Thymic involution,a co‐morbidity factor in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating disease, characterized by extremely rapid loss of motor neurons. Our studies over the last decade have established CD4⁺ T cells as important players in central nervous system maintenance and repair. Those results, together with recent findings that CD4⁺ T cells play a protective role in mouse models of ALS, led us to the current hypothesis that in ALS, a rapid T‐cell malfunction may develop in parallel to the motor neuron dysfunction. Here, we tested this hypothesis by assessing thymic function, which serves as a measure of peripheral T‐cell availability, in an animal model of ALS (mSOD1 [superoxide dismutase] mice; G93A) and in human patients. We found a significant reduction in thymic progenitor‐cell content, and abnormal thymic histology in 3–4‐month‐old mSOD1 mice. In ALS patients, we found a decline in thymic output, manifested in the reduction in blood levels of T‐cell receptor rearrangement excision circles, a non‐invasive measure of thymic function, and demonstrated a restricted T‐cell repertoire. The morbidity of the peripheral immune cells was also manifested in the increase of pro‐apoptotic BAX/BCXL2 expression ratio in peripheral blood mononuclear cells (PBMCs) of these patients. In addition, gene expression screening in the same PBMCs, revealed in the ALS patients a reduction in key genes known to be associated with T‐cell activity, including: CD80, CD86, IFNG and IL18. In light of the reported beneficial role of T cells in animal models of ALS, the present observation of thymic dysfunction, both in human patients and in an animal model, might be a co‐pathological factor in ALS, regardless of the disease aetiology. These findings may lead to the development of novel therapeutic approaches directed at overcoming the thymic defect and T‐cell deficiency.     

Epithelial junctions depend on intercellular trans-interactions between the Na,K-ATPase β₁ subunits

N-Glycans of the Na,K-ATPase β₁ subunit are important for intercellular adhesion in epithelia, suggesting that epithelial junctions depend on N-glycan-mediated interactions between the β₁ subunits of neighboring cells. The level of co-immunoprecipitation of the endogenous β₁ subunit with various YFP-linked β₁ subunits expressed in Madin-Darby canine kidney cells was used to assess β₁-β₁ interactions. The amount of co-precipitated endogenous dog β₁ was greater with dog YFP-β₁ than with rat YFP-β₁, showing that amino acid-mediated interactions are important for β₁-β₁ binding. Co-precipitation of β₁ was also less with the unglycosylated YFP-β₁ than with glycosylated YFP-β_1, indicating a role for N-glycans. Mixing cells expressing dog YFP-β₁ with non-transfected cells increased the amount of co-precipitated β₁, confirming the presence of intercellular (YFP-β₁)-β₁ complexes. Accordingly, disruption of intercellular junctions decreased the amount of co-precipitated β₁ subunits. The decrease in β₁ co-precipitation both with rat YFP-β₁ and unglycosylated YFP-β₁ was associated with decreased detergent stability of junctional proteins and increased paracellular permeability. Reducing N-glycan branching by specific inhibitors increased (YFP-β₁)-β₁ co-precipitation and strengthened intercellular junctions. Therefore, interactions between the β₁ subunits of neighboring cells maintain integrity of intercellular junctions, and alterations in the β₁ subunit N-glycan structure can regulate stability and tightness of intercellular junctions.     

Nuclear proteins acting on mitochondria

An important mechanism in apoptotic regulation is changes in the subcellular distribution of pro- and anti-apoptotic proteins. Among the proteins that change in their localization and may promote apoptosis are nuclear proteins. Several of these nuclear proteins such as p53, Nur77, histone H1.2, and nucleophosmin were reported to accumulate in the cytosol and/or mitochondria and to promote the mitochondrial apoptotic pathway in response to apoptotic stressors. In this review, we will discuss the functions of these and other nuclear proteins in promoting the mitochondrial apoptotic pathway, the mechanisms that regulate their accumulation in the cytosol and/or mitochondria and the potential role of Bax and Bak in this process. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Involvement of ligninolytic enzymes and Fenton-like reaction in humic acid degradation by Trametes sp

Trametes sp. M23, isolated from biosolids compost was found to decompose humic acids (HA). A low N (LN) medium (C/N, 53) provided suitable conditions for HA degradation, whereas in a high N (HN) medium (C/N, 10), HA was not degraded. In the absence of Mn²⁺, HA degradation was similar to that in Mn²⁺-containing medium. In contrast, MnP activity was significantly affected by Mn²⁺. Laccase activity exhibited a negative correlation to HA degradation, while LiP activity was not detected. Thus, ligninolytic enzymes activity could provide only a partial explanation for the HA-degradation mechanism. The decolorization of two dyes, Orange II and Brilliant Blue R250, was also determined. Similar to HA degradation, under LN conditions, decolorization occurred independently of the presence of Mn²⁺. We investigated the possible involvement of a Fenton-like reaction in HA degradation. The addition of DMSO, an OH-radical scavenger, to LN media resulted in a significant decrease in HA bleaching. The rate of extracellular Fe³⁺ reduction was much higher in the LN vs. HN medium. In addition, the rate of reduction was even higher in the presence of HA in the medium. In vitro HA bleaching in non-inoculated media was observed with H₂O₂ amendment to a final concentration of 200 mM (obtained by 50 mM amendments for 4 days) and Fe²⁺ (36 mM). After 4 days of incubation, HA decolorization was similar to the biological treatment. These results support our hypothesis that a Fenton-like reaction is involved in HA degradation by Trametes sp. M23.     

Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with mitochondria in vivo. This supports earlier cell fractionation and microarray-based studies that proposed mMP association with the mitochondrial fraction. Interestingly, a number of mMPs showed a dependency on the mitochondrial Puf3 RNA-binding protein, as well as nonessential proteins of the translocase of the outer membrane (TOM) complex import machinery, for normal colocalization with mitochondria. We examined the specific determinants of ATP2 and OXA1 mRNA localization and found a mutual dependency on the 3' UTR, Puf3, Tom7, and Tom70, but not Tom20, for localization. Tom6 may facilitate the localization of specific mRNAs as OXA1, but not ATP2, mRNA was mislocalized in tom6Δ cells. Interestingly, a substantial fraction of OXA1 and ATP2 RNA granules colocalized with the endoplasmic reticulum (ER) and a deletion in MDM10, which mediates mitochondria-ER tethering, resulted in a significant loss of OXA1 mRNA localization with ER. Finally, neither ATP2 nor OXA1 mRNA targeting was affected by a block in translation initiation, indicating that translation may not be essential for mRNA anchoring. Thus, endogenously expressed mRNAs are targeted to the mitochondria in vivo, and multiple factors contribute to mMP localization.     

Growth Factor-Like Effects Mediated by Muscarinic Receptors in PC12M1 Cells

Rat pheochromocytoma (PC12) cells stably expressing cloned m1 muscarinic acetylcholine receptors (PC12M1) undergo morphological changes when stimulated by muscarinic agonists. These changes, which include the outgrowth of neurite-like processes, are blocked by the muscarinic antagonist atropine and are not observed in PC12 cells. The observed morphological changes, which are independent of RNA and protein synthesis, are blocked by the methylation inhibitor 5'-deoxy-5'-methylthioadenosine, suggesting that methylation plays a role in this process. Analysis of cyclic AMP accumulation and phosphoinositide turnover reveals that both processes are enhanced on activation by muscarinic agonist. Our data suggest, however, that the muscarinic-dependent neurite-like outgrowth processes are not mediated by cyclic AMP, Ca2+, or protein kinase C pathways. The muscarinic-dependent neurite outgrowth effect is enhanced by nerve growth factor, with a resulting increase in both the number of neurite-extending cells and the length of the neurite. In addition, activation of muscarinic receptors in PC12M1 cells stimulates the induction of marker genes for neuronal differentiation. Muscarinic receptors may therefore mediate growth factor-like effects in these cells.     

Knock-down of plasminogen-activator inhibitor-1 enhances expression of E-cadherin and promotes epithelial differentiation of human pancreatic adenocarcinoma cells

High levels of plasminogen activator inhibitor-1 (PAI-1), which is produced by stromal, endothelial, and cancer cells and has multiple complex effects on cancers, correlate with poor cancer prognosis. To more definitively study the role of endogenously produced PAI-1 in human pancreatic adenocarcinoma (PAC) PANC-1 cell line biology, we used anti-PAI-1 shRNA to create stable PAI-1 deficient cells (PD-PANC-1s). PD-PANC-1s exhibited a heterogeneous morphology. While the majority of cells exhibited a cuboidal shape similar to the parental PANC-1 or the vector-infected control cells, numerous large cells with long filopodia and a neuronal-like appearance were observed. Although both Vector-control cells and PD-PANC-1s expressed mRNAs that are characteristic of mesenchymal, neural, and epithelial phenotypes, epithelial marker RNAs were up-regulated (e.g., E-cadherin, 32-fold) whereas mesenchymal marker RNAs were down-regulated (e.g., Thy1, ninefold) in PD-PANC-1s, suggesting mesenchymal-to-epithelial transition. Neural markers exhibited both up- and down-regulation. Immunocytochemistry indicated that epithelial-like PD-PANC-1s expressed E-cadherin and β-catenin in significantly more cells, while neural-like cells exhibited robust expression of organized β-3-tubulin. PAI-1 and E-cadherin were rarely co-expressed in the same cells. Indeed, examination of PAI-1 and E-cadherin mRNAs expression in additional cell lines yielded clear inverse correlation. Indeed, infection of Colo357 PAC cells (that exhibit high expression of E-cadherin) with PAI-1-expressing adenovirus led to a marked decrease in E-cadherin expression and to enhanced migration of cells from clusters. Our results suggest that endogenous PAI-1 suppresses expression of E-cadherin and differentiation in PAC cells in vitro, supporting its negative impact on tumor prognosis.     

Evaluation of the efficacy of the entomopathogenic nematodes Heterorhabditis sp. against sap beetles (Coleoptera: Nitidulidae)

Absract Nitidulid beetles (Coleoptera) are considered serious pests of date palms throughout the world. They attack the ripe fruit, causing it to rot, and damage is reflected in both reduced yield and lower fruit quality. Previous studies demonstrated the susceptibility of larvae of this pest to entomopathogenic nematodes from the genus Heterorhabditidis. In the present study nematode efficacy was evaluated in greenhouse and field. In containers filled with soil, moderate reduction in insect emergence was achieved when the nematodes were applied at concentrations of 25 and 50 IJs/cm². However, the highest concentration (100 IJs/cm²) treatment resulted in a drastic reduction (by 70–90%) in emergence of the beetles. The lowest emergence was achieved by the IS-19 and IS-21 strains (>10%). Efficacy of the IS-19 strain was retained up to 7 days after application at a rate of 100 IJs/cm². When the insect larvae were introduced to the soil 2 weeks after nematode application, the percentage emergence of insects increased by 2–2.5 fold as compared to previous introductions but was still lower than in the control. Insect density per container did not have an effect on efficacy of the nematodes when the strains IS-19 and IS-12 were used. Two field trials were conducted in different sites in Israel. In the first trail, conducted in date palm orchard, four strains of Heterorhabditis sp. were tested. No significant difference in insect emergence was recorded among the various treatments or the control. Whereas in the second trial conducted in a fig orchard, substantial reduction (by 50–70%) in insect emergence was recorded following nematode treatment. Further studies, under natural conditions, are needed to optimize application efficiency and evaluate the commercial utilization of these biological control agents.