Yersinia pseudotuberculosis spatially controls activation and misregulation of host cell Rac1

Yersinia pseudotuberculosis binds host cells and modulates the mammalian Rac1 guanosine triphosphatase (GTPase) at two levels. Activation of Rac1 results from integrin receptor engagement, while misregulation is promoted by translocation of YopE and YopT proteins into target cells. Little is known regarding how these various factors interplay to control Rac1 dynamics. To investigate these competing processes, the localization of Rac1 activation was imaged microscopically using fluorescence resonance energy transfer. In the absence of translocated effectors, bacteria induced activation of the GTPase at the site of bacterial binding. In contrast, the entire cellular pool of Rac1 was inactivated shortly after translocation of YopE RhoGAP. Inactivation required membrane localization of Rac1. The translocated protease YopT had very different effects on Rac1. This protein, which removes the membrane localization site of Rac1, did not inactivate Rac1, but promoted entry of cleaved activated Rac1 molecules into the host cell nucleus, allowing Rac1 to localize with nuclear guanosine nucleotide exchange factors. As was true for YopE, membrane-associated Rac1 was the target for YopT, indicating that the two translocated effectors may compete for the same pool of target protein. Consistent with the observation that YopE inactivation requires membrane localization of Rac1, the presence of YopT in the cell interfered with the action of the YopE RhoGAP. As a result, interaction of target cells with a strain that produces both YopT and YopE resulted in two spatially distinct pools of Rac1: an inactive cytoplasmic pool and an activated nuclear pool. These studies demonstrate that competition between bacterial virulence factors for access to host substrates is controlled by the spatial arrangement of a target protein. In turn, the combined effects of translocated bacterial proteins are to generate pools of a single signaling molecule with distinct localization and activation states in a single cell.     

A three-dimensional finite element stress analysis for tunnel placement and buttons in anterior cruciate ligament reconstructions

This communication reports the results of a three-dimensional finite element (FE) model of stresses in a surgically altered femur and tibia. The model incorporated a novel approach in implementing orthotropic and inhomogeneous bone properties and non-uniform distributed loading. Cortical, cancellous, and subchondral bone of the femur and the tibia were modeled. Mechanical properties for the cortical and cancellous bone were mapped from published data characterizing the anisotropy and inhomogeneity of the bone properties. Mesh adequacy was determined using stress convergence and strain energy error convergence. Qualitatively, the results of the study compare well with experimental principal compressive strains from the literature. With respect to tunnel placement in anterior cruciate ligament reconstruction, the model predicted stress-shielding at the postero-lateral region of the tunnel wall, and increased stress at the postero-medial region of the tunnel wall. The stresses in the cancellous bone beneath the tunnel were, in general, lower than those above the tunnel. Prolonged stress shielding leads to bone resorption of the posterior tunnel wall leading to tunnel enlargement, and possible compromise of the ACL reconstruction. The stresses on the femoral cortex produced from a button-type fixation were noticeable for low levels of loading; the stress levels were very similar in models incorporating bone properties of patients aged 45 and 65. Repeated compression of the femoral cortex at these stress levels may cause microdamage to the cortex eventually resulting in fatigue failure.     

Impact of vitamin E or selenium deficiency on nematode-induced alterations in murine intestinal function

The effects of deficiencies in the antioxidant nutrients, vitamin E and selenium, on the host response to gastrointestinal nematode infection are unknown. The aim of the study was to determine the effect of antioxidant deficiencies on nematode-induced alterations in intestinal function in mice. BALB/c mice were fed control diets or diets deficient in selenium or vitamin E and the response to a secondary challenge inoculation with Heligmosomoides polygyrus was determined. Egg and worm counts were assessed to determine host resistance. Sections of jejunum were mounted in Ussing chambers to measure changes in permeability, absorption, and secretion, or suspended in organ baths to determine smooth muscle contraction. Both selenium and vitamin E deficient diets reduced resistance to helminth infection. Vitamin E, but not selenium, deficiency prevented nematode-induced decreases in glucose absorption and hyper-contractility of smooth muscle. Thus, vitamin E status is an important factor in the physiological response to intestinal nematode infection and may contribute to antioxidant-dependent protective mechanisms in the small intestine.     

Overexpression of the endoplasmic reticulum 60 protein ER-60 downregulates apoB100 secretion by inducing its intracellular degradation via a nonproteasomal pathway: evidence for an ER-60-mediated and pCMB-sensitive intracellular degradative pathway

Co- and posttranslational regulation of apolipoprotein B (apoB) has been postulated to involve degradation by both proteasomal and nonproteasomal pathways; however, nonproteasomal mechanisms of apoB degradation are currently unknown. We have previously demonstrated an intracellular association of newly synthesized apoB with endoplasmic reticulum (ER)-60, an ER-localized protein, possessing both proteolytic and chaperone activities. In the present paper, adenoviral expression vectors containing rat ER-60 cDNA were used to achieve dose- and time-dependent overexpression of ER-60 to investigate its role in apoB100 turnover. Overexpressed ER-60 accumulated in the microsomal lumen of HepG2 cells and was associated with apoB100 in dense lipoprotein particles. Overexpression of ER-60 in HepG2 cells significantly reduced both intracellular and secreted apoB100, with no effect on the secretion of a control protein, albumin. Similar results were obtained in McA-RH7777 rat hepatoma cells. ER-60-stimulated apoB100 degradation and inhibition of apoB100 secretion were sensitive to the protease inhibitor, p-chloromercuribenzoate (pCMB), in a dose-dependent manner but were unaffected by the proteasomal or lysosomal protease inhibitors, N-acetyl-leucinyl-leucinyl-nor-leucinal, E64, and leupeptin. Interestingly, enhanced expression of ER-60 induced apoB100 fragmentation in permeabilized HepG2 cells and resulted in detection of a unique 50 kDa degradation intermediate, a process that could be inhibited by pCMB. Intracellular stability and secretion of apoB100 in primary hamster hepatocytes were also found to be sensitive to pCMB. When taken together, the data suggest an important role for ER-60 in promoting apoB100 degradation via a pCMB-sensitive process in the ER. ER-60 may act directly as a protease or may be involved indirectly as a chaperone/protein factor targeting apoB100 to this nonproteasomal and pCMB-sensitive degradative pathway.     

Sequence analysis and homology modeling of laccase from Pycnoporus cinnabarinus

Industrial effluents of textile, paper, and leather industries contain various toxic dyes as one of the waste material. It imparts major impact on human health as well as environment. The white rot fungus Pycnoporus cinnabarinus Laccase is generally used to degrade these toxic dyes. In order to decipher the mechanism of process by which Laccase degrade dyes, it is essential to know its 3D structure. Homology modeling was performed in presented work, by satisfying Spatial restrains using Modeller Program, which is considered as standard in this field, to generate 3D structure of Laccase in unison, SWISSMODEL web server was also utilized to generate and verify the alternative models. We observed that models created using Modeller stands better on structure evaluation tests. This study can further be used in molecular docking techniques, to understand the interaction of enzyme with its mediators like 2, 2-azinobis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) and Vanillin that are known to enhance the Laccase activity.     

Phosphatidylserine is involved in the ferrichrome-induced plasma membrane trafficking of Arn1 in Saccharomyces cerevisiae

Arn1 is an integral membrane protein that mediates the uptake of ferrichrome, an important nutritional source of iron, in Saccharomyces cerevisiae. In the absence of ferrichrome, Arn1p is sorted directly from the trans-Golgi network to the vacuolar lumen for degradation. In the presence of low levels of ferrichrome, the siderophore binds to a receptor domain on Arn1, triggering the redistribution of Arn1 to the plasma membrane. When extracellular ferrichrome levels are high, Arn1 cycles between the plasma membrane and intracellular vesicles. To further understand the mechanisms of trafficking of Arn1p, we screened 4580 viable yeast deletion mutants for mislocalization of Arn1-GFP using synthetic genetic array technology. We identified over 100 genes required for trans-Golgi network-to-vacuole trafficking of Arn1-GFP and only two genes, SER1 and SER2, required for the ferrichrome-induced plasma membrane trafficking of Arn1-GFP. SER1 and SER2 encode two enzymes of the major serine biosynthetic pathway, and the Arn1 trafficking defect in the ser1Δ strain was corrected with supplemental serine or glycine. Plasma membrane trafficking of Hxt3, a structurally related glucose transporter, was unaffected by SER1 deletion. Serine is required for the synthesis of multiple cellular components, including purines, sphingolipids, and phospholipids, but of these only phosphatidylserine corrected the Arn1 trafficking defects of the ser1Δ strain. Strains with defects in phospholipid synthesis also exhibited alterations in Arn1p trafficking, indicating that the intracellular trafficking of some transporters is dependent on the phospholipid composition of the cellular membranes.     

The effects of manganese on glutamate, dopamine and gamma-aminobutyric acid regulation

Exposure to high levels of manganese (Mn) results in a neurological disorder, termed manganism, which shares a similar phenotype to Parkinson's disease due to the involvement of the basal ganglia circuitry in both. The initial symptoms of manganism are likely due to the involvement of the globus pallidus, a region rich in gamma-aminobutyric acid (GABA) projections, while those of Parkinson's disease are related to the degeneration of the substantia nigra, a dopaminergic nucleus. Additionally, it is known that glutamate regulation is affected by increases in brain Mn levels. As Mn predominantly accumulates in the basal ganglia, it potentially could affect the regulation and interactions of all three neurotransmitters. This review will focus on the circuitry of these neurotransmitters within the basal ganglia and address potential sites for, as well as the temporal relationship, between Mn exposure and changes in the levels of these neurotransmitters. While most research has focused on perturbations in the dopaminergic system, there is evidence to support that early consequences of manganism also include disturbances in GABA regulation as well as glutamatergic-related excitotoxicity. Finally, we suggest that current research focus on the interdependence of these basal ganglial neurochemicals, with a greater emphasis on the GABAergic and glutamatergic systems.     

Very-long-chain secondary alcohols and alkanediols in cuticular waxes of Pisum sativum leaves

In cuticular waxes from leaves of Pisum sativum, 19 secondary alcohols, 10 primary/secondary alkanediols and three secondary/secondary alkanediols were identified by various chemical transformations with product assignment employing GC-MS. The homologous series of C29-C33 secondary alcohols (1.1 microg/cm2) was dominated by hentriacontanol isomers (94%). Only octacosanediols and trace amounts of hexacosanediols (< 1%) were detected in the primary/secondary alkanediol faction (0.7 microg/cm2). The secondary/secondary alkanediols (0.12 microg/cm2) contained a single homologue with chain length C31. All three compound classes showed characteristic isomer distributions with secondary functional groups predominantly located between C-14 and C-16. Based on the isomer compositions, the sequence of biosynthetic steps introducing the hydroxyl functions is discussed.     

The biology of Ku and its potential oncogenic role in cancer

Ku is a heterodimeric protein made up of two subunits, Ku70 and Ku80. It was originally identified as an autoantigen recognized by the sera of patients with autoimmune diseases. It is a highly versatile regulatory protein that has been implicated in multiple nuclear processes, e.g., DNA repair, telomere maintenance and apoptosis. Accordingly, Ku is thought to play a crucial role in maintenance of chromosomal integrity and cell survival. Recent reports suggest that there is a positive relationship between Ku and the development of cancer, making Ku an important candidate target for anticancer drug development. Specifically, prior studies suggest that a delicate balance exists in Ku expression, as overexpression of Ku proteins promotes oncogenic phenotypes, including hyperproliferation and resistance to apoptosis; whereas deficient or low expression of Ku leads to genomic instability and tumorigenesis. Such observations through various experimental models indicate that Ku may act as either a tumor suppressor or an oncoprotein. Hence, understanding the link between the various functions of Ku and the development of cancer in different cell systems may help in the development of novel anticancer therapeutic agents that target Ku. These studies may also increase our understanding of how Ku autoantibodies are generated in autoimmune diseases.