Search

Objective: The increasing prevalence of obesity makes it important to increase the understanding of the maturation and function of the neuronal integrators and regulators of metabolic function. Methods: Behavioral, molecular, and physiological analyses of transgenic mice with Sine oculis 3 (Six3) deleted in mature neurons using the Synapsincre allele. Results: Conditional deletion of the homeodomain transcription factor Six3 in mature neurons causes dwarfism and weakens circadian wheel-running activity rhythms but increases general activity at night, and improves metabolic function, without impacting pubertal onset or fertility in males. The reduced growth in 6-week-old Six3fl/fl:Synapsincre (Six3syn) males correlates with increased somatostatin (SS) expression in the hypothalamus and reduced growth hormone (GH) in the pituitary. In contrast, 12-week-old Six3syn males have increased GH release, despite an increased number of the inhibitory SS neurons in the periventricular nucleus. GH is important in glucose metabolism, muscle function, and bone health. Interestingly, Six3syn males have improved glucose tolerance at 7, 12, and 18 weeks of age, which, in adulthood, is associated with increased % lean mass and increased metabolic rates. Further, 12-week-old Six3syn males have reduced bone mineralization and a lower bone mineral density, indicating that reduced GH levels during early life cause a long-term reduction in bone mineralization. Conclusion: Our study points to the novel role of Six3 in post-proliferative neurons to regulate metabolic function through SS neuron control of GH release.

In mammals, the master circadian clock resides in the suprachiasmatic nucleus (SCN). The SCN is characterized by robust circadian oscillations of clock gene expression and neuronal firing. The synchronization of circadian oscillations among individual cells in the SCN is attributed to intercellular coupling. Previous studies have shown that gap junctions, specifically those composed of connexin-36 (Cx36) subunits, are required for coupling of electrical firing among SCN neurons at a time scale of milliseconds. However, it remains unknown whether Cx36 gap junctions also contribute to coupling of circadian (~24 h) rhythms of clock gene expression. Here, we investigated circadian expression patterns of the clock gene Period 2 (Per2) in the SCN of Cx36-deficient mice using luminometry and single-cell bioluminescence imaging. Surprisingly, we found that synchronization of circadian PER2 expression rhythms is maintained in SCN explants from Cx36-deficient mice. Since Cx36 expression levels change with age, we also tested circadian running-wheel behavior of juvenile (3–4 weeks old) and adult (9–30 weeks old) Cx36-deficient mice. We found that impact of connexin-36 expression on circadian behavior changes greatly during postnatal development. However, consistent with the intact synchrony among SCN cells in cultured explants, Cx36-deficient mice had intact locomotor circadian rhythms, although adults displayed a lengthened period in constant darkness. Our data indicate that even though Cx36 may be required for electrical coupling of SCN cells, it does not affect coupling of molecular clock gene rhythms. Thus, electrical coupling of neurons and coupling of circadian clock gene oscillations can be regulated independently in the SCN.

Background Major depressive disorder is associated with disturbed circadian rhythms. To investigate the causal relationship between mood disorders and circadian clock disruption, previous studies in animal models have employed light/dark manipulations, global mutations of clock genes, or brain area lesions. However, light can impact mood by noncircadian mechanisms; clock genes have pleiotropic, clock-independent functions; and brain lesions not only disrupt cellular circadian rhythms but also destroy cells and eliminate important neuronal connections, including light reception pathways. Thus, a definitive causal role for functioning circadian clocks in mood regulation has not been established. Methods We stereotactically injected viral vectors encoding short hairpin RNA to knock down expression of the essential clock gene Bmal1 into the brain's master circadian pacemaker, the suprachiasmatic nucleus (SCN). Results In these SCN-specific Bmal1 -knockdown (SCN- Bmal1 -KD) mice, circadian rhythms were greatly attenuated in the SCN, while the mice were maintained in a standard light/dark cycle, SCN neurons remained intact, and neuronal connections were undisturbed, including photic inputs. In the learned helplessness paradigm, the SCN- Bmal1 -KD mice were slower to escape, even before exposure to inescapable stress. They also spent more time immobile in the tail suspension test and less time in the lighted section of a light/dark box. The SCN- Bmal1 -KD mice also showed greater weight gain, an abnormal circadian pattern of corticosterone, and an attenuated increase of corticosterone in response to stress. Conclusions Disrupting SCN circadian rhythms is sufficient to cause helplessness, behavioral despair, and anxiety-like behavior in mice, establishing SCN- Bmal1 -KD mice as a new animal model of depression.

Bipolar disorder (BD) is characterized by depression, mania, and circadian rhythm abnormalities. Lithium, a treatment for BD stabilizes mood and increases circadian rhythm amplitude. However, in fibroblasts grown from BD patients, lithium has weak effects on rhythm amplitude compared to healthy controls. To understand the mechanism by which lithium differentially affects rhythm amplitude in BD cells, we investigated the extracellular-signal-regulated kinase (ERK) and related signaling molecules linked to BD and circadian rhythms. In fibroblasts from BD patients, controls and mice, we assessed the contribution of the ERK pathway to lithium-induced circadian rhythm amplification. Protein analyses revealed low phospho-ERK1/2 (p-ERK) content in fibroblasts from BD patients vs. controls. Pharmacological inhibition of ERK1/2 by PD98059 attenuated the rhythm amplification effect of lithium, while inhibition of two related kinases, c-Jun N-terminal kinase (JNK), and P38 did not. Knockdown of the transcription factors CREB and EGR-1, downstream effectors of ERK1/2, reduced baseline rhythm amplitude, but did not alter rhythm amplification by lithium. In contrast, ELK-1 knockdown amplified rhythms, an effect that was not increased further by the addition of lithium, suggesting this transcription factor may regulate the effect of lithium on amplitude. Augmentation of ERK1/2 signaling through DUSP 6 knockdown sensitized NIH3T3 cells to rhythm amplification by lithium. In BD fibroblasts, DUSP 6 knockdown reversed the BD rhythm phenotype, restoring the ability of lithium to increase amplitude in these cells. We conclude that the inability of lithium to regulate circadian rhythms in BD may reflect reduced ERK activity, and signaling through ELK-1.

Bipolar disorder (BD) is associated with mood episodes and low amplitude circadian rhythms. Previously, we demonstrated that fibroblasts grown from BD patients show weaker amplification of circadian rhythms by lithium compared to control cells. Since calcium signals impact upon the circadian clock, and L-type calcium channels (LTCC) have emerged as genetic risk factors for BD, we examined whether loss of function in LTCCs accounts for the attenuated response to lithium in BD cells. We used fluorescent dyes to measure Ca2+ changes in BD and control fibroblasts after lithium treatment, and bioluminescent reporters to measure Per2::luc rhythms in fibroblasts from BD patients, human controls, and mice while pharmacologically or genetically manipulating calcium channels. Longitudinal expression of LTCC genes (CACNA1C, CACNA1D and CACNB3) was then measured over 12–24 h in BD and control cells. Our results indicate that independently of LTCCs, lithium stimulated intracellular Ca2+ less effectively in BD vs. control fibroblasts. In longitudinal studies, pharmacological inhibition of LTCCs or knockdown of CACNA1A, CACNA1C, CACNA1D and CACNB3 altered circadian rhythm amplitude. Diltiazem and knockdown of CACNA1C or CACNA1D eliminated lithium's ability to amplify rhythms. Knockdown of CACNA1A or CACNB3 altered baseline rhythms, but did not affect rhythm amplification by lithium. In human fibroblasts, CACNA1C genotype predicted the amplitude response to lithium, and the expression profiles of CACNA1C, CACNA1D and CACNB3 were altered in BD vs. controls. We conclude that in cells from BD patients, calcium signaling is abnormal, and that LTCCs underlie the failure of lithium to amplify circadian rhythms.

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.

INTRODUCTION: Mental health professionals are gatekeepers of patient confidentiality. Yet, confidentiality held too strictly, by allowing a potentially dangerous driver to assume control of a car, endangers society. Recent court cases have mandated that mental health professionals must warn those who may be potentially harmed by patients. In spite of this, disagreements linger as to whether it is the responsibility of governmental agencies or mental health professionals to decide who is unfit to operate a vehicle because of cognitive impairment. Methods: This article addresses the legally relevant considerations when working with cognitively compromised individuals who operate a motor vehicle. Legal issues surrounding confidentiality, patient rights, foreseeable risk, and the duty to warn and protect are presented in order to understand their relationship to recent court rulings. IMPACT ON INDUSTRY: The impact on the mental health care industry includes not only concerns about increased insurance premiums or costs due to alleged negligence or litigation expenses secondary to failure to ensure the safety of an impaired client under their care. Mental health care providers are aware that the welfare of clients with impairment to cognitive decision making ability may require unique considerations for safety such as ensuring safe and appropriate transportation. Language: en

Questions: Does the addition of cyclic pneumatic soft-tissue compression during the 6-week immobilisation period following fracture of the distal radius result in a faster recovery of muscle strength and joint range of motion? Does it result in a larger recovery of muscle strength and joint range of motion immediately after the immobilisation period (at 6 weeks) or four weeks after the immobilisation period (at 10 weeks)? Design: Randomised controlled trial with concealed allocation and assessor blinding. Participants: 21 patients with fracture of the distal radius. Intervention: The experimental group received cyclic pneumatic soft-tissue compression during the 6-week immobilisation period whereas the control group received usual care. Both groups were instructed to actively make a fist 100 times per day during the 6-week immobilisation period and were given an exercise program during the 4-week post-immobilisation period. Outcome measures: Function was measured as power grip, pinch grip, key grip, and supination strength using dynamometry from Week 1 to 10 as well as wrist flexion/extension and forearm supination/pronation range of motion using goniometry from Week 6 to 10. The outcome measures are presented as a percentage of the intact side. Results: The experimental group improved significantly faster than the control group in muscle strength from Week 1 to 10 (p ≤ 0.001) but not in joint range of motion from Week 6 to 10 (p > 0.05). By Week 6, the experimental group was 12–26% stronger and had 8–14% more range of motion than the control group. By Week 10, the experimental group was 24–29% stronger and had 10–15% more range of motion than the control group. Conclusion: The findings indicate that a larger clinical trial is warranted and should incorporate direct measures of fracture healing. [Challis MJ, Jull GJ, Stanton WR, Welsh MK (2007) Cyclic pneumatic soft-tissue compression enhances recovery following fracture of the distal radius: a randomised controlled trial. Australian Journal of Physiotherapy 53: 247–252]

Luciferase enzymes catalyze the emission of light from a substrate -- a phenomenon known as bioluminescence -- and have been employed as reporters of many biological functions. Luminescent reporters are much dimmer than fluorescent reporters, and therefore provide relatively modest spatial and temporal resolution. Yet, they are generally more sensitive and less toxic, making them particularly useful for long-term longitudinal studies of living cells, tissues and whole animals. Bioluminescence imaging has proven useful for detecting protein-protein interactions, for tracking cells in vivo, and for monitoring the transcriptional and post-transcriptional regulation of specific genes. Recent applications have included longitudinal monitoring of tumor progression in vivo, and monitoring circadian rhythms with single-cell resolution.

Circadian (ca. 24 hr) oscillations in expression of mammalian “clock genes” are found not only in the suprachiasmatic nucleus (SCN), the central circadian pacemaker, but also in peripheral tissues [1]. Under constant conditions in vitro, however, rhythms of peripheral tissue explants [2] or immortalized cells [3] damp partially or completely. It is unknown whether this reflects an inability of peripheral cells to sustain rhythms, as SCN neurons can, or a loss of synchrony among cells. Using bioluminescence imaging of Rat-1 fibroblasts transfected with a Bmal1::luc plasmid and primary fibroblasts dissociated from mPer2Luciferase-SV40 knockin mice, we monitored single-cell circadian rhythms of clock gene expression for 1–2 weeks. We found that single fibroblasts can oscillate robustly and independently with undiminished amplitude and diverse circadian periods. Cells were partially synchronized by medium changes at the start of an experiment, but due to different intrinsic periods, their phases became randomly distributed after several days. Closely spaced cells in the same culture did not have similar phases, implying a lack of functional coupling among cells. Thus, like SCN neurons, single fibroblasts can function as independent circadian oscillators; however, lack of oscillator coupling in dissociated cell cultures leads to a loss of synchrony among individual cells and damping of the ensemble rhythm at the population level.

Individual neurons dissociated from rat suprachiasmatic nucleus can express independently phased circadian firing rhythms in culture. The phases of these rhythms are unperturbed by reversible blockade of neuronal firing lasting 2.5 days, indicating that multiple circadian clocks continue to operate in the absence of conventional synaptic transmission. The possibility remains, however, that these circadian rhythms might depend on some other form of intercellular communication. In the present study, a potential role for gap junctional coupling in SCN cultures was evaluated by introduction of the tracer molecule Neurobiotin into both neurons (n = 98) and astrocytes (n = 10), as well as by immunolabeling for specific connexins, the molecular components of gap junctions. Astrocytes were extensively coupled to each other by connexin43-positive gap junctions, but no evidence was found for coupling of neurons to each other or to astrocytes. These data support the hypothesis that neurons expressing independently phased circadian rhythms in SCN cultures (‘clock cells’) are autonomous, single cell circadian oscillators, but do not exclude a role for glia in synchronizing neuronal clock cells in vivo.

Within the mammalian hypothalamus, the suprachiasmatic nucleus (SCN) contains a circadian clock for timing of diverse neuronal, endocrine, and behavioral rhythms. By culturing cells from neonatal rat SCN on fixed microelectrode arrays, we have been able to record spontaneous action potentials from individual SCN neurons for days or weeks, revealing prominent circadian rhythms in firing rate. Despite abundant functional synapses, circadian rhythms expressed by neurons in the same culture are not synchronized. After reversible blockade of neuronal firing lasting 2.5 days, circadian firing rhythms re-emerge with unaltered phases. These data suggest that the SCN contains a large population of autonomous, single-cell circadian oscillators, and that synapses formed in vitro are neither necessary for operation of these oscillators nor sufficient for synchronizing them.

Objective: The sedative effects of cetirizine (10 mg once daily), diphenhydramine (50 mg three times daily), and placebo, each administered during a 3-day period, were compared with objective measures of sleepiness and performance. Methods: Twelve atopic subjects received each of the three treatments for 3 consecutive days in a double-blind Latin square design. Subjects received either cetirizine at 8:00am and placebo at 3:00pm and 10:00pm; diphenhydramine at 8:00am, 3:00pm, and 10:00pm; or placebo at all three times. Sleepiness was measured on days 1 and 3 with the multiple sleep latency test at 9:00am, 11:00am, 1:00pm, 3:00pm, and 5:00pm. Performance was assessed with a 60-minute simulated assembly line task at 9:30am, 11:30am, 1:30pm, and 3:30pm. Nightly sleep duration was estimated with actigraphy. Results: Compared with placebo and cetirizine, diphenhydramine produced marked impairment only on the first day of drug administration. The multiple sleep latency test and the simulated assembly line task values remained stable across days with cetirizine and placebo but improved with diphenhydramine, resulting in no differences among the three conditions on the third day. Conclusion: Unlike cetirizine, diphenhydramine produced acute impairment of alertness and performance. By the third day of administration, however, this impairment was no longer present, apparently because of development of tolerance to the sedative effects.

The structure of the Escherichia coli thymidylate synthase (TS) covalent inhibitory ternary complex consisting of enzyme, 5-fluoro-2'-deoxyuridylate (FdUMP) and 5,10-methylene tetrahydrofolate (CH2-H4PteGlu) has been determined at 2.5 A resolution using difference Fourier methods. This complex is believed to be a stable structural analog of a true catalytic intermediate. Knowledge of its three-dimensional structure and that for the apo enzyme, also reported here, suggests for the first time how TS may activate dUMP and CH2-H4PteGlu leading to formation of the intermediate and offers additional support for the hypothesis that the substrate and cofactor are linked by a methylene bridge between C-5 of the substrate nucleotide and N-5 of the cofactor. By correlating these structural results with the known stereospecificity of the TS-catalyzed reaction it can be inferred that the catalytic intermediate, once formed, must undergo a conformational isomerization before eliminating across the bond linking C-5 of dUMP to C-11 of the cofactor. The elimination itself may be catalyzed by proton transfer to the cofactor's 5 nitrogen from invariant Asp169 buried deep in the TS active site. The juxtaposition of Asp169 and bound tetrahydrofolate in TS is remarkably reminiscent of binding geometry found in dihydrofolate reductase where a similarly conserved carboxyl group serves as a general acid for protonating the corresponding pyrazine ring nitrogen of dihydrofolate.

Youth with HIV infection experience stress from many sources, including their health, stigma associated with HIV, and economic concerns. 1 Stress experienced by HIV-positive adolescents can have serious negative consequences on their physical and psychosocial health. Exposure to stress is associated with HIV progression, 2,3 and stress frequently results in maladaptive behavioral responses—poor HIV medication adherence, selfmedication through substance use, risky sexual behavior, and other high-risk behaviors. 4 In adults with HIV, stress reduction programs have been shown to improve behavioral, psychological, and immunologic domains. 5 Mindfulness-based stress reduction (MBSR) as developed by Kabat-Zinn 6 is a structured 8- to 10-week program designed to cultivate focused, nonjudgmental awareness of the present moment. 6 Mindfulness-based stress reduction has been used and studied since 1979 in adult populations with heterogeneous chronic illnesses. Weekly MBSR classes provide instruction in mindfulness techniques, such as mindful meditation, mindful yoga, and the body scan, to enhance participants’ appreciation for moment-to-moment awareness instead of attendingtoworriesaboutthefutureorpast.AlthoughMBSRhasbeen studied widely in adults, we are aware of few published studies of MBSR for youth 7-9 and none in HIV-positive youth. We are interested in investigating the potential benefit of MBSR for urban HIV-infected youth. As a first step, we conducted a pilot study to investigate the feasibility (through enrollment and attendance) and acceptability (through participant interview)ofMBSRinanurbanHIV-infectedyouthpopulation.If MBSR is feasible and acceptable in this population, we will plan a larger randomized trial to evaluate MBSR’s effect on psychological and physical well-being and quality of life.

Introduction Acute rejection remains an important cause of graft loss after renal transplantation. It has been suggested that cytokine genotyping may play a predictive role in identifying individuals who are at higher risk of acute rejection with a view to individualizing their immunosuppression. The aim of this study was to investigate any possible associations between acute rejection and certain cytokine polymorphisms. Methods We genotyped 91 cadaveric renal transplant recipients on tacrolimus-based immunosuppression and 84 of their donors. The cytokine polymorphisms studied were the following: tumor necrosis factor (TNF)-α-1032 T/C, TNF-α-865 C/A, TNF-α-859 G/A, interleukin (IL)1-R1-970 C/T, IL-10 haplotype [−1082, −819, −592], and IL-6-174 C/G. Results We found no association between any polymorphism and the incidence of acute rejection. This was true for both the recipient and donor population. Conclusion Cytokine polymorphisms did not influence acute rejection in our study. We conclude that in the modern era of immunosuppression cytokine genotyping is not a significant predictor of acute rejection in renal transplantation.

Maloney and Wandell [Vision Res. 24, 633-640 (1984)] describe a model of the response of a single visual channel to weak test lights. The initial channel response is a linearly filtered version of the stimulus. The filter output is randomly sampled over time. Each time a sample occurs there is some probability--increasing with the magnitude of the sampled response--that a discrete detection event is generated. Maloney and Wandell derive the statistics of the detection events. In this paper we test the hypothesis that the reaction time responses to the presence of a weak test light are initiated at the first detection event. This permits us to extend the application of the model to lights that are slightly above threshold, but still within the linear operating range of the visual system. We test a parameter-free prediction of the model proposed by maloney and Wandell for lights detected by this statistic. The data are in agreement with the prediction.

We describe and test predictions of a model of long-wavelength test sensitivity upon large, uniform backgrounds. The model explains changes in sensitivity in the red-green detection pathways strictly based upon losses of sensitivity in the receptors. We derive the prediction that field mixture data for field-mixtures of μ 1 (fixed) and an addend, μ 2 , must follow the same shape on different intensities of the fixed background, μ 1 . This prediction is not in good agreement with the measurements.