, where both compounds were inhibited by the convulsant, this additivity was absent. Post-freezing wash supernatants were collected and used as a source of putative endogenous factors involved in the above mentioned membrane differences. Addition of a high molecular weight fraction from supernatants to frozen synaptic membranes led to an inhibition of barbiturate and neurosteroid potentiation, as well as a loss of their additive effect. Our results indicate that GABA_A receptor modulation by barbiturates and neurosteroids is affected by synaptic membrane treatment, with a common modulatory site in fresh membranes and separate recognition sites after a freeze-thawing procedure. There may also be endogenous factors involved in overlapping of modulatory sites, which would thus regulate GABA_A receptor functionality by direct interaction with the complex.     

广义隐Markov模型在基因识别中的应用

广义隐Markov模型(GHMM)是基因识别的一种重要模型,但是其计算量比传统的隐Markov模型大得多,以至于不能直 接在基因识别中使用。根据原核生物基因的结构特点,提出了一种高效的简化算法,其计算量是序列长度的线性函数。在此 基础上,构建了针对原核生物基因的识别程序GeneMiner,对实际数据的测试表明,此算法是有效的。