Genome-wide gene expression responses to experimental manipulation of Saccharomyces cerevisiae repressor activator protein 1 (Rap1) expression level.

Precise regulation of transcription in gene expression is critical for all aspects of normal organism form, fitness, and function and even minor alterations in the level, location, and timing of gene expression can result in phenotypic variation within and between species including evolutionary innovations and human disease states. Eukaryotic transcription is regulated by a complex interplay of multiple factors working both at a physical and molecular levels influencing this process. In Saccharomyces cerevisiae, the TF with the greatest number of putative regulatory targets is the essential gene Repressor Activator Protein 1 (RAP1). While much is known about the roles of Rap1 in gene regulation and numerous cellular processes, the response of Rap1 target genes to systematic titration of RAP1 expression level remains unknown. To fill this knowledge gap, we used a strain with a tetracycline-titratable promoter replacing wild-type regulatory sequences of RAP1 to systematically reduce the expression level of RAP1 and followed this with RNA sequencing (RNA-seq) to measure genome-wide gene expression responses. Previous research indicated that Rap1 plays a significant regulatory role in particular groups of genes including telomere-proximal genes, homothallic mating (HM) loci, glycolytic genes, DNA repair genes, and ribosomal protein genes; therefore, we focused our analyses on these groups and downstream targets to determine how they respond to reductions in RAP1 expression level. Overall, despite being known as both an activator and as a repressor of its target genes, we found that Rap1 acts as an activator for more target genes than as a repressor. Additionally, we found that Rap1 functions as an activator of ribosomal protein genes and a repressor for HM loci genes consistent with predictions from the literature. Unexpectedly, we found that Rap1 functions as a repressor of glycolytic enzyme genes contrary to prior reports of it having the opposite effect. We also compared the expression of RAP1 to five different genes related to DNA repair pathway and found that decreasing RAP1 downregulated four of those five genes. Finally, we found no effect of RAP1 depletion on telomere-proximal genes despite its functioning to silence telomeric repeat-containing RNAs. Together our results enrich our understanding of this important transcriptional regulator.

Prophylactic zinc supplementation modulates hippocampal ionic zinc and partially remediates neurological recovery following repetitive mild head injury in mice.

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative condition caused by repetitive mild traumatic brain injury (TBI) that leads to impaired executive functioning, emotional disturbances, and disordered memory, warranting both basic and translational research of potential therapeutic targets. One area of research concerns prophylactic zinc (Zn) supplementation; however, Zn supplementation remains poorly understood. This study explored the effects of Zn supplementation in a mouse model of repetitive mild TBI. Four-week-old male (n = 50) and female (n = 50) C57BL/6J mice consumed tap water or 10 parts per million Zn-supplemented water for eight weeks prior to injury. At 12 weeks of age, mice underwent either five sham procedures or five closed-head injuries spaced apart by 48 h after which they completed behavioral tests. Zinc-supplemented injured mice righted themselves and regained normal ambulatory function as fast as non-injured mice across four out of the five injury days. In contrast, non-supplemented injured mice exhibited impairment in normal ambulation by days 4 and 5. Injury also reduced free, ionic Zn in the dentate gyrus and CA3 region of the hippocampus and Zn supplementation partially remediated this reduction but not to the levels of non-injured mice. There were no structural differences in cortex, hippocampus, striatum, and corpus callosum, suggesting that Zn reduction was not due to macroscopic abnormalities. Overall, these results suggest that Zn may improve short-term and physical neurological recovery, but it may not be sufficient as a single pre-treatment for consequences of repetitive mild TBI such as cognitive impairment. These results further demonstrate the need for additional research documenting the underlying mechanisms of Zn in TBI-related neuropathology.

Perfusion alters free zinc levels in the rodent brain.

BACKGROUND: Fixation of brain tissue is a common practice which allows preservation of tissue and aids in preventing structural and chemical abnormalities. However, fixation procedures may disrupt the levels of biometals such as zinc when compared to tissue that is fresh-frozen. Thus, we sought to determine if any differences in free-zinc levels exist between perfused and fresh-frozen tissue. Zinc is an essential biometal critical for cellular communication and memory and exists in both bound and free forms; the latter playing critical roles in synaptic communication. New method: C57BL/6 J mice were divided into two water types: those given lab water and those given water supplemented with 10 ppm zinc carbonate. Perfusion was carried out with 4% paraformaldehyde on half of the animals in each water group to assess the impact on levels of free Zn as measured through Zinpyr-1 fluorescence. RESULTS: There were significant differences in Zn fluorescence values between Zn-supplemented and lab water groups as well as between perfused and fresh-frozen tissues in the dentate gyrus and CA3 regions of the hippocampus, regions critical in learning & memory. Comparison with existing methods: These results show that when determining a method for euthanasia, any future histological techniques involving assessment of metal content should first be considered. CONCLUSIONS: Researchers must be cautious with the way in which tissue is collected and treated since this can lead to misleading conclusions when linking changes in behavior and relative levels of trace metals.

Preliminary evaluation of the pattern cutting and the ligating loop virtual laparoscopic trainers.

INTRODUCTION: The Fundamentals of Laparoscopic Surgery (FLS) trainer is currently the standard for training and evaluating basic laparoscopic skills. However, its manual scoring system is time-consuming and subjective. The Virtual Basic Laparoscopic Skill Trainer (VBLaST©) is the virtual version of the FLS trainer which allows automatic and real time assessment of skill performance, as well as force feedback. In this study, the VBLaST© pattern cutting (VBLaST-PC©) and ligating loop (VBLaST-LL©) tasks were evaluated as part of a validation study. We hypothesized that performance would be similar on the FLS and VBLaST© trainers, and that subjects with more experience would perform better than those with less experience on both trainers. METHODS: Fifty-five subjects with varying surgical experience were recruited at the Learning Center during the 2013 SAGES annual meeting and were divided into two groups: experts (PGY 5, surgical fellows and surgical attendings) and novices (PGY 1-4). They were asked to perform the PC or the ligating loop task on the FLS and the VBLaST© trainers. Their performance scores for each trainer were calculated and compared. RESULTS: There were no significant differences between the FLS and VBLaST© scores for either the PC or the ligating loop task. Experts' scores were significantly higher than the scores for novices on both trainers. CONCLUSION: This study showed that the subjects' performance on the VBLaST© trainer was similar to the FLS performance for both tasks. Both the VBLaST-PC© and the VBLaST-LL© tasks permitted discrimination between the novice and expert groups. Although concurrent and discriminant validity has been established, further studies to establish convergent and predictive validity are needed. Once validated as a training system for laparoscopic skills, the system is expected to overcome the current limitations of the FLS trainer.

Enhanced zinc consumption causes memory deficits and increased brain levels of zinc.

Zinc deficiency has been shown to impair cognitive functioning, but little work has been done on the effects of elevated zinc. This research examined the effect on memory of raising Sprague-Dawley rats on enhanced levels of zinc (10 ppm ZnCO3; 0.153 mM) in the drinking water for periods of 3 or 9 months, both pre- and postnatally. Controls were raised on lab water. Memory was tested in a series of Morris Water Maze (MWM) experiments, and zinc-treated rats were found to have impairments in both reference and working memory. They were significantly slower to find a stationary platform and showed greater thigmotaxicity, a measure of anxiety. On a working memory task, where the platform was moved each day, zinc-treated animals had longer latencies over both trials and days, swam further from the platform, and showed greater thigmotaxicity. On trials using an Atlantis platform, which remained in one place but was lowered on probe trials, the zinc-treated animals had significantly fewer platform crossings, spent less time in the target quadrant, and did not swim as close to the platform position. They had significantly greater latency on nonprobe trials. Microprobe synchrotron X-ray fluorescence (microSXRF) confirmed that brain zinc levels were increased by adding ZnCO3 to the drinking water. These data show that long-term dietary administration of zinc can lead to impairments in cognitive function.

Dopamine receptor subtype density as a function of age in Aplysia californica.

The age-associated changes in dopamine subtype receptors were examined in Aplysia californica. The density of the subtype receptors D1, D2, D3 and D4 was examined in the ganglia from 4.5-, 6-, 8-, 9- and 12-month animals. Receptor analysis was performed by examining the binding of radiolabeled ligands to the individual subtypes. [3H]SCH23390 and [3H]Clozapine were used to analyze D1 and D4 specific binding. [3H]Quinpirole was used for determining D2 and D3 specific binding. Specific binding was found to be present for all four receptor subtypes. All receptor subtypes showed an increase in density from 4.5 to 6 months. From 6 to 8 months D2 and D3 decreased, while D1 and D4 increased. D4 showed the strongest increase. All four subtypes examined showed decreases from 8 to 12 months. ANOVA results indicated age was a significant factor in the subtype receptor density for all receptor types.

The effect of dopamine receptor blockade on motor behavior in Aplysia californica.

The mammalian D1- and D2-like receptor blockers SCH-23390 and raclopride were used to block receptors in Aplysia californica, and the effect on reflexes and escape behavior was examined. Four groups of 20 young adults were each injected with SCH-23390, raclopride, SCH-23390+raclopride, or seawater. The drug (0.0125 mg/g of body weight) was injected 2 mm anterior to the parapodia. After the injection of either SCH-23390 or SCH-23390+raclopride, there was a significant increase in parapodia opening (P<.001), siphon withdrawal (P<.05), and galloping following tail pinch (P<.01) compared to raclopride-injected or control animals. The data showed that blockade of receptors by SCH-23390, but not raclopride, produced significant changes in motor behavior in A. californica.

Infralimbic muscarinic M1 receptors modulate anxiety-like behaviour and spontaneous working memory in mice.

RATIONALE: Spontaneous working memory and anxiety-like behaviour can be concurrently influenced following kappa 1 opioid agonist or antagonist infusions in the infralimbic (IL) area of the ventromedial prefrontal cortex (vmPFC) in CD-1 mice. OBJECTIVE: The present study sought to evaluate whether acetylcholine (ACh) muscarinic (M) receptor drugs can similarly influence these cognitive-behavioural processes in the IL cortex. METHOD: Anxiety was evaluated in the elevated plusmaze and spontaneous working memory was evaluated in the Y-maze following scopolamine, pirenzepine or McN-A-343 infusion in the IL cortex. RESULTS: In experiment 1, the non-specific muscarinic receptor antagonist, scopolamine, was anxiogenic in trial 1 (5, 10 and 20 nmol), but did not influence behaviour in trial 2 (no-injection) in the elevated plus-maze 24 h later. In week 2, scopolamine disrupted spontaneous working memory in the Y-maze at the highest dose (20 nmol). In experiment 2, pretreatment with the M1 antagonist, pirenzepine, was anxiolytic in trial 1 (5 and 10 nmol), as well as in trial 2 (no-injection) in the elevated plus-maze 24 h later (0.25, 1.25, 2.5, 5 and 10 nmol). In week 2, pirenzepine disrupted spontaneous working memory in the Y-maze (2.5, 5 and 10 nmol). In experiment 3, pretreatment with the M1 agonist, McN-A-343, was anxiogenic in trial 1 (2.5, 5, 10 and 20 nmol), as well as in trial 2 (no-injection) in the elevated plus-maze 24 h later (2.5, 5, 10 and 20 nmol). In week 2, McN-A-343 enhanced spontaneous working memory in the Y-maze (2.5, 5, 10 and 20 nmol). CONCLUSIONS: (1) Enhanced ACh transmission in the vmPFC induces anxiety in challenging environments and enhances spontaneous working memory performance. (2) Blocking or activating postsynaptic M1 receptors in the vmPFC may truncate or exaggerate, respectively, afferent anxiety-relevant information. (3) IL pirenzepine and McN-A-343 exert long-term opposite effects on aversive learning during trial 1 in the elevated plus-maze.

Role of disulfide bridges in the folding, structure and biological activity of omega-conotoxin GVIA.

Omega-Conotoxin GVIA (GVIA), an N-type calcium channel blocker from the cone shell Conus geographus, is a 27 residue polypeptide cross-linked by three disulfide bonds. Here, we report the synthesis, structural analysis by (1)H NMR and bioassay of analogues of GVIA with disulfide bridge deletions and N- and C-terminal truncations. Two analogues that retain the crucial Lys-2 and Tyr-13 residues in loops constrained by two native disulfide bridges were synthesised using orthogonal protection of cysteine residues. In the first analogue, the Cys-15-Cys-26 disulfide bridge was deleted (by replacing the appropriate Cys residues with Ser), while in the second, this disulfide bridge and the eight C-terminal residues were deleted. No activity was detected for either analogue in a rat vas deferens assay, which measures N-type calcium channel activity in sympathetic nerve, and NMR studies showed that this was due to a gross loss of secondary and tertiary structure. Five inactive analogues that were synthesised without orthogonal protection of Cys residues as part of a previous study (Flinn et al. (1995) J. Pept. Sci. 1, 379-384) were also investigated. Three had single disulfide deletions (via Ser substitutions) and two had N- or C-terminal deletions in addition to the disulfide deletion. Peptide mapping and NMR analyses demonstrated that at least four of these analogues had non-native disulfide pairings, which presumably accounts for their lack of activity. The NMR studies also showed that all five analogues had substantially altered tertiary structures, although the backbone chemical shifts and nuclear Overhauser enhancements (NOEs) implied that native-like turn structures persisted in some of these analogues despite the non-native disulfide pairings. This work demonstrates the importance of the disulfides in omega-conotoxin folding and shows that the Cys-15-Cys-26 disulfide is essential for activity in GVIA. The NMR analyses also emphasise that backbone chemical shifts and short- and medium-range NOEs are dictated largely by local secondary structure elements and are not necessarily reliable monitors of the tertiary fold.

Olanzapine for patients with treatment-resistant schizophrenia: a naturalistic case-series outcome study.

OBJECTIVE: This prospective, consecutive case-series outcome investigation evaluates the effectiveness of olanzapine in 16 patients with treatment-resistant schizophrenia. METHOD: Treatment resistance was defined as nonresponsiveness to at least 3 antipsychotic drugs from at least 2 different chemical classes. A minimum baseline score on the Brief Psychiatric Rating Scale (BPRS) of 45 was required for enrolment. Outcome evaluation measures included the BPRS, Global Assessment Scale (GAS), and Abnormal Involuntary Movement Scale (AIMS). RESULTS: Subjects (n = 16) has a mean age of 40 years and mean duration of illness of 16 years. Olanzapine treatment was initiated at 5 mg daily and was increased based on clinical judgement up to a maximum of 40 mg daily. Significant decreases in mean BPRS (P < 0.001) and GAS (P < 0.01) scores were observed at weeks 4, 8, 12, and 16, compared with baseline. Eight of 16 patients responded to olanzapine, as defined by a 20% decrease in BPRS score by week 16. Dyskinetic movements significantly increased at week 4 (P < 0.01) but did not differ from baseline at weeks 8 and 16. CONCLUSION: These results suggest that olanzapine at moderate to high doses may offer an effective treatment for a significant proportion of patients with schizophrenia nonresponsive to multiple trials of conventional antipsychotics. A randomized controlled trial is encouraged to validate these findings, and comparative trials are required to determine when clinicians should consider this approach.

Roles of key functional groups in omega-conotoxin GVIA synthesis, structure and functional assay of selected peptide analogues.

The contributions of various functional groups to the pharmacophore of the N-type calcium-channel blocker, omega-conotoxin GVIA (GVIA), have been investigated using structural and in-vitro functional studies of analogues substituted at one or two positions with non-native residues. In most cases the structure of the analogue was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) were employed to monitor N-type calcium-channel activity. The data provide a more detailed picture of the roles in GVIA structure and activity of the crucial Lys2 and Tyr13, as well as all other positively charged residues, Tyr22, the hydroxyproline residues and the C-terminal amido moiety, many of which were identified as being important for activity in an alanine scan [Lew et al. (1997) J. Biol. Chem. 272, 12014-12023]. Substitutions of Lys2 with nonstandard amino acids and arginine quantified the roles of the length and charge of the Lys side chain. The orientation of the Tyr13 side chain and its hydroxyl moiety was shown to be important by substitution with d-Tyr and the d-form and l-form of the constrained analogue 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid [Tic(OH)]. The roles of the Hyp10 and Hyp21 hydroxyl groups, investigated by proline substitutions, appear to be more structural (as monitored by NMR) than functional, although small decreases in potency were observed in some assays. The reversibility of the channel blockade was also studied, and several analogues with faster wash-out characteristics than native GVIA were identified. Rapid reversibility (as in the case of omega-conotoxin MVIIA) may be beneficial for therapeutic applications. Disubstituted analogues revealed some interesting cooperative effects, which were not predicted from single-residue substitutions. A disubstituted chimera of GVIA and omega-conotoxin MVIIA was more potent than either native molecule. The more detailed description of the GVIA pharmacophore obtained here provides a better basis for the future design of truncated peptide and peptidomimetic analogues.

Meeting the needs of primary care physicians: a guide to content for programs on depression.

OBJECTIVE: The purpose of this article is to identify literature-based content for the design of educational programs on depression for practicing primary care physicians. METHODS: A MEDLINE search was conducted of English-language medical literature published from 1982 through July 1997 for studies describing primary care physicians' knowledge, skills, practice patterns, and perceived barriers related to care of depressed patients. Studies focusing exclusively on residency training and those describing physician practices outside North America were excluded. Of 377 articles identified, forty met inclusion and exclusion criteria. RESULTS: Recommendations for educational content were identified from the literature review. For recognition, educators should prioritize communication skills and strategies for the use of depression questionnaires. For diagnosis, practice interpreting symptoms in the medically ill, strategies for efficient diagnosis, and systematic approaches to assessing suicide risk should be emphasized. For treatment, greater attention to the therapeutic alliance, staged therapy, and strategies for improving medication adherence are indicated. CONCLUSIONS: There is a moderately well developed literature describing self-perceived and observed gaps in the current care for depression in primary care. Addressing the entire list of needs would take more time than practicing physicians are likely to have. An important challenge for educators is to design flexible programs based on individualized needs assessment or, when not possible, to prioritize the most generalizable needs.

Structure-function relationships of omega-conotoxin GVIA. Synthesis, structure, calcium channel binding, and functional assay of alanine-substituted analogues.

The structure-function relationships of the N-type calcium channel blocker, omega-conotoxin GVIA (GVIA), have been elucidated by structural, binding and in vitro and in vivo functional studies of alanine-substituted analogues of the native molecule. Alanine was substituted at all non-bridging positions in the sequence. In most cases the structure of the analogues in aqueous solution was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Replacement of Lys2 and Tyr13 with Ala caused reductions in potency of more than 2 orders of magnitude in three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) and a rat brain membrane binding assay. Replacement of several other residues with Ala (particularly Arg17, Tyr22 and Lys24) resulted in significant reductions in potency (<100-fold) in the functional assays, but not the binding assay. The potencies of the analogues were strongly correlated between the different functional assays but not between the functional assays and the binding assay. Thus, the physiologically relevant assays employed in this study have shown that the high affinity of GVIA for the N-type calcium channel is the result of interactions between the channel binding site and the toxin at more sites than the previously identified Lys2 and Tyr13.

Serotonin levels as a function of age in Aplysia californica.

The neurotransmitter serotonin (5-HT) plays an important role in a number of behaviors in Aplysia californica some of which have been shown to vary with age. We were thus interested in examining the age-dependence of 5-HT in A. californica. Because animals of the same age can have very different weights, and weight alone is reliably known for wild-caught animals, we also examined the variation of 5-HT with weight. Serotonin was measured in the ring and abdominal ganglia combined, in lab-reared animals from 3 to 12 months post-hatch across a wide weight range. Serotonin increased rapidly from 4 to 6 months, and more slowly from 6 to 13 months. Serotonin scaled by soluble ganglion protein increased from 3 to 6-7 months, reached a maximum, and then decreased again. Serotonin, but not scaled 5-HT, increased significantly with weight across the whole weight range. Animals of the same weight, but different ages, had different 5-HT levels, as did young animals of the same age but different weight. Serotonin varied significantly with both age and weight, with the age-dependence being the more significant.

Age dependent changes in serotonin and dopamine receptors in Aplysia californica.

Age related changes in dopaminergic and serotonergic receptors were examined in Aplysia californica. In this study dopamine (DA) and serotonin (5-HT) receptor levels were examined for animals belonging to 4-, 5-, 6-, 8-, 9- and 12-month age groups. Receptors analysis was performed using radio-labeled d-[3H] lysergic acid diethylamide (LSD) as the specific ligand. Specific binding for 5-HT was found to be significantly greater than that for DA in the young (4-month post-hatch) animals. The total DA and 5-HT receptor levels changed significantly with age. Dopamine levels increased from 5.34 fmol/mg of protein at 4 months to 19.11 fmol/mg at 12 months. Serotonin receptor levels increased from 7.35 fmol/mg at 4 months to 20.45 at 12 months.

Synthesis and biological characterization of a series of analogues of omega-conotoxin GVIA.

The 27-residue polypeptide omega-conotoxin GVIA (omega-CgTx), from the venom of the cone shell Conus geographus, blocks N-type neuronal calcium channels. It contains three disulphide bridges. We report here the synthesis and biological characterization of a series of analogues in which one disulphide has been replaced by substitution of appropriate Cys residues with Ser, viz. [Ser1,16]-omega -CgTx, [Ser8,19]-omega-CgTx, [Ser15,26)-omega-CgTx, [Ser16]-omega-CgTx8-27 and [Ser15]-omega-CgTx1-19. All syntheses were conducted manually using either Boc or Fmoc methodology. Deprotected peptides were oxidized to their bridged forms using either aerial oxidation or aqueous dimethyl sulphoxide. Peptides were purified using RP-HPLC, and their purity and identity were checked by RP-HPLC, capillary electrophoresis and mass spectrometry. Inhibition of neuronal N-type calcium channels was assessed as the inhibition of the twitch responses of rat vas deferens stimulated with single electrical pulses at 20 second intervals. None of these analogues was biologically active, suggesting that the disulphides play an important role in maintaining biological activity.

Synthesis and biological characterisation of a series of iberiotoxin analogues.

We report here the synthesis of iberiotoxin (IbTX), a 37-amino acid peptide containing three disulfide bridges, and a series of mono-looped analogues. All syntheses were conducted using Fmoc chemistry. Synthesis of IbTX gave a product which was indistinguishable from a reference sample in both its physico-chemical properties and its biological activity. A series of three mono-looped analogues, in which four of the six cysteines were replaced by alanine, were synthesised to give [Ala7,13,28,33]-IbTX, [Ala13,17,33,35]-IbTX and [Ala7,17,28,35]-IbTX. Oxidation of the linear form of [Ala7,17,28,35]-IbTX to form the Cys13 to Cys33 disulfide bridge proceeded more slowly than that of the other two analogues. None of these analogues was biologically active, indicating that no single loop is the mediator of channel blocking activity.