Pharmacological blockade of serotonin 5-HT₇ receptor reverses working memory deficits in rats by normalizing cortical glutamate neurotransmission

The role of 5-HT₇ receptor has been demonstrated in various animal models of mood disorders; however its function in cognition remains largely speculative. This study evaluates the effects of SB-269970, a selective 5-HT₇ antagonist, in a translational model of working memory deficit and investigates whether it modulates cortical glutamate and/or dopamine neurotransmission in rats. The effect of SB-269970 was evaluated in the delayed non-matching to position task alone or in combination with MK-801, a non-competitive NMDA receptor antagonist, and, in separate experiments, with scopolamine, a non-selective muscarinic antagonist. SB-269970 (10 mg/kg) significantly reversed the deficits induced by MK-801 (0.1 mg/kg) but augmented the deficit induced by scopolamine (0.06 mg/kg). The ability of SB-269970 to modulate MK-801-induced glutamate and dopamine extracellular levels was separately evaluated using biosensor technology and microdialysis in the prefrontal cortex of freely moving rats. SB-269970 normalized MK-801 -induced glutamate but not dopamine extracellular levels in the prefrontal cortex. Rat plasma and brain concentrations of MK-801 were not affected by co-administration of SB-269970, arguing for a pharmacodynamic rather than a pharmacokinetic mechanism. These results indicate that 5-HT₇ receptor antagonists might reverse cognitive deficits associated with NMDA receptor hypofunction by selectively normalizing glutamatergic neurotransmission.     

Prevalence and intensity of Haemobaphes diceraus (Copepoda: Pennellidae) from shiner perch,Cymatogaster aggregata (Embiotocidae)

The prevalence and intensity of the hematophagous pennellid copepod Haemobaphes diceraus were monitored over a 10-mo period in shiner perch Cymatogaster aggregata at Pipers Lagoon, Nanaimo, British Columbia. The prevalence and mean intensity of metamorphosed adult female H. diceraus (n = 421) were 10.0% and 1.2 (+/-0.5 SD), respectively. The majority (97.9%) of infected fish had single infections, reflecting the possibility of intensity-dependent parasite-induced mortality, rejection of additional parasites, or both. Transforming females were detected throughout the year; however, there was no detectable seasonal pattern of colonization. Neither copepodids nor adult males of H. diceraus were observed on the gills of shiner perch, and this was consistent with the hypothesis that an intermediate host harbors these stages. Males of Haemobaphes sp. infected the gills of bay pipefish Syngnathus griseolineatus with a prevalence and mean intensity of 56.0% and 6.8 +/- 3.7, respectively. Transmission of H. diceraus to shiner perch probably occurs in inshore protected areas, where shiner perch ecologically overlap with the probable intermediate host of H. diceraus, the bay pipefish.     

Hepatic regeneration from hematopoietic stem cells

In recent years, numerous investigators have reported novel cellular fates of multipotent stem or progenitor cells. In this review, we discuss the unexpected observations that hematopoietic stem cells can contribute to the hepatocyte lineage in humans and in rodent models of liver disease and regeneration. A key unresolved issue regarding hepatic regeneration from hematopoietic stem cells is whether the mechanism occurs through transdetermination, cell fusion, or other processes. A better understanding of the various stem or progenitor cells of the hepatic lineage may facilitate cellular transplantation approaches for the correction of hepatic function in patients with end-stage liver disease.     

Global Analysis of DNA Methylation Variation in Adipose Tissue from Twins Reveals Links to Disease-Associated Variants in Distal Regulatory Elements

Epigenetic modifications such as DNA methylation play a key role in gene regulation and disease susceptibility. However, little is known about the genome-wide frequency, localization, and function of methylation variation and how it is regulated by genetic and environmental factors. We utilized the Multiple Tissue Human Expression Resource (MuTHER) and generated Illumina 450K adipose methylome data from 648 twins. We found that individual CpGs had low variance and that variability was suppressed in promoters. We noted that DNA methylation variation was highly heritable (h²_median = 0.34) and that shared environmental effects correlated with metabolic phenotype-associated CpGs. Analysis of methylation quantitative-trait loci (metQTL) revealed that 28% of CpGs were associated with nearby SNPs, and when overlapping them with adipose expression quantitative-trait loci (eQTL) from the same individuals, we found that 6% of the loci played a role in regulating both gene expression and DNA methylation. These associations were bidirectional, but there were pronounced negative associations for promoter CpGs. Integration of metQTL with adipose reference epigenomes and disease associations revealed significant enrichment of metQTL overlapping metabolic-trait or disease loci in enhancers (the strongest effects were for high-density lipoprotein cholesterol and body mass index [BMI]). We followed up with the BMI SNP rs713586, a cg01884057 metQTL that overlaps an enhancer upstream of ADCY3, and used bisulphite sequencing to refine this region. Our results showed widespread population invariability yet sequence dependence on adipose DNA methylation but that incorporating maps of regulatory elements aid in linking CpG variation to gene regulation and disease risk in a tissue-dependent manner.     

Genome-wide Screen for Modulation of Hepatic Apolipoprotein A-I (ApoA-I) Secretion

Control of plasma cholesterol levels is a major therapeutic strategy for management of coronary artery disease (CAD). Although reducing LDL cholesterol (LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25–40% reduction in cardiovascular events. Epidemiological studies have shown that a high LDL-c level is not the only risk factor for CAD; low HDL cholesterol (HDL-c) is an independent risk factor for CAD. Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-c that mediates reverse cholesterol transport from tissues to the liver for excretion. Therefore, increasing ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide siRNA screening, targets that regulate hepatocyte ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted enzymes, proteases, G-protein-coupled receptors, metabolic enzymes, ion transporters, and proteins of unknown function. Repression of farnesyltransferase (FNTA) by siRNA and the enzyme inhibitor manumycin A caused elevation of ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.     

Rapid determination of antimicrobial susceptibility patterns ofListeria monocytogenes isolates by the autobac AIS and MIC procedures

A total of 34 isolates ofListeria monocytogenes were tested against ampicillin, cephalothin, chloramphenicol, erythromycin, tetracycline, and penicillin-G using the Autobac 3-h AIS and the Autobac 5-h MIC procedures. The results were compared to susceptibility category interpretations and MICs determined using the Sceptor system. With the Sceptor System, all isolates were interpreted to be moderately susceptible to ampicillin and penicillin-G, and susceptible to the four other antibiotics. With the Autobac AIS, all isolates were interpreted to be susceptible to all the antibiotics except penicillin-G. All but one of the 34 isolates were interpreted to be resistant to penicillin-G with the Autobac AIS test. The remaining isolate was interpreted to be indeterminant. The Autobac AIS test was unsatisfactory for determining the susceptibility ofL. monocytogenes isolates to penicillin-G. The Autobac MIC results correlated well with the MIC results of the Sceptor system provided that the Autobac was programmed as though it were testing enterococci. The Autobac MIC reported penicillin-G MICs in units per milliliter and required the use of a conversion factor to obtain micrograms per milliliter, and did not allow for the testing of erythromycin. The Autobac MIC susceptibility category interpretations must not be used, as they were derived from an outdated susceptibility standard. The Autobac MIC test may be used if the limitations given above are observed.     

Binding of phosphorothioate oligonucleotides with RNase H1 can cause conformational changes in the protein and alter the interactions of RNase H1 with other proteins

We recently found that toxic PS-ASOs can cause P54nrb and PSF nucleolar mislocalization in an RNase H1-dependent manner. To better understand the underlying mechanisms of these observations, here we utilize different biochemical approaches to demonstrate that PS-ASO binding can alter the conformations of the bound proteins, as illustrated using recombinant RNase H1, P54nrb, PSF proteins and various isolated domains. While, in general, binding of PS-ASOs or ASO/RNA duplexes stabilizes the conformations of these proteins, PS-ASO binding may also cause the unfolding of RNase H1, including both the hybrid binding domain and the catalytic domain. The extent of conformational change correlates with the binding affinity of PS-ASOs to the proteins. Consequently, PS-ASO binding to RNase H1 induces the interaction of RNase H1 with P54nrb or PSF in a 2′-modification and sequence dependent manner, and toxic PS-ASOs tend to induce more interactions than non-toxic PS-ASOs. PS-ASO binding also enhances the interaction between P54nrb and PSF. However, the interaction between RNase H1 and P32 protein can be disrupted upon binding of PS-ASOs. Together, these results suggest that stronger binding of PS-ASOs can cause greater conformational changes of the bound proteins, subsequently affecting protein–protein interactions. These observations thus provide deeper understanding of the molecular basis of PS-ASO-induced protein mislocalization or degradation observed in cells and advance our understanding of why some PS-ASOs are cytotoxic.     

Characterization of Kir1.1 channels with the use of a radiolabeled derivative of tertiapin

Inward rectifier potassium channels (Kir) play critical roles in cell physiology. Despite representing the simplest tetrameric potassium channel structures, the pharmacology of this channel family remains largely undeveloped. In this respect, tertiapin (TPN), a 21 amino acid peptide isolated from bee venom, has been reported to inhibit Kir1.1 and Kir3.1/3.4 channels with high affinity by binding to the M1-M2 linker region of these channels. The features of the peptide-channel interaction have been explored electrophysiologically, and these studies have identified ways by which to alter the composition of the peptide without affecting its biological activity. In the present study, the TPN derivative, TPN-Y1/K12/Q13, has been synthesized and radiolabeled to high specific activity with (125)I. TPN-Y1/K12/Q13 and mono-iodo-TPN-Y1/K12/Q13 ([(127)I]TPN-Y1/K12/Q13) inhibit with high affinity rat but not human Kir1.1 channels stably expressed in HEK293 cells. [(125)I]TPN-Y1/K12/Q13 binds in a saturable, time-dependent, and reversible manner to HEK293 cells expressing rat Kir1.1, as well as to membranes derived from these cells, and the pharmacology of the binding reaction is consistent with peptide binding to Kir1.1 channels. Studies using chimeric channels indicate that the differences in TPN sensitivity between rat and human Kir1.1 channels are due to the presence of two nonconserved residues within the M1-M2 linker region. When these results are taken together, they demonstrate that [(125)I]TPN-Y1/K12/Q13 represents the first high specific activity radioligand for studying rat Kir1.1 channels and suggest its utility for identifying other Kir channel modulators.     

Regeneration from intact and sectioned immature embryos of barley (Hordeum vulgare L.): the scutellum exhibits an apico-basal gradient of embryogenic capacity

Immature zygotic embryos from spring barley cv. Dissa were used to induce somatic embryogenenesis. Up to 158 germinated somatic embryos could be recovered per plated zygotic embryo. Critical factors for obtaining a high yield of regenerants were the size of the explant, the level of 2,4-D used for callus induction and the careful division of callus at each subculture. Use of microsections of immature embryos as explants revealed a pronounced gradient of callus formation and embryogenic response across the scutellum. Sections from the scutellar tissue at the coleoptilar end of the embryo gave the most callus and were highly embryogenic. The regeneration response of sectioned explants was comparable to that recovered from intact embryos of similar size.