CRY2 Is Associated with Depression

Background

Abnormalities in the circadian clockwork often characterize patients with major depressive and bipolar disorders. Circadian clock genes are targets of interest in these patients. CRY2 is a circadian gene that participates in regulation of the evening oscillator. This is of interest in mood disorders where a lack of switch from evening to morning oscillators has been postulated.

Principal Findings

We observed a marked diurnal variation in human CRY2 mRNA levels from peripheral blood mononuclear cells and a significant up-regulation (P = 0.020) following one-night total sleep deprivation, a known antidepressant. In depressed bipolar patients, levels of CRY2 mRNA were decreased (P = 0.029) and a complete lack of increase was observed following sleep deprivation. To investigate a possible genetic contribution, we undertook SNP genotyping of the CRY2 gene in two independent population-based samples from Sweden (118 cases and 1011 controls) and Finland (86 cases and 1096 controls). The CRY2 gene was significantly associated with winter depression in both samples (haplotype analysis in Swedish and Finnish samples: OR = 1.8, P = 0.0059 and OR = 1.8, P = 0.00044, respectively).

Conclusions

We propose that a CRY2 locus is associated with vulnerability for depression, and that mechanisms of action involve dysregulation of CRY2 expression.     

The metalloprotease of Listeria monocytogenes is regulated by pH

Listeria monocytogenes is an intracytosolic bacterial pathogen. Among the factors contributing to escape from vacuoles are a phosphatidylcholine phospholipase C (PC-PLC) and a metalloprotease (Mpl). Both enzymes are translocated across the bacterial membrane as inactive proproteins, whose propeptides serve in part to maintain them in association with the bacterium. We have shown that PC-PLC maturation is regulated by Mpl and pH and that Mpl maturation occurs by autocatalysis. In this study, we tested the hypothesis that Mpl activity is pH regulated. To synchronize the effect of pH on bacteria, the cytosolic pH of infected cells was manipulated immediately after radiolabeling de novo-synthesized bacterial proteins. Immunoprecipitation of secreted Mpl from host cell lysates revealed the presence of the propeptide and catalytic domain in samples treated at pH 6.5 but not at pH 7.3. The zymogen was present in small amounts under all conditions. Since proteases often remain associated with their respective propeptide following autocatalysis, we aimed at determining whether pH regulates autocatalysis or secretion of the processed enzyme. For this purpose, we used an Mpl construct that contains a Flag tag at the N terminus of its catalytic domain and antibodies that can distinguish N-terminal and non-N-terminal Flag. By fluorescence microscopy, we observed the Mpl zymogen associated with the bacterium at physiological pH but not following acidification. Mature Mpl was not detected in association with the bacterium at either pH. Using purified proteins, we determined that processing of the PC-PLC propeptide by mature Mpl is also pH sensitive. These results indicate that pH regulates the activity of Mpl on itself and on PC-PLC.     

Posttranslocation chaperone PrsA2 regulates the maturation and secretion of Listeria monocytogenes proprotein virulence factors

PrsA2 is a conserved posttranslocation chaperone and a peptidyl prolyl cis-trans isomerase (PPIase) that contributes to the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the phenotypes associated with a prsA2 mutant is decreased activity of the broad-range phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation is mediated by a metalloprotease (Mpl). The proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface until a decrease in pH triggers their maturation and rapid secretion into the host cell. In this study, we examined the mechanism by which PrsA2 regulates the activity of PC-PLC. We observed that in the absence of PrsA2, the proenzymes are secreted at physiological pH and do not mature upon a decrease in pH. The sensitivity of the prsA2 mutant to cell wall hydrolases was modified. However, no apparent changes in cell wall porosity were detected. Interestingly, synthesis of PC-PLC in the absence of its propeptide lead to the secretion of a fully active enzyme in the cytosol of host cells independent of PrsA2, indicating that neither the propeptide of PC-PLC nor PrsA2 is required for native folding of the catalytic domain, although both influence secretion of the enzyme. Taken together, these results suggest that PrsA2 regulates compartmentalization of Mpl and PC-PLC, possibly by influencing cell wall properties and interacting with the PC-PLC propeptide. Moreover, the ability of these proproteins to respond to a decrease in pH during intracellular growth depends on their localization at the membrane-cell wall interface.     

The Catalytic Activity of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 3 Is Required To Sustain CD4+ CD8+ Thymocyte Survival

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family whose function is largely unknown. Given the central role of MAPKs in T cell development, we hypothesized that ERK3 may regulate thymocyte development. Here we have shown that ERK3 deficiency leads to a 50% reduction in CD4⁺ CD8⁺ (DP) thymocyte number. Analysis of hematopoietic chimeras revealed that the reduction in DP thymocytes is intrinsic to hematopoietic cells. We found that early thymic progenitors seed the Erk3^−/− thymus and can properly differentiate and proliferate to generate DP thymocytes. However, ERK3 deficiency results in a decrease in the DP thymocyte half-life, associated with a higher level of apoptosis. As a consequence, ERK3-deficient DP thymocytes are impaired in their ability to make successful secondary T cell receptor alpha (TCRα) gene rearrangement. Introduction of an already rearranged TCR transgene restores thymic cell number. We further show that knock-in of a catalytically inactive allele of Erk3 fails to rescue the loss of DP thymocytes. Our results uncover a unique role for ERK3, dependent on its kinase activity, during T cell development and show that this atypical MAPK is essential to sustain DP survival during RAG-mediated rearrangements.     

The Non-Classical MAP Kinase ERK3 Controls T Cell Activation

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4⁺ and CD8⁺ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.     

Neuroprotective role of hydralazine in rat spinal cord injury‐attenuation of acrolein‐mediated damage

Acrolein, an α,β‐unsaturated aldehyde and a reactive product of lipid peroxidation, has been suggested as a key factor in neural post‐traumatic secondary injury in spinal cord injury (SCI), mainly based on in vitro and ex vivo evidence. Here, we demonstrate an increase of acrolein up to 300%; the elevation lasted at least 2 weeks in a rat SCI model. More importantly, hydralazine, a known acrolein scavenger can provide neuroprotection when applied systemically. Besides effectively reducing acrolein, hydralazine treatment also resulted in significant amelioration of tissue damage, motor deficits, and neuropathic pain. This effect was further supported by demonstrating the ability of hydralazine to reach spinal cord tissue at a therapeutic level following intraperitoneal application. This suggests that hydralazine is an effective neuroprotective agent not only in vitro, but in a live animal model of SCI as well. Finally, the role of acrolein in SCI was further validated by the fact that acrolein injection into the spinal cord caused significant SCI‐like tissue damage and motor deficits. Taken together, available evidence strongly suggests a critical causal role of acrolein in the pathogenesis of spinal cord trauma. Since acrolein has been linked to a variety of illness and conditions, we believe that acrolein‐scavenging measures have the potential to be expanded significantly ensuring a broad impact on human health.

     

Brucella Omp2a and Omp2b porins: single channel measurements and topology prediction

Brucella usually carry two highly homologous genes (omp2a and omp2b) for porin-like proteins. In several B. abortus biovars the omp2a gene has a large deletion compared to other Brucella omp2's. In this study we have measured Omp2 pore activity in planar bilayers. Omp2b exhibits well-defined trimeric channel activity whilst Omp2a forms monomeric pores of variable size which are smaller than Omp2b. No sequence homology exists between Omp2 and porins of known structure, so hydrophobic moment analysis has been used to model their membrane topology. From this it appears likely that the deletion removes the crucial L3 internal loop.     

Genetic and Pathogenic Characterization of Different Stagonospora sp. Isolated from Bindweed

Genetic variation among 38 isolates of Stagonospora sp. and 10 isolates of Septoria sp. from bindweed was studied using (a) restriction fragment length plymorphism (RFLP) analysis of the internal transcribed spacer (ITS) region, and (b) random amplified polymorphic DNA (RAPD) PCR analysis. RFLP analysis revealed three types of fragment patterns among the isolates. A total of 26 distinct groups, based on common fragment patterns, were identified using cluster analysis of the RAPD-PCR data. When the grouping results of the two methods were compared, the fragment pattern types and clusters were generally in agreement. The degree of pathogenicity of six genetically characterized isolates of Stagonospora sp. was assessed on three ecotypes of field bindweed (Convolvulus arvensis). Disease symptoms were observed with all isolates on all ecotypes, but only Stagonospora convolvuli strain LA39, a potential biocontrol agent, showed a high degree of pathogenicity on all ecotypes. A mixture of two Stagonospora sp. enhanced the mean necrotic leaf area on bindweed from 33.9 and 39.0% (when applied alone) to 64.9% applied together at the same final concentration of 5 X 106 spores ml -1 . Molecular methods were used to identify the two pathogens. Both were present on the same plant when applied together, but never found in the same lesion.     

Intra-crown variation in leaf herbivory and seed production in striped maple,Acer pensylvanicum L. (Aceraceae)

Summary Patterns of spatial variation in leaf herbivory and the effects of this variation on seed production and twig growth were studied in striped maple, Acer pensylvanicum (Aceraceae). Experimental removal of 25% of the leaf area from each of four leaves directly subtending a developing infructescence significantly reduced seed number in that infructescence. When leaf area was removed from leaves neighboring to, but not directly subtending developing infructescences, no reduction in seed production occurred. Together, these results suggest that only photosynthate from leaves directly subtending infructescences contributes to seed production in nearby infructescences. Effects of the experimental removal of leaf area did not persist the second year, suggesting that mobilization of storage products the following spring occurred independent of prior treatment. There was probably little negative impact of leaf herbivores on this plant species during the study year due to (1) low occurrence of localized damage within the crowns of censused trees and (2) delay of leaf area loss until completion of fruit development.