William P. Dubinsky

Identification of serum biomarkers in brain-injured adults: potential for predicting elevated intracranial pressure.

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] <or= 8) and 11 age-, sex- and race-matched volunteers. Two time points-41.5 +/- 4.9 h and 66.3 +/- 6.6 h post-injury-were chosen for the initial screening analysis. Samples were immunodepleted for 12 highly abundant serum proteins, and then labeled with mass-balanced isobaric tags (iTRAQ), and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Identification and quantification of 2455 iTRAQ-labeled peptides that mapped to 160 proteins revealed 31 candidate biomarkers whose serum abundance was altered after injury. Changes in three candidate biomarkers (serum amyloid A, [SAA], c-reactive protein [CRP], retinol binding protein 4 [RBP4]) were verified using independent TBI and healthy volunteer serum samples. Receiver operating characteristic (ROC) curve analysis of CRP and SAA indicated they were robust indicators of injury even at very acute time points. Analysis of serum RBP4 levels at 24-36 h post-injury indicates it may predict subsequent increases in intracranial pressure (ICP) with a sensitivity of 86% and specificity of 88% at 11.6 mug/mL [n = 7, ICP < 20 mm Hg; n = 8, ICP > 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.

Breast Cancer Related Proteins Are Present in Saliva and Are Modulated Secondary to Ductal Carcinoma In Situ of the Breast

Objective: The objective of this study was to determine if protein-by-products secondary to cancer related oncogenes appear in the saliva of breast cancer patients. Methods: Three pooled (n = 10 subjects/pool) stimulated whole saliva specimens from women were analyzed. One pooled specimen was from healthy women, another pooled specimen from women diagnosed with a benign breast tumor and the other one pooled specimen was from women diagnosed with ductal carcinoma in situ (DCIS). Differential expression of proteins was measured by isotopically tagging proteins in the tumor groups and comparing them to the healthy control group. Experimentally, saliva from each of the pooled samples was trypsinized and the peptide digests labeled with the appropriate iTRAQ reagent. Labeled peptides from each of the digests were combined and analyzed by reverse phase (C18) capillary chromatography on an Applied Biosystems QStar LC-MS/MS mass spectrometer equipped with an LC-Packings HPLC. Results: The results of the salivary analyses in this population of patients yielded approximately 130 proteins in the saliva specimens. Forty-nine proteins were differentially expressed between the healthy control pool and the benign and cancer patient groups. Conclusions: 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.

Nitric Oxide-dependent Negative Feedback of PARP-1 trans-Activation of the Inducible Nitric-oxide Synthase Gene

Nitric oxide (NO) participates in a variety of physiologic and pathophysiologic processes in diverse tissues, including the kidney. Although mechanisms for cytokine induction of inducible nitric-oxide synthase (iNOS) have been increasingly clarified, the controls for termination of NO production remain unclear. Because excessive NO production can be cytotoxic to host cells, feedback inhibition of iNOS transcription would represent a means of cytoprotection. Many of the cGMP-independent functions of NO are mediated by S-nitrosylation of cysteine thiols of target proteins. We hypothesized that NO-mediated S-nitrosylation of transcription factors might serve to feedback inhibit their trans-activation potential and deactivate iNOS gene transcription. Transient transfection of murine mesangial cells with iNOS promoter deletion-luciferase constructs revealed the region –915 to –849 to be NO sensitive with respect to IL-1β-induced promoter activity. In vitro DNase I footprinting identified a footprint at –865/–842 in the absence of NO, but not in the presence of endogenous or exogenously delivered NO. Southwestern blotting using this probe coupled with partial peptide sequencing of the protein bands revealed that poly(ADP-ribose) polymerase isoform 1 (PARP-1) bound the probe in a sequence-specific manner. Gel shift/supershift experiments and chromatin immunoprecipitation assay analysis confirmed this binding in vitro and in vivo. Functionally, mutation of the –859/–850 site to prevent PARP-1 binding or PARP-1 knockdown by RNA interference relieved the inhibitory effects of NO on iNOS promoter activity. Biotin-switch assays and co-immunoprecipitation with an anti-nitrocysteine antibody indicated that PARP-1 was S-nitrosylated. We conclude that NO feedback inhibits iNOS gene transcription by S-nitrosylating the trans-activator PARP-1 and decreasing its binding and/or action at the iNOS promoter.

Salivary biomarkers for the detection of malignant tumors that are remote from the oral cavity.

Proteomic analyses by mass spectrometry are propelling the field of medical diagnostics forward at unprecedented rates because of its ability reliably to identify proteins that are at the femtomole level in concentration. These advancements have also benefited biomarker research to the point where saliva is now recognized as an excellent diagnostic medium for the detection of malignant tumors that are remote from the oral cavity. Saliva is easy to collect and may provide diagnostic information about a variety of cancers. In particular, proof-of-principle has been demonstrated for salivary biomarker research. This article reviews the literature, discusses the theories associated with saliva-based tumor diagnostics, and presents the current research focused on the use of saliva as a diagnostic medium for the detection of cancer.

Reconstitution of cAMP-dependent protein kinase regulated renal Na+-H+ exchanger.

SummaryStudies were performed to determine if the Na+−H+ exchanger, solubilized from renal brush border membranes from the rabbit and assayed in reconstituted artificial proteoliposomes, could be regulated by cAMP-dependent protein kinase. Octyl glucoside solubilized renal apical membrane proteins from the rabbit kidney were phosphorylated by incubation with ATP and highly purified catalytic subunit of cAMP-dependent kinase.22Na+ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. cAMP-dependent protein kinase resulted in sustained protein phosphorylation and a concentration-dependent decrease in the amiloride-sensitive component of pH gradient-stimulated sodium uptake. The inhibitory effect of cAMP-dependent protein kinase demonstrated an absolute requirement for ATP and was blocked by the specific protein inhibitor of this kinase. cAMP-dependent protein kinase also inhibited22Na+ uptake in the absence of a pH gradient (pHin 6.0. pHout 6.0) and the inhibitory effect was blocked by the specific inhibitor of the kinase. Solubilized membrane proteins exhibited little endogenous protein kinase or protein phosphatase activity.These studies indicate that Na+−H+ exchange activity of proteoliposomes reconstituted with proteins from renal brush border membranes is inhibited by phosphorylation of selected proteins by cAMP-dependent protein kinase. These findings also indicate that the regulatory components of the Na+−H+ exchanger remain active during the process of solubilization and reconstitution of renal apical membrane proteins.

Isolation of transporting plasma membrane vesicles from bovine tracheal epithelium

A method is described for isolating plasma membrane vesicles from bovine tracheal epithelium. The procedure yields highly purified apical membranes which are enriched 19-fold in the marker enzyme, alkaline phosphatase. Contamination of this fraction by other organelles is minimal. Basolateral membranes isolated from the same preparation have a 4-fold enrichment of (Na+ + K+)-ATPase and a 2-fold reduction in alkaline phosphatase specific activity compared to the starting material. Assays of Na+ uptake by the apical membrane vesicles demonstrate their suitability for transport studies. Transport of Na+ into an intravesicular space was demonstrated by (1) a linear inverse correlation between Na+ uptake and medium osmolarity; (2) complete release of accumulated Na+ by treatment with detergent; and (3) a marked temperature-dependence of Na+ uptake rate. Other features of Na+ transport were (1) inhibition by amiloride; (2) insensitivity to furosemide; and (3) anion-dependence of uptake rate with the following selectivity:SCN- greater than Cl- greater than gluconate-.

Proteomic analysis of saliva for cancer diagnosis

Saliva is fluid that is essential to the function and protection of the oral cavity and contiguous gastrointestinal epithelium. Common functions include cleansing and lubricating of soft and hard tissues, solubilization and bolus formation of food, facilitation of taste perception, mastication and speech. Bicarbonate in saliva protects against acidic challenges from bacterial cariogenic pathogens, while glycoproteins have protective effects against harmful microorganisms. Amylases in saliva aid in digestive function through the breakdown of starch [1]. The aforementioned is the classical definition of saliva that is taught in nearly all medical and dental schools; however, with the recent developments in proteomic technology, the definition may be expanded to include ‘diagnostic fluid’. The field of salivary proteomics is a burgeoning area of research that has expanded our knowledge about the protein composition of saliva. Originally, the majority of the salivary literature was focused upon the relationship between saliva and the structures of the oral cavity, such as the dentition, periodontium, tongue and the oral mucosa. However, with the advent of 2D gel electrophoresis and mass spectrometry technology, the protein catalog of saliva has expanded several hundred-fold [2]. This expansion has revealed proteins that reflect a myriad of functions that are altered in presence of disease [3]. There are serious limitations associated with many current diagnostic methodologies in the field of cancer diagnosis and follow-up treatment. The need to overcome invasive methods of specimen collection and laborintensive analysis is critical. The present diagnostic methodologies frequently yield poorly sensitive or nonspecific results. As a consequence of this public-health need and the advancement of proteomic research, saliva is now being investigated as a potential diagnostic media.

A single proteolytic cleavage within the lower hinge of trastuzumab reduces immune effector function and in vivo efficacy

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.

Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function

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.

A Comparison of the Proteomic Expression in Pooled Saliva Specimens from Individuals Diagnosed with Ductal Carcinoma of the Breast with and without Lymph Node Involvement

Purpose. The objective was to compare the salivary protein profiles of saliva specimens from individuals diagnosed with invasive ductal carcinoma of the breast (IDC) with and without lymph node involvement. Methods. Three pooled saliva specimens from women were analyzed. One pooled specimen was from healthy women; another was from women diagnosed with Stage IIa IDC and a specimen from women diagnosed with Stage IIb. The pooled samples were trypsinized and the peptide digests labeled with the appropriate iTRAQ reagent. Labeled peptides from each of the digests were combined and analyzed by reverse phase capillary chromatography on an LC-MS/MS mass spectrometer. Results. The results yielded approximately 174 differentially expressed proteins in the saliva specimens. There were 55 proteins that were common to both cancer stages in comparison to each other and healthy controls while there were 20 proteins unique to Stage IIa and 28 proteins that were unique to Stage IIb.