Inhibitory Reagents Research Articles

Polymerase-Based Signal Delay for Temporally Regulating DNA Involved Reactions, Programming Dynamic Molecular Systems, and Biomimetic Sensing.

Complex temporal molecular signals play a pivotal role in the intricate biological pathways of living organisms, and cells exhibit the ability to transmit and receive information by intricately managing the temporal dynamics of their signaling molecules. Although biomimetic molecular networks are successfully engineered outside of cells, the capacity to precisely manipulate temporal behaviors remains limited. In this study, the catalysis activity of isothermal DNA polymerase (DNAP) through combined use of molecular dynamics simulation analysis and fluorescence assays is first characterized. DNAP-driven delay in signal strand release ranged from 100 to 102min, which is achieved through new strategies including the introduction of primer overhangs, utilization of inhibitory reagents, and alteration of DNA template lengths. The results provide a deeper insight into the underlying mechanisms of temporal control DNAP-mediated primer extension and DNA strand displacement reactions. Then, the regulated DNAP catalysis reactions are applied in temporal modulation of downstream DNA-involved reactions, the establishment of dynamic molecular signals, and the generation of barcodes for multiplexed detection of target genes. The utility of DNAP-based signal delay as a dynamic DNA nanotechnology extends beyond theoretical concepts and achieves practical applications in the fields of cell-free synthetic biology and bionic sensing.

Emerging Role of IGF-1 in Prostate Cancer: A Promising Biomarker and Therapeutic Target.

Prostate cancer (PCa) is a highly heterogeneous disease driven by gene alterations and microenvironmental influences. Not only enhanced serum IGF-1 but also the activation of IGF-1R and its downstream signaling components has been increasingly recognized to have a vital driving role in the development of PCa. A better understanding of IGF-1/IGF-1R activity and regulation has therefore emerged as an important subject of PCa research. IGF-1/IGF-1R signaling affects diverse biological processes in cancer cells, including promoting survival and renewal, inducing migration and spread, and promoting resistance to radiation and castration. Consequently, inhibitory reagents targeting IGF-1/IGF-1R have been developed to limit cancer development. Multiple agents targeting IGF-1/IGF-1R signaling have shown effects against tumor growth in tumor xenograft models, but further verification of their effectiveness in PCa patients in clinical trials is still needed. Combining androgen deprivation therapy or cytotoxic chemotherapeutics with IGF-1R antagonists based on reliable predictive biomarkers and developing and applying novel agents may provide more desirable outcomes. This review will summarize the contribution of IGF-1 signaling to the development of PCa and highlight the relevance of this signaling axis in potential strategies for cancer therapy.

Role of Signal Transduction Pathways and Transcription Factors in Cartilage and Joint Diseases.

Osteoarthritis and rheumatoid arthritis are common cartilage and joint diseases that globally affect more than 200 million and 20 million people, respectively. Several transcription factors have been implicated in the onset and progression of osteoarthritis, including Runx2, C/EBPβ, HIF2α, Sox4, and Sox11. Interleukin-1 β (IL-1β) leads to osteoarthritis through NF-ĸB, IκBζ, and the Zn2+-ZIP8-MTF1 axis. IL-1, IL-6, and tumor necrosis factor α (TNFα) play a major pathological role in rheumatoid arthritis through NF-ĸB and JAK/STAT pathways. Indeed, inhibitory reagents for IL-1, IL-6, and TNFα provide clinical benefits for rheumatoid arthritis patients. Several growth factors, such as bone morphogenetic protein (BMP), fibroblast growth factor (FGF), parathyroid hormone-related protein (PTHrP), and Indian hedgehog, play roles in regulating chondrocyte proliferation and differentiation. Disruption and excess of these signaling pathways cause genetic disorders in cartilage and skeletal tissues. Fibrodysplasia ossificans progressive, an autosomal genetic disorder characterized by ectopic ossification, is induced by mutant ACVR1. Mechanistic target of rapamycin kinase (mTOR) inhibitors can prevent ectopic ossification induced by ACVR1 mutations. C-type natriuretic peptide is currently the most promising therapy for achondroplasia and related autosomal genetic diseases that manifest severe dwarfism. In these ways, investigation of cartilage and chondrocyte diseases at molecular and cellular levels has enlightened the development of effective therapies. Thus, identification of signaling pathways and transcription factors implicated in these diseases is important.

Blocking Oligomeric Insulin Amyloid Fibrillation via Perylenebisimides Containing Dipeptide Tentacles.

Molecular motifs that could interfere with amyloid fibrillation via non-covalent interactions are vital for aberrant protein aggregation and related human diseases. Mutual aggregation ensues in the presence of these structural motifs, and nucleation on the particle surface leads to inhibition of the fibrillization process. This modular process generates a new generation of inhibitory reagents. Oligomers are the primary toxic species that initiate pathogenic aggregation, leading to toxic β-sheet-rich structures. To inhibit these toxic oligomers, two dipeptide-linked perylenebisimide isomers (PAPAP and APPPA) are developed as selective modulators for insulin fibrillization. Early insulin aggregates are adsorbed onto the modulator surface and stabilize soluble oligomeric aggregates. Fibrillation and inhibition are examined by thioflavin T (ThT) assay in the presence and the absence of both inhibitors PAPAP and APPPA. Conformational modulation using far-UV circular dichroism studies also highlights their role as aggregation inhibitors via reduction of α-helix into β-sheet along with increased random coil contents. Moreover, the inhibitory effects were more pronounced due to the ability of these isomers to undergo varying multiple non-covalent interactions, a performance well beyond all known modulators with respect to selectivity and efficiency, with the more-aggregation-prone derivative due to higher probability of a hydrophobic encounter between the protein and the molecular modulator. These results also lead to the elucidation of an insulin fibril regulating mechanism via selective non-covalent binding and provide fundamental insights into the chemistry of peptide-based probes as tools for developing next-generation therapeutics.

Characterization of an acidophilic α-amylase from Aspergillus niger RBP7 and study of catalytic potential in response to nutritionally important heterogeneous compound

An acidophilic α-amylase from Aspergillus niger RBP7 was purified after solid state fermentation on potato peel substrate. Molecular mass of the purified α-amylase was 37.5 kDa and it exhibited 1.4 mg/ml and 0.992 μ/mol/min Km and Vmax values, respectively. The enzyme was stable in the pH range from 2.0 to 6.0, at high NaCl concentration (3 M) and at temperatures between 40 °C and 70 °C. The enzyme showed an optimal activity at pH 3.0 and at 45 °C. The enzyme was inhibited by Hg2+ and was stable in the presence of different surfactants (Tween 60, Tween 80, and SDS at 1% level) and different inhibitory reagents (β-mercaptoethanol, phenylmethylsulfonyl fluoride, and sodium azide). This acidophilic amylase enzyme can digest heterogeneous food materials, i.e. the mixture of rice, fish, bread and curry with comparable activity to the commercial diastase enzymes available.

BDNF-TrkB Signaling Coupled to nPKCε and cPKCβI Modulate the Phosphorylation of the Exocytotic Protein Munc18-1 During Synaptic Activity at the Neuromuscular Junction.

Munc18-1, a neuron-specific member of the Sec1/Munc18 family, is involved in neurotransmitter release by binding tightly to syntaxin. Munc18-1 is phosphorylated by PKC on Ser-306 and Ser-313 in vitro which reduces the amount of Munc18-1 able to bind syntaxin. We have previously identified that PKC is involved in neurotransmitter release when continuous electrical stimulation imposes a moderate activity on the NMJ and that muscle contraction through TrkB has an important impact on presynaptic PKC isoforms levels, specifically cPKCβI and nPKCε. Therefore, the present study was designed to understand how Munc18-1 phosphorylation is affected by (1) synaptic activity at the neuromuscular junction, (2) nPKCε and cPKCβI isoforms activity, (3) muscle contraction per se, and (4) the BDNF/TrkB signaling in a neuromuscular activity-dependent manner. We performed immunohistochemistry and confocal techniques to evidence the presynaptic location of Munc18-1 in the rat diaphragm muscle. To study synaptic activity, we stimulated the phrenic nerve (1 Hz, 30 min) with or without contraction (abolished by μ-conotoxin GIIIB). Specific inhibitory reagents were used to block nPKCε and cPKCβI activity and to modulate the tropomyosin receptor kinase B (TrkB). Main results obtained from Western blot experiments showed that phosphorylation of Munc18-1 at Ser-313 increases in response to a signaling mechanism initiated by synaptic activity and directly mediated by nPKCε. Otherwise, cPKCβI and TrkB activities work together to prevent this synaptic activity–induced Munc18-1 phosphorylation by a negative regulation of cPKCβI over nPKCε. Therefore, a balance between the activities of these PKC isoforms could be a relevant cue in the regulation of the exocytotic apparatus. The results also demonstrate that muscle contraction prevents the synaptic activity–induced Munc18-1 phosphorylation through a mechanism that opposes the TrkB/cPKCβI/nPKCε signaling.

Efficacy of Vonoprazan-Based Triple Therapy for Helicobacter pylori Eradication: A Multicenter Study and a Review of the Literature.

Eradication therapies for Helicobacter pylori infection are advancing as new acid inhibitory reagents approved. The aim of this study was to assess the efficacy and safety of vonoprazan-based triple treatment. Triple therapy with vonoprazan and two antibiotics (amoxicillin and clarithromycin or metronidazole) received focus in this analysis. We performed a multicenter retrospective study of patients who received vonoprazan-based eradication therapy between February 2015 and February 2016 and conducted a review of the literature. The eradication rate among the 799 patients in our multicenter study was 94.4% (95% confidence interval [CI] 92.6-96.2%) in the per-protocol analysis for first-line treatment (with vonoprazan 20mg, amoxicillin 750mg, and clarithromycin 200 or 400mg, twice a day for 7days) and 97.1% (95% CI 93.0-101.1%) for second-line treatment (with vonoprazan 20mg, amoxicillin 750mg, and metronidazole 250mg, twice a day for 7days). The overall incidence of adverse events was 4.4% in an intention-to-treat analysis with no patients hospitalized. In a literature review, six reports, in which 1380 patients received vonoprazan-based first-line eradication therapy, were included and were all reported by Japanese researchers. The eradication success rates in per-protocol analysis were between 85 and 93%, which was roughly the same among the studies. Vonoprazan-based triple therapy was effective and safe for Helicobacter pylori eradication in real-world experience, confirmed by a multicenter study and a review of the pertinent literature.

Epigallocatechin gallate attenuates amyloid β-induced inflammation and neurotoxicity in EOC 13.31 microglia

Microglia are the primary immune cells that contribute to neuroinflammation by releasing various proinflammatory cytokines and neurotoxins in the brain. Microglia-mediated neuroinflammation is one of the key characteristics of Alzheimer's disease (AD). Therefore, inhibitory reagents that prevent microglial activation may be used as potential therapeutic agents for treating AD. Recently, many studies have been performed to determine the bioactivities of green tea polyphenol epigallocatechin-3-gallate (EGCG), an efficient antioxidant that prevents neuroinflammation. However, limited information is available on the effects of EGCG on microglia-mediated neuroinflammation. In this study, we investigated the inhibitory effects of EGCG on amyloid β (Aβ)-induced microglial activation and neurotoxicity. Our results indicated that EGCG significantly suppressed the expression of tumor necrosis factor α (TNFα), interleukin-1β, interleukin-6, and inducible nitric oxide synthase (iNOS) in Aβ-stimulated EOC 13.31 microglia. EGCG also restored the levels of intracellular antioxidants nuclear erythroid-2 related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), thus inhibiting reactive oxygen species-induced nuclear factor-κB (NF-κB) activation after Aβ treatment. Furthermore, EGCG effectively protected neuro-2a neuronal cells from Aβ-mediated, microglia-induced cytotoxicity by inhibiting mitogen-activated protein kinase-dependent, Aβ-induced release of TNFα. Taken together, our findings suggested that EGCG suppressed Aβ-induced neuroinflammatory response of microglia and protected against indirect neurotoxicity. These results suggest that EGCG is a possible therapeutic agent for preventing Aβ-induced inflammatory neurodegeneration.

Absolute Configurations and NO Inhibitory Activities of Terpenoids from Curcuma longa

Curcuma longa L., belonging to the Zingiberaceae family, is a perennial herb and has been used as a spice and a pigment in the food industry. In the ongoing search for inhibitory reagents of NO production and survey of the chemical composition of natural vegetable foods, the chemical constituents of C. longa used as spice were investigated. This investigation resulted in the isolation of 2 new terpenoids and 14 known analogues. Their structures were established on the basis of the extensive analyses of 1D and 2D NMR spectroscopic data, and the absolute configurations of 1-4 were elucidated by comparison of the calculated and experimental ECD spectra. Among them, compound 1 is a rare norditerpene with an ent-labdane skeleton, and 2 is a skeletally novel sesquiterpene having an eight-membered ring. All of the compounds were found to possess NO inhibitory activities in murine microglial BV-2 cells. The discovery of two new compounds in this chemical investigation further disclosed the chemical composition of C. longa used a food spice, and the bioassay implied that the natural food spice C. longa, containing terpenoids with NO inhibitory activities, may be potentially promotive to human health.

Abstract 463: Evaluation of relative expression of SLC34A2/NaPi-IIb in lung cancer cell lines treated with estrogen and PKC and PKA pathway modulators

Abstract Background: Lung cancer (LC) represents a major health challenge worldwide, being the leading cause of cancer-related deaths in both men and women. SLC34A2 is a member of the solute carrier family and encodes the type IIb sodium phosphate cotransporter (NaPi-IIb). SLC34A2 expression is reduced by ten-fold in LC samples compared to normal lung. Estrogen is a known risk factor for LC in women, and a relationship between its action and SLC34A2/NaPi-IIb super-expression in certain cell lines has been proposed. Furthermore, based on the central role of PKC isoenzymes in carcinogenesis, and on studies showing that administration of cAMP stimulates the NaPi-IIb mRNA expression in adult rats alveolar type II cells, this work aimed to evaluate the correlation between PKA (cAMP mediator), PKC, estrogen signaling pathways and SLC34A2/NaPi-IIb expression levels in human LC cells. Methods: For both LC cell lines studied, A549 and H460, the effects of PKC and PKA activating and inhibitory reagents (PMA, calphostin C, dB cAMP and KT 5720) were tested in relation to SLC34A2/NaPi-IIb expression. Additionally, a dose-response curve of 17-beta-estradiol was performed for the expression of SLC34A2/NaPi-IIb in A549 cell line. SLC34A2/NaPi-IIb gene expression was analyzed by qRT-PCR, using SYBR Green PCR Master Mix as the detection system. Relative quantification of gene expression was performed by calculating the delta-delta Ct using two housekeeping genes: GAPDH and Beta-Actin. Findings: In our analysis we found that PKC and PKA did not influence the expression of SLC34A2/NaPi-IIb in LC cell line evaluated. There was, however, a considerable increase in the expression of this gene when treated with calphostin C, probably by a mechanism independent of its classic role in the PKC inhibition. There was also a trend of decreased SLC34A2/NaPi-IIb gene expression in LC cell line following the treatment with 17 beta-estradiol, and SLC34A2/NaPi-IIb - which expression is already reduced in LC - undergoes a greater reduction in its relative expression. Interpretation: We believe that maintaining the reduced expression of SLC34A2/NaPi-IIb should provide benefits to LC cells. Calphostin C induces apoptosis in several tumor cell lines by mechanisms not yet fully elucidated. Thus, we suggest a possible involvement of SLC34A2/NaPi-IIb in this pro-apoptotic pathway, which corroborates the fact that LC tumors present a reduced SLC34A2/NaPi-IIb expression compared to normal lung. Likewise, based on our findings, we believe that the inclusion of selective Estrogen receptor modulators and aromatase inhibitors in the routine clinical practice of LC might help to control the disease that is still the leading cause of cancer-related deaths worldwide. Citation Format: Murilo F. Cerri, Lucas C d Rezende, Marcela F. Paes, Klesia P. Madeira, Renata D. Daltoe, Nayara G. Tessarollo, Ian V. Silva, Leticia B a Rangel. Evaluation of relative expression of SLC34A2/NaPi-IIb in lung cancer cell lines treated with estrogen and PKC and PKA pathway modulators. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 463. doi:10.1158/1538-7445.AM2014-463

Sesquiterpenes inhibiting the microglial activation from Laurus nobilis.

The inhibitory reagents to inhibit the activation of microglial cells may be potentially useful for the treatment of neurodegenerative diseases. The leaves of the plant Laurus nobilis belonging to the family Lauraceae, namely, bay leaves, have been used as a popular spice, and their extract showed moderate inhibition on microglial activation. A further phytochemical investigation of the leaves led to the isolation of two new (1, 2) and eight known (3-10) sesquiterpenes. Their structures were elucidated on the basis of extensive 1D and 2D NMR (HMQC, HMBC, (1)H-(1)H COSY, and NOESY) spectroscopic data analyses and Chem3D modeling. The following biological studies disclosed that these isolated compounds showed inhibitory activities on LPS-induced microglial activation. The results of our phytochemical investigation, including two new sesquiterpenes (1 and 2) and the first report of two compounds (3 and 4) from this species, further revealed the chemical composition of bay leaves as a popular spice, and the biological studies implied that bay leaves, containing bioactive substances with the inhibition of microglial activation, were potentially beneficial to human health.

Xenoantibody response to porcine islet cell transplantation using GTKO, CD55, CD59, and fucosyltransferase multiple transgenic donors

Promising developments in porcine islet xenotransplantation could resolve the donor pancreas shortage for patients with type 1 diabetes. Using α1,3-galactosyltransferase gene knockout (GTKO) donor pigs with multiple transgenes should extend xenoislet survival via reducing complement activation, thrombus formation, and the requirement for exogenous immune suppression. Studying the xenoantibody response to GTKO/hCD55/hCD59/hHT islets in the pig-to-baboon model, and comparing it with previously analyzed responses, would allow the development of inhibitory reagents capable of targeting conserved idiotypic regions. We generated IgM heavy and light chain gene libraries from 10 untreated baboons and three baboons at 28days following transplantation of GTKO/hCD55/hCD59/hHT pig neonatal islet cell clusters with immunosuppression. Flow cytometry was used to confirm the induction of a xenoantibody response. IgM germline gene usage was compared pre- and post-transplant. Homology modeling was used to compare the structure of xenoantibodies elicited after transplantation of GTKO/hCD55/hCD59/hHT pig islets with those induced by GTKO and wild-type pig endothelial cells without further genetic modification. IgM xenoantibodies that bind to GTKO pig cells and wild-type pig cells were induced after transplantation. These anti-non-Gal antibodies were encoded by the IGHV3-66*02 (Δ28%) and IGKV1-12*02 (Δ25%) alleles, for the immunoglobulin heavy and light chains, respectively. IGHV3-66 is 86.7% similar to IGHV3-21 which was elicited by rhesus monkeys in response to GTKO endothelial cells. Heavy chain genes most similar to IGHV3-66 were found to utilize the IGHJ4 gene in 85% of V-D regions analyzed. However, unlike the wild-type response, a consensus complementary determining region 3 was not identified. Additional genetic modifications in transgenic GTKO pigs do not substantially modify the structure of the restricted group of anti-non-Gal xenoantibodies that mediate induced xenoantibody responses with or without immunosuppression. The use of this information to develop new therapeutic agents to target this restricted response will likely be beneficial for long-term islet cell survival and for developing targeted immunosuppressive regimens with less toxicity.

A reporter gene system for screening inhibitors of Wnt signaling pathway

Graphical abstractAbnormal activation of canonical Wnt signaling has been associated with various types of cancer. Inhibitory reagents targeting the Wnt signaling have great potential to inhibit the growth of relevant tumors. Here we generated a cell-based screening strategy for identification of antagonists of the Wnt/β-catenin signaling pathway. Stable expression wnt3a was generated in HEK293 cell line, which harbors dual-luciferase reporters. The Wnt signaling in the stably transfected cell line was proved to be very sensitive to (−)-epigallocatechin-3-gallate (EGCG) and lithium chloride (LiCl) treatment, respectively. Natural compounds were screened and a couple of novel inhibitory modulators of the Wnt signaling pathway were obtained.

Mutually Exclusive Cytoplasmic Dynein Regulation by NudE-Lis1 and Dynactin

Cytoplasmic dynein is responsible for a wide range of cellular roles. How this single motor protein performs so many functions has remained a major outstanding question for many years. Part of the answer is thought to lie in the diversity of dynein regulators, but how the effects of these factors are coordinated in vivo remains unexplored. We previously found NudE to bind dynein through its light chain 8 (LC8) and intermediate chain (IC) subunits (1), the latter of which also mediates the dynein-dynactin interaction (2). We report here that NudE and dynactin bind to a common region within the IC, and compete for this site. We find LC8 to bind to a novel sequence within NudE, without detectably affecting the dynein-NudE interaction. We further find that commonly used dynein inhibitory reagents have broad effects on the interaction of dynein with its regulatory factors. Together these results reveal an unanticipated mechanism for preventing dual regulation of individual dynein molecules, and identify the IC as a nexus for regulatory interactions within the dynein complex.

High‐throughput evaluation of Janus Kinase‐STAT and MAPK pathways in normal peripheral whole blood cells using 96 deep well plates by flow cytometry

When performing high‐throughput, flow cytometric screening of the patterns by which treated cells induce phosphorylated proteins involved in signaling pathways, tedious preparation of peripheral blood mononuclear cells (PBMC) was usually required. However, we have developed a Whole Blood Lyse/Fix Buffer System. This system allows for simultaneous red blood cell lysis and the fixation of leukocytes human whole blood. The cells can be stained for cell surface markers and intracellular cell signaling molecules and subsequently analyzed by flow cytometry. This eliminated the need for preparing PBMC. We have recently extended the application of this Whole Blood Lyse/Fix Buffer System to process cell samples in 96‐deep‐well plates. This method greatly facilitates the ease of preparing multiple human whole blood samples for cell signaling flow analysis. Whole blood stimulation, fixation, permeabilization, staining, and acquisition were done in deep well plates. Using deep well plates, we were able to evaluate the dose responses and time courses of STAT1, STAT3, STAT5, STAT6, Erk1/2, and P38 phosphorylation expressed by human whole blood cells that received various treatments. Inhibitory reagents, such as MEK and JAK inhibitors, were also applied to look at their differential effects on the phosphorylation status of intracellular signaling molecules that can be modified by the targeted kinases.

Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line

Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for "immediate-early 1") protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

Leukocyte Function-associated Antigen 1-mediated Adhesion Stability Is Dynamically Regulated through Affinity and Valency during Bond Formation with Intercellular Adhesion Molecule-1

Neutrophil rolling and transition to arrest on inflamed endothelium are dynamically regulated by the affinity of the beta(2) integrin CD11a/CD18 (leukocyte function associated antigen 1 (LFA-1)) for binding intercellular adhesion molecule (ICAM)-1. Conformational shifts are thought to regulate molecular affinity and adhesion stability. Also critical to adhesion efficiency is membrane redistribution of active LFA-1 into dense submicron clusters where multimeric interactions occur. We examined the influences of affinity and dimerization of LFA-1 on LFA-1/ICAM-1 binding by engineering a cell-free model in which two recombinant LFA-1 heterodimers are bound to respective Fab domains of an antibody attached to latex microspheres. Binding of monomeric and dimeric ICAM-1 to dimeric LFA-1 was measured in real time by fluorescence flow cytometry. ICAM-1 dissociation kinetics were measured while LFA-1 affinity was dynamically shifted by the addition of allosteric small molecules. High affinity LFA-1 dissociated 10-fold faster when bound to monomeric compared with dimeric ICAM-1, corresponding to bond lifetimes of 25 and 330 s, respectively. Downshifting LFA-1 into an intermediate affinity state with the small molecule I domain allosteric inhibitor IC487475 decreased the difference in dissociation rates between monomeric and dimeric ICAM-1 to 4-fold. When LFA-1 was shifted into the low affinity state by lovastatin, both monomeric and dimeric ICAM-1 dissociated in less than 1 s, and the dissociation rates were within 50% of each other. These data reveal the respective importance of LFA-1 affinity and proximity in tuning bond lifetime with ICAM-1 and demonstrate a nonlinear increase in the bond lifetime of the dimer versus the monomer at higher affinity.

A Ca(2+)-sensing receptor modulates shark rectal gland function.

The elasmobranch Squalus acanthias controls plasma osmolality and extracellular fluid volume by secreting a hypertonic fluid from its rectal gland. Because we found a correlation between extracellular Ca(2+) concentration and changes in cytosolic Ca(2+) ([Ca(2+)](i)), we sought the possible presence of a calcium-sensing receptor in rectal gland artery and tubules. Cytosolic Ca(2+) of both tissues responded to the addition of external Ca(2+) (0.8-5.3 mmol l(-1)) in a linear fashion. Spermine, Gd(3+) and Ni(2+), known agonists of the calcium-sensing receptor, increased [Ca(2+)](i). To assess the participation of inositol triphosphate (IP(3)) generation, sarcoplasmic/endoplasmic reticulum (SR/ER) Ca(2+) depletion, and activation of store-operated Ca(2+) entry, we utilized thapsigargin and ryanodine to deplete Ca(2+) SR/ER stores and the inhibitory reagents TMB-8 and 2-APB to block IP(3) receptors. In each case, these agents inhibited the [Ca(2+)](i) response to agonist stimulation by approximately 50 %. Blockade of L-channels with nifedipine had no significant effect. Increases in ionic strength are known to inhibit the calcium-sensing receptor. We postulate that the CaSR stimulates Ca(2+)-mediated constriction of the rectal gland artery and diminishes cyclic AMP-mediated salt secretion in rectal gland tubules during non-feeding conditions. When the shark ingests sea water and fish, an increase in blood and interstitial fluid ionic strength inhibits the activity of the calcium-sensing receptor, relaxing the rectal gland artery and permitting salt secretion by the rectal gland tubules.

K562 erythroleukemic cells are equipped with multiple mechanisms of resistance to lysis by complement.

Resistance of tumor cells to lysis by complement is generally attributed to several protective mechanisms. The relative impact of these mechanisms in the same tumor cell, however, has not been assessed yet. We have analyzed the interaction of the human erythroleukemia tumor cell line K562 with human complement. K562 cells express the membrane complement regulatory proteins CD59, CD55 and CD46. As shown here for the first time, K562 also spontaneously release the soluble regulators C1 inhibitor, factor H, and soluble CD59. Complement resistance of K562 cells is augmented upon treatment with PMA, TNF or even with sublytic complement. Unlike TNF and sublytic complement, PMA enhanced the expression of membrane-bound CD55 and CD59 and led to increased secretion of soluble CD59. In addition, we show that complement-resistant K562 cells express a membrane-associated proteolytic activity, higher than the complement-sensitive K562/S cells. Treatment of complement-resistant K562 cells with serine protease inhibitors enhance their sensitivity to complement-mediated lysis. Inhibitors of protein kinase C (PKC) also sensitize K562 cells to complement lysis, implicating PKC-mediated signaling in cell resistance to complement. Neutralization of the CD55 and CD59 but not of CD46 regulatory activity with specific antibodies significantly increases complement-mediated K562 cell lysis. Treatment of K562 cells with a mixture of inhibitory reagents results in a significant additive enhancing effect on complement-mediated lysis of K562. In conclusion, K562 cells resist a complement attack by concomitantly using multiple molecular evasion strategies. Future attempts in antibody-based tumor therapy should include strategies to interfere with those resistance mechanisms.

The critical role of IL-12 and the IL-12R beta 2 subunit in the generation of pathogenic autoreactive Th1 cells.

Experimental Allergic Encephalomyelitis (EAE) is a demyelinating disease of the central nervous system which is an animal model for the human autoimmune disease, multiple sclerosis. EAE is mediated by CD4+ T cells and the T cells responsible for disease induction produce Th1 cytokines. IL-12 produced by monocytes and dendritic cells is the most critical factor which influences the development and differentiation of pathogenic autoreactive Th1 cells. Here, we review our recent studies on the critical contributions of IL-12 and the IL-12R beta 2 subunit to the generation of autoreactive effector cells which mediate EAE. In addition, we discuss the potential contribution of IL-18 to the upregulation of the IL-12/IL-12R beta 2 pathway and the contribution of the suppressor cytokines, IL-4 and IL-10, in downregulating this pathway. Collectively, our studies demonstrate that the IL-12/IL-12R beta 2 pathway is a critical intermediary in the process of Th1 differentiation which can be both positively or negatively regulated. This pathway remains an attractive immunotherapeutic target for blockade of function with inhibitory reagents or downregulation by Th2 cytokines.