Regulation of Contractility by Adenosine A\u3csub\u3e1\u3c/sub\u3e and A\u3csub\u3e2A\u3c/sub\u3e Receptors in the Murine Heart: Role of Protein Phosphatase 2A: A Dissertation

Adenosine is a nucleoside that plays an important role in the regulation of contractility in the heart. Adenosine receptors are G-protein coupled and those implicated in regulation of contractility are presumed to act via modulating the activity of adenylyl cyclase and cAMP content of cardiomyocytes. Adenosine A1 receptors (A1R) reduce the contractile response of the myocardium to β-adrenergic stimulation. This is known as anti adrenergic action. The A2A adenosine receptor (A2AR) has the opposite effect of increasing contractile responsiveness of the myocardium. The A2AR also appears to attenuate the effects of A1R. The effects of these receptors have been primarily studied in the rat heart and with the utilization of cardiomyocyte preparations. With the increasing use of receptor knockout murine models and murine models of various pathological states, it is of importance to comprehensively study the effects of adenosine receptors on regulation of contractility in the murine heart. The following studies examine the adenosinergic regulation of myocardial contractility in isolated murine hearts. In addition, adenosinergic control of contractility is examined in hearts isolated from A2AR knockout animals. Responses to adenosinergic stimulation in murine isolated hearts are found to be comparable to those observed in the rat, with A1R exhibiting an anti adrenergic action and A2AR conversely enhancing contractility. A significant part of the A2AR effect was found to occur via inhibition of the A1R antiadrenergic action. A part of the anti adrenergic action of A1R has previously been shown to be the result of protein phosphatase 2A activation and localization to membranes. Additional experiments in the present study examine the effect of adenosinergic signaling on PP2A in myocardial extracts from wild type and A2AR knockout hearts. A2AR activation was found to decrease the activity of PP2A and enhance localization of the active enzyme to the cytosol; away from its presumed sites of action. In the A2AR knockout the response to A1R activation was enhanced compared with the wild type and basal PP2A activity was reduced. It is concluded that A2AR modulation of PP2A activity may account for the attenuation of the A1R effect by A2AR observed in the contractile studies

Complete enzyme set for chlorophyll biosynthesis in Escherichia coli

Chlorophylls are essential cofactors for photosynthesis, which sustains global food chains and oxygen production. Billions of tons of chlorophylls are synthesized annually, yet full understanding of chlorophyll biosynthesis has been hindered by the lack of characterization of the Mg-protoporphyrin IX monomethyl ester oxidative cyclase step, which confers the distinctive green color of these pigments. We demonstrate cyclase activity using heterologously expressed enzyme. Next, we assemble a genetic module that encodes the complete chlorophyll biosynthetic pathway and show that it functions in Escherichia coli. Expression of 12 genes converts endogenous protoporphyrin IX into chlorophyll a, turning E. coli cells green. Our results delineate a minimum set of enzymes required to make chlorophyll and establish a platform for engineering photosynthesis in a heterotrophic model organism

PufQ regulates porphyrin flux at the haem/bacteriochlorophyll branchpoint of tetrapyrrole biosynthesis via interactions with ferrochelatase

Facultative phototrophs such as Rhodobacter sphaeroides can switch between heterotrophic and photosynthetic growth. This transition is governed by oxygen tension and involves the large-scale production of bacteriochlorophyll, which shares a biosynthetic pathway with haem up to protoporphyrin IX. Here, the pathways diverge with the insertion of Fe(2+) or Mg(2+) into protoporphyrin by ferrochelatase or magnesium chelatase, respectively. Tight regulation of this branchpoint is essential, but the mechanisms for switching between respiratory and photosynthetic growth are poorly understood. We show that PufQ governs the haem/bacteriochlorophyll switch; pufQ is found within the oxygen-regulated pufQBALMX operon encoding the reaction centre-light harvesting photosystem complex. A pufQ deletion strain synthesises low levels of bacteriochlorophyll and accumulates the biosynthetic precursor coproporphyrinogen III; a suppressor mutant of this strain harbours a mutation in the hemH gene encoding ferrochelatase, substantially reducing ferrochelatase activity. FLAG-immunoprecipitation experiments retrieve a ferrochelatase-PufQ-carotenoid complex, proposed to regulate the haem/bacteriochlorophyll branchpoint by directing porphyrin flux towards bacteriochlorophyll production under oxygen-limiting conditions. The co-location of pufQ and the photosystem genes in the same operon ensures that switching of tetrapyrrole metabolism towards bacteriochlorophyll is coordinated with the production of reaction centre and light harvesting polypeptides. This article is protected by copyright. All rights reserved

A paralog of a bacteriochlorophyll biosynthesis enzyme catalyzes the formation of 1,2-dihydro-carotenoids in green sulfur bacteria

Chlorobaculum tepidum, a green sulfur bacterium, utilizes chlorobactene as its major carotenoid, and this organism also accumulates a reduced form of this monocyclic pigment, 1',2'-dihydrochlorobactene. The protein catalyzing this reduction is the last unidentified enzyme in the biosynthetic pathways for all of the green sulfur bacterial pigments used for photosynthesis. The genome of Chlorobaculum tepidum contains two paralogous genes encoding members of the FixC family of flavoproteins: bchP, that has been shown to encode an enzyme of bacteriochlorophyll biosynthesis; and bchO, for which a function has not been assigned. Here we demonstrate that a bchO mutant is unable to synthesize 1',2'-dihydrochlorobactene, and when bchO is heterologously expressed in a neurosporene-producing mutant of the purple bacterium, Rhodobactersphaeroides, the encoded protein is able to catalyze the formation of 1,2-dihydroneurosporene, the major carotenoid of the only other organism reported to synthesize 1,2-dihydrocarotenoids, Blastochloris viridis Identification of this enzyme completes the pathways for the synthesis of photosynthetic pigments in Chlorobiaceae, and accordingly and consistent with its role in carotenoid biosynthesis, we propose to rename the gene, cruI Notably, the absence of cruI in Blastochloris viridis indicates that a second 1,2-carotenoid reductase, which is structurally unrelated to CruI (BchO), must exist in nature. The evolution of this carotenoid reductase in green sulfur bacteria is discussed herein

A mini review of giant invasive sacral schwannoma

Primary giant invasive sacral schwannoma is a rare disease. Its uncommon location and aggressive growth behavior with eroding bone and compressing adjacent structures may lead to misdiagnosis. Awareness of this rare entity will help pathologists to differentiate it from other tumors possibly seen at this location. In this review, we summarize its clinical features, histopathology, differential diagnoses, treatment, and prognosis

Contractile effects of adenosine A1 and A2A receptors in isolated murine hearts

The adenosine A1 receptor (A1R) inhibits beta-adrenergic-induced contractile effects (antiadrenergic action), and the adenosine A2A receptor (A2AR) both opposes the A1R action and enhances contractility in the heart. This study investigated the A1R and A2AR function in beta-adrenergic-stimulated, isolated wild-type and A2AR knockout murine hearts. Constant flow and pressure perfused preparations were employed, and the maximal rate of left ventricular pressure (LVP) development (+dp/dt(max)) was used as an index of cardiac function. A1R activation with 2-chloro-N6-cyclopentyladenosine (CCPA) resulted in a 27% reduction in contractile response to the beta-adrenergic agonist isoproterenol (ISO). Stimulation of A2AR with 2-P(2-carboxyethyl)phenethyl-amino-5\u27-N-ethylcarboxyamidoadenosine (CGS-21680) attenuated this antiadrenergic effect, resulting in a partial (constant flow preparation) or complete (constant pressure preparation) restoration of the ISO contractile response. These effects of A2AR were absent in knockout hearts. Up to 63% of the A2AR influence was estimated to be mediated through its inhibition of the A1R antiadrenergic effect, with the remainder being the direct contractile effect. Further experiments examined the effects of A2AR activation and associated vasodilation with low-flow ischemia in the absence of beta-adrenergic stimulation. A2AR activation reduced by 5% the depression of contractile function caused by the flow reduction and also increased contractile performance over a wide range of perfusion flows. This effect was prevented by the A2AR antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]et hyl)phenol (ZM-241385). It is concluded that in the murine heart, A1R and A2AR modulate the response to beta-adrenergic stimulation with A2AR, attenuating the effects of A1R and also increasing contractility directly. In addition, A2AR supports myocardial contractility in a setting of low-flow ischemia

A Tale of Two Reductases: Extending the Bacteriochlorophyll Biosynthetic Pathway in <i>E. coli</i>

<div><p>The creation of a synthetic microbe that can harvest energy from sunlight to drive its metabolic processes is an attractive approach to the economically viable biosynthetic production of target compounds. Our aim is to design and engineer a genetically tractable non-photosynthetic microbe to produce light-harvesting molecules. Previously we created a modular, multienzyme system for the heterologous production of intermediates of the bacteriochlorophyll (BChl) pathway in <i>E. coli</i>. In this report we extend this pathway to include a substrate promiscuous 8-vinyl reductase that can accept multiple intermediates of BChl biosynthesis. We present an informative comparative analysis of homologues of 8-vinyl reductase from the model photosynthetic organisms <i>Rhodobacter sphaeroides</i> and <i>Chlorobaculum tepidum</i>. The first purification of the enzymes leads to their detailed biochemical and biophysical characterization. The data obtained reveal that the two 8-vinyl reductases are substrate promiscuous, capable of reducing the C8-vinyl group of Mg protoporphyrin IX, Mg protoporphyrin IX methylester, and divinyl protochlorophyllide. However, activity is dependent upon the presence of chelated Mg²⁺ in the porphyrin ring, with no activity against non-Mg²⁺ chelated intermediates observed. Additionally, CD analyses reveal that the two 8-vinyl reductases appear to bind the same substrate in a different fashion. Furthermore, we discover that the different rates of reaction of the two 8-vinyl reductases both <i>in vitro</i>, and <i>in vivo</i> as part of our engineered system, results in the suitability of only one of the homologues for our BChl pathway in <i>E. coli</i>. Our results offer the first insights into the different functionalities of homologous 8-vinyl reductases. This study also takes us one step closer to the creation of a nonphotosynthetic microbe that is capable of harvesting energy from sunlight for the biosynthesis of molecules of choice.</p></div

Bacteriochlorophyll pathway intermediates produced by <i>E. coli</i> cells expressing various combinations of genes from the Heme and BChl pathways as detected by HPLC.

<p><i>E. coli</i> cells expressing HemA-F and the magnesium chelatase complex BchSID produce P^IX and MgP^IX. Addition of the methyl transferase BchM results in production of both P^IX ME and MgP^IX ME. Expression with the divinyl reductase <i>CT</i>BciA in the presence and absence of BchM leads to the production of mono-vinyl forms of pathway intermediates. <i>RS</i>BciA is not active in our <i>in vivo</i> system. Abbreviations: P^IX - protoporphyrin IX, MgP^IX - Mg-protoporphyrin IX, P^IXME - protoporphyrin IX methylester, MgP^IXME - Mg-protoporphyrin IX methylester, mvP^IX - mono-vinyl protoporphyrin IX, mvMgPIX - mono-vinyl Mg-protoporphyrin IX, mvP^IXME - mono-vinyl protoporphyrin IX methylester, mvMgP^IXME - mono-vinyl Mg-protoporphyrin IX methylester, ND – none detected.</p

Reaction efficiency as a measure of percent conversion of divinyl-protochlorophyllide to mono-vinyl protochlorophyllide by 8-vinyl reductase.

<p>Purified <i>CT</i>BciA reduces greater than 85% DVP to mono-vinyl form in 1.5 hours (black bar). Purified <i>RS</i>BciA acts more slowly, reaching 100% conversion of divinyl to mono-vinyl in 18 hours (hashed bars). Attempts to improve reaction efficiency of <i>RS</i>BciA by addition of crude cell lysate to the reaction vessel actually reduced the rate of reaction as well as the overall conversion to less than 80% in 18 hours (white bars). Error bars are calculated from reactions carried out in duplicate.</p