Dysfunctional cellular lipid metabolism contributes to common chronic human diseases, including type 2 diabetes, obesity, fatty liver disease and diabetic cardiomyopathy. How cells balance lipid storage and mitochondrial oxidative capacity is poorly understood. Here we identify the lipid droplet protein Perilipin 5 as a catecholamine-triggered interaction partner of PGC-1α. We report that during catecholamine-stimulated lipolysis, Perilipin 5 is phosphorylated by protein kinase A and forms transcriptional complexes with PGC-1α and SIRT1 in the nucleus. Perilipin 5 promotes PGC-1α co-activator function by disinhibiting SIRT1 deacetylase activity. We show by gain-and-loss of function studies in cells that nuclear Perilipin 5 promotes transcription of genes that mediate mitochondrial biogenesis and oxidative function. We propose that Perilipin 5 is an important molecular link that couples the coordinated catecholamine activation of the PKA pathway and of lipid droplet lipolysis with transcriptional regulation to promote efficient fatty acid catabolism and prevent mitochondrial dysfunction.

IntroductionRecent studies reported that human IgG antibodies are susceptible to specific proteolytic cleavage in their lower hinge region, and the hinge cleavage results in a loss of Fc-mediated effector functions. Trastuzumab is a humanized IgG1 therapeutic monoclonal antibody for the treatment of HER2-overexpressing breast cancers, and its mechanisms of action consist of inhibition of HER2 signaling and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC). The objective of this study is to investigate the potential effect of proteinase hinge cleavage on the efficacy of trastuzumab using both a breast cancer cell culture method and an in vivo mouse xenograft tumor model.MethodsTrastuzumab antibody was incubated with a panel of human matrix metalloproteinases, and proteolytic cleavage in the lower hinge region was detected using both western blotting and mass spectrometry. Single hinge cleaved trastuzumab (scIgG-T) was purified and evaluated for its ability to mediate ADCC and inhibition of breast cancer cell proliferation in vitro as well as anti-tumor efficacy in the mouse xenograft tumor model. Infiltrated immune cells were detected in tumor tissues by immunohistochemistry.ResultsscIgG-T retains HER2 antigen binding activity and inhibits HER2-mediated downstream signaling and cell proliferation in vitro when compared with the intact trastuzumab. However, scIgG-T lost Fc-mediated ADCC activity in vitro, and had significantly reduced anti-tumor efficacy in a mouse xenograft tumor model. Immunohistochemistry showed reduced immune cell infiltration in tumor tissues treated with scIgG-T when compared with those treated with the intact trastuzumab, which is consistent with the decreased ADCC mediated by scIgG-T in vitro.ConclusionTrastuzumab can be cleaved by matrix metalloproteinases within the lower hinge. scIgG-T exhibited a significantly reduced anti-tumor efficacy in vivo due to the weakened immune effector function such as ADCC. The results suggest that the lower hinge cleavage of trastuzumab can occur in the tumor microenvironment where matrix metalloproteinases often have high levels of expression and scIgG-T might compromise its anti-tumor efficacy in the clinic. However, further studies are needed to validate these hypotheses in the clinical setting.

The study suggests that saliva is a fluid suffused with solubilized by-products of oncogenic expression and that these proteins may be modulated secondary to DCIS. Additionally, there may be salivary protein profiles that are unique to both DCIS and fibroadenoma tumors.

Brain injury biomarkers may have clinical utility in stratifying injury severity level, predicting adverse secondary events or outcomes, and monitoring the effectiveness of therapeutic interventions. As a biomarker source, serum offers several advantages over cerebrospinal fluid (CSF), including ease of accessibility and reduced risk to the patient. We screened pooled serum samples obtained from 11 severely injured traumatic brain injury (TBI) patients (Glasgow Coma Scale [GCS] 25 mm Hg). Our results support the use of serum as a source for discovery of TBI biomarkers, and indicate that serum biomarkers may have utility for predicting secondary pathologies (e.g., elevated ICP) associated with TBI.

Lactate accumulates in human erythrocytes stored at 4 degrees C in the presence of glucose. Efflux of lactate exhibits an activation energy of 22kcal/mole and is markedly stimulated with increasing medium pH. Lactate influx into erythrocytes that were depleted of intracellular lactate by incubation at 37 degrees at pH 8.0 was stimulated by decreasing medium pH. Under appropriate conditions the pH-dependent lactate flux was insensitive to 4-acet-amido-4'-isothiocyano-2,2'-disulfonic stilbene or 4,4'-diisothiocyano-2,2'-disulfonic stilbene, inhibitors of the inorganic anion channel, while, e.g., inorganic phosphate transport was fully sensitive. These experiments as well as measurements of H+ movements associated with lactate fluxes demonstrate that lactate transport takes place via a specific monocarboxylate transporter (distinct from the inorganic ion channel) by a H+-lactate symport mechanism.

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PLAC1 is a trophoblast-specific gene that maps to a locus on the X-chromosome important to placental development. We have previously shown that PLAC1 gene expression is linked to trophoblast differentiation. The objective of this study was to define the localization of the PLAC1 polypeptide as a prerequisite to understanding its function. Polyclonal antibodies specific for the putative PLAC1 polypeptide were generated. The subcellular localization of PLAC1 in the trophoblast was examined by immunohistochemical analysis of human placenta complemented by immunoblot analysis of subcellular fractions. Brightfield immunohistochemical analysis of placental tissue indicated that the PLAC1 protein localizes to the differentiated syncytiotrophoblast in the apical region of the cell. Deconvlution immunofluorescence microscopy confirmed localization to the apical region of the syncytiotrophoblast. Its distribution included both intracellular compartments as well as loci in close association with the maternal-facing, microvillous brush border membrane (MVM). These findings were supported by immunoblot analysis of subcellular fractions. A 30 kDa band was associated with the microsomal fraction of placental lysates but not the mitochondrial, nuclear, or soluble fractions, suggesting PLAC1 is targeted to a membrane location. Plasma membranes were obtained from the fetal-facing, basal surface (BM) and the maternal-facing, MVM of the syncytiotrophoblast membrane. PLAC1 immunoreactivity was only detected in membrane fractions derived from the apical MVM consistent with immunohistochemical analyses. These data demonstrate that the PLAC1 protein is restricted primarily to the differentiated trophoblast, localizing to intracellular membranous compartment(s) in the apical region of the syncytiotrophoblast and associated with its apical, microvillous membrane surface.

Hicks K, O'Neil RG, Dubinsky WS, Brown RC. TRPC-mediated actin-myosin contraction is critical for BBB disruption following hypoxic stress. Am J Physiol Cell Physiol 298: C1583-C1593, 2010. First published February 17, 2010 doi:10.1152/ajpcell.00458.2009.-Hypoxia-induced disruption of the blood-brain barrier (BBB) is the result of many different mechanisms, including alterations to the cytoskeleton. In this study, we identified actin-binding proteins involved in cytoskeletal dynamics with quantitative proteomics and assessed changes in subcellular localization of two proteins involved in actin polymerization [vasodilator-stimulated phosphoprotein (VASP)] and cytoskeleton-plasma membrane cross-linking (moesin). We found significant redistribution of both VASP and moesin to the cytoskeletal and membrane fractions of BBB endothelial cells after 1-h hypoxic stress. We also investigated activation of actin-myosin contraction through assessment of phosphorylated myosin light chain (pMLC) with confocal microscopy. Hypoxia caused a rapid and transient increase in pMLC. Blocking MLC phosphorylation through inhibition of myosin light chain kinase (MLCK) with ML-7 prevented hypoxiainduced BBB disruption and relocalization of the tight junction protein ZO-1. Finally, we implicate the transient receptor potential (TRP)C family of channels in mediating these events since blockade of TRPC channels and the associated calcium influx with SKF-96365 prevents hypoxia-induced permeability changes and the phosphorylation of MLC needed for actin-myosin contraction. These data suggest that hypoxic stress triggers alterations to cytoskeletal structure that contribute to BBB disruption and that calcium influx through TRPC channels contributes to these events. cation channels; endothelial cell; stroke; blood-brain barrier; calcium; transient receptor potential C channels BREAKDOWN of the blood-brain barrier (BBB) contributes to edema formation, infarct size, and brain damage following ischemic stroke (3,10,36,39). There are many factors contributing to BBB disruption in ischemia, including generation of oxygen radicals (24, 56, 58), nitric oxide (22, 42), production of vascular endothelial growth factor (67, 69), and changes in intracellular calcium (5,26,35). Under normal conditions, the integrity of the BBB is maintained by tight junction complexes between adjacent brain capillary endothelial cells (2). Changes in BBB permeability are correlated with changes in tight junction structure (33,45,51,59). After hypoxia, this increased permeability is associated with disruptions in the subcellular localization of the tight junction proteins zonula occludens-1 (ZO-1) (43) and occludin (5).A major contributing factor to disruption of the BBB after hypoxia is contraction of the actin-myosin cytoskeleton (23). A recent study demonstrated that inhibition of actin-myosin contraction protected the BBB after hypoxic stress (34); hypoxiainduced BBB disruption was prevented by inhibition of myosin light chain kinase (MLCK), by inhibition of NADPH-oxida...