Altered regulation of phosphatidylinositol 3-kinase signaling in cathepsin D-deficient brain

Cathepsin D (CD) is an essential lysosomal protease and mice lacking this enzyme exhibit neuropathology similar to that observed in brains of patients with neuronal ceroid lipofuscinosces (NCL/Batten disease), a group of autosomal recessive pediatric neurodegenerative diseases. CD-deficient (CD-/-) brains exhibit a dramatic induction of autophagic stress as defined by the aberrant accumulation of autophagosomes, which is concomitant with markers of apoptosis. However, the signaling abnormalities which lead to CD deficiency-induced neurodegeneration are poorly defined. Since phosphatidylinositol-3 kinase (PI3-K) is known to regulate both apoptosis and autophagy, PI3-K-mediated signaling events were assessed in CD-/- brain at P14 and P25-26. Compared to WT littermate controls, CD-/- cortical neurons exhibited a widespread decrease in phosphorylation of Akt (inactivation) and GSK3beta (disinhibition) at P25-26, while levels of total Akt and GSK3beta remained unchanged. This P25-26-specific decrease in phosphorylation of Akt and GSK-3beta in CD-/- brain coincided temporally with markers of apoptosis but followed the induction of autophagic stress observed at both P14 and P25-26. In addition, levels and/or activation of mTOR and Beclin were not affected by CD deficiency, suggesting that the accumulation of autophagosomes is not due to an increased synthesis of autophagosomes but rather from an inhibition of autophagosome recycling, due most likely to a compromise in lysosome function. Together these observations indicate a pronounced decrease in pro-survival PI3-K signaling in CD-/- brain that may contribute to autophagic stress-induced and apoptotic neuropathology.     

Dedicator of cytokinesis 8 is disrupted in two patients with mental retardation and developmental disabilities

We have identified disruptions in the dedicator of cytokinesis 8 gene, DOCK8, in two unrelated patients with mental retardation (MR). In one patient, a male with MR and no speech, we mapped a genomic deletion of approximately 230 kb in subtelomeric 9p. In the second patient, a female with mental retardation and ectodermal dysplasia and a balanced translocation, t(X;9) (q13.1;p24), we mapped the 9p24 breakpoint to a region overlapping with the centromeric end of the 230-kb subtelomeric deletion. We characterized the DOCK8 gene from the critical 9p deletion region and determined that the longest isoform of the DOCK8 gene is truncated in both patients. Furthermore, the DOCK8 gene is expressed in several human tissues, including adult and fetal brain. Recently, a role for DOCK8 in processes that affect the organization of filamentous actin has been suggested. Several genes influencing the actin cytoskeleton have been implicated in human cognitive function and thus a possibility exists that the rare mutations in the DOCK8 gene may contribute to some cases of autosomal dominant mental retardation.     

Blueberry-Enriched Diet Protects Rat Heart from Ischemic Damage

Objectives

to assess the cardioprotective properties of a blueberry enriched diet (BD).

Background

Reactive oxygen species (ROS) play a major role in ischemia-related myocardial injury. The attempts to use synthetic antioxidants to block the detrimental effects of ROS have produced mixed or negative results precipitating the interest in natural products. Blueberries are readily available product with the highest antioxidant capacity among fruits and vegetables.

Methods and Results

Following 3-mo of BD or a regular control diet (CD), the threshold for mitochondrial permeability transition (t_MPT) was measured in isolated cardiomyocytes obtained from young male Fischer-344 rats. Compared to CD, BD resulted in a 24% increase (p<0.001) of ROS indexed t_MPT. The remaining animals were subjected to a permanent ligation of the left descending coronary artery. 24 hrs later resulting myocardial infarction (MI) in rats on BD was 22% less than in CD rats (p<0.01). Significantly less TUNEL(+) cardiomyocytes (2% vs 9%) and 40% less inflammation cells were observed in the myocardial area at risk of BD compared to CD rats (p<0.01). In the subgroup of rats, after coronary ligation the original diet was either continued or switched to the opposite one, and cardiac remodeling and MI expansion were followed by serial echocardiography for 10 weeks. Measurements suggested that continuation of BD or its withdrawal after MI attenuated or accelerated rates of post MI cardiac remodeling and MI expansion.

Conclusion

A blueberry-enriched diet protected the myocardium from induced ischemic damage and demonstrated the potential to attenuate the development of post MI chronic heart failure.     

Psychological Distress, Depression, Anxiety, and Burnout among International Humanitarian Aid Workers: A Longitudinal Study

Background

International humanitarian aid workers providing care in emergencies are subjected to numerous chronic and traumatic stressors.

Objectives

To examine consequences of such experiences on aid workers'' mental health and how the impact is influenced by moderating variables.

Methodology

We conducted a longitudinal study in a sample of international non-governmental organizations. Study outcomes included anxiety, depression, burnout, and life and job satisfaction. We performed bivariate regression analyses at three time points. We fitted generalized estimating equation multivariable regression models for the longitudinal analyses.

Results

Study participants from 19 NGOs were assessed at three time points: 212 participated at pre-deployment; 169 (80%) post-deployment; and 154 (73%) within 3–6 months after deployment. Prior to deployment, 12 (3.8%) participants reported anxiety symptoms, compared to 20 (11.8%) at post-deployment (p = 0·0027); 22 (10.4%) reported depression symptoms, compared to 33 (19.5%) at post-deployment (p = 0·0117) and 31 (20.1%) at follow-up (p = .00083). History of mental illness (adjusted odds ratio [AOR] 4.2; 95% confidence interval [CI] 1·45–12·50) contributed to an increased risk for anxiety. The experience of extraordinary stress was a contributor to increased risk for burnout depersonalization (AOR 1.5; 95% CI 1.17–1.83). Higher levels of chronic stress exposure during deployment were contributors to an increased risk for depression (AOR 1·1; 95% CI 1·02–1.20) comparing post- versus pre-deployment, and increased risk for burnout emotional exhaustion (AOR 1.1; 95% CI 1.04–1.19). Social support was associated with lower levels of depression (AOR 0·9; 95% CI 0·84–0·95), psychological distress (AOR = 0.9; [CI] 0.85–0.97), burnout lack of personal accomplishment (AOR 0·95; 95% CI 0·91–0·98), and greater life satisfaction (p = 0.0213).

Conclusions

When recruiting and preparing aid workers for deployment, organizations should consider history of mental illness and take steps to decrease chronic stressors, and strengthen social support networks.     

Pneumococcal pili are composed of protofilaments exposing adhesive clusters of Rrg A

Pili have been identified on the cell surface of Streptococcus pneumoniae, a major cause of morbidity and mortality worldwide. In contrast to Gram-negative bacteria, little is known about the structure of native pili in Gram-positive species and their role in pathogenicity. Triple immunoelectron microscopy of the elongated structure showed that purified pili contained RrgB as the major compound, followed by clustered RrgA and individual RrgC molecules on the pilus surface. The arrangement of gold particles displayed a uniform distribution of anti-RrgB antibodies along the whole pilus, forming a backbone structure. Antibodies against RrgA were found along the filament as particulate aggregates of 2-3 units, often co-localised with single RrgC subunits. Structural analysis using cryo electron microscopy and data obtained from freeze drying/metal shadowing technique showed that pili are oligomeric appendages formed by at least two protofilaments arranged in a coiled-coil, compact superstructure of various diameters. Using extracellular matrix proteins in an enzyme-linked immunosorbent assay, ancillary RrgA was identified as the major adhesin of the pilus. Combining the structural and functional data, a model emerges where the pilus RrgB backbone serves as a carrier for surface located adhesive clusters of RrgA that facilitates the interaction with the host.     

Substrate specificity of sugar transport by rabbit SGLT1: single-molecule atomic force microscopy versus transport studies

In the apical membrane of epithelial cells from the small intestine and the kidney, the high-affinity Na+/d-glucose cotransporter SGLT1 plays a crucial role in selective sugar absorption and reabsorption. How sugars are selected at the molecular level is, however, poorly understood. Here atomic force microscopy (AFM) was employed to investigate the substrate specificity of rbSGLT1 on the single-molecule level, while competitive-uptake assays with isotope-labeled sugars were performed in the study of the stereospecificity of the overall transport. rbSGLT1-transfected Chinese hamster ovary (CHO) cells were used for both approaches. Evidence of binding of d-glucose to the extracellular surface of rbSGLT1 could be obtained using AFM tips carrying 1-thio-d-glucose coupled at the C1 position to a PEG linker via a vinylsulfon group. Competition experiments with monosaccharides in solution revealed the following selectivity ranking of binding: 2-deoxy-d-glucose >or= 6-deoxy-d-glucose > d-glucose > d-galactose >or= alpha-methyl glucoside; 3-deoxy-d-glucose, d-xylose, and l-glucose did not measurably affect binding. These results were different from those of competitive alpha-methyl glucoside transport assays, where the ranking of inhibition was as follows: d-glucose > d-galactose > 6-deoxy-d-glucose; no uptake inhibition by d-xylose, 3-deoxy-d-glucose, 2-deoxy-d-glucose, or l-glucose was observed. Taken together, these results suggest that the substrate specificity of SGLT1 is determined by different recognition sites: one possibly located at the surface of the transporter and others located close to or within the translocation pathway.     

Binding of netrin-4 to laminin short arms regulates basement membrane assembly

Netrins were first identified as neural guidance molecules, acting through receptors that are members of the DCC and UNC-5 family. All netrins share structural homology to the laminin N-terminal domains and the laminin epidermal growth factor-like domains of laminin short arms. Laminins use these domains to self-assemble into complex networks. Here we demonstrate that netrin-4 is a component of basement membranes and is integrated into the laminin polymer via interactions with the laminin gamma1 andgamma3 short arms. The binding is mediated through the laminin N-terminal domain of netrin-4. In contrast to netrin-4, other members of the netrin family do not bind to these laminin short arms. Moreover, a truncated form of netrin-4 completely inhibits laminin-111 self-assembly in vitro, and full-length netrin-4 can partially disrupt laminin self-interactions. When added to explant cultures, netrin-4 retards salivary gland branching morphogenesis.     

Cell proliferation and STAT6 pathways are negatively regulated in T cells by STAT1 and suppressors of cytokine signaling

Suppressor of cytokine signaling (SOCS) proteins have emerged as important regulators of cytokine signals in lymphocytes. In this study, we have investigated regulation of SOCS expression and their role in Th cell growth and differentiation. We show that SOCS genes are constitutively expressed in naive Th cells, albeit at low levels, and are differentially induced by Ag and Th-polarizing cytokines. Whereas cytokines up-regulate expression of SOCS1, SOCS2, SOCS3, and cytokine-induced Src homology 2 protein, Ags induce down-regulation of SOCS3 within 48 h of Th cell activation and concomitantly up-regulate SOCS1, SOCS2, and cytokine-induced Src homology 2 protein expression. We further show that STAT1 signals play major roles in inducing SOCS expression in Th cells and that induction of SOCS expression by IL-4, IL-12, or IFN-gamma is compromised in STAT1-deficient primary Th cells. Surprisingly, IL-4 is a potent inducer of STAT1 activation in Th2 but not Th1 cells, and SOCS1 or SOCS3 expression is dramatically reduced in STAT1(-/-) Th2 cells. To our knowledge, this is the first report of IL-4-induced STAT1 activation in Th cells, and suggests that its induction of SOCS, may in part, regulate IL-4 functions in Th2 cells. In fact, overexpression of SOCS1 in Th2 cells represses STAT6 activation and profoundly inhibits IL-4-induced proliferation, while depletion of SOCS1 by an anti-sense SOCS1 cDNA construct enhances cell proliferation and induces constitutive activation of STAT6 in Th2 cells. These results are consistent with a model where IL-4 has dual effects on differentiating T cells: it simulates proliferation/differentiation through STAT6 and autoregulates its effects on Th2 growth and effector functions via STAT1-dependent up-regulation of SOCS proteins.