Leukocyte migration across the endothelial lining is a critical step in the body's response to infection and inflammation. The homophilic interaction between endothelial PECAM and leukocyte PECAM is essential for this process. The molecular events that are triggered in the endothelial cell by PECAM engagement have been well characterized; however, the function of leukocyte PECAM remains to be elucidated. To study this, we first blocked leukocyte transmigration using anti-PECAM Ab and then specifically activated leukocyte PECAM. This was sufficient to overcome the block and promote transmigration, suggesting an active signaling role for leukocyte PECAM. Consistent with this, we found that ligation of leukocyte PECAM induces phosphorylation of two tyrosine residues on its cytoplasmic tail. By performing RNA interference-rescue experiments, we demonstrate that these phosphorylation events are indispensable for transendothelial migration. Finally, we show that leukocyte PECAM translocates to a detergent-resistant membrane (DRM) during transmigration. PECAM localized in DRMs displays reduced phosphorylation and does not support transmigration. Together, these data support a model whereby engagement of leukocyte PECAM induces its transient tyrosine phosphorylation and induction of downstream signals that drive transmigration. These signals are then downregulated following PECAM translocation to DRMs.

Reporter genes are widely used in biology and only a limited number are available. We present a new reporter gene for the localization of mammalian cells and transgenic tissues based on detection of the bglA (SYNbglA) gene of Caldocellum saccharolyticum that encodes a thermophilic beta-glucosidase.

Lymphocyte development and function are regulated by tyrosine kinase and G-protein coupled receptors. Each of these classes of receptors activates phosphoinositide 3-kinase (PI3K). In this chapter, we summarize current understanding of how PI3K contributes to key aspects of the adaptive immune system.

In Drosophila melanogaster, gustatory receptor genes (Grs) encode putative G-protein-coupled receptors (GPCRs) that are expressed in gustatory receptor neurons (GRNs). One of the Gr genes, Gr5a, encodes a receptor for trehalose that is expressed in a subset of GRNs. Although a role for the G protein, Gsα, has been shown in Gr5a-expressing taste neurons, there is the residual responses to trehalose in Gsα mutants which could suggest additional transduction mechanisms. Expression and genetic analysis of the heterotrimeric G-protein subunit, Gq, shown here suggest involvement of this Gα subunit in trehalose perception in Drosophila. A green fluorescent protein reporter of Gq expression is detected in gustatory neurons in the labellum, tarsal segments, and wing margins. Animals heterozygous for dgq mutations and RNA interference-mediated knockdown of dgq showed reduced responses to trehalose in the proboscis extension reflex assay and feeding behavior assay. These defects were rescued by targeted expression of the wild-type dgqα transgene in the GRNs. These data together with observations from other mutants in phospholipid signaling provide insights into the mechanisms of taste transduction in Drosophila.

T cells develop in the thymus and are critical for adaptive immunity. Natural killer (NK) lymphocytes constitute an essential component of the innate immune system in tumor surveillance, reproduction, and defense against microbes and viruses. Here, we show that the transcription factor Bcl11b was expressed in all T cell compartments and was indispensable for T lineage development. When Bcl11b was deleted, T cells from all developmental stages acquired NK cell properties and concomitantly lost or decreased T cell-associated gene expression. These induced T-to-natural killer (ITNK) cells, which were morphologically and genetically similar to conventional NK cells, killed tumor cells in vitro, and effectively prevented tumor metastasis in vivo. Therefore, ITNKs may represent a new cell source for cell-based therapies.

In vitro antibody generation technologies have now been available for two decades. Research reagents prepared via phage display are becoming available and several recent studies have demonstrated that these technologies are now sufficiently advanced to facilitate generation of a comprehensive renewable resource of antibodies for any protein encoded by the approximately 22,500 human protein-coding genes. Antibody selection in vitro offers properties not available in animal-based antibody generation methods. By adjusting the biochemical milieu during selection, it is possible to control the antigen conformation recognized, the antibody affinity or unwanted cross-reactivity. For larger-scale antibody generation projects, the handling, transport and storage logistics and bacterial production offer cost benefits. Because the DNA sequence encoding the antibody is available, modifications, such as site-specific in vivo biotinylation and multimerization, are only a cloning step away. This opinion article summarizes opportunities for the generation of antibodies for proteome research using in vitro technologies.

This is the concluding article in a series of 3 studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). We used viral synaptophysin-enhanced green fluorescent protein expression in thalamic neurons and reconstructions of biocytin-labeled cortical neurons in TC slices to quantify the number and distribution of boutons from the ventral posterior medial (VPM) and posteromedial (POm) nuclei potentially innervating dendritic arbors of excitatory neurons located in layers (L)2-6 of a cortical column in rat somatosensory cortex. We found that 1) all types of excitatory neurons potentially receive substantial TC input (90-580 boutons per neuron); 2) pyramidal neurons in L3-L6 receive dual TC input from both VPM and POm that is potentially of equal magnitude for thick-tufted L5 pyramidal neurons (ca. 300 boutons each from VPM and POm); 3) L3, L4, and L5 pyramidal neurons have multiple (2-4) subcellular TC innervation domains that match the dendritic compartments of pyramidal cells; and 4) a subtype of thick-tufted L5 pyramidal neurons has an additional VPM innervation domain in L4. The multiple subcellular TC innervation domains of L5 pyramidal neurons may partly explain their specific action potential patterns observed in vivo. We conclude that the substantial potential TC innervation of all excitatory neuron types in a cortical column constitutes an anatomical basis for the initial near-simultaneous representation of a sensory stimulus in different neuron types.

Th cell functional subsets have unique transcriptional programs that form the molecular basis for T cell differentiation and functions. T follicular helper (TFH) cells have emerged as the main providers of T cell help to B cells during the germinal center (GC) reaction, where B cells undergo selection events through competition for Ag and for access to GC T cell-mediated prosurvival and differentiation signals. Because T cell help is one limiting factor for GC B cells, the molecular mechanisms controlling TFH cell abundance and functionality are central to the GC reaction and generation of long-term humoral immunity. Two signaling pathways are absolutely critical for TFH cells: phosphoinositide-3 kinase pathway and the signaling lymphocyte activation molecule-associated protein. In this review, the molecular mechanisms constituting the signaling network in TFH cells will be explored.

MicroRNAs 125a and 125b are predicted to be able to bind to the B lymphocyte-induced maturation protein-1 (BLIMP-1) and IFN regulatory protein-4 (IRF-4) transcription factors, which are essential for plasma cell differentiation. A computational survey of the human and mouse genomes revealed that miR-125a and miR-125b are members of a multigene family located in paralogous clusters. The miR-125a cluster on chromosome 19 in humans includes miR-99b and let-7e, whereas the miR-125b cluster on chromosome 21 includes miR-99a and miR-let-7c. Our analysis of the expression profiles for these six miRs during B lineage differentiation indicated that mature miR-125a, miR-125b, miR-99b and let-7e transcripts are preferentially expressed by the actively dividing centroblasts in germinal centers (GC). However, miR-99b and let-7e are not predicted to bind BLIMP-1 or IRF-4 transcripts, and binding to the untranslated region of BLIMP-1 and IRF-4 messenger RNAs could be confirmed only for miR-125b. When the effect of miR-125b over-expression on terminal B cell differentiation was evaluated in an LPS-responsive B cell line, the induction of BLIMP-1 expression and IgM secretion was inhibited in this model system. Furthermore, miR-125b over-expression inhibited the differentiation of primary B cells and compromised the survival of cultured myeloma cells. These findings suggest that miR-125b promotes B lymphocyte diversification in GC by inhibiting premature utilization of essential transcription factors for plasma cell differentiation.

Schizophrenia is a multifactorial complex disease with a large impact on society. Many hypotheses have been proposed over the years to explain its causes, and genomics and functional genomic approaches may shed light on the reason behind these controversies and discrepancies. We give an overview of several approaches that have been used to identify the genetic causes and molecular phenotypes of the disease. We focus on a recent microarray analysis by Torkamani and colleagues on the evolution of regulatory networks in normal and schizophrenic brains. Combining the conclusion of that study with the prevalent hypotheses of schizophrenia, we suggest that the schizophrenic brain might resemble a juvenile brain.

An open chromatin architecture devoid of compact chromatin is thought to be associated with pluripotency in embryonic stem cells. Establishing this distinct epigenetic state may also be required for somatic cell reprogramming. However, there has been little direct examination of global structural domains of chromatin during the founding and loss of pluripotency that occurs in preimplantation mouse development. Here, we used electron spectroscopic imaging to examine large-scale chromatin structural changes during the transition from one-cell to early postimplantation stage embryos. In one-cell embryos chromatin was extensively dispersed with no noticeable accumulation at the nuclear envelope. Major changes were observed from one-cell to two-cell stage embryos, where chromatin became confined to discrete blocks of compaction and with an increased concentration at the nuclear envelope. In eight-cell embryos and pluripotent epiblast cells, chromatin was primarily distributed as an extended meshwork of uncompacted fibres and was indistinguishable from chromatin organization in embryonic stem cells. In contrast, lineage-committed trophectoderm and primitive endoderm cells, and the stem cell lines derived from these tissues, displayed higher levels of chromatin compaction, suggesting an association between developmental potential and chromatin organisation. We examined this association in vivo and found that deletion of Oct4, a factor required for pluripotency, caused the formation of large blocks of compact chromatin in putative epiblast cells. Together, these studies show that an open chromatin architecture is established in the embryonic lineages during development and is sufficient to distinguish pluripotent cells from tissue-restricted progenitor cells.

A unique property of the mammalian embryo is that stem cells can be derived from its early tissue lineages. These lineages will give rise to the fetus as well as essential extraembryonic tissues. Understanding how chromatin regulation participates in establishment of these lineages in the embryo and their derived stem cells provides insight that will critically inform our understanding of embryogenesis and stem cell biology. Here, we compare the genomewide location of active and repressive histone modifications in embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm stem cells from the mouse. Our results show that the active modification H3K4me3 has a similar role in the three stem cell types, but the repressive modification H3K27me3 varies in abundance and genomewide distribution. Thus, alternative mechanisms mediate transcriptional repression in stem cells from the embryo. In addition, using carrier chromatin immunoprecipitation we show that bivalent histone domains seen in embryonic stem cells exist in pluripotent cells of the early embryo. However, the epigenetic status of extraembryonic progenitor cells in the embryo did not entirely reflect the extraembryonic stem cell lines. These studies indicate that histone modification mechanisms may differ between early embryo lineages and emphasize the importance of examining in vivo and in vitro progenitor cells.

Epidemiological studies have shown an association between statin use and a decreased risk of dementia. However, the mechanism by which this beneficial effect is brought about is unclear. In the context of Alzheimer's disease, at least three possibilities have been studied; reduction in amyloid beta peptide (Abeta) production, the promotion of alpha-secretase cleavage and positive effects on neurite outgrowth. By investigating the effects of mevalonate pathway blockade on neurite outgrowth using real-time imaging, we found that rather than promote the production of neurite extensions, inhibition rapidly induced cell rounding. Crucially, neurite-like structures were generated through the persistence of cell-cell and cell-substrate adhesions and not through a mechanism of positive outgrowth. This effect can be strikingly enhanced by the over-expression of human amyloid precursor protein and is isoprenoid rather than cholesterol dependent.

Polycomb Repressor Complexes (PRCs) are important regulators of embryogenesis. In embryonic stem (ES) cells many genes that regulate subsequent stages in development are enriched at their promoters for PRC1, PRC2 and Ser 5-phosphorylated RNA Polymerase II (RNAP), and contain domains of 'bivalent' chromatin (enriched for H3K4me3; histone H3 di- or trimethylated at Lys 4 and H3K27me3; histone H3 trimethylated at Lys 27). Loss of individual PRC components in ES cells can lead to gene de-repression and to unscheduled differentiation. Here we show that Jarid2 is a novel subunit of PRC2 that is required for the co-recruitment of PRC1 and RNAP to genes that regulate development in ES cells. Jarid2-deficient ES cells showed reduced H3K4me2/me3 and H3K27me3 marking and PRC1/PRC2 recruitment, and did not efficiently establish Ser 5-phosporylated RNAP at target genes. ES cells lacking Jarid2, in contrast to previously characterized PRC1 and PRC2 mutants, did not inappropriately express PRC2 target genes. Instead, they show a severely compromised capacity for successful differentiation towards neural or mesodermal fates and failed to correctly initiate lineage-specific gene expression in vitro. Collectively, these data indicate that transcriptional priming of bivalent genes in pluripotent ES cells is Jarid2-dependent, and suggests that priming is critical for subsequent multi-lineage differentiation.

Adaptive behavior in dynamic environments critically depends on the ability to learn rapidly and flexibly from the outcomes of prior choices. In social environments, facial expressions of emotion often serve as performance feedback and thereby guide declarative learning. Abundant evidence implicates beta-noradrenergic signaling in the modulatory influence of emotion on declarative learning. It is currently unclear whether a similar mechanism also mediates a guidance of declarative learning by social-emotional feedback administered in the form of facial expressions. We therefore conducted a double-blind randomized placebo-controlled trial to test the effects of a 40-mg single oral dose of the nonspecific beta-noradrenergic antagonist propranolol in a behavioral task that required gradual declarative learning of item-category associations from either social-emotional (happy vs. angry faces) or nonsocial (green vs. red color signals) trial-by-trial feedback. As predicted on the basis of our previous experiments, learning from social-emotional feedback was more effective than learning from nonsocial feedback in placebo-treated subjects. This advantage of social-emotional over nonsocial feedback was abolished by propranolol treatment. Propranolol had no effect on learning during the nonsocial feedback condition. Our findings suggest that a facilitation of declarative learning by social-emotional feedback critically involves signaling via beta-noradrenergic receptors.

Autoantibodies to gastric cellular antigens and glycoproteins including mucins and Lewis X and Y antigens have been implicated in the induction of autoimmune gastritis. Monoclonal antibody D10 (D10 MAb) recognizes a highly conserved mucin expressed in the foregut of mammals and other vertebrates. The objective of this study was to biochemically characterize the autoantigen identified by D10 MAb and examine its autoimmunogenicity in the mouse. Characterization of the mucin autoantigen was undertaken following purification, by amino acid and carbohydrate analyses, deglycosylation, SDS-PAGE, and immunoblotting using D10 MAb. Autoimmune reactivity and specificity of D10 MAb were validated by immunohistochemistry and ELISA using mouse tissue. Induction of autoimmune gastritis was investigated following immunization of mice with D10 MAb-reactive heterologous mucin. D10 MAb was shown to be a murine anti-mucin autoantibody with a unique pattern of immunohistochemical staining of Brunner's glands of the duodenum and the cardiac glands, mucous neck cells, and pyloric glands of the stomach from inbred Balb/c mice in patterns identical to that previously reported in human tissue. Amino acid and carbohydrate analysis of purified D10 mucin reflected a compositional profile of a typical mucin molecule. Confirmation that D10 MAb recognizes a mucin was also provided by demonstration that the carbohydrate epitope resides on a high molecular weight (>1x10(6)Da), high-density (>1.40 g/mL) molecule comprised of greater than 60% carbohydrate. Mice immunized with D10 MAb-reactive, purified, heterologous mucin produced autoantibodies of identical specificity to the original D10 MAb. These data demonstrate the autoimmunogenic properties of a novel foregut mucin and raise the potential of anti-mucin autoantibodies in the induction of autoimmune gastritis.

Size-dependent protein segregation at the cell-cell contact interface has been suggested to be critical for regulation of lymphocyte function. We investigated the role of ligand dimensions in regulation of mouse NK-cell activation and inhibition. Elongated forms of H60a, a mouse NKG2D ligand, were generated and expressed stably in the RMA cell line. RMA cells expressing the normal size H60a were lysed efficiently by both freshly isolated and IL-2 stimulated C57BL/6 mouse-derived NK cells; however the level of lysis decreased as the H60a ligand size increased. Importantly, H60a elongation did not affect NKG2D binding, as determined by soluble NKG2D tetramer staining, and by examining NK-cell target cell conjugate formation. CHO cells are efficient at activating NK cells from C57BL/6 mice, and expression of a single chain form of H-2K(b), a ligand for the mouse inhibitory receptor Ly49C, strongly inhibited such activation of Ly49C/I positive NK cells. Elongation of H-2K(b) resulted in decreased inhibition of both lysis and IFN-gamma production by NK cells. These results establish that small ligand dimensions are important for both NK-cell activation and inhibition, and suggest that there are shared features between the mechanisms of receptor triggering on different types of lymphocytes.

Wallerian degeneration of the CNS is accompanied by axonal dystrophy or swelling. To understand the mechanisms by which swellings arise, we studied their spatiotemporal dynamics, ultrastructure, composition, and the conditions that affect their formation in vivo and ex vivo. In contrast to peripheral nerve axons, lesioned optic nerve (ON) axons in vivo developed focal swellings asynchronously within 6 hours, long before there is any axon fragmentation. Axons in ON, spinal cord dorsal column, and corpus callosum all showed marked gradients with more swellings in proximal regions of their distal stumps early after lesion. Time-lapse imaging of a validated ex vivo system showed that multiple focal swellings arise after around 1 hour close to the injury site, followed by anterograde wave-like progression on continuous ON axon stumps. Swellings were largely stable but occasionally seemed to fuse with neighboring swellings. Their ultrastructural appearances resembled disease-associated spheroids. Although accumulation of axonal markers suggested transport deficits, large accumulations of mitochondria were not observed. Early swelling formation was decreased in Wld gene-expressing rodents and by removing extracellular calcium. Several pharmacologic agents that inhibit axon loss in vitro and/or in vivo also prevented early formation of axonal spheroids in acute ON explants. Because axonal swellings are hallmarks of many neurodegenerative conditions, these data suggest that they are a manifestation of Wallerian-like degeneration in some cases. Thus, Wallerian-like degeneration may be a more common component mechanism in CNS diseases than previously thought.

Crossing plants of the same species but different ploidies can have dramatic effects on seed growth, but little is known about the alterations to transcriptional programmes responsible for this. Parental genomic imbalance particularly affects proliferation of the endosperm, with an increased ratio of paternally to maternally contributed genomes ('paternal excess') associated with overproliferation, while maternal excess inhibits endosperm growth. One interpretation is that interploidy crosses disrupt the balance in the seed of active copies of parentally imprinted genes. This is supported by the observation that mutations in imprinted FIS-class genes of Arabidopsis thaliana share many features of the paternal excess phenotype. Here we investigated gene expression underlying parent-of-origin effects in Arabidopsis through transcriptional profiling of siliques generated by interploidy crosses and FIS-class mutants.

Nuclear architecture and chromatin reorganization have recently been shown to orchestrate gene expression and act as key players in developmental pathways. To investigate how regulatory elements in the mouse CD8 gene locus are arranged in space and in relation to each other, three-dimensional fluorescence in situ hybridization and chromosome conformation capture techniques were employed to monitor the repositioning of the locus in relation to its subchromosomal territory and to identify long-range interactions between the different elements during development. Our data demonstrate that CD8 gene expression in murine lymphocytes is accompanied by the relocation of the locus outside its subchromosomal territory. Similar observations in the CD4 locus point to a rather general phenomenon during T cell development. Furthermore, we show that this relocation of the CD8 gene locus is associated with a clustering of regulatory elements forming a tight active chromatin hub in CD8-expressing cells. In contrast, in nonexpressing cells, the gene remains close to the main body of its chromosomal domain and the regulatory elements appear not to interact with each other.

Alzheimer's disease is the most common neurodegenerative disease of the central nervous system characterized by a progressive loss in memory and deterioration of cognitive functions. In this study the transgenic mouse TgCRND8, which encodes a mutant form of the amyloid precursor protein 695 with both the Swedish and Indiana mutations and develops extracellular amyloid beta-peptide deposits as early as 2-3 months, was investigated. Extract from eight brain regions (cortex, frontal cortex, cerebellum, hippocampus, olfactory bulb, pons, midbrain and striatum) were studied using (1)H NMR spectroscopy. Analysis of the NMR spectra discriminated control from APP695 tissues in hippocampus, cortex, frontal cortex, midbrain and cerebellum, with hippocampal and cortical region being most affected. The analysis of the corresponding loading plots for these brain regions indicated a decrease in N-acetyl-L-aspartate, glutamate, glutamine, taurine (exception hippocampus), gamma-amino butyric acid, choline and phosphocholine (combined resonances), creatine, phosphocreatine and succinate in hippocampus, cortex, frontal cortex (exception gamma-amino butyric acid) and midbrain of affected animals. An increase in lactate, aspartate, glycine (except in midbrain) and other amino acids including alanine (exception frontal cortex), leucine, iso-leucine, valine and water soluble free fatty acids (0.8-0.9 and 1.2-1.3 ppm) were observed in the TgCRND8 mice. Our findings demonstrate that the perturbations in metabolism are more widespread and include the cerebellum and midbrain. Furthermore, metabolic perturbations are associated with a wide range of metabolites which could improve the diagnosis and monitoring of the progression of Alzheimer's disease.

Two major subsets of rat NK cells can be distinguished based on their expression of the Ly49s3 or the NKR-P1B lectin-like receptor. Ly49s3(+) NK cells, but not NKR-P1B(+) NK cells, express a wide range of Ly49 receptors. Here, we have examined differences between these two subsets in their expression of certain NK cell-associated molecules as well as their responses to cytokines. A microarray analysis suggested several differentially expressed genes, including preferential expression of NKG2A/C receptors by NKR-P1B(+) NK cells. This was confirmed by staining with tetramers of RT.BM1, the putative ligand of CD94/NKG2, indicating that Ly49 and CD94/NKG2 receptors separate into distinct NK cell compartments. Further, expression of CD25 by Ly49s3(+) NK cells was associated with more rapid proliferation in response to IL-2 as compared with NKR-P1B(+) NK cells. Thus, certain inflammatory situations may preferentially expand the Ly49s3(+) NK cells. Moreover, freshly isolated Ly49s3(+) and NKR-P1B(+) NK cells produce similar amounts of cytokines, and a minor Ly49s3(-)NKR-P1B(-) double-negative NK subset appears to be hyporesponsive based on its significantly lower IFN-gamma production. Collectively, our data demonstrate divergent profiles of NKR-P1B(+) and Ly49s3(+) NK cells, indicating distinct tasks in vivo.

Oxytocin (OT) is becoming increasingly established as a prosocial neuropeptide in humans with therapeutic potential in treatment of social, cognitive, and mood disorders. However, the potential of OT as a general facilitator of human learning and empathy is unclear. The current double-blind experiments on healthy adult male volunteers investigated first whether treatment with intranasal OT enhanced learning performance on a feedback-guided item-category association task where either social (smiling and angry faces) or nonsocial (green and red lights) reinforcers were used, and second whether it increased either cognitive or emotional empathy measured by the Multifaceted Empathy Test. Further experiments investigated whether OT-sensitive behavioral components required a normal functional amygdala. Results in control groups showed that learning performance was improved when social rather than nonsocial reinforcement was used. Intranasal OT potentiated this social reinforcement advantage and greatly increased emotional, but not cognitive, empathy in response to both positive and negative valence stimuli. Interestingly, after OT treatment, emotional empathy responses in men were raised to levels similar to those found in untreated women. Two patients with selective bilateral damage to the amygdala (monozygotic twins with congenital Urbach-Wiethe disease) were impaired on both OT-sensitive aspects of these learning and empathy tasks, but performed normally on nonsocially reinforced learning and cognitive empathy. Overall these findings provide the first demonstration that OT can facilitate amygdala-dependent, socially reinforced learning and emotional empathy in men.

Nitric oxide (NO) mediates intercellular signaling through activation of its receptor, soluble guanylyl cyclase (sGC), leading to elevation of intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels. Through this signal transduction pathway, NO regulates a diverse range of physiological effects, from vasodilatation and platelet disaggregation to synaptic plasticity. Measurement of sGC activity has traditionally been carried out using end-point assays of cGMP accumulation or by transfection of cells with "detector" proteins such as fluorescent proteins coupled to cGMP binding domains or cyclic nucleotide gated channels. Here we report a simpler approach: the use of a fluorescently labeled substrate analog, mant-GTP (2'-O-(N-methylanthraniloyl) guanosine 5'-triphosphate), which gives an increase in emission intensity after enzymatic cyclization to mant-cGMP. Activation of purified recombinant sGC by NO led to a rapid rise in fluorescence intensity within seconds, reaching a maximal 1.6- to 1.8-fold increase above basal levels. The sGC inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), eliminated the fluorescence increase due to NO, and the synergistic activator of sGC, BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine), increased the rate at which the maximal fluorescence increase was attained. High-performance liquid chromatography (HPLC) confirmed the formation of mant-cGMP product. This real-time assay allows the progress of purified sGC activation to be quantified precisely and, with refinement, could be optimized for use in a cellular environment.