Faculty Bibliography

The authors recently reported a novel role for autophagy in late-stage quality control of a secreted protein, apolipoprotein-B(100) (apoB). Hepatocytes assemble this protein with triglycerides, cholesterol and other lipids into macromolecular complexes called lipoproteins. In what appears to be a normal response to diets rich in polyunsaturated fatty acids, which are readily peroxidized, apoB comes into contact with lipid peroxides in or after the Golgi apparatus. The protein becomes oxidatively damaged, aggregates, and is diverted out of the secretory pathway by autophagosomes, which deliver it to lysosomes for destruction. ApoB secretory control via autophagosomes is likely a key component of normal and pathological regulation of plasma lipoprotein levels, as well as a means for remarkably late-stage quality control of a secreted protein

Candida parapsilosis has emerged as a common cause of invasive fungal infection, especially in Latin America and in the neonatal setting. C. parapsilosis is part of a closely related group of organisms that includes the species Candida orthopsilosis and Candida metapsilosis. All three species show elevated MICs for the new echinocandin class drugs caspofungin, micafungin, and anidulafungin relative to other Candida species. Despite potential impacts on therapy, the mechanism behind this reduced echinocandin susceptibility has not been determined. In this report, we investigated the role of a naturally occurring Pro-to-Ala substitution at amino acid position 660 (P660A), immediately distal to the highly conserved hot spot 1 region of Fks1p, in the reduced-echinocandin-susceptibility phenotype. Kinetic inhibition studies demonstrated that glucan synthase from the C. parapsilosis group was 1 to 2 logs less sensitive to echinocandin drugs than the reference enzyme from C. albicans. Furthermore, clinical isolates of C. albicans and C. glabrata which harbor mutations at this equivalent position also showed comparable 2-log decreases in target enzyme sensitivity, which correlated with increased MICs. These mutations also resulted in 2.4- to 18.8-fold-reduced V(max) values relative to those for the wild-type enzyme, consistent with kinetic parameters obtained for C. parapsilosis group enzymes. Finally, the importance of the P660A substitution for intrinsic resistance was confirmed by engineering an equivalent P647A mutation into Fks1p of Saccharomyces cerevisiae. The mutant glucan synthase displayed characteristic 2-log decreases in sensitivity to the echinocandin drugs. Overall, these data firmly indicate that a naturally occurring P660A substitution in Fks1p from the C. parapsilosis group accounts for the reduced susceptibility phenotype.

OBJECTIVE: Subjects with diabetes experience an increased risk of myocardial infarction and cardiac failure compared with nondiabetic age-matched individuals. The receptor for advanced glycation end products (RAGE) is upregulated in diabetic tissues. In this study, we tested the hypothesis that RAGE affected ischemia/reperfusion (I/R) injury in the diabetic myocardium. In diabetic rat hearts, expression of RAGE and its ligands was enhanced and localized particularly to both endothelial cells and mononuclear phagocytes. RESEARCH DESIGN AND METHODS: To specifically dissect the impact of RAGE, homozygous RAGE-null mice and transgenic (Tg) mice expressing cytoplasmic domain-deleted RAGE (DN RAGE), in which RAGE-dependent signal transduction was deficient in endothelial cells or mononuclear phagocytes, were rendered diabetic with streptozotocin. Isolated perfused hearts were subjected to I/R. RESULTS: Diabetic RAGE-null mice were significantly protected from the adverse impact of I/R injury in the heart, as indicated by decreased release of LDH and lower glycoxidation products carboxymethyl-lysine (CML) and pentosidine, improved functional recovery, and increased ATP. In diabetic Tg mice expressing DN RAGE in endothelial cells or mononuclear phagocytes, markers of ischemic injury and CML were significantly reduced, and levels of ATP were increased in heart tissue compared with littermate diabetic controls. Furthermore, key markers of apoptosis, caspase-3 activity and cytochrome c release, were reduced in the hearts of diabetic RAGE-modified mice compared with wild-type diabetic littermates in I/R. CONCLUSIONS: These findings demonstrate novel and key roles for RAGE in I/R injury in the diabetic heart

The calcineurin pathway is a critical signal transduction pathway in fungi that mediates growth, morphology, stress responses, and pathogenicity. The importance of the calcineurin pathway in fungal physiology creates an opportunity for the development of new antifungal therapies that target this critical signaling pathway. In this study, we examined the role of the zinc finger transcription factor Crz1 homolog (CrzA) in the physiology and pathogenicity of the opportunistic human fungal pathogen Aspergillus fumigatus. Genetic replacement of the crzA locus in A. fumigatus resulted in a strain with significant defects in conidial germination, polarized hyphal growth, cell wall structure, and asexual development that are similar to but with differences from defects seen in the A. fumigatus DeltacnaA (calcineurin A) strain. Like the DeltacnaA strain, the DeltacrzA strain was incapable of causing disease in an experimental persistently neutropenic inhalational murine model of invasive pulmonary aspergillosis. Our results suggest that CrzA is an important downstream effector of calcineurin that controls morphology in A. fumigatus, but additional downstream effectors that mediate calcineurin signal transduction are likely present in this opportunistic fungal pathogen. In addition, the importance of CrzA to the production of disease is critical, and thus CrzA is an attractive fungus-specific antifungal target for the treatment of invasive aspergillosis.

Health-care safety management has recently been highlighted for patient safety. However, specialist-based risks in clinical settings have hardly been discussed in Japan so far. A review of dermatological legal claims may delineate these risks. This study examined court precedents from the databases "Courts in Japan" and LEX/DB. Thirty-four dermatology-related civil cases were found from 1968-2006. Of the 34 cases, 32 (94%) were judged and two (6%) were retried. Of these 32 cases, 11 (34%) were appealed to higher courts. Among the 34 litigations, the defendants of eight (23%) were dermatology specialists, 20 (59%) were non-dermatologists and six (18%) of unknown specialty. The defendants' negligence was determined at either level in court in 25 of the 34 cases. The negligence in these 25 cases was categorized into five groups: (i) delayed diagnosis (none); (ii) complication during diagnosis procedure (one, 4%); (iii) inappropriate treatment (nine, 36%); (iv) complication during treatment procedure (10, 40%); and (v) insufficient informed consent (five, 20%). The present study may help to improve strategies for health-care safety management in the dermatological field in Japan.

Neutrophil influx is an early inflammatory response that is essential for the clearance of bacteria and cellular debris during cutaneous wounding. A non-invasive real-time fluorescence imaging technique was developed to examine the kinetics of enhanced green fluorescence protein-polymorphonuclear leukocyte (EGFP-PMN) influx within a wound. We hypothesized that infection or systemic availability would directly regulate the dynamics of EGFP-PMN recruitment and the efficiency of wound closure. Neutrophil recruitment increased dramatically over the first 24 hours from 10(6) at 4 hours up to a maximum of 5 x 10(6) EGFP-PMNs at 18 hours. A high rate of EGFP-PMN turnover was evidenced by approximately 80% decrease in EGFP signal within 6 hours. In response to wound colonization by Staphylococcus aureus or injection of GM-CSF, systemic PMNs increased twofold above saline control. This correlated with an increase in EGFP-PMN recruitment up to approximately 10(7) within the wound. Despite this effect by these distinct inflammatory drivers, wound closure occurred at a rate similar to the saline-treated control group. In summary, a non-invasive fluorescence-based imaging approach combined with genetic labeling of neutrophils provides a dynamic inner view of inflammation and the kinetics of neutrophil infiltration into the wounded skin over extended durations

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that causes cardiac contractile dysfunction, whereas inactivation of MIF improves cardiac function in experimental animal models of sepsis. We used cultured cardiomyocytes to determine whether MIF-induced contractile dysfunction was mediated in part by myocyte apoptosis and to identify MIF-activated intracellular signaling pathways in this process. MIF treatment significantly increased myocyte apoptosis in a dose-dependent manner to 15.5+/-3.9% and 26.0+/-7.1% TUNEL positive nuclei (20 and 30 ng/ml MIF for 24h) vs control (3.7+/-0.9%). This effect was attenuated by inactivation of MIF with the chemical inhibitor, ISO-1. MIF-induced cleavage of caspase 3 and reduction of Bcl-xL/Bax were similarly attenuated by ISO-1 pre-treatment. MIF stimulated the rapid, transient phosphorylation of stress kinases, p38MAPK and JNK. Thus, MIF induces cardiomyocyte apoptosis by activating stress kinases and mitochondria-associated apoptotic mechanisms, whereas inactivation of MIF pro-inflammatory activity improves cardiomyocyte survival

Early embryos of some metazoans polarize radially to facilitate critical patterning events such as gastrulation and asymmetric cell division; however, little is known about how radial polarity is established. Early embryos of Caenorhabditis elegans polarize radially when cell contacts restrict the polarity protein PAR-6 to contact-free cell surfaces, where PAR-6 regulates gastrulation movements. We have identified a Rho guanosine triphosphatase activating protein (RhoGAP), PAC-1, which mediates C. elegans radial polarity and gastrulation by excluding PAR-6 from contacted cell surfaces. We show that PAC-1 is recruited to cell contacts, and we suggest that PAC-1 controls radial polarity by restricting active CDC-42 to contact-free surfaces, where CDC-42 binds and recruits PAR-6. Thus, PAC-1 provides a dynamic molecular link between cell contacts and PAR proteins that polarizes embryos radially

Neurodegenerative diseases such as Parkinson's disease (PD), frontal lobe dementia (FLD) and diffuse Lewy-body dementia (DLBD) have diverse neuropathologic features. Here we report that serotonin fibers are dystrophic in the brains of individuals with these three diseases. In neuropathologically normal (control) brains (n=3), serotonin axons immunoreactive (IR) with antibodies against the serotonin transporter (5-HTT) protein were widely distributed in cortex (entorhinal and dorsolateral prefrontal), hippocampus and rostral brainstem. 5-HTT-IR fibers-of-passage appeared thick, smooth, and unbranched in medial forebrain bundle, medial lemniscus and cortex white matter. The terminal branches were fine, highly branched and varicose in substantia nigra, hippocampus and cortical gray matter. In the diseased brains, however, 5-HTT-IR fibers in the forebrain were reduced in number and were frequently bulbous, splayed, tightly clustered and enlarged. Morphometric analysis revealed significant differences in the size distribution of the 5-HTT-IR profiles in dorsolateral prefrontal area between neurodegenerative diseases and controls. Our observations provide direct morphologic evidence for degeneration of human serotonergic axons in the brains of patients with neurodegenerative diseases despite the limited size (n=3 slices for each region (3) from each brain (4), total slices was n=36) and the lack of extensive clinical characterization of the analyzed cohort. This is the first report of dystrophic 5-HTT-IR axons in postmortem human tissue

Integrins play a role in fibroblast growth factor (FGF) signaling through cross-talk with FGF receptors (FGFRs), but the mechanism underlying the cross-talk is unknown. We discovered that FGF1 directly bound to soluble and cell-surface integrin alphavbeta3 (K(D) about 1 microm). Antagonists to alphavbeta3 (monoclonal antibody 7E3 and cyclic RGDfV) blocked this interaction. alphavbeta3 was the predominant, if not the only, integrin that bound to FGF1, because FGF1 bound only weakly to several beta1 integrins tested. We presented evidence that the CYDMKTTC sequence (the specificity loop) within the ligand-binding site of beta3 plays a role in FGF1 binding. We found that the integrin-binding site of FGF1 overlaps with the heparin-binding site but is distinct from the FGFR-binding site using docking simulation and mutagenesis. We identified an FGF1 mutant (R50E) that was defective in integrin binding but still bound to heparin and FGFR. R50E was defective in inducing DNA synthesis, cell proliferation, cell migration, and chemotaxis, suggesting that the direct integrin binding to FGF1 is critical for FGF signaling. Nevertheless, R50E induced phosphorylation of FGFR1 and FRS2alpha and activation of AKT and ERK1/2. These results suggest that the defect in R50E in FGF signaling is not in the initial activation of FGF signaling pathway components, but in the later steps in FGF signaling. We propose that R50E is a useful tool to identify the role of integrins in FGF signaling