Infrared spectroscopic analysis of tumor pathology

Infrared spectra of normal and malignant breast tissues were measured in the 600 cm(-1) to 4000 cm(-1) region. The measured spectroscopic features which are the spectroscopic fingerprints of the tissues contain the vital information about the malignant and normal tissues. Fourier Transform Infrared (FTIR) data on 25 cases of infiterating ductal carcinoma of breast with different grades of malignancy from patients of different age groups were analyzed. The samples were taken from the tumor sections of the tissue removed during surgery. Infrared spectra demonstrate significant spectral differences between the normal and the cancerous breast tissues. In particular changes in frequency and intensity in the spectra of protein, nucleic acid and glycogen vibrational modes as well as the band intensity ratios for lipid/proteins, protein/nucleic acids, protein/glycogen were observed. This allows to make a qualitative and semi quantitative evaluation of the changes in proliferation activity from normal to diseased tissue. It was evident that the sample to sample or patient to patient variations were small and the spectral differences between normal and diseased tissues were reproducible. The findings establish a framework for additional studies, which may enable us to establish a relation of the diseased state with its infrared spectra.     

A reporter gene assay for screening of PDE4 subtype selective inhibitors

Phosphodiesterase (PDE) constitutes a superfamily of enzymes that catalyze the hydrolysis of cAMP and cGMP into their corresponding monophosphates and play an important role in diverse physiological functions. The present study provides a process for identifying PDE4 subtypes selective inhibitors using a reporter gene assay. Stable recombinant HEK-293 cell lines expressing high levels of PDE4A4B, PDE4B2A, and PDE4D3 subtypes individually were generated. Transient transfection of pCRE-Luc plasmid, harboring luciferase reporter gene under the control of cAMP response element (CRE)-binding sequence, into these stable recombinant cell lines followed by treatment with PDE4 inhibitor, resulted in a dose dependent increase in luciferase activity. This methods provide a novel, simple and sensitive assay for high throughput screening of PDE4 subtype selective inhibitors for treatment of asthma and COPD.     

The tumor suppressor BAP1 cooperates with BRAFV600E to promote tumor formation in cutaneous melanoma

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk of mesothelioma and melanocytic tumors. Here, we show that Bap1 deletion in melanocytes cooperates with the constitutively active, oncogenic form of BRAF (BRAF^V600E) and UV to cause melanoma in mice, albeit at very low frequency. In addition, Bap1‐null melanoma cells derived from mouse tumors are more aggressive and colonize and grow at distant sites more than their wild‐type counterparts. Molecularly, Bap1‐null melanoma cell lines have increased DNA damage measured by γH2aX and hyperubiquitination of histone H2a. Therapeutically, these Bap1‐null tumors are completely responsive to BRAF‐ and MEK‐targeted therapies. Therefore, BAP1 functions as a tumor suppressor and limits tumor progression in melanoma.     

Structural Evidence for a Sequential Release Mechanism for Activation of Heterotrimeric G Proteins

Heptahelical G-protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors couple to heterotrimeric G proteins to relay extracellular signals to intracellular signaling networks, but the molecular mechanism underlying guanosine 5′-diphosphate (GDP) release by the G protein α-subunit is not well understood. Amino acid substitutions in the conserved α5 helix of G_i, which extends from the C-terminal region to the nucleotide-binding pocket, cause dramatic increases in basal (receptor-independent) GDP release rates. For example, mutant Gα_i1-T329A shows an 18-fold increase in basal GDP release rate and, when expressed in culture, it causes a significant decrease in forskolin-stimulated cAMP accumulation. The crystal structure of Gα_i1-T329A·GDP shows substantial conformational rearrangement of the switch I region and additional striking alterations of side chains lining the catalytic pocket that disrupt the Mg⁺² coordination sphere and dislodge bound Mg⁺². We propose a “sequential release” mechanism whereby a transient conformational change in the α5 helix alters switch I to induce GDP release. Interestingly, this mechanistic model for heterotrimeric G protein activation is similar to that suggested for the activation of the plant small G protein Rop4 by RopGEF8.     

Online HPLC-DPPH method for antioxidant activity of Picrorhiza kurroa Royle ex Benth. and characterization of kutkoside by ultra-performance LC-electrospray ionization quadrupole time-of-flight mass spectrometry

Picrorhiza kurroa Royle ex Benth., is widely used in the Indian systems of medicine for the treatment of various liver ailments. Since, the role of oxidative stress in the pathogenesis of liver injury has become generally recognized, in present study the free radical scavenging effect of P. kurroa was assessed by on-line HPLC-DPPH and colorimetric DPPH methods. The comparative study on antioxidant activity of P. kurroa extracts by both methods revealed that colorimetric method showed very less free radical scavenging effect while HPLC-DPPH method showed high activity. Further, the kutkoside, an important ingredient of a potent hepatoprotective formulation "kutkin/picroliv" was investigated for its chemical composition by ultra-performance liquid chromatography coupled with diode array detection/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-DAD/ESI-QTOF-MS). Kutkoside was considered to be a single compound and reported as picroside-II or kutkoside, however, present investigation illustrated that kutkoside is a mixture of iridoid glycosides namely, picroside II, picroside IV and 6-ferulloylcatalpol.     

Nucleobindin 1 Is a Calcium-regulated Guanine Nucleotide Dissociation Inhibitor of Gαi1

Nucleobindin 1 (NUCB1) is a widely expressed multidomain calcium-binding protein whose precise physiological and biochemical functions are not well understood. We engineered and heterologously expressed a soluble form of NUCB1 (sNUCB1) and characterized its biophysical and biochemical properties. We show that sNUCB1 exists as a dimer in solution and that each monomer binds two divalent calcium cations. Calcium binding causes conformational changes in sNUCB1 as judged by circular dichroism and fluorescence spectroscopy experiments. Earlier reports suggested that NUCB1 might interact with heterotrimeric G protein α subunits. We show that dimeric calcium-free sNUCB1 binds to expressed Gα_i1 and that calcium binding inhibits the interaction. The binding of sNUCB1 to Gα_i1 inhibits its basal rate of GDP release and slows its rate and extent of GTPγS uptake. Additionally, our tissue culture experiments show that sNUCB1 prevents receptor-mediated Gα_i-dependent inhibition of adenylyl cyclase. Thus, we conclude that sNUCB1 is a calcium-dependent guanine nucleotide dissociation inhibitor (GDI) for Gα_i1. To our knowledge, sNUCB1 is the first example of a calcium-dependent GDI for heterotrimeric G proteins. We also show that the mechanism of GDI activity of sNUCB1 is unique and does not arise from the consensus GoLoco motif found in RGS proteins. We propose that cytoplasmic NUCB1 might function to regulate heterotrimeric G protein trafficking and G protein-coupled receptor-mediated signal transduction pathways.     

Early-age-related changes in proteostasis augment immunopathogenesis of sepsis and acute lung injury

Background

The decline of proteasomal activity is known to be associated with the age-related disorders but the early events involved in this process are not apparent. To address this, we investigated the early-age-related (pediatric vs. adult) mechanisms that augment immunopathogenesis of sepsis and acute lung injury.

Methodology/Principal Findings

The 3-weeks (pediatric) and 6-months (adult) old C57BL/6 mice were selected as the study groups. Mice were subjected to 1×20 cecal ligation and puncture (CLP) mediated sepsis or intratracheal Psuedomonas aeruginosa (Pa)-LPS induced acute lung injury (ALI).We observed a significant increase in basal levels of pro-inflammatory cytokine, IL-6 and neutrophil activity marker, myeloperoxidase (MPO) in the adult mice compared to the pediatric indicating the age-related constitutive increase in inflammatory response. Next, we found that age-related decrease in PSMB6 (proteasomal subunit) expression in adult mice results in accumulation of ubiquitinated proteins that triggers the unfolded protein response (UPR). We identified that Pa-LPS induced activation of UPR modifier, p97/VCP (valosin-containing protein) in the adult mice lungs correlates with increase in Pa-LPS induced NFκB levels. Moreover, we observed a constitutive increase in p-eIF2α indicating a protective ER stress response to accumulation of ubiquitinated-proteins. We used MG-132 treatment of HBE cells as an in vitro model to standardize the efficacy of salubrinal (inhibitor of eIF2α de-phosphorylation) in controlling the accumulation of ubiquitinated proteins and the NFκB levels. Finally, we evaluated the therapeutic efficacy of salubrinal to correct proteostasis-imbalance in the adult mice based on its ability to control CLP induced IL-6 secretion or recruitment of pro-inflammatory cells.

Conclusions/Significance

Our data demonstrate the critical role of early-age-related proteostasis-imbalance as a novel mechanism that augments the NFκB mediated inflammation in sepsis and ALI. Moreover, our data suggest the therapeutic efficacy of salubrinal in restraining NFκB mediated inflammation in the adult or older subjects.