The effect of linker type and recognition peptide conjugation chemistry on tissue affinity and cytotoxicity of charged polyacrylamide.

To increase colonoscopy competence in ambiguous situations (e.g. the existence of flat polyps), an explicit in situ (at real time) diagnosis at the molecular level is required. We have previously shown that the affinity of fluorescent cationic polyacrylamide (Flu-CPAA) to malignant regions in the colon mucosa can be improved by conjugating the recognition peptide EPPT1 to the polymer backbone (to form Flu-CPAA-Pep). Using another recognition peptide, namely VRPMPLQ, we elucidated in the present study the effect of linker type and conjugating methods on Flu-CPAA-VRPMPLQ cytotoxicity and on its affinity to cell lines as well as human colorectal cancer (CRC) biopsies. In order to derive the relationship between the response variable and the experimental factors in a minimal set of experiments, a computerized statistical design of experiment (DoE) strategy was implemented. Data were collected in a six-factor factorial design to study the effect of experimental factors (independent variables) on the ability of the Flu-CPAA polymers to bind specifically to the colon cancer cell lines or the human biopsies (the response). It was found that the presence of VRPMPLQ on the Flu-CPAA improved the polymer's affinity to the human CRC biopsies and to the colon cancer cell lines representing stage B in the Duke severity staging system. The cytotoxicity of Flu-CPAA with high charge density was reduced after conjugated with VRPMPLQ. The replacement of Ahx linker by PEG linker of similar length did not affect the affinity to the human biopsies, nor did it affect cytotoxicity. However, elongating the PEG linker reduced the in vitro affinity to the colon cancer cell lines and to human CRC biopsies. Changing the conjugation method from condensation (amide bond formation) to the click conjugation method did not affect the affinity properties of the polymers. It did reduce, however, the polymer cytotoxicity. We suggest that Flu-CPAA-Pep, with the VRPMPLQ peptide as a recognition moiety, could serve for early diagnosis and screening of CRC patients during endoscopic procedures.

PKCδ null mutations in a mouse model of osteoarthritis alter osteoarthritic pain independently of joint pathology by augmenting NGF/TrkA-induced axonal outgrowth.

OBJECTIVES: A key clinical paradox in osteoarthritis (OA), a prevalent age-related joint disorder characterised by cartilage degeneration and debilitating pain, is that the severity of joint pain does not strictly correlate with radiographic and histological defects in joint tissues. Here, we determined whether protein kinase Cδ (PKCδ), a key mediator of cartilage degeneration, is critical to the mechanism by which OA develops from an asymptomatic joint-degenerative condition to a painful disease. METHODS: OA was induced in 10-week-old PKCδ null (PKCδ-/-) and wild-type mice by destabilisation of the medial meniscus (DMM) followed by comprehensive examination of the histology, molecular pathways and knee-pain-related-behaviours in mice, and comparisons with human biopsies. RESULTS: In the DMM model, the loss of PKCδ expression prevented cartilage degeneration but exacerbated OA-associated hyperalgesia. Cartilage preservation corresponded with reduced levels of inflammatory cytokines and of cartilage-degrading enzymes in the joints of PKCδ-deficient DMM mice. Hyperalgesia was associated with stimulation of nerve growth factor (NGF) by fibroblast-like synovial cells and with increased synovial angiogenesis. Results from tissue specimens of patients with symptomatic OA strikingly resembled our findings from the OA animal model. In PKCδ null mice, increases in sensory neuron distribution in knee OA synovium and activation of the NGF-tropomyosin receptor kinase (TrkA) axis in innervating dorsal root ganglia were highly correlated with knee OA hyperalgesia. CONCLUSIONS: Increased distribution of synovial sensory neurons in the joints, and augmentation of NGF/TrkA signalling, causes OA hyperalgesia independently of cartilage preservation.

Using antibodies against P2Y and P2X receptors in purinergic signaling research.

The broad expression pattern of the G protein-coupled P2Y receptors has demonstrated that these receptors are fundamental determinants in many physiological responses, including neuromodulation, vasodilation, inflammation, and cell migration. P2Y receptors couple either G(q) or G(i) upon activation, thereby activating different signaling pathways. Ionotropic ATP (P2X) receptors bind extracellular nucleotides, a signal which is transduced within the P2X protein complex into a cation channel opening, which usually leads to intracellular calcium concentration elevation. As such, this family of proteins initiates or shapes several cellular processes including synaptic transmission, gene expression, proliferation, migration, and apoptosis. The ever-growing range of applications for antibodies in the last 30 years attests to their major role in medicine and biological research. Antibodies have been used as therapeutic tools in cancer and inflammatory diseases, as diagnostic reagents (flow cytometry, ELISA, and immunohistochemistry, to name a few applications), and in widespread use in biological research, including Western blot, immunoprecipitation, and ELISPOT. In this article, we will showcase several of the advances that scientists around the world have achieved using the line of antibodies developed at Alomone Labs for P2Y and P2X receptors.

A novel mutation in the HCN4 gene causes symptomatic sinus bradycardia in Moroccan Jews.

OBJECTIVES: to conduct a clinical, genetic, and functional analysis of 3 unrelated families with familial sinus bradycardia (FSB). BACKGROUND: mutations in the hyperpolarization-activated nucleotide-gated channel (HCN4) are known to be associated with FSB. METHODS AND RESULTS: three males of Moroccan Jewish descent were hospitalized: 1 survived an out-of-hospital cardiac arrest and 2 presented with weakness and presyncopal events. All 3 had significant sinus bradycardia, also found in other first-degree relatives, with a segregation suggesting autosomal-dominant inheritance. All had normal response to exercise and normal heart structure. Sequencing of the HCN4 gene in all patients revealed a C to T transition at nucleotide position 1,454, which resulted in an alanine to valine change (A485V) in the ion channel pore found in most of their bradycardiac relatives, but not in 150 controls. Functional expression of the mutated ion channel in Xenopus oocytes and in human embryonic kidney 293 cells revealed profoundly reduced function and synthesis of the mutant channel compared to wild-type. CONCLUSIONS: we describe a new mutation in the HCN4 gene causing symptomatic FSB in 3 unrelated individuals of similar ethnic background that may indicate unexplained FSB in this ethnic group. This profound functional defect is consistent with the symptomatic phenotype.

Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by GalphaiGDP and Gbetagamma.

G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gbetagamma following activation of Gi/o proteins via G protein-coupled receptors (GPCRs). Although Galphai subunits do not activate GIRKs, they interact with the channels and regulate the gating pattern of the neuronal heterotetrameric GIRK1/2 channel (composed of GIRK1 and GIRK2 subunits) expressed in Xenopus oocytes. Coexpressed Galphai3 decreases the basal activity (Ibasal) and increases the extent of activation by purified or coexpressed Gbegagamma. Here we show that this regulation is exerted by the 'inactive' GDP-bound Galphai3GDP and involves the formation of Galphai3betagamma heterotrimers, by a mechanism distinct from mere sequestration of Gbetagamma 'away' from the channel. The regulation of basal and Gbetagamma-evoked current was produced by the 'constitutively inactive' mutant of Galphai3, Galphai3G203A, which strongly binds Gbetagamma, but not by the 'constitutively active' mutant, Galphai3Q204L, or by Gbetagamma-scavenging proteins. Furthermore, regulation by Galphai3G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified Gbetagamma enhanced the binding of Galphai3GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a 'classical' Gbetagamma effector, showing low Ibasal and strong Gbetagamma-dependent activation. Expression of Galphai3G203A did not affect either Ibasal or Gbetagamma-induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large Ibasal and was poorly activated by Gbegagamma. Expression of Galphai3GDP reduced Ibasal and restored the ability of Gbetagamma to activate GIRK1*, like in GIRK1/2. Transferring the unique distal segment of the C terminus of GIRK1 to GIRK2 rendered the latter functionally similar to GIRK1*. These results demonstrate that GIRK1 containing channels are regulated by both Galphai3GDP and Gbetagamma, while GIRK2 is a Gbetagamma-effector insensitive to Galphai3GDP.

Point mutation in the HCN4 cardiac ion channel pore affecting synthesis, trafficking, and functional expression is associated with familial asymptomatic sinus bradycardia.

BACKGROUND: The hyperpolarization-activated nucleotide-gated channel--HCN4 plays a major role in the diastolic depolarization of sinus atrial node cells. Mutant HCN4 channels have been found to be associated with inherited sinus bradycardia. METHODS AND RESULTS: Sixteen members of a family with sinus bradycardia were evaluated. Evaluation included a clinical questionnaire, 12-lead ECGs, Holter monitoring, echocardiography, and treadmill exercise testing. Eight family members (5 males) were classified as affected. All affected family members were asymptomatic with normal exercise capacity during long-term follow-up. Electrophysiological testing performed on 2 affected family members confirmed significant isolated sinus node dysfunction. Segregation analysis suggested autosomal-dominant inheritance. Direct sequencing of the exons encoding HCN4 revealed a missense mutation, G480R, in the ion channel pore domain in all affected family members. Function analysis, including expression of HCN4 wild-type and G480R in Xenopus oocytes and human embryonic kidney 293 cells, revealed that mutant channels were activated at more negative voltages compared with wild-type channels. Synthesis and expression of the wild-type and mutant HCN4 channel on the plasma membrane tested in human embryonic kidney 293 cells using biotinylation and Western blot analysis demonstrated a reduction in synthesis and a trafficking defect in mutant compared with wild-type channels. CONCLUSIONS: We describe an inherited, autosomal-dominant form of sinus node dysfunction caused by a missense mutation in the HCN4 ion channel pore. Despite its critical location, this mutation carries a favorable prognosis without the need for pacemaker implantation during long-term follow-up.

Galphai3 primes the G protein-activated K+ channels for activation by coexpressed Gbetagamma in intact Xenopus oocytes.

G protein-activated K+ channels (GIRK) mediate postsynaptic inhibitory effects of neurotransmitters in the atrium and in the brain by coupling to G protein-coupled receptors (GPCRs). In neurotransmitter-dependent GIRK signalling, Gbetagamma is released from the heterotrimeric Galphabetagamma complex upon GPCR activation, activating the channel and attenuating its rectification. Now it becomes clear that Galpha is more than a mere Gbetagamma donor. We have proposed that Galphai3-GDP regulates GIRK gating, keeping its basal activity low but priming (predisposing) the channel for activation by agonist in intact cells, and by Gbetagamma in excised patches. Here we have further investigated GIRK priming by Galphai3 using a model in which the channel was activated by coexpression of Gbetagamma, and the currents were measured in intact Xenopus oocytes using the two-electrode voltage clamp technique. This method enables the bypass of GPCR activation during examination of the regulation of the channel in intact cells. Using this method, we further characterize the priming phenomenon. We tested and excluded the possibility that our estimates of priming are affected by artifacts caused by series resistance or large K+ fluxes. We demonstrate that both Galphai3 and membrane-attached Gbetagamma scavenger protein, m-phosducin, reduce the basal channel activity. However, Galphai3 allows robust channel activation by coexpressed Gbetagamma, in sharp contrast to m-phosducin, which causes a substantial reduction in the total Gbetagamma-induced current. Furthermore, Galphai3 also does not impair the Gbetagamma-dependent attenuation of the channel rectification, in contrast to m-phosducin, which prevents this Gbetagamma-induced modulation. The Galphai3-induced enhancement of direct activation of GIRK by Gbetagamma, demonstrated here for the first time in intact cells, strongly supports the hypothesis that Galphai regulates GIRK gating under physiological conditions.