Extracellular esterase activity as an indicator of endoplasmic reticulum calcium depletion

Abstract

Context: Endoplasmic reticulum (ER) calcium depletion is associated with diverse diseases, including cardiac, hepatic, and neurologic diseases.

Objective: The aim of the present study was to identify and characterize an endogenous protein that could be used to monitor ER calcium depletion comparably to a previously described exogenous reporter protein.

Materials and methods: The use of a selective esterase-fluorescein diester pair allowed for carboxylesterase activity in extracellular fluid to be measured using a fluorescent readout. Cell culture media from three different cell lines, rat plasma, and human serum all possess quantifiable amounts of esterase activity.

Results: Fluorescence produced by the interaction of carboxylesterases with a fluorescein diester substrate tracks with pharmacological and physiological inducers of ER calcium depletion. The fluorescence measured for in vitro and in vivo samples were consistent with ER calcium depletion being the trigger for increased esterase activity.

Discussion: Decreased luminal ER calcium causes ER resident esterases to be released from the cell, and, when assessed concurrently with other disease biomarkers, these esterases may provide insight into the role of ER calcium homeostasis in human diseases.

Conclusion: Our results indicate that carboxylesterases are putative markers of ER calcium dysfunction.     

Antiproliferative Activity of Kinetin Riboside on HCT-15 Colon Cancer Cell Line

Cytokinins and cytokinin nucleosides are purine derivatives with potential anticancer activity both in vitro and in vivo. N⁶-furfuryladenosine (kinetin riboside, KR) displays antiproliferative and apoptogenic activity against various human cancer cell lines and has recently been shown to suppress tumor growth in murine xenograft models of human leukemia and melanoma. In this study, we demonstrate that KR is able to inhibit the proliferation in HCT-15 human colon cancer cells in a dose-dependent manner with a concentration of 2.5 μM, which causes 50% inhibition of cell viability. The cell cycle analysis by flow cytometry showed that KR arrested cell cycle progression in the S Phase by blocking through G₂/M and G₀/G₁ phase in HCT-15 colon cells. Moreover, suppression of clonogenic activity occurs after exposure to KR at a concentration of 2.5 μM for HCT-15.     

Synthesis and Antimalarial Activity of Some New 1,2-Dioxolane Derivatives

The synthesis of 1,2-dioxolane derivatives in two different acetophenone series, as simplified models of natural coumarins is described. 2-Acetyl-3-acetoxy-4-(3-hydroperoxy-3-methylbut-1-enyl)phenyl acetate and 2-acetyl-5-acetoxy-4-(3-hydroperoxy-3-methylbut-1-enyl) phenyl acetate synthons are used as precursors to these structures. In vitro antimalarial activity of the 1,2-dioxolane derivatives has been investigated.     

Evidence that TNF-β (lymphotoxin α) can activate the inflammatory environment in human chondrocytes

Introduction

Inflammatory cytokines play a key role in the pathogenesis of joint diseases such as rheumatoid arthritis (RA). Current therapies target mainly tumor necrosis factor α (TNF-α) as this has proven benefits. However, a large number of patients do not respond to or become resistant to anti-TNF-α therapy. While the role of TNF-α in RA is quite evident, the role of TNF-β, also called lymphotoxin-α (LT-α), is unclear. In this study we investigated whether TNF-β and its receptor play a role in chondrocytes in the inflammatory environment.

Methods

An in vitro model of primary human chondrocytes was used to study TNF-β-mediated inflammatory signaling.

Results

Cytokine-induced inflammation enhances TNF-β and TNF-β-receptor expression in primary human chondrocytes accompanied by the up-regulation of inflammatory (cyclooxygenase-2), matrix degrading (matrix metalloproteinase-9 and -13) and apoptotic (p53, cleaved caspase-3) signaling pathways, all known to be regulated by NF-κB. In contrast, anti-TNF-β, similar to the natural NF-κB inhibitor (curcumin, diferuloylmethane) or the knockdown of NF-κB by using antisense oligonucleotides (ASO), suppressed IL-1β-induced NF-κB activation and its translocation to the nucleus, and abolished the pro-inflammatory and apoptotic effects of IL-1β. This highlights, at least in part, the crucial role of NF-κB in TNF-β-induced-inflammation in cartilage, similar to that expected for TNF-α. Finally, the adhesiveness between TNF-β-expressing T-lymphocytes and the responding chondrocytes was significantly enhanced through a TNF-β-induced inflammatory microenvironment.

Conclusions

These results suggest for the first time that TNF-β is involved in microenvironment inflammation in chondrocytes during RA parallel to TNF-α, resulting in the up-regulation of NF-κB signaling and activation of pro-inflammatory activity.     

A Critical Function of Mad2l2 in Primordial Germ Cell Development of Mice

The development of primordial germ cells (PGCs) involves several waves of epigenetic reprogramming. A major step is following specification and involves the transition from the stably suppressive histone modification H3K9me2 to the more flexible, still repressive H3K27me3, while PGCs are arrested in G2 phase of their cycle. The significance and underlying molecular mechanism of this transition were so far unknown. Here, we generated mutant mice for the Mad2l2 (Mad2B, Rev7) gene product, and found that they are infertile in both males and females. We demonstrated that Mad2l2 is essential for PGC, but not somatic development. PGCs were specified normally in Mad2l2^−/− embryos, but became eliminated by apoptosis during the subsequent phase of epigenetic reprogramming. A majority of knockout PGCs failed to arrest in the G2 phase, and did not switch from a H3K9me2 to a H3K27me3 configuration. By the analysis of transfected fibroblasts we found that the interaction of Mad2l2 with the histone methyltransferases G9a and GLP lead to a downregulation of H3K9me2. The inhibitory binding of Mad2l2 to Cyclin dependent kinase 1 (Cdk1) could arrest the cell cycle in the G2 phase, and also allowed another histone methyltransferase, Ezh2, to upregulate H3K27me3. Together, these results demonstrate the potential of Mad2l2 in the regulation of both cell cycle and the epigenetic status. The function of Mad2l2 is essential in PGCs, and thus of high relevance for fertility.     

Theoretical study on cooperative effects between X⋯N and X⋯Carbene halogen bonds (X = F,Cl,Br and I)

Quantum chemical calculations are performed to study the interplay between halogen?nitrogen and halogen?carbene interactions in NCX?NCX?CH₂ complexes, where X?=?F, Cl, Br and I. Molecular geometries and interaction energies of dyads and triads are investigated at the MP2/aug-cc-pVTZ level of theory. It is found that the X?N and X?C_carbene interaction energies in the triads are larger than those in the dyads, indicating that both the halogen bonding interactions are enhanced. The estimated values of cooperative energy E _coop are all negative with much larger E _coop in absolute value for the systems including iodine. The nature of halogen bond interactions of the complexes is analyzed using parameters derived from the quantum theory atoms in molecules methodology and energy decomposition analysis.

Insights into the strength and nature of carbene···halogen bond interactions: a theoretical perspective

Halogen-bonding, a noncovalent interaction between a halogen atom X in one molecule and a negative site in another, plays critical roles in fields as diverse as molecular biology, drug design and material engineering. In this work, we have examined the strength and origin of halogen bonds between carbene CH₂ and XCCY molecules, where X?=?Cl, Br, I, and Y?=?H, F, COF, COOH, CF₃, NO₂, CN, NH₂, CH₃, OH. These calculations have been carried out using M06-2X, MP2 and CCSD(T) methods, through analyses of surface electrostatic potentials V _S(r) and intermolecular interaction energies. Not surprisingly, the strength of the halogen bonds in the CH₂···XCCY complexes depend on the polarizability of the halogen X and the electron-withdrawing power of the Y group. It is revealed that for a given carbene···X interaction, the electrostatic term is slightly larger (i.e., more negative) than the dispersion term. Comparing the data for the chlorine, bromine and iodine substituted CH₂···XCCY systems, it can be seen that both the polarization and dispersion components of the interaction energy increase with increasing halogen size. One can see that increasing the size and positive nature of a halogen’s σ-hole markedly enhances the electrostatic contribution of the halogen-bonding interaction.

A Proline/Arginine-Rich End Leucine-Rich Repeat Protein (PRELP) Variant Is Uniquely Expressed in Chronic Lymphocytic Leukemia Cells

Proline/arginine-rich end leucine-rich repeat protein (PRELP) belongs to the small leucine-rich proteoglycan (SLRP) family, normally expressed in extracellular matrix of collagen-rich tissues. We have previously reported on another SLRP, fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL). PRELP is structurally similar to FMOD with adjacent localization on chromosome 1 (1q32.1). As cluster-upregulation of genes may occur in malignancies, the aim of our study was to analyze PRELP expression in CLL. PRELP was expressed (RT-PCR) in all CLL patients (30/30), as well as in some patients with mantle cell lymphoma (3/5), but not in healthy donor leukocytes (0/20) or tumor samples from other hematological malignancies (0/35). PRELP was also detected in CLL cell-lines (4/4) but not in cell-lines from other hematological tumors (0/9). PRELP protein was detected in all CLL samples but not in normal leukocytes. Deglycosylation experiments revealed a CLL-unique 38 kDa core protein, with an intact signal peptide. This 38 kDa protein was, in contrast to the normal 55 kDa size, not detected in serum which, in combination with the uncleaved signal peptide, suggests cellular retention. The unique expression of a 38 kDa PRELP in CLL cells may suggest involvement in the pathobiology of CLL and merits further studies.     

Added Value of HER-2 Amplification Testing by Multiplex Ligation-Dependent Probe Amplification in Invasive Breast Cancer

Background

HER-2 is a prognostic and predictive marker, but as yet no technique is perfectly able to identify patients likely to benefit from HER-2 targeted therapies. We aimed to prospectively assess the added value of first-line co-testing by IHC, and multiplex ligation-dependent probe amplification (MLPA) and chromogenic in situ hybridization (CISH).

Methods

As local validation, HER-2 MLPA and CISH were compared in 99 breast cancers. Next, we reviewed 937 invasive breast cancers, from 4 Dutch pathology laboratories, that were prospectively assessed for HER-2 by IHC and MLPA (and CISH in selected cases).

Results

The validation study demonstrated 100% concordance between CISH and MLPA, if both methods were assessable and conclusive (81.8% of cases). Significant variation regarding percentages IHC 0/1+ and 2+ cases was observed between the laboratories (p<0.0001). Overall concordance between IHC and MLPA/CISH was 98.1% (575/586) (Kappa = 0.94). Of the IHC 3+ cases, 6.7% failed to reveal gene amplification, whereas 0.8% of the IHC 0/1+ cases demonstrated gene amplification. Results remained discordant after retrospective review in 3/11 discordant cases. In the remaining 8 cases the original IHC score was incorrect or adapted after repeated IHC staining.

Conclusions

MLPA is a low-cost and quantitative high-throughput technique with near perfect concordance with CISH. The use of MLPA in routinely co-testing all breast cancers may reduce HER-2 testing variation between laboratories, may serve as quality control for IHC, will reveal IHC 0/1+ patients with gene amplification, likely responsive to trastuzumab, and identify IHC 3+ cases without gene amplification that may respond less well.     

Pentavalent Single-Domain Antibodies Reduce Campylobacter jejuni Motility and Colonization in Chickens

Campylobacter jejuni is the leading cause of bacterial foodborne illness in the world, with symptoms ranging from acute diarrhea to severe neurological disorders. Contaminated poultry meat is a major source of C. jejuni infection, and therefore, strategies to reduce this organism in poultry, are expected to reduce the incidence of Campylobacter-associated diseases. We have investigated whether oral administration of C. jejuni-specific single-domain antibodies would reduce bacterial colonization levels in chickens. Llama single-domain antibodies specific for C. jejuni were isolated from a phage display library generated from the heavy chain IgG variable domain repertoire of a llama immunized with C. jejuni flagella. Two flagella-specific single-domain antibodies were pentamerized to yield high avidity antibodies capable of multivalent binding to the target antigen. When administered orally to C. jejuni-infected two-day old chicks, the pentabodies significantly reduced C. jejuni colonization in the ceca. In vitro, the motility of the bacteria was also reduced in the presence of the flagella-specific pentabodies, suggesting the mechanism of action is through either direct interference with flagellar motility or antibody-mediated aggregation. Fluorescent microscopy and Western blot analyses revealed specific binding of the anti-flagella pentabodies to the C. jejuni flagellin.