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Magnuson [J. Acoust. Soc. Am. 137, 1481-1492 (2015)] makes claims for Interactive Activation (IA) models and against Adaptive Resonance Theory (ART) models of speech perception. Magnuson also presents simulations that claim to show that the TRACE model can simulate phonemic restoration, which was an explanatory target of the cARTWORD ART model. The theoretical analysis and review herein show that these claims are incorrect. More generally, the TRACE and cARTWORD models illustrate two diametrically opposed types of neural models of speech and language. The TRACE model embodies core assumptions with no analog in known brain processes. The cARTWORD model defines a hierarchy of cortical processing regions whose networks embody cells in laminar cortical circuits as part of the paradigm of laminar computing. cARTWORD further develops ART speech and language models that were introduced in the 1970s. It builds upon Item-Order-Rank working memories, which activate learned list chunks that unitize sequences to represent phonemes, syllables, and words. Psychophysical and neurophysiological data support Item-Order-Rank mechanisms and contradict TRACE representations of time, temporal order, silence, and top-down processing that exhibit many anomalous properties, including hallucinations of non-occurring future phonemes. Computer simulations of the TRACE model are presented that demonstrate these failures.

We describe a large Lebanese family with two affected members, a young female proband and her male cousin, who had multisystem involvement including profound global developmental delay, severe hypotonia and weakness, respiratory insufficiency, blindness, and lactic acidemia-findings consistent with an underlying mitochondrial disorder. Whole-exome sequencing was performed on DNA from the proband and both parents. The proband and her cousin carried compound heterozygous mutations in the PMPCA gene that encodes for α-mitochondrial processing peptidase (α-MPP), a protein likely involved in the processing of mitochondrial proteins. The variants were located close to and postulated to affect the substrate binding glycine-rich loop of the α-MPP protein. Functional assays including immunofluorescence and western blot analysis on patient's fibroblasts revealed that these variants reduced α-MPP levels and impaired frataxin production and processing. We further determined that those defects could be rescued through the expression of exogenous wild-type PMPCA cDNA. Our findings link defective α-MPP protein to a severe mitochondrial disease.

Background: The aim of this study was to compare the outcomes after subretinal recombinant tissue plasminogen activator (rtPA) treatment for subretinal hemorrhages (SRH), subpigment epithelial hemorrhages (SPH), and combined subretinal and subpigment epithelial hemorrhages (CH)., Methods: An observational analysis of patients treated with subretinal rtPA was performed. The primary endpoint was the assessment of visual improvement (best-corrected visual acuity, BCVA) after surgery. Secondary endpoints were evaluation of the maximal hemorrhage diameter (MHD) and central macular thickness (CMT) measured by spectral domain optical coherence tomography., Results: From a total of 83 eyes included in the study, 19 eyes showed SRH, 11 eyes SPH, and 53 eyes CH. For SRH and CH, the mean BCVA, MHD, and CMT improved significantly (p < 0.05). For patients with SPH, both the mean MHD and CMT decreased significantly (p < 0.05), whereas the mean BCVA improved only slightly after surgery (p = 0.28)., Conclusion: Vitrectomy combined with subretinal rtPA injection and gas or air tamponade has a strong functional and anatomical effect on both SRH and CH and also seems to slightly improve the anatomical outcome in SPH., (© 2016 S. Karger AG, Basel.)

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome associated with ribosomal protein (RP) gene mutations. Recent studies have also demonstrated an increased risk of cancer predisposition among DBA patients. In this study, we report the formation of soft tissue sarcoma in the Rpl5 and Rps24 heterozygous mice. Our observation suggests that even though one wild-type allele of the Rpl5 or Rps24 gene prevents anemia in these mice, it still predisposes them to cancer development.

How are sequences of events that are temporarily stored in a cognitive working memory unitized, or chunked, through learning? Such sequential learning is needed by the brain in order to enable language, spatial understanding, and motor skills to develop. In particular, how does the brain learn categories, or list chunks, that become selectively tuned to different temporal sequences of items in lists of variable length as they are stored in working memory, and how does this learning process occur in real time? The present article introduces a neural model that simulates learning of such list chunks. In this model, sequences of items are temporarily stored in an Item-and-Order, or competitive queuing, working memory before learning categorizes them using a categorization network, called a Masking Field, which is a self-similar, multiple-scale, recurrent on-center off-surround network that can weigh the evidence for variable-length sequences of items as they are stored in the working memory through time. A Masking Field hereby activates the learned list chunks that represent the most predictive item groupings at any time, while suppressing less predictive chunks. In a network with a given number of input items, all possible ordered sets of these item sequences, up to a fixed length, can be learned with unsupervised or supervised learning. The self-similar multiple-scale properties of Masking Fields interacting with an Item-and-Order working memory provide a natural explanation of George Miller's Magical Number Seven and Nelson Cowan's Magical Number Four. The article explains why linguistic, spatial, and action event sequences may all be stored by Item-and-Order working memories that obey similar design principles, and thus how the current results may apply across modalities. Item-and-Order properties may readily be extended to Item-Order-Rank working memories in which the same item can be stored in multiple list positions, or ranks, as in the list ABADBD. Comparisons with other models, including TRACE, MERGE, and TISK, are made.

It is well established that the secretion of thrombospondin-1 (TSP-1) by activated stromal cells and its accumulation in the tumor microenvironment during dysplasia inhibits primary tumor growth through inhibition of angiogenesis. This inhibitory function of TSP-1 is actuated either by inhibiting MMP9 activation and the release of VEGF from extracellular matrix or by an interaction with CD36 on the surface of endothelial cells resulting in an increase in apoptosis. In contrast, several published articles have also shown that as tumor cells become more invasive and enter the early stage of carcinoma, they up-regulate TSP-1 expression, which may promote invasion and migration. In our in vivo studies using the polyoma middle T antigen (PyT) transgenic mouse model of breast cancer, we observed that the absence of TSP-1 significantly increased the growth of primary tumors, but delayed metastasis to the lungs. In this study, we propose a mechanism for the promigratory function of TSP-1 in mouse mammary tumor cells in vitro . We demonstrate the correlations between expression of TSP-1 and its receptor integrin α3β1, which is considered a promigratory protein in cancer cells. In addition we propose that binding of TSP-1 to integrin α3β1 is important for mediating actin filament polymerization and therefore, cell motility. These findings can help explain the dual functionality of TSP-1 in cancer progression.

Curiosity Driven Modular Incremental Slow Feature Analysis (CD-MISFA;) is a recently introduced model of intrinsically-motivated invariance learning. Artificial curiosity enables the orderly formation of multiple stable sensory representations to simplify the agent's complex sensory input. We discuss computational properties of the CD-MISFA model itself as well as neurophysiological analogs fulfilling similar functional roles. CD-MISFA combines 1. unsupervised representation learning through the slowness principle, 2. generation of an intrinsic reward signal through learning progress of the developing features, and 3. balancing of exploration and exploitation to maximize learning progress and quickly learn multiple feature sets for perceptual simplification. Experimental results on synthetic observations and on the iCub robot show that the intrinsic value system is essential for representation learning. Representations are typically explored and learned in order from least to most costly, as predicted by the theory of curiosity.

Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology.

Mechanisms governing the distinct temporal dynamics that characterize post-natal angiogenesis and lymphangiogenesis elicited by cutaneous wounds and inflammation remain unclear. RhoB, a stress-induced small GTPase, modulates cellular responses to growth factors, genotoxic stress and neoplastic transformation. Here we show, using RhoB null mice, that loss of RhoB decreases pathological angiogenesis in the ischaemic retina and reduces angiogenesis in response to cutaneous wounding, but enhances lymphangiogenesis following both dermal wounding and inflammatory challenge. We link these unique and opposing roles of RhoB in blood versus lymphatic vasculatures to the RhoB-mediated differential regulation of sprouting and proliferation in primary human blood versus lymphatic endothelial cells. We demonstrate that nuclear RhoB-GTP controls expression of distinct gene sets in each endothelial lineage by regulating VEZF1-mediated transcription. Finally, we identify a small-molecule inhibitor of VEZF1-DNA interaction that recapitulates RhoB loss in ischaemic retinopathy. Our findings establish the first intra-endothelial molecular pathway governing the phased response of angiogenesis and lymphangiogenesis following injury.

How are laminar circuits of neocortex organized to generate conscious speech and language percepts? How does the brain restore information that is occluded by noise, or absent from an acoustic signal, by integrating contextual information over many milliseconds to disambiguate noise-occluded acoustical signals? How are speech and language heard in the correct temporal order, despite the influence of contexts that may occur many milliseconds before or after each perceived word? A neural model describes key mechanisms in forming conscious speech percepts, and quantitatively simulates a critical example of contextual disambiguation of speech and language; namely, phonemic restoration. Here, a phoneme deleted from a speech stream is perceptually restored when it is replaced by broadband noise, even when the disambiguating context occurs after the phoneme was presented. The model describes how the laminar circuits within a hierarchy of cortical processing stages may interact to generate a conscious speech percept that is embodied by a resonant wave of activation that occurs between acoustic features, acoustic item chunks, and list chunks. Chunk-mediated gating allows speech to be heard in the correct temporal order, even when what is heard depends upon future context., (© 2011 Acoustical Society of America)

CD36 plays a critical role in the inhibition of angiogenesis through binding to the type 1 repeats of thrombospondin-1 (TSP-1) and activating Fyn tyrosine kinase and MAPK pathways. Here, we reveal a novel association of CD36 with VEGFR-2 and spleen tyrosine kinase (Syk). We also address the correlation between the expression of CD36 and Syk by demonstrating that overexpression of CD36 in HUVECs up-regulates endogenous Syk expression. We also define a new role for TSP-1 and CD36 in the activation of the VEGFR-2 signaling pathway that requires Syk. Our findings also identify a role for Syk as a stimulator of VEGF-A-induced angiogenesis by increasing phosphorylation of Y1175 in VEGFR-2, which is a major tyrosine for promoting VEGF-A-induced endothelial cell migration. Together, these studies introduce a new signaling pathway for TSP-1, CD36, and Syk, and address the role of these proteins in regulating the angiogenic switch.

RhoB is an early-response gene whose expression is elevated by multiple cellular stresses; this gene plays an important role in cancer, macrophage motility, and apoptosis. These factors are essential for the onset of type 1 diabetes mellitus and related complications. This study explores the role of RhoB in β-cell depletion and hyperglycemia-associated complications and tests whether the pleiotropic effect of statins on glycemic control is RhoB dependent. We induced β-cell depletion in RhoB(+/+), RhoB(+/-), and RhoB(-/-) mice with streptozotocin (STZ). Diabetic status was assessed by glucose tolerance and pancreatic islet loss. RhoB(-/-) mice showed a significant reduction in the severity of STZ-induced diabetes; only 13% of the STZ-treated RhoB-null animals became hyperglycemic, as opposed to 61% of the wild-type controls. Diabetes-related complications, such as wound healing rate and onset of nephropathy, were also assessed. Hyperglycemic RhoB(-/-) mice had fewer signs of nephropathy and showed faster wound healing than RhoB(+/+) animals. After assessing the diabetic status of mice treated simultaneously with STZ and simvastatin, we conclude that the effect of statins in improving glycemic control is RhoB independent. We propose that RhoB is a modifier of diabetes, important for the induction of β-cell loss. Suppression of RhoB expression may have potential application in the treatment of diabetes and associated complications., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Vascular endothelial growth factor (VEGF) is a well-established stimulator of vascular permeability and angiogenesis, whereas thrombospondin-1 (TSP-1) is a potent angiogenic inhibitor. In this study, we have found that the TSP-1 receptors CD36 and beta1 integrin associate with the VEGF receptor 2 (VEGFR2). The coclustering of receptors that regulate angiogenesis may provide the endothelial cell with a platform for integration of positive and negative signals in the plane of the membrane. Thus, this complex may represent a molecular switch that regulates angiogenesis and determines endothelial cell behavior. In this context, physiological levels of TSP-1 appear to support VEGFR2 function on both the cellular and tissue level, because phosphorylation of VEGFR2 and vascular permeability in response to VEGF are decreased in TSP-1-null mice and isolated endothelial cells. A therapeutic agent based on the antiangiogenic domain of TSP-1, designated 3TSR (for three TSP-1 type 1 repeats), has significant antiangiogenic and antitumor efficacy. Systemic treatment of wild-type mice with 3TSR significantly decreased VEGF-induced permeability. Consistent with this result, VEGF-stimulated phosphorylation of VEGFR2 was also significantly decreased in lung extracts from 3TSR-treated mice. Moreover, 3TSR significantly decreased VEGF-stimulated VEGFR2 phosphorylation in human dermal microvascular endothelial cells in culture. Taken together, the results indicate that TSP-1 and 3TSR modulate the function of VEGFR2.

Thrombospondin-1 (TSP-1) has been proposed to have both pro-metastatic and anti-metastatic properties. To elucidate its role in breast cancer metastasis, we compared tumor progression in the polyomavirus middle T antigen (Pyt) transgenic mouse and the TSP-1-null Pyt transgenic mouse. We characterized the tumors in these mice at 45, 60 and 90 days of age. Tumor size, areas of necrosis, macrophage infiltration, levels of active and total TGF-beta, vessel morphology, and lung and blood metastasis were measured in these mice. Mammary tumors were larger in the TSP-1-null mouse, and vessels were larger, but fewer in number in these tumors. The level of total TGF-beta was significantly higher in the Pyt tumors at 90 days of age. Importantly, significantly fewer metastases were observed in the lungs of the TSP-1-null/Pyt mouse. Primary Pyt tumor cells were more migratory than TSP-1-null Pyt tumor cells on collagen. Treatment of Pyt mice with recombinant proteins that contain the type-1 repeats of TSP-1 resulted in decreased primary tumor growth and metastasis. Sequences that are involved in CD36 binding and those required for TGF-beta activation mediated the inhibition of primary tumor growth. Thus, TSP-1 in the mammary tumor microenvironment inhibits angiogenesis and tumor growth, but promotes metastasis to the lung in the Pyt transgenic mouse. The ability of TSP-1 to support metastasis correlates with its ability to promote tumor cell migration.

Bacterial diarrheagenic heat-stable enterotoxins induce colon cancer cell cytostasis by targeting guanylyl cyclase C (GCC) signaling. Anticancer actions of these toxins are mediated by cyclic guanosine 3',5'-monophosphate (cGMP)-dependent influx of Ca2+ through cyclic nucleotide-gated channels. However, prolonged stimulation of GCC produces resistance in tumor cells to heat-stable enterotoxin-induced cytostasis. Resistance reflects rapid (tachyphylaxis) and slow (bradyphylaxis) mechanisms of desensitization induced by cGMP. Tachyphylaxis is mediated by cGMP-dependent protein kinase, which limits the conductance of cyclic nucleotide-gated channels, reducing the influx of Ca2+ propagating the antiproliferative signal from the membrane to the nucleus. In contrast, bradyphylaxis is mediated by cGMP-dependent allosteric activation of phosphodiesterase 5, which shapes the amplitude and duration of heat-stable enterotoxin-dependent cyclic nucleotide accumulation required for cytostasis. Importantly, interruption of tachyphylaxis and bradyphylaxis restores cancer cell cytostasis induced by heat-stable enterotoxins. Thus, regimens that incorporate cytostatic bacterial enterotoxins and inhibitors of cGMP-mediated desensitization offer a previously unrecognized therapeutic paradigm for treatment and prevention of colorectal cancer.

Guanylyl cyclase C and accumulation of cGMP induced by bacterial heat-stable enterotoxins (STs) promote colon cancer cell cytostasis, serving as a tumor suppressor in intestine. Conversely, capacitative calcium entry through store-operated calcium channels (SOCs) is a key signaling mechanism that promotes colon cancer cell proliferation. The present study revealed that proliferative signaling by capacitative calcium entry through SOCs opposes and is reciprocally coupled to cytostasis mediated by guanylyl cyclase C in T84 human colon carcinoma cells. Elimination of capacitative calcium entry employing 2-aminoethoxydiphenylborate (2-APB), a selective inhibitor of SOCs, potentiated cytostasis induced by ST. Opposition of ST-induced cytostasis by capacitative calcium entry reflects reciprocal inhibition of guanylyl cyclase C signaling. Calcium entry through SOCs induced by the calcium-ATPase inhibitor thapsigargin or the receptor agonists UTP or carbachol inhibited guanylyl cyclase C-dependent cGMP accumulation. This effect was mimicked by the calcium ionophore ionomycin and blocked by 2-APB and intracellular 1,2-bis(o-amino-5,5'-dibromophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), a chelator of calcium. Moreover, regulation by capacitative calcium entry reflected ligand-dependent sensitization of guanylyl cyclase C to inhibition by that cation. Although basal catalytic activity was refractory, ST-stimulated guanylyl cyclase C was inhibited by calcium, which antagonized binding of magnesium to allosteric sites required for receptor-effector coupling. These observations demonstrate that reciprocal regulation of guanylyl cyclase C signaling by capacitative calcium entry through SOCs represents one limb of a coordinated mechanism balancing colon cancer cell proliferation and cytostasis. They suggest that combining guanylyl cyclase C agonists and SOC inhibitors offers a novel paradigm for cGMP-directed therapy and prevention for colorectal tumors.

Calcium and guanosine-3',5'-cyclic monophosphate (cGMP) are second messenger molecules that regulate opposing physiological functions, reflected in the reciprocal regulation of their intracellular concentrations, in many systems. Indeed, cGMP and Ca2+ constitute discrete points of integration between multiple cell signaling cascades in both convergent and parallel pathways. This chapter describes the molecular mechanisms regulating intracellular Ca2+ and cGMP, and their integration in specific cellular responses.

While keratinocytes go through the terminal differentiation and move toward the outer layers of epidermis, multiple proteins become sequentially incorporated into the cornified cell envelope. We have identified through yeast two-hybrid screening a novel protein, periphilin, interacting with periplakin, which is known as a precursor of the cornified cell envelope. Periphilin gene at chromosome 12q12 gives rise to multiple alternatively spliced transcripts. A monoclonal antibody detected the keratinocyte-specific periphilin isoform in undifferentiated keratinocytes in speckle-type nuclear granules and at the nuclear membrane, but in differentiated keratinocytes periphilin localized to the cell periphery and at cell-cell junctions, colocalizing there with periplakin. From cultured keratinocytes, periphilin was solubilized only after urea extraction, indicating the highly insoluble character of this protein. The nuclear localization, mediated through the N-terminal sequences of periphilin protein, is a prerequisite for the formation of insoluble complexes. Although the globular N terminus of periphilin was necessary for the interaction with the periplakin tail, the keratinocyte-specific C terminus was responsible for the homodimerization. The C-terminal helical domain, composed of multiple heptad repeats, serves as a substrate for cross-linking by transglutaminases but also was specifically cleaved by caspase-5 in vitro. In conclusion, the localization pattern and insolubility of periphilin indicate that this novel protein is potentially involved in epithelial differentiation and contributes to epidermal integrity and barrier formation.

The purpose of the present study was to characterize different beta-adrenoceptors (beta-ARs) and determine their role in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The beta-AR subtypes in the opossum IAS were investigated by functional in vitro, radioligand binding, Western blot, and reverse transcription-polymerase chain reaction (RT-PCR) studies. ZD 7114 [(S)-4-[2-hydroxy-3-phenoxypropylaminoethoxy]-N-(2-methoxyethyl)phenoxyacetamide], a selective beta(3)-AR agonist, caused a potent and concentration-dependent relaxation of the IAS smooth muscle that was antagonized by the beta(3)-AR antagonist SR 59230A [1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride]. Conversely, the IAS smooth muscle relaxation caused by beta(1)- and beta(2)-AR agonists (xamoterol and procaterol, respectively) was selectively antagonized by their respective antagonists CGP 20712 [(+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate salt] and ICI 118551. Saturation binding of [(125)I]iodocyanopindolol to beta-AR subtypes revealed the presence of a high-affinity site (K(d1) = 96.4 +/- 8.7 pM; B(max1) = 12.5 +/- 0.6 fmol/mg protein) and a low-affinity site (K(d2) = 1.96 +/- 1.7 nM; B(max2) = 58.7 +/- 4.3 fmol/mg protein). Competition binding with selective beta-AR antagonists revealed that the high-affinity site correspond to beta(1)/beta(2)-AR and the low affinity site to beta(3)-AR. Receptor binding data suggest the predominant presence of beta(3)-AR over beta(1)/beta(2)-AR. Western blot studies identified beta(1)-, beta(2)-, and beta(3)-AR subtypes. The presence of beta(1)-, beta(2)-, and beta(3)-ARs was further demonstrated by mRNA analysis using RT-PCR. The studies demonstrate a comprehensive functional and molecular characterization of beta(1)-, beta(2)-, and beta(3)-ARs in IAS smooth muscle. These studies may have important implications in anorectal and other gastrointestinal motility disorders.

Plectin, desmoplakin, and the 230-kDa bullous pemphigoid antigen (BPAG1), members of the plakin family of proteins, are multifunctional cytolinkers, connecting the cytoskeletal structures to the cell adhesion complexes. Envoplakin and periplakin are components of the cornified envelope, but less is known about their role in tissues other than the stratified epithelium. Our tissue-wide survey utilizing RT-PCR revealed that periplakin, like plectin and desmoplakin, has a wide tissue distribution, but envoplakin expression is limited to certain tissues only, and BPAG1 is clearly specific for epidermal keratinocytes. Plectin, desmoplakin and BPAG1 are known to bind to the intermediate filaments through their C-terminal domains. The short C-terminal domain of periplakin is composed only of the linker domain, a region highly homologous between the plakin proteins. Here we demonstrate, through the use of yeast two-hybrid assay, a specific interaction of the periplakin linker domain with keratin 8 and vimentin. Co-expression of each plakin linker domain with keratin 8 revealed that periplakin and BPAG1 linkers co-localize with keratin signals in HaCaT cells, plectin and desmoplakin linkers were detected both in the nucleus and in cytoplasm together with the overexpressed keratin 8, while envoplakin linker localized independently into the nucleus. These results suggest that, in spite of its high homology and structural similarity with envoplakin, periplakin is functionally closer to the well-characterized plakin proteins plectin and desmoplakin, and thus may function tissue-wide as a scaffolding protein in intermediate filament assembly.

Nitric oxide (NO), the principal endogenous ligand for soluble guanylate cyclase (sGC), stimulates that enzyme and accumulation of intracellular cGMP, which mediates many of the (patho) physiological effects of NO. Previous studies demonstrated that 2-substituted adenine nucleotides, including 2-methylthioATP (2MeSATP) and 2-chloroATP (2ClATP), allosterically inhibit guanylate cyclase C, the membrane-bound receptor for the Escherichia coli heat-stable enterotoxin in the intestine. The present study examined the effects of 2-substituted adenine nucleotides on crude and purified sGC. 2-Substituted nucleotides inhibited basal and NO-activated crude and purified sGC, when Mg2+ served as the substrate cation cofactor. Similarly, 2-substituted adenine nucleotides inhibited those enzymes when Mn2+, which activates sGC in a ligand-independent fashion, served as the substrate cation cofactor. Inhibition of sGC by 2-substituted nucleotides was associated with a decrease in Vmax, consistent with a noncompetitive mechanism. In contrast to guanylate cyclase C, 2-substituted nucleotides inhibited sGC by a guanine nucleotide-independent mechanism. These studies demonstrate that 2-substituted adenine nucleotides allosterically inhibit basal and ligand-stimulated sGC. They support the suggestion that allosteric inhibition by adenine nucleotides is a general characteristic of the family of guanylate cyclases. This allosteric inhibition is mediated by direct interaction of adenine nucleotides with sGC, likely at the catalytic domain in a region outside the substrate-binding site.

Cyclic GMP (cGMP) and Ca(2+) regulate opposing mechanisms in (patho)physiological processes reflected in the reciprocal regulation of their intracellular concentrations. Although mechanisms by which cGMP regulates [Ca(2+)](i) have been described, those by which Ca(2+) regulates [cGMP](i) are less well understood. In the present study, Ca(2+) inhibited purified sGC activated by sodium nitroprusside (SNP), a precursor of nitric oxide (NO), employing Mg-GTP as substrate in a concentration-dependent fashion, but was without effect on basal enzyme activity. Ca(2+) inhibited sGC stimulated by protoporphyrin IX or YC-1 suggesting that inhibition was not NO-dependent. In contrast, Ca(2+) was without effect on sGC activated by SNP employing Mn-GTP as substrate, demonstrating that inhibition did not reflect displacement of heme from sGC. Ligand activation of sGC unmasked negative allosteric sites of high (K(i) similar 10(-7) M) and low (K(i) approximately 10(-5) M) affinity for Ca(2+) that mediated noncompetitive and uncompetitive inhibition, respectively. Free Mg(2+) in excess of substrate did not alter the concentration-response relationship of Ca(2+) inhibition at high affinity sites, but produced a rightward shift in that relationship at low affinity sites. Similarly, Ca(2+) inhibition at high affinity sites was noncompetitive, whereas inhibition at low affinity sites was competitive, with respect to free Mg(2+). Purified sGC specifically bound (45)Ca(2+) in the presence of a 1000-fold excess of Mg(2+) and in the absence of activating ligands. These data suggest that sGC is a constitutive Ca(2+) binding protein whose allosteric function is conditionally dependent upon ligand activation.

Guanylyl cyclases are a family of enzymes that catalyze the conversion of GTP to cGMP. The family comprises both membrane-bound and soluble isoforms that are expressed in nearly all cell types. They are regulated by diverse extracellular agonists that include peptide hormones, bacterial toxins, and free radicals, as well as intracellular molecules, such as calcium and adenine nucleotides. Stimulation of guanylyl cyclases and the resultant accumulation of cGMP regulates complex signaling cascades through immediate downstream effectors, including cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cyclic nucleotide-gated ion channels. Guanylyl cyclases and cGMP-mediated signaling cascades play a central role in the regulation of diverse (patho)physiological processes, including vascular smooth muscle motility, intestinal fluid and electrolyte homeostasis, and retinal phototransduction. Topics addressed in this review include the structure and chromosomal localization of the genes for guanylyl cyclases, structure and function of the members of the guanylyl cyclase family, molecular mechanisms regulating enzymatic activity, and molecular sequences coupling ligand binding to catalytic activity. A brief overview is presented of the downstream events controlled by guanylyl cyclases, including the effectors that are regulated by cGMP and the role that guanylyl cyclases play in cell physiology and pathophysiology.