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.

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

Intronic elements flanking the splice-site consensus sequences are thought to play a role in pre-mRNA splicing. However, the generality of this role, the catalog of effective sequences, and the mechanisms involved are still lacking. Using molecular genetic tests, we first showed that the approximately 50-nt intronic flanking sequences of exons beyond the splice-site consensus are generally important for splicing. We then went on to characterize exon flank sequences on a genomic scale. The G+C content of flanks displayed a bimodal distribution reflecting an exaggeration of this base composition in flanks relative to the gene as a whole. We divided all exons into two classes according to their flank G+C content and used computational and statistical methods to define pentamers of high relative abundance and phylogenetic conservation in exon flanks. Upstream pentamers were often common to the two classes, whereas downstream pentamers were totally different. Upstream and downstream pentamers were often identical around low G+C exons, and in contrast, were often complementary around high G+C exons. In agreement with this complementarity, predicted base pairing was more frequent between the flanks of high G+C exons. Pseudo exons did not exhibit this behavior, but rather tended to form base pairs between flanks and exon bodies. We conclude that most exons require signals in their immediate flanks for efficient splicing. G+C content is a sequence feature correlated with many genetic and genomic attributes. We speculate that there may be different mechanisms for splice site recognition depending on G+C content.

Reported effects of estrogen administration on tyrosine hydroxylase (TH) gene expression are confusing. Therefore, we studied the mechanism of regulation of TH transcription by estrogen with different estradiol receptor (ER) subtypes. PC12 cells, transiently co-transfected with expression vector for ERalpha or ERbeta, and luciferase gene under control of the TH promoter, were treated with 17 beta-estradiol (E2). E2 doubled luciferase activity with ERalpha; however, it was decreased with ERbeta. Mapping the TH promoter showed that the putative half estrogen response element (ERE) motif at - 675, as well as the activation protein 1 motif at - 205, were not required for response to E2 with either ER. The specificity protein 1/early growth response gene 1 (Egr 1) motif was required for the E2-elicited response with ERbeta, but not with ERalpha. Deletion of the cyclic AMP/Ca2+ response element (CRE/CaRE) nearly abolished E2-triggered responses with either ER. Further analysis revealed an imperfect canonical putative ERE overlapping with CRE/CaRE and Nurr1 response element. Oligonucleotides spanning this ERE displayed binding to ER, Cyclic AMP Response Element Binding Protein (CREB) and other proteins. Moreover, E2 attenuated the increase in TH transcription seen with cyclic AMP analogs. Thus, TH is transcriptionally regulated by estradiol in opposite directions depending on ER subtype. The overlapping ERE and CRE/CaRE may integrate interactions elicited by various regulators of TH transcription including cAMP and estrogens.

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

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

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