Nuclear envelope dispersion triggered by deregulated Cdk5 precedes neuronal death

Nuclear fragmentation is a common feature in many neurodegenerative diseases, including Alzheimer's disease (AD). In this study, we show that nuclear lamina dispersion is an early and irreversible trigger for cell death initiated by deregulated Cdk5, rather than a consequence of apoptosis. Cyclin-dependent kinase 5 (Cdk5) activity is significantly increased in AD and contributes to all three hallmarks: neurotoxic amyloid-β (Aβ), neurofibrillary tangles (NFT), and extensive cell death. Using Aβ and glutamate as the neurotoxic stimuli, we show that deregulated Cdk5 induces nuclear lamina dispersion by direct phosphorylation of lamin A and lamin B1 in neuronal cells and primary cortical neurons. Phosphorylation-resistant mutants of lamins confer resistance to nuclear dispersion and cell death on neurotoxic stimulation, highlighting this as a major mechanism for neuronal death. Rapid alteration of lamin localization pattern and nuclear membrane change are further supported by in vivo data using an AD mouse model. After p25 induction, the pattern of lamin localization was significantly altered, preceding neuronal death, suggesting that it is an early pathological event in p25-inducible transgenic mice. Importantly, lamin dispersion is coupled with Cdk5 nuclear localization, which is highly neurotoxic. Inhibition of nuclear dispersion rescues neuronal cells from cell death, underscoring the significance of this event to Cdk5-mediated neurotoxicity.     

Unique properties of muscularis mucosae smooth muscle in guinea pig urinary bladder

The muscularis mucosae, a type of smooth muscle located between the urothelium and the urinary bladder detrusor, has been described, although its properties and role in bladder function have not been characterized. Here, using mucosal tissue strips isolated from guinea pig urinary bladders, we identified spontaneous phasic contractions (SPCs) that appear to originate in the muscularis mucosae. This smooth muscle layer exhibited Ca(2+) waves and flashes, but localized Ca(2+) events (Ca(2+) sparks, purinergic receptor-mediated transients) were not detected. Ca(2+) flashes, often in bursts, occurred with a frequency (～5.7/min) similar to that of SPCs (～4/min), suggesting that SPCs are triggered by bursts of Ca(2+) flashes. The force generated by a single mucosal SPC represented the maximal force of the strip, whereas a single detrusor SPC was ～3% of maximal force of the detrusor strip. Electrical field stimulation (0.5-50 Hz) evoked force transients in isolated detrusor and mucosal strips. Inhibition of cholinergic receptors significantly decreased force in detrusor and mucosal strips (at higher frequencies). Concurrent inhibition of purinergic and cholinergic receptors nearly abolished evoked responses in detrusor and mucosae. Mucosal SPCs were unaffected by blocking small-conductance Ca(2+)-activated K(+) (SK) channels with apamin and were unchanged by blocking large-conductance Ca(2+)-activated K(+) (BK) channels with iberiotoxin (IbTX), indicating that SK and BK channels play a much smaller role in regulating muscularis mucosae SPCs than they do in regulating detrusor SPCs. Consistent with this, BK channel current density in myocytes from muscularis mucosae was ～20% of that in detrusor myocytes. These findings indicate that the muscularis mucosae in guinea pig represents a second smooth muscle compartment that is physiologically and pharmacologically distinct from the detrusor and may contribute to the overall contractile properties of the urinary bladder.     

Malaria sporozoite antigen‐directed genome‐wide response in transgenic Drosophila

Malaria kills a million people annually. Understanding the relationship between a causative parasite, Plasmodium falciparum, and the mosquito vector might suggest novel prevention approaches. We created and transformed into Drosophila two genes encoding, thrombospondin‐related adhesive protein (TRAP) and circumsporozoite protein (CSP), found on the cell surface of Plasmodium sporozoites. To understand a model insect's response, we induced these proteins separately and together, performing whole genome microarray analysis measuring gene expression changes. Gene ontology classification of responding genes reveals that TRAP and CSP strongly and differentially influence Drosophila genes involved with cell motility and gene regulation, respectively; however, the most striking effects are on the immune system. While immune‐related genes are but modestly elevated compared with responses to sepsis, there is a marked repression of the Toll pathway. This suggests: (1) how Plasmodium infection of the mosquito might use TRAP and CSP to modulate the host insect's physiology to promote sporozoite survival and transmission to man and (2) that approaches to elevate expression of the mosquito's Toll pathway might lead to novel methods of malaria prevention. genesis 47:196–203, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of Six Bacteriophages of Serratia liquefaciens CP6 Isolated from the Sugar Beet Phytosphere

Six phages (ΦCP6-1 to ΦCP6-6) that are commonly found in the phytosphere of sugar beet (Beta vulgaris var. Amethyst) were investigated, and their relative impacts on their host (Serratia liquefaciens CP6) were compared. There were fundamental differences between the two most abundant predators of CP6 (ΦCP6-1 and ΦCP6-4). Like ΦCP6-2 and ΦCP6-5, ΦCP6-1 belonged to the family Siphoviridae, while ΦCP6-4 exhibited the morphology of the family Podoviridae. The other phages were members of the family Myoviridae. DNA-DNA cross-hybridization revealed that ΦCP6-1 and ΦCP6-4 had little common DNA, although all of the other phages exhibited some genetic similarity. Like ΦCP6-2, ΦCP6-3, and ΦCP6-5, ΦCP6-1 was capable of forming a lysogenic association with its host, while ΦCP6-4 and ΦCP6-6 appeared to be entirely virulent. Single-step growth curve experiments revealed that ΦCP6-4 had a much shorter latent period and a smaller burst size than ΦCP6-1. Also, ΦCP6-1 could transduce a number of host chromosomal markers with transfer frequencies of 2.9 × 10⁻⁹ to 3.9 × 10⁻⁷, whereas ΦCP6-4 could not transduce S. liquefaciens CP6 genes. When viewed in the context of the strikingly different temporal niches of these phages, our data provide an insight into how bacteriophage interactions with their hosts might reflect the natural ecology of bacteriophages. Our data also illustrate how the potential for gene transfer changes over time in an environment that supports several different phages.     

RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain

Amyloid-beta peptide (Abeta) interacts with the vasculature to influence Abeta levels in the brain and cerebral blood flow, providing a means of amplifying the Abeta-induced cellular stress underlying neuronal dysfunction and dementia. Systemic Abeta infusion and studies in genetically manipulated mice show that Abeta interaction with receptor for advanced glycation end products (RAGE)-bearing cells in the vessel wall results in transport of Abeta across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-1), the latter mediating Abeta-induced vasoconstriction. Inhibition of RAGE-ligand interaction suppresses accumulation of Abeta in brain parenchyma in a mouse transgenic model. These findings suggest that vascular RAGE is a target for inhibiting pathogenic consequences of Abeta-vascular interactions, including development of cerebral amyloidosis.     

Association between Gender,Process of Care Measures,and Outcomes in ACS in India: Results from the Detection and Management of Coronary Heart Disease (DEMAT) Registry

Background

Studies from high-income countries have shown that women receive less aggressive diagnostics and treatment than men in acute coronary syndromes (ACS), though their short-term mortality does not appear to differ from men. Data on gender differences in ACS presentation, management, and outcomes are sparse in India.

Methods and Results

The Detection and Management of Coronary Heart Disease (DEMAT) Registry collected data from 1,565 suspected ACS patients (334 women; 1,231 men) from ten tertiary care centers throughout India between 2007–2008. We evaluated gender differences in presentation, in-hospital and discharge management, and 30-day death and major adverse cardiovascular event (MACE; death, re-hospitalization, and cardiac arrest) rates. Women were less likely to present with STEMI than men (38% vs. 55%, p<0.001). Overall inpatient diagnostics and treatment patterns were similar between men and women after adjustment for potential confounders. Optimal discharge management with aspirin, clopidogrel, beta-blockers, and statin therapy was lower for women than men, (58% vs. 65%, p = 0.03), but these differences were attenuated after adjustment (OR = 0.86 (0.62, 1.19)). Neither the outcome of 30-day mortality (OR = 1.40 (0.62, 3.16)) nor MACE (OR = 1.00 (0.67, 1.48)) differed significantly between men and women after adjustment.

Conclusions

ACS in-hospital management, discharge management, and 30-day outcomes did not significantly differ between genders in the DEMAT registry, though consistently higher treatment rates and lower event rates in men compared to women were seen. These findings underscore the importance of further investigation of gender differences in cardiovascular care in India.     

BRCTx is a novel, highly conserved RAD18-interacting protein

The BRCT domain is a highly conserved module found in many proteins that participate in DNA damage checkpoint regulation, DNA repair, and cell cycle control. Here we describe the cloning, characterization, and targeted mutagenesis of Brctx, a novel gene with a BRCT motif. Brctx was found to be expressed ubiquitously in adult tissues and during development, with the highest levels found in testis. Brctx-deficient mice develop normally, show no pathological abnormalities, and are fertile. BRCTx binds to the C terminus of hRAD18 in yeast two-hybrid and immunoprecipitation assays and colocalizes with this protein in the nucleus. Despite this, Brctx-deficient murine embryonic fibroblasts (MEFs) do not show overt sensitivity to DNA-damaging agents. MEFs from Brctx-deficient embryos grow at a similar rate to wild-type MEF CD4/CD8 expressions, and the cell cycle parameters of thymocytes from wild-type and Brctx knockout animals are indistinguishable. Intriguingly, the BRCT domain of BRCTx is responsible for mediating its localization to the nucleus and centrosome in interphase cells. We conclude that, although highly conserved, Brctx is not essential for the above-mentioned processes and may be redundant.     

Function and regulation of Alx4 in limb development: complex genetic interactions with Gli3 and Shh

The role of the aristaless-related homeobox gene Alx4 in antero-posterior (AP-) patterning of the developing vertebrate limb has remained somewhat elusive. Polydactyly of Alx4 mutant mice is known to be accompanied by ectopic anterior expression of genes like Shh, Fgf4 and 5'Hoxd. We reported previously that polydactyly in Alx4 mutant mice requires SHH signaling, but we now show that in early Alx4-/- limb buds the anterior ectopic expression of Fgf4 and Hoxd13, and therefore disruption of AP-patterning, occurs independently of SHH signaling. To better understand how Alx4 functions in the pathways that regulate AP-patterning, we also studied genomic regulatory sequences that are capable of directing expression of a reporter gene in a pattern corresponding to endogenous Alx4 expression in anterior limb bud mesenchyme. We observed, as expected for authentic Alx4 expression, expansion of reporter construct expression in a Shh-/- background. Total lack of reporter expression in a Gli3-/- background confirms the existence of Gli3-dependent and -independent Alx4 expression in the limb bud. Apparently, these two modules of Alx4 expression are linked to dissimilar functions.