Three new isoforms of Caenorhabditis elegans UNC-89 containing MLCK-like protein kinase domains.

In Caenorhabditis elegans, the gene unc-89 is required for A-band organization of striated muscle. In mammals, a likely homolog of UNC-89, called obscurin, has been described and found to be localized at both the M-lines and Z-discs of striated muscle. Here, we show that the coding sequence for unc-89 is larger than originally thought, and that the gene encodes at least four major isoforms: UNC-89-A (original isoform, 732 kDa), UNC-89-B (potentially 900 kDa), and UNC-89-C and UNC-89-D (each 156 kDa).

Cooperative prosurvival activity by ERK and Akt in human alveolar macrophages is dependent on high levels of acid ceramidase activity.

Human alveolar macrophages are unique in that they have an extended life span in contrast to precursor monocytes. In evaluating the role of sphingolipids in alveolar macrophage survival, we found high levels of sphingosine, but not sphingosine-1-phosphate. Sphingosine is generated by the action of ceramidase(s) on ceramide, and alveolar macrophages have high constitutive levels of acid ceramidase mRNA, protein, and activity.

Adenovirus vectors activate survival pathways in lung epithelial cells.

Airway epithelial cells are often the sites of targeted adenovirus vector delivery. Activation of the host inflammatory response and modulation of signal transduction pathways by adenovirus vectors have been previously documented, including activation of MAP kinases and phosphatidylinositol 3-kinase (PI3-kinase). The effect of activation of these pathways by adenovirus vectors on cell survival has not been examined.

Caenorhabditis elegans UNC-98, a C2H2 Zn finger protein, is a novel partner of UNC-97/PINCH in muscle adhesion complexes.

To further understand the assembly and maintenance of the muscle contractile apparatus, we have identified a new protein, UNC-98, in the muscle of Caenorhabditis elegans. unc-98 mutants display reduced motility and a characteristic defect in muscle structure. We show that the major defect in the mutant muscle is in the M-lines and dense bodies (Z-line analogs).

Redox factor-1 contributes to the regulation of progression from G0/G1 to S by PDGF in vascular smooth muscle cells.

Redox factor-1 (Ref-1/APE), a multifunctional DNA base excision repair and redox regulation enzyme, plays an important role in oxidative signaling, transcription factor regulation, and cell cycle control. We hypothesized that Ref-1 plays a regulatory role in smooth muscle cell (SMC) proliferation induced by PDGF. Ref-1 antisense oligodeoxynucleotides (AODN), which diminished the level of Ref-1 protein in SMCs by approximately 50%, inhibited PDGF-BB (composed of the homodimer of B-polypeptide chain)-induced [3H]thymidine incorporation compared with control oligodeoxynucleotides.

Transgenic zebrafish model of neurodegeneration.

In Alzheimer's disease (AD), the microtubule-associated protein, tau, is compromised in its normal association with microtubules and forms into paired helical filaments (PHF) that are the hallmark cytoskeletal pathology of the disease. Several posttranslational modifications of tau including phosphorylation have been implicated in AD pathogenesis. In addition, and importantly, mutations in the genes encoding human tau have recently been implicated in a variety of hereditary dementias, collectively termed frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17).

First-generation adenovirus vectors shorten survival time in a murine model of sepsis.

Adverse immunological reactions to adenoviral vectors have significantly impacted the utility of this virus for treating genetic and environmentally induced diseases. In this study, we evaluate the effect of adenoviral vectors on an animal model of sepsis. Systemic delivery of first-generation adenoviral vectors to septic mice (cecal ligation and puncture) resulted in a shortened survival time.

Titins in C.elegans with unusual features: coiled-coil domains, novel regulation of kinase activity and two new possible elastic regions.

We report that there are previously unrecognized proteins in Caenorhabditis elegans that are similar to the giant muscle proteins called titins, and these are encoded by a single approximately 90kb gene. The gene structure was predicted by GeneMark.hmm and then experimentally verified.

Bronchoalveolar fluid is not a major hindrance to virus-mediated gene therapy in cystic fibrosis.

Successfully targeting the airway epithelium is essential for gene therapy of some pulmonary diseases. However, the airway epithelium is resistant to virus-mediated gene transfer with commonly used vectors. Vectors that interact with endogenously expressed receptors on the apical surface significantly increase gene transfer efficiency.

Phosphatidylinositol 3-kinase activity negatively regulates stability of cyclooxygenase 2 mRNA.

Human alveolar macrophages have both lipopolysaccharide (LPS)-induced and constitutive phosphatidylinositol 3-kinase (PI3K) activity. We observed that blocking PI3K activity increased release of prostaglandin E2 after LPS exposure, and increasing PI3K activity (interleukin-13) decreased release of prostaglandin E2 after LPS exposure. This was not because of an effect of PI3K on phospholipase 2 activity.

Oxidant-mediated increases in redox factor-1 nuclear protein and activator protein-1 DNA binding in asbestos-treated macrophages.

Alveolar macrophages have been implicated in the pathogenesis of a number of acute and chronic lung disorders. We have previously shown that normal human alveolar macrophages exhibit decreased DNA binding activity of the transcription factor, AP-1, compared with monocytes. Furthermore, this decrease in AP-1 DNA binding appears to be due to a decrease in the redox active protein, redox factor (Ref)-1.

Ceramide regulates lipopolysaccharide-induced phosphatidylinositol 3-kinase and Akt activity in human alveolar macrophages.

The phosphatidylinositol (PI) 3-kinase pathway is an important regulator of cell survival. In human alveolar macrophages, we found that LPS activates PI 3-kinase and its downstream effector, Akt. LPS exposure of alveolar macrophages also results in the generation of ceramide.

Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox.

High molecular weight homologues of gp91phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for "dual oxidase" because they have both a peroxidase homology domain and a gp91phox domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals.

GM-CSF increases AP-1 DNA binding and Ref-1 amounts in human alveolar macrophages.

Alveolar macrophages have been implicated in the pathogenesis of a number of acute and chronic lung disorders. A characteristic feature of many of the chronic lung diseases is that the types of macrophages in the lung change, and in most instances, the cells resemble monocyte-like cells. We have previously shown that normal human alveolar macrophages have a decreased capacity to express protein kinase C (PKC)-induced DNA binding activity of the transcription factor activator protein (AP)-1 compared with monocytes.

Lipopolysaccharide activates Akt in human alveolar macrophages resulting in nuclear accumulation and transcriptional activity of beta-catenin.

Exposure of human alveolar macrophages to bacterial LPS results in activation of a number of signal transduction pathways. An early event after the alveolar macrophage comes in contact with LPS is activation of the phosphatidylinositol 3 kinase (PI 3-kinase). This study evaluates the downstream effects of that activation.

Phosphorylation of human tau protein by microtubule-associated kinases: GSK3beta and cdk5 are key participants.

Microtubules (MTs), primarily composed of alpha and beta tubulin polymers, must often work in concert with microtubule-associated proteins (MAPs) in order to modulate their functional demands. In a mature brain neuron, one of the key MAPs that resides primarily in the axonal compartment is the tau protein. Tau, in the adult human brain, is a set of six protein isoforms, whose binding affinity to MTs can be modulated by phosphorylation.

Protein kinase C zeta plays a central role in activation of the p42/44 mitogen-activated protein kinase by endotoxin in alveolar macrophages.

Human alveolar macrophages respond to endotoxin (LPS) by activation of a number of mitogen-activated protein kinase pathways, including the p42/44 (extracellular signal-related kinase (ERK)) kinase pathway. In this study, we evaluated the role of the atypical protein kinase C (PKC) isoform, PKC zeta, in LPS-induced activation of the ERK kinase pathway. Kinase activity assays showed that LPS activates PKC zeta, mitogen-activated protein/ERK kinase (MEK, the upstream activator of ERK), and ERK.

Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123.

To detect intracellular oxidant formation during reoxygenation of anoxic endothelium, the oxidant-sensing fluorescent probes, 2',7'-dichlorodihydrofluorescein diacetate, dihydrorhodamine 123, or 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate were added to human umbilical vein endothelial cells during reoxygenation. None of these fluorescent probes were able to differentiate the controls from the reoxygenated cells in the confocal microscope. However, dihydrofluorescein diacetate demonstrated fluorescence of linear structures, consistent with mitochondria, in reoxygenated endothelium.

Regulation of tau phosphorylation in microtubule fractions by apolipoprotein E.

In Alzheimer's disease (AD), a family of proteins collectively named tau are displaced from their normal association with microtubules and are found in in a hyperphosphorylated state deposited into paired helical filaments (PHFs). PHFs are the hallmark cytoskeletal pathology of the disease, and the degree of PHF pathology correlates with the clinical severity of AD. Certain apolipoprotein E (apoE) isoforms have been identified as either risk or protective factors for AD, and one of the proposed mechanisms involves an interaction and potentially modulatory effects on tau hyperphosphorylation by the different apoE isoforms.

Human endothelial nitric oxide synthase gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation after balloon injury in rats.

Background: Loss of endothelial NO production after arterial injury may contribute to restenosis, characterized by neointima formation and elastic recoil. Adenovirus-mediated transfer of the gene encoding NO synthase (NOS) in balloon-injured arteries may restore NO production and inhibit neointima formation.

Methods And Results: After balloon injury, rat carotid arteries were transduced with 3x10(10) pfu/mL recombinant adenovirus carrying the human endothelial constitutive NOS cDNA (AdCMVceNOS, n=8) or no cDNA (AdRR5, n=8).

A critique of the World Resources Institute's report "Pesticides and the immune system: the public health risks".

A recent World Resources Institute (WRI) report concluded that pesticides are a likely cause of immune suppression for millions of people throughout the world. The gravity of this conclusion motivated us to review the scientific evidence cited in the report. The predominant human evidence came from cross-sectional studies conducted in the former Soviet Union.

Comparison of three methods used for assessment of pain in dogs.

Objective: To investigate the reliability of 3 scales used for assessment of pain in dogs.

Design: Prospective study.

Animals: 50 dogs that had surgery.

Aging and lymphocyte subsets in the spleen and peripheral blood of the Sprague-Dawley rat.

This study was undertaken to determine the effects of aging on lymphocyte subsets in the peripheral blood and spleens of Sprague-Dawley rats. Rats aged 3,13 and 26 months were used in the study. Analyses of dual labeled lymphocytes from the 26 month animals show decreases in the numbers of lymphocytes due to decreased cellularity (spleen) or reduced lymphocyte percentages within the total white blood cell population (peripheral blood).