Prediction and validation of a mechanism to control the threshold for inhibitory synaptic plasticity

Synaptic plasticity, neuronal activity‐dependent sustained alteration of the efficacy of synaptic transmission, underlies learning and memory. Activation of positive‐feedback signaling pathways by an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) has been implicated in synaptic plasticity. However, the mechanism that determines the [Ca²⁺]_i threshold for inducing synaptic plasticity is elusive. Here, we developed a kinetic simulation model of inhibitory synaptic plasticity in the cerebellum, and systematically analyzed the behavior of intricate molecular networks composed of protein kinases, phosphatases, etc. The simulation showed that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII), which is essential for the induction of synaptic plasticity, was persistently activated or suppressed in response to different combinations of stimuli. The sustained CaMKII activation depended on synergistic actions of two positive‐feedback reactions, CaMKII autophosphorylation and CaMKII‐mediated inhibition of a CaM‐dependent phosphodiesterase, PDE1. The simulation predicted that PDE1‐mediated feedforward inhibition of CaMKII predominantly controls the Ca²⁺ threshold, which was confirmed by electrophysiological experiments in primary cerebellar cultures. Thus, combined application of simulation and experiments revealed that the Ca²⁺ threshold for the cerebellar inhibitory synaptic plasticity is primarily determined by PDE1.     

Abnormally High Levels of Virus-Infected IFN-γ+CCR4+CD4+CD25+ T Cells in a Retrovirus-Associated Neuroinflammatory Disorder

Background

Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL). The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified.

Principal Findings

Here, we demonstrate that CD4⁺CD25⁺CCR4⁺ T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2), Th17, and regulatory T (Treg) cells in healthy individuals, we demonstrate that IFN-γ production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4⁺CD25⁺CCR4⁺ T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-γ-producing CD4⁺CD25⁺CCR4⁺Foxp3⁻ T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity.

Conclusions

We have defined a unique T cell subset—IFN-γ⁺CCR4⁺CD4⁺CD25⁺ T cells—that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system.     

Identification of FABP7 in fibroblastic reticular cells of mouse lymph nodes

Fatty acids and their metabolites regulate immune cell function. The present study was undertaken to examine the detailed distribution of fatty acid binding proteins (FABPs), the cytosolic chaperones of fatty acids, in mouse peripheral immune organs. Using immunohistochemistry, FABP7 was localized to the alpha-smooth muscle actin (SMA)⁺ fibroblastic reticular cells, which construct the stromal reticula in the T cell areas of the peripheral lymph nodes and spleen. Immunoelectron microscopy showed that FABP7⁺ cells enclosed the collagen fibers, forming a conduit system, which transport lymph and associated low-molecular-mass proteins. In contrast, FABP5⁺ cells were distributed throughout the lymph node and contained well-developed lysosome and phagocytic materials within the cytoplasm. The mesenteric lymph nodes of FABP7 knockout mice showed normal histological features, but the percentage of CD4⁺ cells was significantly increased compared with that in wild-type mice. These data indicate that FABP7 may be involved in T cell homeostasis, possibly by modulating lipid metabolism in fibroblastic reticular cells within the peripheral lymph nodes.     

Arabidopsis Qa-SNARE SYP2 proteins localized to different subcellular regions function redundantly in vacuolar protein sorting and plant development

SYP2 proteins are a sub-family of Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) that may be responsible for protein trafficking between pre-vacuolar compartments (PVC) and vacuoles. Arabidopsis thaliana SYP22/VAM3/SGR3 and SYP21/PEP12 proteins function independently, but are both reported to be essential for male gametophytic viability. Here, we systematically examined the redundancy of three SYP2 paralogs (i.e. SYP21, 22 and 23) using a Col-0 ecotype harboring a SYP2 paralog (SYP23/PLP) that lacked a transmembrane domain. Surprisingly, no visible phenotypes were observed, even in the double knockout syp21/pep12 syp23/plp. Deficiency of either SYP21/PEP12 or SYP23/PLP in the syp22 background resulted in a defect in vacuolar protein sorting, characterized by abnormal accumulation of protein precursors in seeds. SYP21/PEP12 knockdown enhanced the syp22 phenotype (i.e. semi-dwarfism, poor leaf vein development and abnormal development of myrosin cells), and additional knockout of SYP23/PLP further aggravated the phenotype. A GFP-SYP23/PLP fusion localized to the cytosol, but not to the PVC or vacuolar membrane, where SYP21/PEP12 or SYP22/VAM3, respectively, were localized. Immunoprecipitation analysis showed that SYP23/PLP interacted with the vacuolar Qb- and Qc-SNAREs, VTI11 and SYP5, respectively, suggesting that SYP23/PLP is able to form a SNARE complex anchoring the membrane. Unexpectedly, we found that expression of multiple copies of a genomic fragment of SYP23/PLP suppressed the abnormal syp22-3 phenotype. Thus, SYP2 proteins, including cytosolic SYP23/PLP, appear to function redundantly in vacuolar trafficking and plant development.     

Serum metabolomics as a novel diagnostic approach for pancreatic cancer

Pancreatic cancer is one of the leading causes of cancer-related death, and there is currently little hope of a cure because there are no effective biomarkers for its early detection. Therefore, the search for novel biomarkers that would allow the early detection of pancreatic cancer is ongoing. In this study, the differences between the metabolomes of pancreatic cancer patients with Stage III, Stage IVa, or Stage IVb disease (n = 20) and healthy volunteers (n = 9) were evaluated by metabolomics, which is the endpoint of the Omics cascade and therefore the last step in the cascade before the phenotype. In our experimental conditions using gas chromatography mass spectrometry (GC/MS), a total of 60 metabolites were detected in serum, and the levels of 18 of the 60 metabolites were significantly changed in pancreatic cancer patients compared with those in healthy volunteers. Then, Principal Component Analysis (PCA), which is a basic form of Multiple Classification Analysis, was performed, and the PCA scores plots based on the 60 metabolites highlighted the metabolomic differences between the pancreatic cancer patients and healthy volunteers. The differences between different stages of pancreatic cancer were also assessed by Partial Least Squares Discriminant Analysis (PLS-DA), which is one of Multiple Classification Analysis, and we found that it was possible to discriminate among the Stage III, Stage IVa, and Stage IVb groups. In addition, values of the 9 metabolites in 1 Stage I pancreatic cancer patient were similar to those obtained from the Stage III, Stage IVa, and Stage IVb pancreatic cancer patients. Our findings will aid the discovery of novel biomarkers that allow the early detection of pancreatic cancer by metabolomic approaches.     

Structural features of human initiation factor 4E,studied by X-ray crystal analyses and molecular dynamics simulations

The structural features of human eIF4E were investigated by X-ray crystal analyses of its cap analog (m(7)GTP and m(7)GpppA) complexes and molecular dynamics (MD) simulations of cap-free and cap-bound eIF4Es, as well as the cap-bound Ser209-phosphorylated eIF4E. Crystal structure analyses at 2.0 A resolution revealed that the molecule forms a temple-bell-shaped surface of eight antiparallel beta-structures, three alpha-helices and ten loop structures, where the N-terminal region corresponds to the handle of the bell. This concave backbone provides a scaffold for the mRNA cap-recognition pocket consisting of three receiving parts for the 5'-terminal m(7)G base, the triphosphate, and the second nucleotide. The m(7)G base is sandwiched between the two aromatic side-chains of Trp102 and Trp56. The two (m(7)G)NH-O (Glu103 carboxy group) hydrogen bonds stabilize the stacking interaction. The basic residues of Arg157 and Lys162 and water molecules construct a binding pocket for the triphosphate moiety, where a universal hydrogen-bonding network is formed. The flexible C-terminal loop region unobserved in the m(7)GTP complex was clearly observed in the m(7)GpppA complex, as a result of the fixation of this loop by the interaction with the adenosine moiety, indicating the function of this loop as a receiving pocket for the second nucleotide. On the other hand, MD simulation in an aqueous solution system revealed that the cap-binding pocket, especially its C-terminal loop structure, is flexible in the cap-free eIF4E, and the entrance of the cap-binding pocket becomes narrow, although the depth is relatively unchanged. SDS-PAGE analyses showed that this structural instability is highly related to the fast degradation of cap-free eIF4E, compared with cap-bound or 4E-BP/cap-bound eIF4E, indicating the conferment of structural stability of eIF4E by the binary or ternary complex formation. MD simulation of m(7)GpppA-bound Ser209-phosphorylated eIF4E showed that the size of the cap-binding entrance is dependent on the ionization state in the Ser209 phosphorylation, which is associated with the regulatory function through the switching on/off of eIF4E phosphorylation.     

C-terminal KDEL sequence of a KDEL-tailed cysteine proteinase (sulfhydryl-endopeptidase) is involved in formation of KDEL vesicle and in efficient vacuolar transport of sulfhydryl-endopeptidase

Sulfhydryl-endopeptidase (SH-EP) is a papain-type vacuolar proteinase expressed in cotyledons of germinated Vigna mungo seeds, and the enzyme possesses a C-terminal propeptide containing KDEL tail, an endoplasmic reticulum retention signal for soluble proteins. SH-EP is transported to vacuoles via a KDEL vesicle (KV) through a Golgi complex-independent route. To see the function of the KDEL sequence of SH-EP, wild-type SH-EP and its KDEL deletion mutant (SH-EPDeltaKDEL) were heterologously expressed in Arabidopsis and in cultured tobacco Bright Yellow 2 cells, and their intracellular transport pathways and localizations were analyzed. A combination of the results from analyses for transformed Arabidopsis and tobacco (Nicotiana tabacum) cells indicated that wild-type SH-EP is packed into KV-like vesicles through the KDEL sequence and is transported to vacuoles in the cells of transformants. In contrast, KV was not formed/induced in the cells expressing SH-EPDeltaKDEL, and the mutant protein was mainly secreted. Therefore, the C-terminal KDEL sequence of the KDEL-tailed cysteine proteinase is thought to be involved in the formation of KV, and in the efficient vacuolar transport of the proteins through KV.     

Cloning of structural gene of Deinococcus radiodurans UV-endonuclease beta

To characterize its enzymic property we cloned and sequenced the gene of Deinococcus radiodurans encoding UV-endonuclease beta, an alternative enzyme to UvrABC repairing damaged DNA. Amino acid substitutions were found in UV-sensitive mutants. The putative amino acid sequence had some similarity with those of eukaryotic UV-endonucleases and with a sequence found in a protein data base of Bacillus subtilis.     

A new bipyrrole and some phenolic constituents in prunes (Prunus domestica L.) and their oxygen radical absorbance capacity (ORAC)

Isolation and structural elucidation of prune constituents were performed and total 10 compounds were determined by NMR and MS analyses. A novel compound was identified to be 2-(5-hydroxymethyl-2',5'-dioxo-2',3',4',5'-tetrahydro-1'H-1,3'-bipyrrole)carbaldehyde, and 7 phenolic compounds were isolated from prunes for the first time. In addition, antioxidant activity of them was evaluated on the basis of the oxygen radical absorbance capacity (ORAC).     

MDM2, an introduction

The murine double minute 2 (mdm2) gene encodes a negative regulator of the p53 tumor suppressor. Amplification of mdm2 or increased expression by unknown mechanisms occurs in many tumors. Thus, increased levels of MDM2 would inactivate the apoptotic and cell cycle arrest functions of p53, as do deletion or mutation of p53, common events in the genesis of many kinds of tumors. MDM2 functions as an E3 ubiquitin ligase to degrade p53. MDM2 also binds another tumor suppressor, ARF. This interaction sequesters MDM2 in the nucleolus away from p53, thus activating p53. Many additional MDM2 interacting proteins have been identified. Functions of MDM2 independent of p53 have also been identified. This article is an introduction to MDM2, its structure and biological functions, as well as its relationship to its binding partners.