Faculty Bibliography

The 70S ribosome and its complement of factors required for initiation of translation in E. coli were purified separately and reassembled in vitro with GDPNP, producing a stable initiation complex (IC) stalled after 70S assembly. We have obtained a cryo-EM reconstruction of the IC showing IF2*GDPNP at the intersubunit cleft of the 70S ribosome. IF2*GDPNP contacts the 30S and 50S subunits as well as fMet-tRNA(fMet). IF2 here adopts a conformation radically different from that seen in the recent crystal structure of IF2. The C-terminal domain of IF2 binds to the single-stranded portion of fMet-tRNA(fMet), thereby forcing the tRNA into a novel orientation at the P site. The GTP binding domain of IF2 binds to the GTPase-associated center of the 50S subunit in a manner similar to EF-G and EF-Tu. Additionally, we present evidence for the localization of IF1, IF3, one C-terminal domain of L7/L12, and the N-terminal domain of IF2 in the initiation complex

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.

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.

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