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We studied the inotropic responses to intravenous infusions of norepinephrine in nine conscious chronically instrumented dogs before and early (2-4 weeks) in the development of perinephritic hypertension; seven conscious dogs were studied later (approximately 14 weeks), during a more stable phase of hypertension. perinephritic hypertension was associated with a 24% increase in left ventricular (LV) mass during developing hypertension; no further increase was seen during the stable hypertension phase. LV end-systolic stress was increased early (p less than 0.01) but was normalized later. The LV end-systolic stress-volume relation demonstrated an enhanced contractile response to norepinephrine during developing hypertension, which returned toward control later in the course of stable hypertension. The LV dP/dt responses to norepinephrine (0.4 microgram/kg/min) were significantly greater during developing hypertension (7,509 +/- 337 mm Hg/sec, p less than 0.05) compared with the control period (4,737 +/- 286 mm Hg/sec) and returned toward the control value during stable hypertension (5,168 +/- 465 mm Hg/sec). The enhanced inotropic responses to norepinephrine in developing hypertension were preserved in the presence of ganglionic blockade, suggesting that the augmentation was not mediated via reflex mechanisms. These physiological responses were associated with an increase in beta-adrenergic receptor density, but no significant change in basal or maximal adenylate cyclase stimulation occurred during developing hypertension. Thus, in contrast to prior studies in anesthetized animals, the inotropic response to beta-adrenergic stimulation is not depressed in conscious dogs but is enhanced selectively during the development of hypertension and maintained during stable hypertension.

Summary In order to lower departmental costs in an ophthalmological outpatient department by reducing wastage, the stability of available chlorine at levels of 280 ppm and 560 ppm in litre solutions of sodium dichloroisocyanu rate was investigated over a three-week period. There was no significant decay in available chlorine at these levels in solutions kept at 20°C. Sodium dichloroisocyanu rate may be prepared on a weekly instead of a daily basis with an annual saving of £1200 to £1400. In busy ophthalmology outpatient depart­ ments, the tonometer prism is the most likely potential source for patient to patient spread of inapparent infection. Consultations may be separated by 15 minutes or less, and since good practice dictates the frequent use of the tonometer, a rapid effective disinfection pro­ cedure is essential. In 1985 following an adenovirus outbreak and with HIV infections in prospect, two of us (KM'C and VD) made recommendations to this end based on the available chlorine in a solution of sodium dichloroisocyanu rate

Left ventricular hypertrophy (LVH) develops as a compensatory response to myocardial dysfunction due to diverse causes, but is nonetheless a major risk factor for premature cardiovascular morbidity and mortality. It is thus unclear if regressing LVH is beneficial or may worsen patient outcome. To evaluate the effects of LVH regression, we developed a transgenic mouse model in which the expression of a familial hypertrophic cardiomyopathy (FHC)-inducing mutation (R403Q alpha-MHC) can be regulated in a temporal and dose-dependent manner. In this model, transgene expression can be shut off by feeding with a tetracycline analogue (doxycycline). Serial echocardiography and histology studies were performed in a cohort of mice expressing the FHC mutant (“gene-on”) and in wildtype (WT) littermates. A second cohort of WT and 403/+ mice was randomised to placebo or doxycycline (“gene off”) from 6 (Dox6) or 20 weeks (Dox20) and evaluated at 40 weeks of age. Compared to WT littermates, “gene on” 403/+ mice showed increased LV mass, LV end-diastolic diameter (LVDD) and left atrial diameter (LAD), and reduced fractional shortening (LVFS), with changes evident from 12 weeks of age. LV sections from 403/+ mice showed typical features of FHC: myofibre disarray and interstitial fibrosis. LV mass, LV function and myocardial histology were unchanged in both male and female placebo- vs Dox6 or Dox20 mice at 40 weeks (Table 1 ). Thus, consistent with the major LV thickening in FHC humans occurring in adolescence, overexpression of R403Q for only 6 weeks is sufficient to trigger the complete LVH phenotypic response. Moreover, switching off the genetic trigger for LVH in 403/+ mice at 6 weeks (prior to overt disease manifestation) or 20 weeks (established disease) does not induce regression of LVH or exacerbate contractile dysfunction. Interventions to induce LVH regression may, therefore, need to be directed at downstream factors in hypertrophic pathways. Table 1. Echo data for male WT and 403/+ mice aged 40 weeks

Background & Aims Despite high morbidity and mortality of alcoholic liver disease worldwide, the molecular mechanisms underlying alcohol-induced liver cell death are not fully understood. Transglutaminase 2 (TG2) is a cross-linking enzyme implicated in apoptosis. TG2 levels and activity are increased in association with various types of liver injury. However, how TG2 induces hepatic apoptosis is not known. Methods Human hepatic cells or primary hepatocytes from rats or TG2 +/+ and TG2 −/− mice were treated with ethanol. Mice were administered anti-Fas antibody or alcohol. Liver sections were prepared from patients with alcoholic steatohepatitis. Changes in TG2 levels, Sp1 cross-linking and its activities, expression of hepatocyte growth factor receptor, c-Met, and hepatic apoptosis were measured. Results Ethanol induced apoptosis in hepatic cells, enhanced activity and nuclear accumulation of TG2 as well as accumulation of cross-linked and inactivated Sp1, and reduced expression of the Sp1-responsive gene, c-Met . These effects were rescued by TG2 knockdown, restoration of functional Sp1, or addition of hepatocyte growth factor, whereas apoptosis was reproduced by Sp1 knockdown or TG2 overexpression. Compared with TG2 +/+ mice, TG2 −/− mice showed markedly reduced hepatocyte apoptosis and Sp1 cross-linking following ethanol or anti-Fas treatment. Treatment of TG2 +/+ mice with the TG2 inhibitors putrescine or cystamine blocked anti-Fas–induced hepatic apoptosis and Sp1 silencing. Moreover, enhanced expression of cross-linked Sp1 and TG2 was evident in hepatocyte nuclei of patients with alcoholic steatohepatitis. Conclusions TG2 induces hepatocyte apoptosis via Sp1 cross-linking and inactivation, with resultant inhibition of the expression of c-Met required for hepatic cell viability.

Previously, we showed that perturbations of protein transmembrane helices are manifested as one of three types of noncanonical structures (wide turns, tight turns, and kinks), which, compared with alpha-helices, are evident by distinctive Calpha(i)--Calpha(x) distances. In this study, we report the analysis of more than 3000 transmembrane helices in 244 crystal structures from which we identified 70 wide turns (29 proline- and 41 nonproline-induced). Based on differences in the Calpha(i)--Calpha(i)(-4) and Calpha(i)--Calpha(i)(-5) profiles, we show that wide turns can be subclassified into three distinct subclasses (W(1), W(2), and W(3)) that differ with regard to the number and position of backbone i --i-5 H-bonds formed N-terminal to the perturbing or signature proline or nonproline residue. Although wide turns generally produce changes in helical direction of 20 degrees to 30 degrees and a lateral shift in the helical axis, some of the W(3) subclass are associated with changes of5 degrees . We also show that the distinct architectural features of wide turns allow the carbonyl bond of the i-4th residue, which is located on the widened loop of a wide turn, to be directed away from the helical axis. This provides regions of flexibility within helical regions allowing, for example, unique opportunities for interhelical H-bonding, including interactions with glycine zipper motifs, and for ion and cofactor binding. Furthermore, differences in wide-turn subtype usage by related protein family members, such as the G-protein-coupled receptors rhodopsin and the beta2-adrenergic receptor, can significantly affect the orientation and position of residues critical for ligand binding and receptor activation.

Recent studies have demonstrated that phosphorylation of atrial natriuretic factor (ANF) (99-126) in vitro modulates the bioactivity of this hormone. The potential physiological relevance of this observation was revealed in latter studies showing that endogenous proANF can be 32PO4-biosynthetically labeled by primary cultured atrial myocytes and by atrial appendage explants. The site and extent of proANF phosphorylation were different, however, in these two model systems. Whereas proANF extracted from atrial explants was phosphorylated on the bioactive, carboxy (C)-terminal portion of the molecule [ANF(99-126)], cultured atrial myocytes phosphorylated proANF on the amino (N)-terminal portion of the prohormone molecule [ANF(1-98)]. It was the goal of this study, therefore, to determine whether the bioactive region of proANF, ANF(99-126), is phosphorylated in vivo. ProANF was obtained by acid extraction of isolated rat atrial secretory granules followed by purification using reverse phase-HPLC. Analysis of purified 125I-labeled proANF by isoelectric focusing (IEF) revealed two bands with isoelectric points of 5.3 and 5.0. The more acidic band comigrated on IEF gels with 32PO4-biosynthetically labeled proANF obtained from primary cultures of atrial myocytes, suggesting that this species of proANF represented endogenously phosphorylated proANF. The more acidic band accounted for only 15-25% of the total proANF found in the mature atrial secretory granule. The phosphorylation state of ANF(99-126) produced by thrombin cleavage of secretory granule proANF was examined using three complementary methods: 1) cation-exchange HPLC, 2) amino-terminal amino acid sequence analysis and 3) anti-ANF(99-105) antibody immunoreactivity. Evidence from these three independent approaches indicated that proANF is not phosphorylated on the C-terminal portion of the molecule in vivo. Therefore, phosphorylation is not a physiological regulator of ANF(99-126) bioactivity.

The G-protein involved in alpha 1-adrenergic receptor signaling was identified using two different approaches. First, purified rat liver membranes were incubated with [alpha-32P]GTP in the absence or presence of the adrenergic agonist (-)-epinephrine, or in the presence of GTP. After UV irradiation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography, covalent labeling of a number of proteins was apparent and could be blocked by unlabeled GTP. In the preparation treated with (-)-epinephrine alone, labeling of a 74-kDa species was markedly enhanced. Enhanced labeling of 40-50-kDa species was also observed. Labeling of the 74-kDa protein was also evident in similarly treated membranes prepared from FRTL-5 thyroid cells, which contain abundant alpha 1-adrenergic receptors, but not in those prepared from turkey erythrocytes or NIH 3T3 fibroblasts, which are essentially devoid of alpha 1-receptors. Second, alpha 1-agonist-receptor-G-protein ternary complex formation was induced by incubating purified rat liver membranes with (-)-epinephrine. Rauwolscine (10(-7) M) and (+/-)-propranolol (10(-6) M) were included to prevent activation of alpha 2- and beta-adrenergic receptors by (-)-epinephrine. The ternary complex of hormone, receptor, and G-protein was solubilized, partially purified using heparin- and wheat germ agglutinin-agarose, and reconstituted into phospholipid vesicles. The vesicles displayed agonist-stimulated guanosine 5'-O-3-thiotriphosphate (GTP gamma S) binding that was blocked by phentolamine (10(-4) M). By contrast, stimulation of GTP gamma S binding was not evident when the vesicles were incubated with the beta-agonist, isoproterenol. Incubation of the vesicles with [alpha-32P]GTP or [alpha-32P]azido-GTP in the presence of (-)-epinephrine, followed by photolysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography, resulted in the covalent labeling of a 74-kDa protein. Labeling of this protein could be blocked by preincubation with phentolamine or unlabeled GTP. These findings provide direct evidence for the coupling of the alpha 1-adrenergic receptor to a previously uncharacterized G-protein (termed Gh), which has an apparent molecular mass of approximately 74 kDa.

The heart is richly innervated by sympathetic nerves, and both acute and chronic regulation of cardiac function via sympathetically released catecholamines acting on cardiomyocyte adrenergic receptors (ARs), is critical for circulatory homeostasis. Cardiomyocytes express alpha 1A- and alpha 1B-, and beta 1- and beta 2-AR subtypes, which are all members of the G-protein-coupled receptor superfamily that signal via interaction with heterotrimeric G-proteins. Cardiac function - both inotropy and chronotropy - is regulated predominantly by beta 1-AR. Activation of alpha 1-ARs also results in increased contractility, as well as changes in the electrophysiological properties and metabolic responses of the heart. Nonetheless, there is little evidence that cardiac alpha 1-ARs play a major functional role under normal physiological conditions. In pathological settings, alpha 1-ARs may function in a compensatory fashion to maintain cardiac inotropy when the beta-AR system is downregulated and uncoupled from G-proteins and effectors. In addition, as we consider here, recent evidence from clinical studies and from genetically engineered animal models indicates that alpha 1-ARs are importantly involved in both developmental cardiomyocyte growth, as well as pathological hypertrophy. In the presence of pressure overload or with myocardial infarction, activation of alpha 1-ARs, particularly the alpha 1A-subtype, also appears to produce important pro-survival effects at the level of the cardiomyocyte, and to protect against maladaptive cardiac remodelling and decompensation to heart failure.

Background G-CSF is a novel therapy for ischemic heart disease (IHD), but its safety and efficacy in acute or ‘no option’ refractory IHD (R-IHD) remains unclear. Also, the few completed studies generally did not adapt G-CSF to IHD patients but merely administered G-CSF according to usual hematological practice. To address these issues we evaluated the safety and efficacy of G-CSF therapy in 20 patients (18 males, 2 females, mean age 62.4) with R-IHD. Three approaches were incorporated to enhance the efficacy of G-CSF in R-IHD: Repeated, controlled myocardial ischemia was induced during G-CSF therapy to activate myocardial chemokine expression as a homing signal for mobilized angioblasts; Two cycles of G-CSF were administered to enhance the likelihood of clinically meaningful angiogenesis; During the second cycle of G-CSF an intracoronary infusion of CD133+ cells was performed. Methods After baseline cardiac assessment (CA) (angina questionnaire, exercise stress test (EST), nuclear Sestamibi (MIBI) and dobutamine stress echocardiographic (DSE) imaging), patients received open-label G-CSF commencing at 10μg/kg s/c for 5 days, with an EST on days 4 and 6 (to facilitate angioblast trafficking). After 3 months CA and the same regimen of G-CSF +ESTs were repeated but in addition, leukapheresis and a randomized double blinded intracoronary infusion of CD133+ or unselected cells were performed. Final CA was 3 months thereafter. Results Eight events fulfilled pre-specified adverse event (AE) criteria, but despite the poor outlook and co-morbidities of R-IHD patients there were no deaths or AEs resulting in long-term sequelae. Administration of 2 cycles of G-CSF resulted in step-wise improvements in angina frequency, quality of life, EST performance and Duke treadmill score (all p < 0.005). Pre-specified MIBI and DSE results trended towards improvement (p > 0.1), while a composite post hoc computation indicated improved ischemia/perfusion compared to baseline (p = 0.025). Intracoronary infusion of CD133+ or unselected cells was safe but neither enhanced the response to G-CSF. Conclusions Administering G-CSF to R-IHD patients may be performed safely and shows promise as a therapy for debilitating angina. A phase II/III study of this therapy is warranted.

Store-operated Ca 2+ entry was investigated in isolated mouse sinoatrial nodes (SAN) dissected from right atria and loaded with Ca 2+ indicators. Incubation of the SAN in Ca 2+ -free solution caused a substantial decrease in resting intracellular Ca 2+ concentration ([Ca 2+ ] i ) and stopped pacemaker activity. Reintroduction of Ca 2+ in the presence of cyclopiazonic acid (CPA), a sarcoplasmic reticulum Ca 2+ pump inhibitor, led to sustained elevation of [Ca 2+ ] i , a characteristic of store-operated Ca 2+ channel (SOCC) activity. Two SOCC antagonists, Gd 3+ and SKF-96365, inhibited 72±8% and 65±8% of this Ca 2+ influx, respectively. SKF-96365 also reduced the spontaneous pacemaker rate to 27±4% of control in the presence of CPA. Because members of the transient receptor potential canonical (TRPC) gene family may encode SOCCs, we used RT-PCR to examine mRNA expression of the 7 known mammalian TRPC isoforms. Transcripts for TRPC1, 2, 3, 4, 6, and 7, but not TRPC5, were detected. Immunohistochemistry using anti-TRPC1, 3, 4, and 6 antibodies revealed positive labeling in the SAN region and single pacemaker cells. These results indicate that mouse SAN exhibits store-operated Ca 2+ activity which may be attributable to TRPC expression, and suggest that SOCCs may be involved in regulating pacemaker firing rate.

Background & Aims: Mallory body (MB) inclusions are a characteristic feature of several liver disorders and share similarities with cytoplasmic inclusions observed in neural diseases and myopathies. MBs consist primarily of keratins 8 and 18 (K8/K18), require a K8-greater-than-K18 ratio for their formation, and contain glutamine-lysine cross-links generated by transglutaminase (TG). We hypothesized that protein transamidation is essential for MB formation. Methods: Because TG2 is the most abundant hepatocyte TG, we tested our hypothesis using TG2−/− and their wild-type counterpart mice fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), an established MB inducer. Keratin cross-linking was further examined using recombinant proteins or transgenic mice that overexpress K8 or K18. Results: TG2−/− livers have markedly reduced TG2 activity as compared with TG2+/+ livers. The DDC-fed TG2−/− mice have dramatic decreases in MB formation and liver hypertrophy response as contrasted with DDC-fed TG2+/+ mice. Despite similar hepatocellular damage, TG2−/− mice had more gallstones, jaundice, and ductal proliferation than wild-type mice. Inhibition of MB formation in TG2−/− mice was associated with marked attenuation of ubiquitination and K8-containing protein cross-linking. MB formation and resolution paralleled the generation then disappearance of cross-linked K8, respectively. K8 is a preferential TG2 substrate when compared to K18, as examined in vitro or in DDC-fed transgenic mice that overexpress K8 or K18. Conclusions: We demonstrate an essential role for TG2 in determining injury-mediated liver enlargement and the necessity of K8 and TG2 for generating cross-linked keratins and MBs. The role of TG in inclusion formation might extend to nonkeratin intermediate filament protein-related diseases.

Severe thrombocytopenia in association with G-CSF therapy is extremely rare. Here we report a case of profound thrombocytopenia in a 57-year-old male with refractory cardiac ischemia, who received G-CSF during an angiogenesis trial. After 5 days of G-CSF therapy (10 μg/kg/day) the platelet count fell progressively to a nadir of 5 × 109/L. The patient received steroid, immunoglobulin and platelet support and recovered without sequelae. Subsequent investigations suggested an underlying immune-mediated thrombocytopenia, which we hypothesize was exacerbated by G-CSF therapy. Am. J. Hematol., 2007. © 2006 Wiley-Liss, Inc.

Transglutaminases are a family of calcium- and thiol-dependent acyl transferases that catalyze the formation of an amide bond between the γ-carboxamide groups of peptide-bound glutamine residues and the primary amino groups in various compounds, including the ε-amino group of lysines in certain proteins. As a result, these enzymes effect posttranslational modification of proteins by amine incorporation, or stabilization of protein assemblies by their cross-linking; such actions profoundly influence critical biological processes such as blood clotting and protection from infection and dehydration by establishing the barrier function of skin. In addition, transglutaminases have other more diverse actions, including involvement in signaling by the superfamily of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) in one of three ways: (i) through actions as guanosine triphosphate–binding proteins that activate intracellular effectors, such as phospholipase C; (ii) by cross-linking GPCR monomers to enhance signaling as a result of covalent dimer formation; or (iii) by interacting with an apparent growth inhibitory orphan GPCR, GPR56, to limit metastatic spread of melanoma cells. The implications of these receptor-coupled actions of transglutaminases are discussed.

Adrenergic receptors are critical mediators of sympaiMolecular Cardiology Program, Vihysiological responses. Activated by the neurotransmitter and neurohormone, norepinephrine and epinephrine, released from sympathetic nerve endings and the adrenal medulla, respectively, they play a central role in this evolutionarily ancient defense system that regulates many physiological functions, including those involved in circulatory, metabolic, respiratory, and central nervous system homeostasis. In addition, alterations in the regulation and molecular structure of adrenergic receptors have been implicated in a variety of diseases, and drugs targeting these receptors are important and widely used therapeutics. The molecular cloning of the first adrenergic receptor in 1986 revealed structural homology with the functionally related rhodopsin visual transduction system—a finding that led to the realization that these receptors formed a new superfamily of proteins, now known as G protein-coupled receptors. Since that time, a plethora of structure-function studies have provided major insights into the molecular determinants of adrenergic receptor ligand-binding, activation, and signaling, many of which are relevant not only to the adrenergic receptor family but also, more generally, to the broader superfamily of G protein-coupled receptors. These advances in our understanding of adrenergic receptor activation, regulation, and functioning are reviewed in this chapter.

This Perspective focuses on the alpha(1D)-adrenergic receptor (AR), the often neglected sibling of the alpha(1)-AR family. This neglect is due in part to its poor cell-surface expression. However, it has recently been shown that dimerization of the alpha(1D)-AR with either the alpha(1B)-AR or the beta(2)-AR increases alpha(1D)-AR cell-surface expression, and in this issue of Molecular Pharmacology, Hague et al. (p. 45) demonstrate that dimerization of the alpha(1D)-AR with the alpha(1B)-AR not only leads to increased cell-surface expression but also results in the formation of a novel functional entity.

We previously reported that transgenic (TG) mice with cardiac-restricted alpha(1A)-adrenergic receptor (alpha(1A)-AR)-overexpression showed enhanced contractility, but no hypertrophy. Since chronic inotropic enhancement may be deleterious, we investigated if long-term, cardiac function and longevity are compromised. alpha(1A)-TG mice, but not their non-TG littermates (NTLs), showed progressive loss of left ventricular (LV) hypercontractility (dP/dt(max): 14,567+/-603 to 11,610+/-915 mmHg/s, P0.05, A1A1 line: 170-fold overexpression; and 13,625+/-826 to 8322+/-682 mmHg/s, respectively, P0.05, A1A4 line: 112-fold overexpression, at 2 and 6 months, respectively). Both TG lines developed LV fibrosis, but not LV dilatation or hypertrophy, despite activation of hypertrophic signaling pathways. Microarray and real time RT-PCR analyses revealed activation of matricellular protein genes, including those for thrombospondin 1, connective tissue growth factor and tenascin C, but not transforming growth factor beta1. Life-span was markedly shortened (mean age at death: 155 days, A1A1 line; 224 days, A1A4 line compared with NTLs:300 days). Telemetric electrocardiography revealed that death in the alpha(1A)-AR TG mice was due to cardiac standstill preceded by a progressive diminution in QRS amplitude, but not by arrhythmias. The QRS changes and sudden death could be mimicked by alpha(1)-AR activation, and reversed preterminally by alpha(1)-AR blockade, suggesting a relationship to stress- or activity-associated catecholamine release. Thus, long-term augmentation of cardiac alpha(1A)-adrenergic drive leads to premature death and progressive LV fibrosis with reactivation of matricellular protein genes. To our knowledge this is the first evidence in vivo for a role of the alpha(1A)-AR in ventricular fibrosis and in pathological cardiac remodeling.

Improving HIV/AIDS prevention efforts, especially those focused on young people, is a key challenge in the fight against the pandemic. Since the mid-1990s the Henry J. Kaiser Family Foundation has partnered with U.S.-based media companies on comprehensive HIV/AIDS campaigns. This paper outlines the unique public education model that it is using, and how Kaiser is expanding this work globally by working with some of the largest media companies in the world to undertake new initiatives in Russia and India.

Proteolytic processing is a primary means of biological control. Exopeptidases use terminal anchoring interactions to restrict cleavage at peptide substrate N or C termini. In contrast, internal peptide bond targeting by endopeptidases is through context-driven recognition. Angiotensin I-converting enzyme (ACE), a zinc metalloproteinase, has tandem duplicate catalytic domains, N- and C-terminal, each of which is a dual specificity enzyme with exo- and endocarboxypeptidase activities. The mechanisms by which ACE evolved from its endopeptidase ancestors as a dual specificity enzyme have not been defined. Based on kinetic studies of wild-type and mutant forms of the C-terminal catalytic domain of human ACE and of the ACE substrates angiotensin I, substance P, and bradykinin, as well as considerations of the ACE x-ray structure, we provide evidence that the acquisition of its exopeptidase activity is due to novel evolutionary specializations. These involve not only interactions between the S(2)' subsite cognate for the C-terminal substrate P(2)' side chain, acting in concert with carboxylate-docking interactions with Lys(1087) and Tyr(1096), but also electrostatic selection against a cationic C-terminal substrate carboxylate. With a blocked C terminus, substrate side chain interactions are dominant in cleavage site selection. In the evolution of obligate exopeptidases from endopeptidase ancestors, mutations that destroy context-driven peptide bond targeting are likely to have followed the acquisition of terminal docking interactions. Evolutionary intermediates between endopeptidases and obligate exopeptidases could therefore have been dual specificity proteinases like ACE.

A range of 3,6,6-trisubstituted nortropane derivatives based upon 6 β -phenyltropane-3 β ,6 α -diol have been synthesised from 6 β -hydroxytropinone, including some novel related tricyclic hemi-ketal and tricyclic ketal compounds. Derivatives were assessed for pharmacological affinity and selectivity at α 1 -adrenergic receptors, and 6 β -phenyl-8-azabicyclo[3.2.1]octan-3-spiro-2′-(1′,3′-dioxolane)-6-ol, a novel lead compound selective for the alpha 1D -adrenergic receptor, is reported.

Publisher Summary α1-Adrenergic receptors (α1-ARs) are members of the biogenic amine family of G protein-coupled receptors (GPCRs). Like other members of the GPCR superfamily, the largest family of membrane receptors and possibly the largest gene family in the human genome, α1-ARs are long, single-chain polypeptides. Three subtypes have been identified by molecular cloning in several species, including humans. In addition to these subtypes, a putative fourth subtype, the α1L-AR, which displays low affinity for prazosin and other α1-antagonists, has been identified by functional and not molecular studies. This subtype is postulated to mediate the contraction of prostate smooth muscle, but it remains unclear if it is a distinct subtype or merely a functional variant of one of the cloned α1subtypes—such as the α1A splice variants—because all display some of the characteristics of an α1L subtype when expressed in heterologous cell systems. Another molecular variant of the α1A-AR is a coding region polymorphism that involves an arginine to cysteine substitution in an arginine-rich region of the C-terminal tail.

Angiotensin I-converting enzyme (ACE) inhibitors are thought to lower blood pressure in hypertensive patients, mainly by decreasing angiotensin II (Ang II) formation. Chymase, a human mast cell protease, has recently been proposed to play a role in blood pressure regulation because of its Ang II-forming activity. Here we show that the predominant chymase mRNA species in the mouse aorta are those for types 4 and 5 isoforms, and that both are efficient Ang II-forming enzymes. Evaluation of ACE-dependent and ACE-independent Ang II-forming pathways in mast cell-deficient (Kit(w)/Kit(w-v)) mice and their mast cell-sufficient littermate (MC(+/+)) controls revealed that, in contrast to the latter, Kit(w)/Kit(w-v) mice fail to express chymase mRNAs in the vasculature and have almost no ACE-independent Ang II-forming activity in either isolated blood vessels or homogenates. Moreover, in MC(+/+) but not in Kit(w)/Kit(w-v) mice, a contribution of ACE-independent Ang II generation to blood pressure regulation was evident by a 1.6-fold greater maximal reduction in mean arterial pressure with acute ACE inhibition plus AT(1) receptor blockade than with ACE inhibition alone. Thus, mast cells are the source of the vascular ACE-independent pathway, and the antihypertensive benefit of combining ACE inhibitor therapy with AT(1) receptor antagonist therapy is most likely due to negation of chymase-catalyzed Ang II generation.

Covalent posttranslational protein modifications by eukaryotic transglutaminases proceed by a kinetic pathway of acylation and deacylation. Ammonia is released as the acylenzyme is formed, whereas the cross-linked product is released later in the deacylation step. Superposition of the active sites of transglutaminase type 2 (TG2) and the structurally related cysteine protease, papain, indicates that in the formation of tetrahedral intermediates, the backbone nitrogen of the catalytic Cys-277 and the Nε1 nitrogen of Trp-241 of TG2 could contribute to transition-state stabilization. The importance of this Trp-241 side chain was demonstrated by examining the kinetics of dansylcadaverine incorporation into a model peptide. Although substitution of the Trp-241 side chain with Ala or Gly had only a small effect on the Michaelis constant K m (1.5-fold increase), it caused a >300-fold lowering of the catalytic rate constant k cat . The wild-type and mutant TG2-catalyzed release of ammonia showed kinetics similar to the kinetics for the formation of cross-linked product, indicating that transition-state stabilization in the acylation step was rate-limiting. In papain, a Gln residue is at the position of TG2-Trp-241. The conservation of Trp-241 in all eukaryotic transglutaminases and the finding that W241Q-TG2 had a much lower k cat than wild-type enzyme suggest evolutionary specialization in the use of the indole group. This notion is further supported by the observation that transition-state-stabilizing side chains of Tyr and His that operate in some serine and metalloproteases only partially substituted for Trp.

Tissue transglutaminase (tTG) is a multifunctional enzyme that catalyzes peptide cross-linking and polyamination reactions, and also is a signal-transducing GTPase. tTG protein content and enzymatic activity are upregulated in the brain in Huntington's disease and in other neurological diseases and conditions. Since mouse models are currently being used to study the role of tTG in Huntington's disease and other neurodegenerative diseases, it is critical that the level of its expression in the mouse forebrain be determined. In contrast to human forebrain where tTG is abundant, tTG can only be detected in mouse forebrain by immunoblotting a GTP-binding-enriched protein fraction. tTG mRNA content and transamidating activity are approximately 70% lower in mouse than in human forebrain. However, tTG contributes to the majority of transglutaminase activity within mouse forebrain. Thus, although tTG is expressed at lower levels in mouse compared with human forebrain, it likely plays important roles in neuronal function.

One of the promising methods of protein structure prediction involves the use of amino acid sequence-derived patterns. Here we report on the creation of non-degenerate motif descriptors derived through data mining of training sets of residues taken from the transmembrane-spanning segments of polytopic proteins. These residues correspond to short regions in which there is a deviation from the regular alpha-helical character (i.e. pi-helices, 3(10)-helices and kinks). A 'search engine' derived from these motif descriptors correctly identifies, and discriminates amongst instances of the above 'non-canonical' helical motifs contained in the SwissProt/TrEMBL database of protein primary structures. Our results suggest that deviations from alpha-helicity are encoded locally in sequence patterns only about 7-9 residues long and can be determined in silico directly from the amino acid sequence. Delineation of such variations in helical habit is critical to understanding the complex structure-function relationships of polytopic proteins and for drug discovery. The success of our current methodology foretells development of similar prediction tools capable of identifying other structural motifs from sequence alone. The method described here has been implemented and is available on the World Wide Web at http://cbcsrv.watson.ibm.com/Ttkw.html.

A peptide contained in the venom of the predatory marine snail Conus tulipa, rho-TIA, has previously been shown to possess alpha1-adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, rho-TIA inhibited alpha1-adrenoreceptor-mediated increases in cytosolic Ca2+ concentration that were triggered by norepinephrine, but did not affect presynaptic alpha2-adrenoreceptor-mediated responses. In radioligand binding assays using [125I]HEAT, rho-TIA displayed slightly greater potency at the alpha 1B than at the alpha 1A or alpha 1D subtypes. Moreover, although it did not affect the rate of association for [3H]prazosin binding to the alpha 1B-adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by rho-TIA. N-terminally truncated analogs of rho-TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of rho-TIA (Arg4). An alanine walk of rho-TIA confirmed the importance of Arg4 for activity and revealed a number of other residues clustered around Arg4 that contribute to the potency of rho-TIA. The unique allosteric antagonism of rho-TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive alpha1-adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.

Ischemia and simulated ischemic conditions cause intracellular Ca2+ overload in the myocardium. The relationship between ischemia injury and Ca2+ overload has not been fully characterized. The aim of the present study was to investigate the expression and characteristics of PLC isozymes in myocardial infarction-induced cardiac remodeling and heart failure. In normal rat heart tissue, PLC-delta1 (about 44 ng/mg of heart tissue) was most abundant isozymes compared to PLC-gamma1 (6.8 ng/mg) and PLC-beta1 (0.4 ng/mg). In ischemic heart and hypoxic neonatal cardiomyocytes, PLC-delta1, but not PLC-beta1 and PLC-gamma1, was selectively degraded, a response that could be inhibited by the calpain inhibitor, calpastatin, and by the caspase inhibitor, zVAD-fmk. Overexpression of the PLC-delta1 in hypoxic neonatal cardiomyocytes rescued intracellular Ca2+ overload by ischemic conditions. In the border zone and scar region of infarcted myocardium, and in hypoxic neonatal cardiomyocytes, the selective degradation of PLC-delta1 by the calcium sensitive proteases may play important roles in intracellular Ca2+ regulations under the ischemic conditions. It is suggested that PLC isozyme-changes may contribute to the alterations in calcium homeostasis in myocardial ischemia.

We showed previously that Phe(303) in transmembrane segment (TM) VI of the alpha(1B)-adrenergic receptor (alpha(1B)-AR), a residue conserved in many G protein-coupled receptors (GPCRs), is critically involved in coupling agonist binding with TM helical movement and G protein activation. Here the equivalent residue, Phe(282), in the beta(2)-AR was evaluated by mutation to glycine, asparagine, alanine, or leucine. Except for F282N, which exhibits attenuated basal and maximal isoproterenol stimulation, the Phe(282) mutants display varying degrees of constitutive activity (F282LF282AF282G), and as shown by the results of substituted cysteine accessibility method (SCAM) studies, induce movement of endogenous cysteine(s) into the water-accessible ligand-binding pocket. For F282A, movement is confined to Cys(285) in TMVI, whereas F282L induces movement of both Cys(285) in TMVI and Cys(327) in TMVII. Further, engineered cysteine-sensor studies indicate that F282L causes movement of TMVI, both above and below an apparent kink-inducing TMVI proline (Pro(288)), whereas that due to F282A is confined to the domain below Pro(288). A plausible interpretation of these data is that receptor activation involves rigid body movement of TMVI which, because of its Pro(288)-induced kink, acts as a pivot to transduce and amplify the agonist-induced conformational change in the upper domain, to a change in the lower domain required for productive receptor-G protein coupling.

Transglutaminase 2 (TG2) is a distinctive member of the family of Ca 2+ -dependent enzymes recognized mostly by their abilities to catalyze the posttranslational crosslinking of proteins. TG2 uniquely binds and hydrolyzes GTP; binding GTP inhibits its crosslinking activity but allows it to function in signal transduction (hence the G h designation). The core domain of TG2 (residues 139–471, rat) comprises the papain-like catalytic triad and the GTP-binding domain (residues 159–173) and contains almost all of the conserved tryptophans of the protein. Examining point mutations at Trp positions 180, 241, 278, 332, and 337 showed that, upon binding 2′-(or 3′)- O -( N -methylanthraniloyl)GTP (mantGTP), the Phe-332 mutant was the weakest (35% less than wild type) in resonance energy transfer from the protein (λ exc, max = 290 nm) to the mant fluorophore (λ em = 444 nm) and had a reduced affinity for mantGTP. Trp-332, situated near the catalytic center and the nucleotide-binding area of TG2, may be part of the allosteric relay machinery that transmits negative effector signals from nucleotide binding to the active center of TG2. A most important observation was that, whereas no enzyme activity could be detected when Trp-241 was replaced with Ala or Gln, partial preservation of catalytic activity was seen with substitutions by Tyr > Phe > His. The results indicate that Trp-241 is essential for catalysis, possibly by stabilizing the transition states by H-bonding, quadrupole–ion, or van der Waals interactions. This contrasts with the evolutionarily related papain family of cysteine proteases, which uses Gln-19 (papain) for stabilizing the transition state.

We showed previously that Phe(303) in transmembrane segment (TM) VI of the alpha(1B)-adrenergic receptor, a highly conserved residue in G-protein-coupled receptors (GPCRs), is critically involved in receptor-activation and G-protein-coupling [Chen, S. H., Lin, F., Xu, M., Hwa, J., and Graham, R. M. (2000) EMBO J. 19, 4265-4271]. Here, we show that saturation mutagenesis of Phe(303) results in a series of mutants with different levels of constitutive activity for inositol phosphate (IP) signaling. Mutants F303G and F303N showed neither basal nor agonist-stimulated IP turnover, whereas F303A displayed increased basal activity but an attenuated maximal response to (-)-epinephrine-stimulation. F303L, on the other hand, showed all features of a typical constitutively active GPCR with markedly increased basal activity and increased potency and efficacy of agonist-stimulated IP signaling. All mutants displayed higher agonist-binding affinities than the wild-type receptor, and by thermal stability studies, those able to signal showed increased susceptibility to inactivation with an order of sensitivity (F303L > F303A > WT) directly related to their degree of constitutive activity. Using the substituted cysteine accessibility method (SCAM) and equilibrium binding studies, we further show that the F303A and F303L mutants result in TM helical movements that differ in accordance with their degree of constitutive activity. These findings, therefore, confirm and extend our previous data implicating Phe(303) as a key residue coupling TM helical movements to G-protein-activation.

Transient Regenerative Potential of the Neonatal Mouse Heart Porello et al Science . 2011;331:1078–1080 The regenerative capacity of adult human hearts after infarction seems vestigial at best, perhaps because of the poor proliferative capacity of terminally differentiated cardiomyocytes and desecration of the local environment that would otherwise be conducive of stem cell-mediated repair or loss of stem cells, or both. In a recent edition of the journal Science , Porello et al set a new benchmark in this field by showing that the hearts of neonatal mice have, in contrast, an impressive regenerative capacity after surgical resection, albeit one that is lost almost immediately as cardiomyocytes exit the cell cycle and mature. In a technical tour de force, Porello et al1 report that day 1 neonatal mice subjected to resection of the apex of the left ventricle (amounting to ∼15% of the ventricular myocardium) not only survive but also show complete regeneration of the myocardium and restoration of cardiac function. Furthermore, they provide evidence that the predominant mechanism involved is not differentiation of stem cells, but transient dedifferentiation and proliferation of cardiomyocytes in both the border and remote zones of the heart. In contrast, resection of a lesser amount of apical myocardium in day 7 neonates, at a time after terminal differentiation of cardiomyocytes has already taken place, resulted in markedly increased mortality and failure of myocardial regeneration, with wound repair by scar formation only. It has long been known that in adult mammals, the heart is one of the least regenerative organs in the body. As a result, repair after massive tissue injury, as occurs with myocardial infarction, is limited to replacement of the myocardium by dense collagenous scar tissue. This impairs contractile function, resulting in adverse remodelling and, in severe cases, ventricular dilation, heart failure, and death. To explore if intracardiac …

Activation of the α 1A -adrenergic receptor (α 1A -AR)/Gq pathway has been implicated as a critical trigger for the development of cardiac hypertrophy. However, direct evidence from in vivo studies is still lacking. To address this issue, transgenic mice with cardiac-targeted overexpression of the α 1A -AR (4- to 170-fold) were generated, using the rodent α-myosin heavy chain promoter. Heterozygous animals displayed marked enhancement of cardiac contractility, evident from increases in dP/dt max (80%, P max /LVP inst (76%, P max :dP/dt min (104%, P P max –end-diastolic volume relationship provided load-independent evidence of a primary increase in contractility. Blood pressure and heart rate were largely unchanged, and there was a small increase in (−)norepinephrine-stimulated, but not basal, phospholipase C activity. Increased contractility was directly related to the level of receptor overexpression and could be completely reversed by acute α 1A - but not β-AR blockade. Despite the robust changes in contractility, transgenic animals displayed no morphological, histological, or echocardiographic evidence of left ventricular hypertrophy. In addition, apart from an increase in atrial natriuretic factor mRNA, expression of other hypertrophy-associated genes was unchanged. To our knowledge, these data provide the first in vivo evidence for an inotropic action of the α 1A -AR.

Six young men diagnosed with systolic hypertension had normal carotid pressure wave contours, normal synthesized aortic pressure wave contours and normal diastolic and mean pressures in upper limb arteries. Elevated brachial systolic pressure was caused by a high narrow systolic peak of the pressure wave. This was attributed to amplification of the pressure wave between the ascending aorta and upper limb (radial and brachial) arteries that is associated with attainment of full body length and very distensible arteries. These young men were not truly hypertensive. Exaggeration of the upper limb systolic peak represented an extreme of the normal pressure wave pattern in youth, where amplification is greater than in childhood or in older subjects. This phenomenon accounts for the rapid increase in systolic pressure between the ages of 5 and 20 years, and the relative plateau in systolic pressure between the ages of 20 and 45 years that is seen in population studies.

Clostridium difficile is a causative agent of nosocomially acquired antibiotic-associated diarrhea and pseudomembranous colitis ([3][1]). Typing schemes include serogrouping, pyrolysis mass spectroscopy, restriction endonuclease analysis, whole-cell protein profiling, arbitrarily primed PCR, pulsed

Based on both in vivo and in vitro studies, we have shown previously that the intracellular domain of a membrane-bound isoform of the growth factor, neuregulin, regulates proteolytic release of its extracellular domain ErbB receptor-activating ligand. To investigate the mechanism(s) involved in this regulation, a series of intracellular domain mutants were constructed and tested for susceptibility to proteolytic processing after transient transfection in COS-7 cells. These studies revealed that regulation of extracellular domain cleavage by the intracellular domain is sequence-specific and involves three distinct 30–60-residue segments. The presence of any two of these three segments is both necessary and sufficient for proteolytic processing, and resistance to proteolysis is not due to an alteration in cellular localization or transport. Evidence was also obtained that regulation of extracellular domain processing involves initial intracellular domain dimerization. Thus, with expression of a construct encoding only the intracellular domain, dimerization could be demonstrated in cross-linking experiments. Furthermore, resistance to proteolytic processing of a construct lacking a large portion of the intracellular domain was rescued with a chimera, in which the intracellular domain was replaced with a spontaneously dimerizing Fc fragment. Taken together these studies indicate that intracellular domain interactions are critically involved in the spacial and temporal control of growth and development by membrane-bound neuregulin isoforms.

Neuregulins are a multi-isoform family of growth factors that activate members of the erbB family of receptor tyrosine kinases. The membrane-anchored isoforms contain the receptor-activating ligand in their extracellular domain, a single membrane-spanning region, and a long cytoplasmic tail. To evaluate the potential biological role of the intracellular domain of the membrane-anchored neuregulin isoforms, we used a domain-specific gene disruption approach to produce a mouse line in which only the region of the neuregulin gene encoding almost the entire intracellular domain was disrupted. Consistent with previous reports in which all neuregulin isoforms were disrupted, the resulting homozygous neuregulin mutants died at E10.5 of circulatory failure and displayed defects in neural and cardiac development. To further understand these in vivo observations, we evaluated a similarly truncated neuregulin construct after transient expression in COS-7 cells. This cytoplasmic tail-deleted mutant, unlike wild-type neuregulin isoforms, was resistant to proteolytic release of its extracellular-domain ligand, a process required for erbB receptor activation. Thus, proteolytic processing of the membrane-bound neuregulin isoforms involved in cranial ganglia and heart embryogenesis is likely developmentally regulated and is critically controlled by their intracellular domain. This observation indicates that erbB receptor activation by membrane-bound neuregulins most likely involves a unique temporally and spatially regulated “inside-out” signaling process that is critical for processing and release of the extracellular-domain ligand.

Transglutaminase type 2 (TG2) has been reported to be a candidate gene for maturity onset diabetes of the young (MODY) because three different mutations that impair TG2 transamidase activity have been found in 3 families with MODY. TG2 null (TG2(-/-)) mice have been reported to be glucose intolerant and have impaired glucose-stimulated insulin secretion (GSIS). Here we rigorously evaluated the role of TG2 in glucose metabolism using independently generated murine models of genetic TG2 disruption, which show no compensatory enhanced expression of other TGs in pancreatic islets or other tissues. First, we subjected chow- or fat-fed congenic SV129 or C57BL/6 wild type (WT) and TG2(-/-) littermates, to oral glucose gavage. Blood glucose and serum insulin levels were similar for both genotypes. Pancreatic islets isolated from these animals and analysed in vitro for GSIS and cholinergic potentiation of GSIS, showed no significant difference between genotypes. Results from intraperitoneal glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were similar for both genotypes. Second, we directly investigated the role of TG2 transamidase activity in insulin secretion using a coisogenic model that expresses a mutant form of TG2 (TG2(R579A)), which is constitutively active for transamidase activity. Intraperitoneal GTTs and ITTs revealed no significant differences between WT and TG2(R579A/R579A) mice. Given that neither deletion nor constitutive activation of TG2 transamidase activity altered basal responses, or responses to a glucose or insulin challenge, our data indicate that glucose homeostasis in mice is TG2 independent, and question a link between TG2 and diabetes.

Paul Korner's life's work centered on unraveling the sympatho-adrenal control of the circulation and applying this knowledge to understanding the pathogenesis of hypertension and to improving the management of cardiovascular diseases. He made major contributions as Foundation Professor of Physiology at the University of New South Wales (1960–8), as the first Scandrett Professor of Cardiology at the University of Sydney and the Royal Prince Alfred Hospital (1968–74), and as Director of the Baker Institute for Medical Research (1975–90). After retirement in 1990, he undertook his last major work, writing an influential single-author text, Essential Hypertension and it Causes.