The brown clock: circadian rhythms in stramenopiles

Circadian clocks allow organisms to anticipate environmental changes associated with the diurnal light/dark cycle. Circadian oscillators have been described in plants and green algae, cyanobacteria, animals and fungi, however, little is known about the circadian clocks of photosynthetic eukaryotes outside the green lineage. Stramenopiles are a diverse group of secondary endosymbionts whose plastid originated from a red alga. Photosynthetic stramenopiles, which include diatoms and brown algae, play key roles in biogeochemical cycles and are important components of marine ecosystems. Genome annotation efforts indicated the presence of a novel type of oscillator in these organisms and the first circadian clock component in a stramenopile has been recently discovered. This review summarizes the phenotypic characterization of circadian rhythms in stramenopiles and current efforts to determine the mechanisms of this ‘brown clock’. The elucidation of this brown clock will enable a deeper understanding of the role of self-sustained oscillations in the adaptation to life in marine environments.     

Amiloride does not alter NaCl avoidance in Fischer-344 rats

Fischer-344 (F-344) rats differ from other common rat strains in that they fail to show any preference for NaCl at any concentration in two- bottle preference tests. Because 100 microM amiloride partially blocks the NaCl-evoked chorda tympani (CT) response in electrophysiological studies, we tested NaCl preference (0.068-0.273 M) in F-344 rats with and without 100 microM amiloride solution as the solvent. A third group was tested with unadulterated NaCl solutions following CT transection. Amiloride had no significant effect on the NaCl preference-aversion function, whereas CT transection significantly reduced NaCl avoidance. These results suggest that the amiloride-sensitive component of the NaCl response is not necessary for F-344 rats to display avoidance of NaCl, but the entire CT input is.      

The coelomycetous genera <Emphasis Type="Italic">Chaetomella</Emphasis> and <Emphasis Type="Italic">Pilidium</Emphasis> represent a newly discovered lineage of inoperculate discomycetes

The coelomycetous genera Chaetomella and Pilidium were determined to be closely related to each other, yet are recognized as distinct genera based on both morphological observations and rDNA sequence analyses. Analyses of the SSU and LSU of the nuclear ribosomal RNA genes suggest that Chaetomella and Pilidium along with Sphaerographium tenuirostrum and Synchaetomella lunatospora constitute a distinct lineage within the ascomycetes that is allied with the Leotiomycetes. Chaetomella and Pilidium both produce black pycnidia generally opening by a raphe, acropleurogenous conidiogenous cells, and non-septate, hyaline, usually fusiform to falcate, rarely ellipsoid, conidia. Pycnidia of Pilidium are smooth while those of Chaetomella have setae of various types. A Hainesia sporodochial synanamorph was observed in cultures of C. oblonga and C. raphigera similar to H. lythri, the synanamorph of P. concavum. Specimens of C. oblonga, type species of Chaetomella, C. acutiseta, C. circinoseta, C. raphigera, Pilidium acerinum, type species of Pilidium, and P. concavum were examined. Diagnoses and illustrations are provided for these species along with a key to the accepted species in both genera. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Effects of mammary engorgement and feed withdrawal on microvascular function in lactating goat mammary glands

The responses of the mammary microvasculature in lactating goats (n = 8) during feed withdrawal (18-20 h) and mammary engorgement (26-28 h of milk accumulation) were compared using an indicator-dilution technique with FITC-albumin and [(14)C]sucrose as the intravascular and diffusible indicators, respectively. Feed withdrawal and mammary engorgement caused a 50-60% decrease in mammary arterial flow and in the permeability-surface area product (PS) values for sucrose. Only feed withdrawal increased the mean transit time [from 17.3 to 30.0 s, SE of the difference (SED) = 2.16, P < 0.01] of FITC-albumin, whereas only mammary engorgement reduced sucrose extraction (0.63 to 0.51, SED = 0.04, P < 0.05). Mammary engorgement also caused a substantial reduction in the sucrose-accessible extravascular space from 92 to 44 ml (SED = 15.2, P < 0.01). In a separate experiment using five goats, milking after mammary engorgement did not immediately restore arterial flow or sucrose extraction, indicating that the effect of milk accumulation was not mediated simply via increased intramammary pressure. In conclusion, feed withdrawal resulted in slower flow in the capillary bed but apparently no change in capillary recruitment, whereas mammary engorgement caused capillary derecruitment.     

Evolution of recombination in eutherian mammals: insights into mechanisms that affect recombination rates and crossover interference

Recombination allows faithful chromosomal segregation during meiosis and contributes to the production of new heritable allelic variants that are essential for the maintenance of genetic diversity. Therefore, an appreciation of how this variation is created and maintained is of critical importance to our understanding of biodiversity and evolutionary change. Here, we analysed the recombination features from species representing the major eutherian taxonomic groups Afrotheria, Rodentia, Primates and Carnivora to better understand the dynamics of mammalian recombination. Our results suggest a phylogenetic component in recombination rates (RRs), which appears to be directional, strongly punctuated and subject to selection. Species that diversified earlier in the evolutionary tree have lower RRs than those from more derived phylogenetic branches. Furthermore, chromosome-specific recombination maps in distantly related taxa show that crossover interference is especially weak in the species with highest RRs detected thus far, the tiger. This is the first example of a mammalian species exhibiting such low levels of crossover interference, highlighting the uniqueness of this species and its relevance for the study of the mechanisms controlling crossover formation, distribution and resolution.     

Distribution of alcohol dehydrogenase mRNA in the rat central nervous system. Consequences for brain ethanol and retinoid metabolism.

The localization of alcohol dehydrogenase (ADH) in brain regions would demonstrate active ethanol metabolism in brain during alcohol consumption, which would be a new basis to explain the effects of ethanol in the central nervous system. Tissue sections from several regions of adult rat brain were examined by in situ hybridization to detect the expression of genes encoding ADH1 and ADH4, enzymes highly active with ethanol and retinol. ADH1 mRNA was found in the granular and Purkinje cell layers of cerebellum, in the pyramidal and granule cells of the hippocampal formation and in some cell types of cerebral cortex. ADH4 expression was detected in the Purkinje cells, in the pyramidal and granule cells of the hippocampal formation and in the pyramidal cells of cerebral cortex. High levels of ADH1 and ADH4 mRNAs were detected in the CNS epithelial and vascular tissues: leptomeninges, choroid plexus, ependymocytes of ventricle walls, and endothelium of brain vessels. Histochemical methods detected ADH activity in rodent cerebellar slices, while Western-blot analysis showed ADH4 protein in homogenates from several brain regions. In consequence, small but significant levels of ethanol metabolism can take place in distinct areas of the CNS following alcohol consumption, which could be related to brain damage caused by a local accumulation of acetaldehyde. Moreover, the involvement of ADH in the synthesis of retinoic acid suggests a role for the enzyme in the regulation of adult brain functions. The impairment of retinol oxidation by competitive inhibition of ADH in the presence of ethanol may be an additional origin of CNS abnormalities caused by ethanol.     

Synergistic effect of IL-2, IL-12, and IL-18 on thymocyte apoptosis and Th1/Th2 cytokine expression

In the periphery, IL-18 synergistically induces the expression of the Th1 cytokine IFN-gamma in the presence of IL-12 and the Th2 cytokines IL-5 and IL-13 in the presence of IL-2. Although the expression of these cytokines has been described in the thymus, their role in thymic development and function remains uncertain. We report here that freshly isolated thymocytes from C57BL/6 and BALB/c mice stimulated in vitro with IL-2-plus-IL-18 or IL-12-plus-IL-18 produce large amounts of IFN-gamma and IL-13. Analysis of the thymic subsets, CD4(-)CD8(-) (DN), CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) revealed that IL-18 in combination with IL-2 or IL-12 induces IFN-gamma and IL-13 preferentially from DN cells. Moreover, DN2 and DN3 thymocytes contained more IFN-gamma(+) cells than cells in the later stage of maturation. Additionally, IL-18 in combination with IL-2 induces CCR4 (Th2-associated) and CCR5 (Th1-associated) gene expression. In contrast, IL-18-plus-IL-12 specifically induced CCR5 expression. The IL-2-plus-IL-18 or IL-12-plus-IL-18 effect on IFN-gamma and IL-13 expression is dependent on Stat4 and NF-kappaB but independent of Stat6, T-bet, or NFAT. Furthermore, IL-12-plus-IL-18 induces significant thymocyte apoptosis when expressed in vivo or in vitro, and this effect is exacerbated in the absence of IFN-gamma. IL-12-plus-IL-18-stimulated thymocytes can also induce IA-IE expression on cortical and medullary thymic epithelial cells in an IFN-gamma-dependent manner. Thus, the combination of IL-2, IL-12, and IL-18 can induce phenotypic and functional changes in thymocytes that may alter migration, differentiation, and cell death of immature T cells inside the thymus and potentially affect the Th1/Th2 bias in peripheral immune compartments.     

Topologies of a substrate protein bound to the chaperonin GroEL

The chaperonin GroEL assists polypeptide folding through sequential steps of binding nonnative protein in the central cavity of an open ring, via hydrophobic surfaces of its apical domains, followed by encapsulation in a hydrophilic cavity. To examine the binding state, we have classified a large data set of GroEL binary complexes with nonnative malate dehydrogenase (MDH), imaged by cryo-electron microscopy, to sort them into homogeneous subsets. The resulting electron density maps show MDH associated in several characteristic binding topologies either deep inside the cavity or at its inlet, contacting three to four consecutive GroEL apical domains. Consistent with visualization of bound polypeptide distributed over many parts of the central cavity, disulfide crosslinking could be carried out between a cysteine in a bound substrate protein and cysteines substituted anywhere inside GroEL. Finally, substrate binding induced adjustments in GroEL itself, observed mainly as clustering together of apical domains around sites of substrate binding.     

The influence of genetic and environmental factors among MDMA users in cognitive performance

This study is aimed to clarify the association between MDMA cumulative use and cognitive dysfunction, and the potential role of candidate genetic polymorphisms in explaining individual differences in the cognitive effects of MDMA. Gene polymorphisms related to reduced serotonin function, poor competency of executive control and memory consolidation systems, and high enzymatic activity linked to bioactivation of MDMA to neurotoxic metabolites may contribute to explain variations in the cognitive impact of MDMA across regular users of this drug. Sixty ecstasy polydrug users, 110 cannabis users and 93 non-drug users were assessed using cognitive measures of Verbal Memory (California Verbal Learning Test, CVLT), Visual Memory (Rey-Osterrieth Complex Figure Test, ROCFT), Semantic Fluency, and Perceptual Attention (Symbol Digit Modalities Test, SDMT). Participants were also genotyped for polymorphisms within the 5HTT, 5HTR2A, COMT, CYP2D6, BDNF, and GRIN2B genes using polymerase chain reaction and TaqMan polymerase assays. Lifetime cumulative MDMA use was significantly associated with poorer performance on visuospatial memory and perceptual attention. Heavy MDMA users (>100 tablets lifetime use) interacted with candidate gene polymorphisms in explaining individual differences in cognitive performance between MDMA users and controls. MDMA users carrying COMT val/val and SERT s/s had poorer performance than paired controls on visuospatial attention and memory, and MDMA users with CYP2D6 ultra-rapid metabolizers performed worse than controls on semantic fluency. Both MDMA lifetime use and gene-related individual differences influence cognitive dysfunction in ecstasy users.     

Novel Approach to Simulate Sleep Apnea Patients for Evaluating Positive Pressure Therapy Devices

Bench testing is a useful method to characterize the response of different automatic positive airway pressure (APAP) devices under well-controlled conditions. However, previous models did not consider the diversity of obstructive sleep apnea (OSA) patients’ characteristics and phenotypes. The objective of this proof-of-concept study was to design a new bench test for realistically simulating an OSA patient’s night, and to implement a one-night example of a typical female phenotype for comparing responses to several currently-available APAP devices. We developed a novel approach aimed at replicating a typical night of sleep which includes different disturbed breathing events, disease severities, sleep/wake phases, body postures and respiratory artefacts. The simulated female OSA patient example that we implemented included periods of wake, light sleep and deep sleep with positional changes and was connected to ten different APAP devices. Flow and pressure readings were recorded; each device was tested twice. The new approach for simulating female OSA patients effectively combined a wide variety of disturbed breathing patterns to mimic the response of a predefined patient type. There were marked differences in response between devices; only three were able to overcome flow limitation to normalize breathing, and only five devices were associated with a residual apnea-hypopnea index of <5/h. In conclusion, bench tests can be designed to simulate specific patient characteristics, and typical stages of sleep, body position, and wake. Each APAP device behaved differently when exposed to this controlled model of a female OSA patient, and should lead to further understanding of OSA treatment.