Faculty Bibliography

Despite their location in the aqueous extracellular environment, a number of secreted proteins carry hydrophobic lipid modifications. These modifications include glycosylphosphatidylinositol, cholesterol, and both saturated and unsaturated fatty acids, and they are attached in the secretory pathway by different classes of enzymes. Lipid attachments make crucial contributions to protein function in vivo through a diverse array of mechanisms. They can promote protein maturation and secretion, membrane tethering, targeting to specific membrane subdomains, or receptor binding and activation. Additionally, secretion of lipid-modified morphogens of the Wnt and Hh families requires dedicated accessory proteins and may involve their packaging into lipoprotein particles for long-range transport

Signal transduction along the Ras/MAPK pathway has been generally thought to take place at the plasma membrane. It is now evident that the plasma membrane is not the only platform capable of Ras/MAPK signal induction. Fusion of Ras with green fluorescent protein and the development of genetically encoded fluorescent probes for Ras activation have revealed signaling events on a variety of intracellular membranes including endosomes, the Golgi apparatus and the endoplasmic reticulum. Thus, the Ras/MAPK pathway is spatially compartmentalized within cells and this may afford greater complexity of signal output

OBJECTIVES: Several Leishmania strains with episomal expression of green fluorescent protein (GFP) require constant drug pressure for its continuous expression and hence limit its use in ex vivo or in vivo systems. The aim of this study was to alleviate this problem by stably integrating the GFP gene into the parasite genome, so as to use these transfectants for ex vivo and in vivo drug screening. METHODS: The GFP gene was integrated downstream of the 18S ribosomal promoter region of Leishmania donovani. After initial selection, GFP-expressing parasites-both sodium stibogluconate (SAG)-susceptible (2001) and -resistant (2039) isolates-were grown without adding G418. The infectivity of these transfectants to macrophages (J774.1) as well as to hamsters was checked. The ex vivo screening assay was standardized using standard antileishmanial drugs. RESULTS: A constitutive and enhanced expression of GFP in promastigote and amastigote stages was achieved for approximately 12 months without any need for drug pressure. These transfectants were highly infective to macrophage cell lines as well as to hamsters, as observed by fluorescence microscopy and flow cytometry (FACS). GFP-tagged promastigotes as well as intracellular amastigotes were found to be highly susceptible to miltefosine, amphotericin B and pentamidine, in a concentration-dependent manner. SAG was inactive against the GFP-promastigotes, as well as SAG-resistant intracellular amastigotes, correlating well with earlier reports. CONCLUSIONS: The GFP-transfectants were found to be suitable for FACS-based ex vivo screening assays. They were also infective to hamsters up to day 60 post-infection.

Epithelial-mesenchymal transition (EMT) is essential for organogenesis and is triggered during carcinoma progression to an invasive state. Transforming growth factor-beta (TGF-beta) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT. The SNAIL1-SMAD3/4 complex was targeted to the gene promoters of CAR, a tight-junction protein, and E-cadherin during TGF-beta-driven EMT in breast epithelial cells. SNAIL1 and SMAD3/4 acted as co-repressors of CAR, occludin, claudin-3 and E-cadherin promoters in transfected cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA inhibited repression of CAR and occludin during EMT. Moreover, loss of CAR and E-cadherin correlated with nuclear co-expression of SNAIL1 and SMAD3/4 in a mouse model of breast carcinoma and at the invasive fronts of human breast cancer. We propose that activation of a SNAIL1-SMAD3/4 transcriptional complex represents a mechanism of gene repression during EMT

OBJECTIVES/OBJECTIVE:This study sought to compare contrast-enhanced anatomic imaging and contrast-enhanced tissue characterization (delayed-enhancement cardiac magnetic resonance [DE-CMR]) for left ventricular (LV) thrombus detection. BACKGROUND:Contrast echocardiography (echo) detects LV thrombus based on anatomic appearance, whereas DE-CMR imaging detects thrombus based on tissue characteristics. Although DE-CMR has been validated as an accurate technique for thrombus, its utility compared with contrast echo is unknown. METHODS:Multimodality imaging was performed in 121 patients at high risk for thrombus due to myocardial infarction or heart failure. Imaging included 3 anatomic imaging techniques for thrombus detection (contrast echo, noncontrast echo, cine-CMR) and a reference of DE-CMR tissue characterization. LV structural parameters were quantified to identify markers for thrombus and predictors of additive utility of contrast-enhanced thrombus imaging. RESULTS:Twenty-four patients had thrombus by DE-CMR. Patients with thrombus had larger infarcts (by DE-CMR), more aneurysms, and lower LV ejection fraction (by CMR and echo) than those without thrombus. Contrast echo nearly doubled sensitivity (61% vs. 33%, p < 0.05) and yielded improved accuracy (92% vs. 82%, p < 0.01) versus noncontrast echo. Patients who derived incremental diagnostic utility from DE-CMR had lower LV ejection fraction versus those in whom noncontrast echo alone accurately assessed thrombus (35 +/- 9% vs. 42 +/- 14%, p < 0.01), with a similar trend for patients who derived incremental benefit from contrast echo (p = 0.08). Contrast echo and cine-CMR closely agreed on the diagnosis of thrombus (kappa = 0.79, p < 0.001). Thrombus prevalence was lower by contrast echo than DE-CMR (p < 0.05). Thrombus detected by DE-CMR but not by contrast echo was more likely to be mural in shape or, when apical, small in volume (p < 0.05). CONCLUSIONS:Echo contrast in high-risk patients markedly improves detection of LV thrombus, but does not detect a substantial number of thrombi identified by DE-CMR tissue characterization. Thrombi detected by DE-CMR but not by contrast echo are typically mural in shape or small in volume.

The development of nervous system connectivity depends upon the arborization of dendritic fields and the stabilization of dendritic spine synapses. It is well established that neuronal activity and the neurotrophin BDNF modulate these correlated processes. However, the downstream mechanisms by which these extrinsic signals regulate dendritic development and spine stabilization are less well known. Here we report that a substrate of BDNF signaling, the Ankyrin Repeat-rich Membrane Spanning (ARMS) protein or Kidins220, plays a critical role in the branching of cortical and hippocampal dendrites and in the turnover of cortical spines. In the barrel somatosensory cortex and the dentate gyrus, regions where ARMS/Kidins220 is highly expressed, no difference in the complexity of dendritic arbors was observed in 1-month-old adolescent ARMS/Kidins220(+/-) mice compared to wild-type littermates. However, at 3 months of age, young adult ARMS/Kidins220(+/-) mice exhibited decreased dendritic complexity. This suggests that ARMS/Kidins220 does not play a significant role in the initial formation of dendrites but, rather, is involved in the refinement or stabilization of the arbors later in development. In addition, at 1 month of age, the rate of spine elimination was higher in ARMS/Kidins220(+/-) mice than in wild-type mice, suggesting that ARMS/Kidins220(+/-) levels regulate spine stability. Taken together, these data suggest that ARMS/Kidins220 is important for the growth of dendritic arbors and spine stability during an activity- and BDNF-dependent period of development. (c) 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009

A rapid ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometric (UPLC-ESI-MS/MS) method is developed for the qualitative identification of constituents in the flower buds of seven Lonicera species. The optimal condition of separation and detection were achieved on an AcQuity UPLC BEH C18 column with a gradient elution with acetonitrile and 0.1% acetic acid within 17 min. Among the 33 constituents detected, 6 caffeoylquinic acids (including caffeic acid), 8 flavonoids and 8 iridoid glycosides were characterized based on their fragmentation patterns in collision-induced dissociation (CID) experiments and/or by comparison with standard compounds. In addition, to statistically establish the correlation and discrimination of the Lonicera species, principle component analysis (PCA) was applied in this study. Lonicera samples were divided into well-defined groups directly related to their species based on PCA in terms of the log transformed relative contents of the major caffeoylquinic acids (including caffeic acid) as the variables. All of results indicated that the method presented here is able to classify the sample species and to reveal characteristic details of the chemical constituents of different Lonicera species.

Contrast-enhanced MRI of atherosclerosis can provide valuable additional information on a patient's disease state. As a result of the interactions of HDL with atherosclerotic plaque and the flexibility of its reconstitution, it is a versatile candidate for the delivery of contrast-generating materials to this pathogenic lesion. We herein discuss the reports of HDL modified with gadolinium to act as an MRI contrast agent for atherosclerosis. Furthermore, HDL has been modified with fluorophores and nanocrystals, allowing it to act as a contrast agent for fluorescent imaging techniques and for computed tomography. Such modified HDL has been found to be macrophage specific, and, therefore, can provide macrophage density information via noninvasive MRI. As such, modified HDL is currently a valuable contrast agent for probing preclinical atherosclerosis. Future developments may allow the application of this particle to further diseases and pathological or physiological processes in both preclinical models as well as in patients.

Porokeratotic eccrine ostial and dermal duct nevus (PEODDN) is a rare disorder of keratinization involving the intraepidermal eccrine duct (acrosyringium). We detail two examples of this unique clinicopathological entity--one with a more typical clinical presentation and one with a solitary lesion and late adult onset. In addition, we discuss the distinctive histologic and immunohistochemical findings and review the literature