Sample sizes for the exact test of 'no interaction' in 2 X 2 X 2 tables

The exact two-sided test of the hypothesis of 'no interaction' in a 2 X 2 X 2 table with fixed totals of rows X columns is considered. A method of approximating the power of the test, based on the limiting conditional distribution of a cell entry (Godambe and Harkness, 1975, Communications in Statistics 4, 699-709), is introduced and found to provide a close fit. This method can be utilized to calculate the sample sizes required to detect interaction effects with a pregiven power.     

利用基因与环境互作控制家蚕性别

S伴、K05、伴1、S1、D01 5个蚕品种,在胚胎期对高温干燥的催青条件表现敏感性,农 4、新九、限抗对高温干燥催青条件有较高的耐受能力,当用新九的雌与S伴、K05、伴1、S1、D01的雄蛾杂交时,其杂种一代的蚕卵,在高温干燥条件下催青,雌蚕因在胚胎发育过程中对环境条件表现敏感而不能孵化,雄蚕则能正常孵化,因而利用基因与环境的相互作用可以控制家蚕的性别。雌蚕高温干燥条件的敏感性,主要发生在胚胎发育至后期阶段。对S伴等5个温敏性品种的亲缘关系进行RAPD分析,经统计计算,相似率在63.04%~76.47%之间。 Abstract:The silkworm embryo of the varieties,sBan,K05,Ban1,S1 and D05 was sensitive to the high temperature and low humidity incubating condition,while that of Nong4,Xinjiu and Xiankang was more endurable.By crossing the female Xinjiu with the male of sensitive varieties,such as sBaa,K05,Ban1,S1 or D05,their female progenies were sensitive to the high temperature and low humidity during incubation and could not hatch,while the males could hatch normally.Experiment showed that silkworm sex could be controlled by the interaction between gene and environment. The sensitive period was at late stage during the embryo development.The RAPD analysis showed that the similarity index of the five temperature-sensitive varieties of sBan,K05,Ban1,S1 and D05 was 63.04-76.47%.     

Evaluation of the CTSL2 Gene as a Candidate Gene For Alopecia X in Pomeranians and Keeshonden

Alopecia X is a noninflammatory, progressive, bilateral symmetric alopecia in dogs. The disease is mainly found in Nordic breeds. The breed predisposition and a strong familial accumulation suggest a hereditary background. We analyzed the cathepsin L2 gene (CTSL2) as a candidate for alopecia X. The comparative sequencing of 14 affected and 18 control animals revealed ten polymorphisms; however, none of these polymorphisms affected the coding sequence. Haplotype analysis did not reveal an association of one particular CTSL2 haplotype with the disease phenotype; therefore, we conclude that the CTSL2 gene is probably not the causative gene for alopecia X.     

Involvement of P2X and P2Y receptors in microglial activation in vivo

Microglial cells are the primary immune effector cells in the brain. Extracellular ATP, e.g., released after brain injury, may initiate microglial activation via stimulation of purinergic receptors. In the rat nucleus accumbens (NAc), the involvement of P2X and P2Y receptors in the generation of microglial reaction in vivo was investigated. A stab wound in the NAc increased immunoreactivity (IR) for P2X_1,2,4,7 and P2Y_1,2,4,6,12 receptors on microglial cells when visualized with confocal laser scanning microscopy. A prominent immunolabeling of P2X₇ receptors with antibodies directed against the ecto- or endodomain was found on Griffonia simplicifolia isolectin-B4-positive cells. Additionally, the P2X₇ receptor was colocalized with active caspase 3 but not with the anti-apoptotic marker pAkt. Four days after local application of the agonists α,βmeATP, ADPβS, 2MeSATP, and BzATP, an increase in OX 42- and G. simplicifolia isolectin-IR was observed around the stab wound, quantified both densitometrically and by counting the number of ramified and activated microglial cells, whereas UTPγS appeared to be ineffective. The P2 receptor antagonists PPADS and BBG decreased the injury-induced increase of these IRs when given alone and in addition inhibited the agonist effects. Further, the intra-accumbally applied P2X₇ receptor agonist BzATP induced an increase in the number of caspase-3-positive cells. These results indicate that ATP, acting via different P2X and P2Y receptors, is a signaling molecule in microglial cell activation after injury in vivo. The up-regulation of P2X₇-IR after injury suggests that this receptor is involved in apoptotic rather than proliferative effects.     

Analysis of the DRD4 Gene Polymorphism in Populations of Russia and Neighboring Countries

Allele and genotype frequencies of the VNTR polymorphism in the third exon of humanDRD4 gene were determined in 544 individuals living in Russia (Russians, Bashkirs, Tatars, and Mordovians) and in the neighboring countries (Kazakhs and Ukrainians). The data obtained were compared with the allele frequency distribution patterns reported for the populations of Eurasia. Similarly to other Eurasian populations, in our population samples R4 allele was prevalent (64 to 87%). The frequency of this allele in the populations of Western Europe constitute 61 to 71%, while in the populations of Asia it varies from 74 to 96%. In this respect, the populations studied occupied the intermediate position. In the samples examined the R7 allele frequency decreased from 7% in Ukrainians to 1% in Bashkirs, while in Kazakhs and Mordovians the allele was absent. This finding was consistent with theR7 allele distribution pattern in the populations of Eurasia, characterized by higher frequency in the West and lower frequency or absence of the allele in the East. In the group of 22 Eurasian populations, the R7 allele frequency negatively correlated with the frequency of the R4 allele (r = -0.86 at P < 0.001). Unlike the R4 and R7 alleles, the frequency of which changed in the eastward direction, the R2 allele frequency distribution displayed slightly expressed latitudinal increase southwards. The DRD4 genotype distribution deviated from the equilibrium in most of the samples examined. In some samples, statistically significant increase of the R2/R2homozygotes frequency was demonstrated. One of the possible explanations of this phenomenon is assortative mating with respect to phenotypic (behavioral) allele manifestation. The data obtained can serve as the basis for the investigation of the possible role of the DRD4 alleles as the risk factors for the development of alcoholism and other types of addictions.     

Snf2 Family Gene Distribution in Higher Plant Genomes Reveals DRD1 Expansion and Diversification in the Tomato Genome

As part of large protein complexes, Snf2 family ATPases are responsible for energy supply during chromatin remodeling, but the precise mechanism of action of many of these proteins is largely unknown. They influence many processes in plants, such as the response to environmental stress. This analysis is the first comprehensive study of Snf2 family ATPases in plants. We here present a comparative analysis of 1159 candidate plant Snf2 genes in 33 complete and annotated plant genomes, including two green algae. The number of Snf2 ATPases shows considerable variation across plant genomes (17-63 genes). The DRD1, Rad5/16 and Snf2 subfamily members occur most often. Detailed analysis of the plant-specific DRD1 subfamily in related plant genomes shows the occurrence of a complex series of evolutionary events. Notably tomato carries unexpected gene expansions of DRD1 gene members. Most of these genes are expressed in tomato, although at low levels and with distinct tissue or organ specificity. In contrast, the Snf2 subfamily genes tend to be expressed constitutively in tomato. The results underpin and extend the Snf2 subfamily classification, which could help to determine the various functional roles of Snf2 ATPases and to target environmental stress tolerance and yield in future breeding.     

P2X7-Receptor Pathway Involvement in the Anti-Inflammatory Activity of Medicinal Plants

Medicinal plants used in European folk medicine attached to Lamiales, Gentianales or Asterales orders are used to treat inflammatory disorders. Many targets have been identified but to date, implication of purinergic receptor P2X7 activation has not yet been investigated. We managed to evaluate the protective effect on P2X7 activation by plant extracts used as anti-inflammatory in European folk medicine by the YO-PRO-1 uptake dye in vitro bioassay. Results revealed that among our selected plants, species from Scrophularia and Plantago genus were able to decrease significantly P2X7 activation (>50 % at 0.1 and 1 μg/mL). UPLC/MS, dereplication and metabolomic analysis of Scrophularia extracts, allowed us to identify the cinnamoyl-iridoid harpagoside as putative inhibitor of P2X7 activation. These results open a new research field regarding the anti-inflammatory mechanism of cinnamoyl-iridoids bearing plants, which may involve the P2X7 receptor.     

Parenting Characteristics in the Home Environment and Adolescent Overweight: A Latent Class Analysis

Parenting style and parental support and modeling of physical activity and healthy dietary intake have been linked to youth weight status, although findings have been inconsistent across studies. Furthermore, little is known about how these factors co‐occur, and the influence of the coexistence of these factors on adolescents' weight. This article examines the relationship between the co‐occurrence of various parenting characteristics and adolescents' weight status. Data are from Project EAT (eating among teens), a population‐based study of 4,746 diverse adolescents. Theoretical and latent class groupings of parenting styles and parenting practices were created. Regression analyses examined the relationship between the created variables and adolescents' BMI. Having an authoritarian mother was associated with higher BMI in sons. The co‐occurrence of an authoritarian mother and neglectful father was associated with higher BMI for sons. Daughters' whose fathers did not model or encourage healthy behaviors reported higher BMIs. The co‐occurrence of neither parent modeling healthy behaviors was associated with higher BMIs for sons, and incongruent parental modeling and encouraging of healthy behaviors was associated with higher BMIs in daughters. Although, further research into the complex dynamics of the home environment is needed, findings indicate that authoritarian parenting style is associated with higher adolescent weight status and incongruent parenting styles and practices between mothers and fathers are associated with higher adolescent weight status.     

Involvement of the purinergic P2X7 receptor in the formation of multinucleated giant cells

Multinucleated giant cells (MGC), a hallmark of chronic inflammatory reactions, remain an enigma of cell biology. There is evidence implicating the purinergic P2X7 receptor in the fusion process leading to MGC. To investigate this, we used HEK 293 cells stably transfected with either 1) the full-length rat P2X7 receptor (P2X7 cells), 2) a rat P2X7 receptor lacking the C-terminal domain (P2X7TC), or 3) a mock vector, and rat alveolar macrophages (MA) expressing the native receptor. P2X7 cells cultured in serum-free medium formed increased numbers of MGC and displayed a higher fusion index compared with mock transfectants. Stimulation of P2X7 pore-forming activity in P2X7 cells by polymyxin B (PMB) further increased significantly the formation of MGC. Conversely, blockers of P2X-receptors including oxidized ATP, brilliant blue G, and pyridoxal phosphate-6-azophenyl-2'-4'-disulfonic acid inhibited significantly MGC formation in both unstimulated and PMB-stimulated P2X7-transfected cells. In contrast, cells transfected with the truncated P2X7TC were devoid of pore-forming activity, did not respond to PMB stimulation, and failed to form enhanced numbers of MGC, thus behaving as mock transfectants. As found for P2X7-transfected cells, PMB also potentiated dose-dependently the formation of multinucleated MA by rat alveolar MA. Pretreatment with oxidized ATP abrogated the PMB stimulatory effects. Together, these data demonstrate unequivocally the participation of P2X7 receptor in the process of MGC formation. Our study also provides evidence suggesting that stimulation of the P2X7 receptor pathway in MA may mediate increased formation of MGC during chronic inflammatory reactions.     

A Novel Interaction Between Aging and ER Overload in a Protein Conformational Dementia

Intraneuronal deposition of aggregated proteins in tauopathies, Parkinson disease, or familial encephalopathy with neuroserpin inclusion bodies (FENIB) leads to impaired protein homeostasis (proteostasis). FENIB represents a conformational dementia, caused by intraneuronal polymerization of mutant variants of the serine protease inhibitor neuroserpin. In contrast to the aggregation process, the kinetic relationship between neuronal proteostasis and aggregation are poorly understood. To address aggregate formation dynamics, we studied FENIB in Caenorhabditis elegans and mice. Point mutations causing FENIB also result in aggregation of the neuroserpin homolog SRP-2 most likely within the ER lumen in worms, recapitulating morphological and biochemical features of the human disease. Intriguingly, we identified conserved protein quality control pathways to modulate protein aggregation both in worms and mice. Specifically, downregulation of the unfolded protein response (UPR) pathways in the worm favors mutant SRP-2 accumulation, while mice overexpressing a polymerizing mutant of neuroserpin undergo transient induction of the UPR in young but not in aged mice. Thus, we find that perturbations of proteostasis through impairment of the heat shock response or altered UPR signaling enhance neuroserpin accumulation in vivo. Moreover, accumulation of neuroserpin polymers in mice is associated with an age-related induction of the UPR suggesting a novel interaction between aging and ER overload. These data suggest that targets aimed at increasing UPR capacity in neurons are valuable tools for therapeutic intervention.     

Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus

Although originally cloned from rat brain, the P2X7 receptor has only recently been localized in neurones, and functional responses mediated by these neuronal P2X7 receptors (P2X7 R) are largely unknown. Here we studied the effect of P2X7 R activation on the release of neurotransmitters from superfused rat hippocampal slices. ATP (1-30 mm) and other ATP analogues elicited concentration-dependent [3 H]GABA outflow, with the following rank order of potency: benzoylbenzoylATP (BzATP) > ATP > ADP. PPADS, the non-selective P2-receptor antagonist (3-30 microm), Brilliant blue G (1-100 nm) the P2X7 -selective antagonist and Zn2+ (0.1-30 microm) inhibited, whereas lack of Mg2+ potentiated the response by ATP. In situ hybridization revealed that P2X7 R mRNA is expressed in the neurones of the cell body layers in the hippocampus. P2X7 R immunoreactivity was found in excitatory synaptic terminals in CA1 and CA3 region targeting the dendrites of pyramidal cells and parvalbumin labelled structures. ATP (3-30 microm) and BzATP (0.6-6 microm) elicited concentration-dependent [14 C]glutamate efflux, and blockade of the kainate receptor-mediated transmission by CNQX (10-100 microm) and gadolinium (100 microm), decreased ATP evoked [3 H]GABA efflux. The Na+ channel blocker TTX (1 microm), low temperature (12 degrees C), and the GABA uptake blocker nipecotic acid (1 mm) prevented ATP-induced [3 H]GABA efflux. Brilliant blue G and PPADS also reduced electrical field stimulation-induced [3 H]GABA efflux. In conclusion, P2X7 Rs are localized to the excitatory terminals in the hippocampus, and their activation regulates the release of glutamate and GABA from themselves and from their target cells.     

Involvement of P2X and P2U receptors in the constrictor effect of ATP on the pancreatic vascular bed

The effects of ATP on the pancreatic vascular bed were studied on the isolated rat pancreas perfused at a constant pressure so as any change in the vascular tone induces a modification in the flow rate. This study was performed in two different experimental conditions: 1) In the presence of indomethacin, inhibiting the cyclo-oxygenase and prostacyclin (PGI2) synthesis, ATP (which acts on vasodilatator P2Y receptors and vasoconstrictor P2X and P2U receptors) was used at a concentration (165 microM) which did not modify per se the vascular flow rate. With indomethacin, ATP induced a slight but significant and long lasting decrease in the flow rate. This effect is different from that induced by the stimulation of P2X receptors; it is comparable to that induced by the activation of P2U receptors. 2) In the presence of 2,2'pyridylisatogen tosilate (PIT) used at two different concentrations, the first (5 microM) inhibiting the P2Y effects on insulin secreting B cells and pancreatic vessels, the second (25 microM) inhibiting the P2X effects on pancreatic vessels. The effects of ATP are different according to the concentration of PIT. In both cases, ATP induced only a vasoconstriction. However, the kinetics of the flow rate is totally different: in the presence of 5 microM PIT, an immediate and drastic vasoconstriction was observed, followed by a long lasting vasoconstriction of lesser magnitude, which can be ascribed to P2X and P2U receptor activation, respectively. This hypothesis was confirmed by the results in the presence of PIT at 25 microM. At this concentration this compound completely suppressed the drastic and transient vasoconstriction, so that only a progressive and long lasting vasoconstriction of the P2U type could be observed. From these results, it can be concluded that: 1) PGI2 plays a part in the vasodilatator effects of ATP. 2) At the concentrations used, PIT does not block the vasoconstriction induced by P2U receptors. 3) The effects of ATP on pancreatic vessels is dependent on the balance between its vasodilator effect due to the activation of P2Y receptors and its vasoconstrictor effect which involves two types of receptors: P2X and P2U.     

A global survey of haplotype frequencies and linkage disequilibrium at the DRD2 locus

A four-site haplotype system at the dopamine D2 receptor locus (DRD2) has been studied in a global sample of 28 distinct populations. The haplotype system spans about 25 kb, encompassing the coding region of the gene. The four individual markers include three TaqI restriction site polymorphisms (RSPs) – TaqI “A”, “B”, and “D” sites – and one dinucleotide short tandem repeat polymorphism (STRP). All four of the marker systems are polymorphic in all regions of the world and in most individual populations. The haplotype system shows the highest average heterozygosity in Africa, a slightly lower average heterozygosity in Europe, and the lowest average heterozygosities in East Asia and the Americas. Across all populations, 20 of the 48 possible haplotypes reached a frequency of at least 5% in at least one population sample. However, no single population had more than six haplotypes reaching that frequency. In general, African populations had more haplotypes present in each population and more haplotypes occurring at a frequency of at least 5% in that population. Permutation tests for significance of overall disequilibrium (all sites considered simultaneously) were highly significant (P<0.001) in all 28 populations. Except for three African samples, the pairwise disequilibrium between the outermost RSP markers, TaqI “B” and “A”, was highly significant with D’ values greater than 0.8; in two of those exceptions the RSP marker was not polymorphic. Except for those same two African populations, the 16-repeat allele at the STRP also showed highly significant disequilibrium with the TaqI “B” site in all populations, with D’ values usually greater than 0.7. Only four haplotypes account for more than 70% of all chromosomes in virtually all non-African populations, and two of those haplotypes account for more than 70% of all chromosomes in most East Asian and Amerindian populations. A new measure of the amount of overall disequilibrium shows least disequilibrium in African populations, somewhat more in European populations, and the greatest amount in East Asian and Amerindian populations. This pattern seems best explained by random genetic drift with low levels of recombination, a low mutation rate at the STRP, and essentially no recurrent mutation at the RSP sites, all in conjunction with an “Out of Africa” model for recent human evolution. Received: 14 January 1998 / Accepted 19 March 1998     

Probing the Interaction Between KCNE2 and KCNQ1 in Their Transmembrane Regions

KCNE1-KCNE5 are single membrane-spanning proteins that associate with voltage-gated potassium channels to diversify their function. Other than the KCNQ1/KCNE1 complex, little is known about how KCNE proteins work. We focus on KCNE2, which associates with KCNQ1 to form K channels critical for gastric acid secretion in parietal cells. We use cysteine (Cys)-scanning mutagenesis to probe the functional role of residues along the KCNE2 transmembrane domain (TMD) in modulating KCNQ1 function. There is an α-helical periodicity in how Cys substitutions along the KCNE2 TMD perturb KCNQ1 pore conductance/ion selectivity. However, positions where Cys substitutions perturb KCNQ1 gating kinetics cluster to the extracellular end and cytoplasmic half of the KCNE2 TMD. This is the first systematic perturbation analysis of a KCNE TMD. We propose that the KCNE2 TMD adopts an α-helical secondary structure with one face making intimate contact with the KCNQ1 pore domain, while the contacts with the KCNQ1 voltage-sensing domain appear more dynamic.     

BST-2和Vpu相互作用抑制剂筛选模型的建立

骨髓基质细胞抗原2(Bone marrow stromal cell antigen 2,BST-2,又称Tetherin、CD317、HM1.24)是宿主先天性免疫应答的组成部分,可抑制人类免疫缺陷病毒1(Human immunodeficiency virus 1,HIV-1)的释放,HIV-1的辅助蛋白Vpu可通过跨膜区与BST-2的跨膜区产生相互作用,进而将其降解,下调其在细胞表面的数量,拮抗BST-2的抗病毒功能。本研究将海肾荧光素酶(Renilla luciferase,Rluc)与BST-2的N端连接,增强型黄色荧光蛋白(Enhanced yellow fluorescent protein,EYFP)与Vpu的C端连接,分别构建质粒RB和VE,使两种融合蛋白在细胞内共表达,产生生物发光共振能量转移(Bioluninescence resonance energy transfer,BRET)信号,进而建立稳定双表达细胞系,以BST-2和Vpu的跨膜区相互作用为靶点,应用BRET技术,建立两种蛋白相互作用抑制剂的筛选模型,以期通过BRET信号变化筛选出相互作用抑制剂,发展新型的艾滋病治疗手段。