Faculty Bibliography

The P mRNA of the viruses belonging to the subfamily Paramyxovirinae possesses a unique property of giving rise to several accessory proteins by a process that involves the utilization of overlapping open reading frames (the C proteins) and by an "RNA-editing" mechanism (the V proteins). Although these proteins are considered accessory, numerous studies have highlighted the importance of these proteins in virus transcription and interferon signaling, including our previous observation on the role of human parainfluenza virus type 3 (HPIV 3) C protein in the transcription of viral genome (Malur et al., Virus Res. 99:199-204, 2004). In this report, we have addressed its role in interferon signaling by generating a stable cell line, L-C6, by using the lentiviral expression system which expresses HPIV 3 C protein. The L-C6 cells were efficient in abrogating both alpha and gamma interferon-induced antiviral states and demonstrated a drastic reduction in the formation of gamma-activated factor complexes in the cell extracts. Western blot analysis subsequently revealed a defect in the phosphorylation of STAT 1 in these cells. Taken together, our results indicate that HPIV 3 C protein is capable of counteracting the interferon signaling pathway by specifically inhibiting the activation of STAT 1.

Barth syndrome (BTHS) is a multisystem disorder of individuals who carry mutations in tafazzin, a putative phospholipid acyltransferase. We investigated the hypothesis that BTHS is caused by specific impairment of the mitochondrial lipid metabolism. The fatty acid composition of all major mitochondrial phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL), changed in lymphoblasts from BTHS patients. These changes were most extensive in CL and least extensive in PE. The complementary nature of the fatty acid alterations in CL and PC suggested that fatty acid transfer between these two lipids was inhibited in BTHS. Fluorescence staining and electron microscopy showed abnormal proliferation of mitochondria in BTHS lymphoblasts. The mitochondrial membrane potential, monitored with the fluorescence probe JC-1, was reduced in BTHS lymphoblasts. However, mitochondrial ATP formation of permeabilized lymphoblasts remained unaffected in BTHS. The data suggest that phospholipid abnormalities of BTHS mitochondria led to partial uncoupling of oxidative phosphorylation and that lymphoblasts compensated for this deficiency by expanding the mitochondrial compartment.Laboratory Investigation (2005) 85, 823-830, advance online publication, 4 April 2005; doi:10.1038/labinvest.3700274

Neurotrophin receptor trafficking plays an important role in directing cellular communication in developing as well as mature neurons. However, little is known about the requirements for intracellular localization of the neurotrophin receptors in neurons. To isolate the subcellular membrane compartments containing the Trk neurotrophin receptor, we performed biochemical subcellular fractionation experiments using primary cortical neurons and rat PC12 pheochromocytoma cells. By differential centrifugation and density gradient centrifugation, we have isolated Trk-bearing compartments, suggesting distinct membranous localization of Trk receptors. A number of Trk-interacting proteins, such as GIPC and dynein light chain Tctex-1 were found in these fractions. Additionally, membranes enriched in phosphorylated activated forms of Trk receptors were found upon ligand treatment in primary neurons and PC12 cells. Interestingly, density gradient centrifugation experiments showed that Trk receptors from PC12 cells are present in heavy membrane fractions, while Trk from primary neurons are fractionated in lighter membrane fractions. These results suggest that the intracellular membrane localization of Trk can differ according to cell type. Taken together, these biochemical approaches allowed separation of distinct Trk-bearing membrane pools, which may be involved in different functions of neurotrophin receptor signaling and trafficking

Ankylosing spondylitis is an inflammatory disease of unknown etiology that affects an estimated 350,000 persons in the United States and 600,000 in Europe, primarily Caucasian males in the second through fourth decades of life. Worldwide, the prevalence is 0.9%. Genetic linkage to HLA-B27 has been established. Ankylosing spondylitis primarily affects the axial skeleton and is characterized by inflammation and fusion of the sacroiliac joints, spine, and hips. The resultant deformity leads to severe functional impairment in approximately 30% of patients. Orthopaedic management primarily involves correction of hip deformity through total hip arthroplasty and, less frequently, correction of spinal deformity with spine osteotomy. Closing wedge osteotomies have the lowest incidence of complications. Whether patients with ankylosing spondylitis are at increased risk for heterotopic ossification remains controversial, but comparison with age- and sex-matched counterparts suggests no dramatically higher risk. Because of the high rate of missed fractures and complications after minor trauma in patients with ankylosing spondylitis, plain radiographs are usually not sufficient for evaluation. Thorough patient assessment should include a comprehensive history, physical examination, and laboratory studies

Brain hemorrhage is a severe complication of both neoplastic and nonneoplastic brain disease. Mice deficient in the alpha(v)beta8 integrin display defective brain vessel formation resulting in hemorrhage and perinatal death, but the mechanism of brain hemorrhage is unknown. Because the alpha(v)beta8 integrin is expressed by astrocytes and not expressed by endothelium, paracrine interactions between astrocytes and endothelial cells could contribute to the maintenance of brain vessel integrity. We have investigated the mechanisms underlying astrocytic-endothelial paracrine signaling and have found that integrin-mediated activation of transforming growth factor (TGF)-beta by astrocytes influences endothelial cell function. Thus, we identified the integrin alpha(v)beta8 in human perivascular glial cell processes surrounding developing blood vessels. Human astrocytic alpha(v)beta8 was a major cell surface receptor for latent TGF-beta, and alpha(v)beta8-dependent activation of TGF-beta was the major mechanism of TGF-beta activation in primary cultures of astrocytes or freshly dissociated fetal brain cells. This activation of TGF-beta was sufficient to inhibit endothelial migration in fibrin gels and to alter expression of genes affecting proteolytic and angiogenic pathways. Taken together, our data suggest that astrocytic alpha(v)beta8 acts as a central regulator of brain vessel homeostasis through regulation of TGF-beta activation and expression of TGF-beta-responsive genes that promote vessel differentiation and stabilization, most notably plasminogen activator inhibitor-1 and thrombospondin-1

BACKGROUND AND PURPOSE: In contrast to tissue-type plasminogen activator (tPA), vampire bat (Desmodus rotundus) salivary plasminogen activator (desmoteplase [DSPA]) does not promote excitotoxic injury when injected directly into the brain. We have compared the excitotoxic effects of intravenously delivered tPA and DSPA and determined whether DSPA can antagonize the neurotoxic and calcium enhancing effects of tPA. METHODS: The brain striatal region of wild-type c57 Black 6 mice was stereotaxically injected with N-methyl-d-Aspartate (NMDA); 24 hour later, mice received an intravenous injection of tPA or DSPA (10 mg/kg) and lesion size was assessed after 24 hours. Cell death and calcium mobilization studies were performed using cultures of primary murine cortical neurons. RESULTS: NMDA-mediated injury was increased after intravenous administration of tPA, whereas no additional toxicity was seen after administration of DSPA. Unlike DSPA, tPA enhanced NMDA-induced cell death and the NMDA-mediated increase in intracellular calcium levels in vitro. Moreover, the enhancing effects of tPA were blocked by DSPA. CONCLUSIONS: Intravenous administration of tPA promotes excitotoxic injury, raising the possibility that leakage of tPA from the vasculature into the parenchyma contributes to brain damage. The lack of such toxicity by DSPA further encourages its use as a thrombolytic agent in the treatment of ischemic stroke.

Dermal fibroblasts actively contribute to wound healing by migrating to the wound, synthesizing extracellular matrices, and generating mechanical forces within the wound to initiate wound contraction. Fibroblast-seeded collagen gels provide an in vitro model to study wound contraction. The authors are evaluating the role of the adrenergic signaling system in cutaneous wound repair and recently found that beta2-adrenergic receptor (beta2-AR) activation markedly decreases keratinocyte migration, an essential step in wound reepithelialization. Because the beta2-ARs are also expressed on dermal fibroblasts, a study was initiated to determine the effects of beta-adrenergic agonists on dermal fibroblast-mediated collagen gel contraction. A beta-agonist (isoproterenol) delayed gel contraction in a dose-dependent manner. A beta2-AR specific antagonist (ICI 118,551) prevented the delay, indicating that the beta2-AR alone mediated the delay. The active cyclic adenosine monophosphate (cAMP) analog also delayed collagen gel contraction, whereas an inactive cAMP analog partially prevented the delay, suggesting that the mechanism for beta-AR agonist-mediated delay was partly cAMP-dependent. Identifying and characterizing agents that modulate wound contraction improves understanding of the wound healing process and could result in novel therapeutic strategies for preventing unwanted wound contraction in burn and trauma patients

We have developed an efficient, sensitive and specific method for the detection of phosphopeptides present in peptide mixtures by MALDI Q-TOF mass spectrometry. Use of the MALDI Q-TOF enables selection of phosphopeptides and characterization by collision-induced dissociation of the phosphopeptides performed on the same sample spot. However, this type of experiment has been limited by low ionization efficiency of phosphopeptides in positive ion mode while selecting precursor ions of phosphopeptides. Our method entails neutralizing negative charges on acidic groups of nonphosphorylated peptides by methyl esterification prior to mass spectrometry in positive and negative ion modes. Methyl esterification significantly increases the relative signal intensity generated by phosphopeptides in negative ion mode compared with positive ion mode, and greatly increases selectivity for phosphopeptides by suppressing the signal intensity generated by acidic peptides in negative ion mode. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of ss-casein and a-s-casein. We also identified 10 phosphopeptides containing five phosphorylation sites from an in-gel tryptic digest of 100 fmol of an in vitro autophosphorylated fibroblast growth factor receptor kinase domain, and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides

Hearts with overexpression of anchored lipoprotein lipase (LpL) by cardiomyocytes (hLpL(GPI) mice) develop a lipotoxic cardiomyopathy. To characterize cardiac fatty acid (FA) and triglyceride (TG) metabolism in these mice and to determine whether changes in lipid metabolism precede cardiac dysfunction, hearts from young mice were perfused in Langendorff mode with [14C]palmitate. In hLpL(GPI) hearts, FA uptake and oxidation were decreased by 59 and 82%, respectively. This suggests reliance on an alternative energy source, such as TG. Indeed, these hearts oxidized 88% more TG. Hearts from young hLpL(GPI) mice also had greater uptake of intravenously injected cholesteryl ester-labeled Intralipid and VLDL. To determine whether perfusion of normal hearts would mimic the metabolic alterations found in hLpL(GPI) mouse hearts, wild-type hearts were perfused with [14C]palmitate and either human VLDL or Intralipid (0.4 mM TG). Both sources of TG reduced [14C]palmitate uptake (48% with VLDL and 45% with Intralipid) and FA oxidation (71% with VLDL and 65% with Intralipid). Addition of either heparin or LpL inhibitor P407 to Intralipid-containing perfusate restored [14C]palmitate uptake and confirmed that Intralipid inhibition requires local LpL. Our data demonstrate that reduced FA uptake and oxidation occur before mechanical dysfunction in hLpL(GPI) lipotoxicity. This physiology is reproduced with perfusion of hearts with TG-containing particles. Together, the results demonstrate that cardiac uptake of TG-derived FA reduces utilization of albumin-FA