Nucleotide Messenger Research Articles

Nucleotide Messenger Signaling of Staphylococci in Responding to Nitric Oxide - Releasing Biomaterials.

Nitric oxide (NO) releasing biomaterials are a promising approach against medical device associated microbial infection. In contrast to the bacteria-killing effects of NO at high concentrations, NO at low concentrations serves as an important signaling molecule to inhibit biofilm formation or disperse mature biofilms by regulating the intracellular nucleotide second messenger signaling network such as cyclic dimeric guanosine monophosphate (c-di-GMP) for many Gram-negative bacterial strains. However, Gram-positive staphylococcal bacteria are the most commonly diagnosed microbial infections on indwelling devices, but much less is known about the nucleotide messengers and their response to NO as well as the mechanism by which NO inhibits biofilm formation. This study investigated the cyclic nucleotide second messengers c-di-GMP, cyclic dimeric adenosine monophosphate (c-di-AMP), and cyclic adenosine monophosphate (cAMP) in both Staphylococcus aureus (S. aureus) Newman D2C and Staphylococcus epidermidis (S. epidermidis) RP62A after incubating with S-nitroso-N-acetylpenicillamine (SNAP, NO donor) impregnated polyurethane (PU) films. Results demonstrated that NO release from the polymer films significantly reduced the c-di-GMP levels in S. aureus planktonic and sessile cells, and these bacteria showed inhibited biofilm formation. However, the effect of NO release on c-di-GMP in S. epidermidis was weak, but rather, S. epidermidis showed significant reduction in c-di-AMP levels in response to NO release and also showed reduced biofilm formation. Results strongly suggest that NO regulates the nucleotide second messenger signaling network in different ways for these two bacteria, but for both bacteria, these changes in signaling affect the formations of biofilms. These findings provide cues to understand the mechanism of Staphylococcus biofilm inhibition by NO and suggest novel targets for antibiofilm interventions.

Phosphodiesterase 7 as a therapeutic target – Where are we now?

Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes that hydrolyse the intracellular second messengers cAMP and cGMP to their inactive forms 5’AMP and 5’GMP. Some members of the PDE family display specificity towards a single cyclic nucleotide messenger, and PDE4, PDE7, and PDE8 specifically hydrolyse cAMP. While the role of PDE4 and its use as a therapeutic target have been well studied, less is known about PDE7 and PDE8. This review aims to collate the present knowledge on human PDE7 and outline its potential use as a therapeutic target. Human PDE7 exists as two isoforms PDE7A and PDE7B that display different expression patterns but are predominantly found in the central nervous system, immune cells, and lymphoid tissue. As a result, PDE7 is thought to play a role in T cell activation and proliferation, inflammation, and regulate several physiological processes in the central nervous system, such as neurogenesis, synaptogenesis, and long-term memory formation. Increased expression and activity of PDE7 has been detected in several disease states, including neurodegenerative diseases such as Parkinson's, Alzheimer's and Huntington's disease, autoimmune diseases such as multiple sclerosis and COPD, and several types of cancer. Early studies have shown that administration of PDE7 inhibitors may ameliorate the clinical state of these diseases. Targeting PDE7 may therefore provide a novel therapeutic strategy for targeting a broad range of disease and possibly provide a complementary alternative to inhibitors of other cAMP-selective PDEs, such as PDE4, which are severely limited by their side-effects.

Light dependent synthesis of a nucleotide second messenger controls the motility of a spirochete bacterium

Nucleotide second messengers are universally crucial factors for the signal transduction of various organisms. In prokaryotes, cyclic nucleotide messengers are involved in the bacterial life cycle and in functions such as virulence and biofilm formation, mainly via gene regulation. Here, we show that the swimming motility of the soil bacterium Leptospira kobayashii is rapidly modulated by light stimulation. Analysis of a loss-of-photoresponsivity mutant obtained by transposon random mutagenesis identified the novel sensory gene, and its expression in Escherichia coli through codon optimization elucidated the light-dependent synthesis of cyclic adenosine monophosphate (cAMP). GFP labeling showed the localization of the photoresponsive enzyme at the cell poles where flagellar motors reside. These findings suggest a new role for cAMP in rapidly controlling the flagella-dependent motility of Leptospira and highlight the global distribution of the newly discovered photoactivated cyclase among diverse microbial species.

The nucleotide messenger (p)ppGpp is an anti-inducer of the purine synthesis transcription regulator PurR in Bacillus.

The nucleotide messenger (p)ppGpp allows bacteria to adapt to fluctuating environments by reprogramming the transcriptome. Despite its well-recognized role in gene regulation, (p)ppGpp is only known to directly affect transcription in Proteobacteria by binding to the RNA polymerase. Here, we reveal a different mechanism of gene regulation by (p)ppGpp in Firmicutes: (p)ppGpp directly binds to the transcription factor PurR to downregulate purine biosynthesis gene expression upon amino acid starvation. We first identified PurR as a receptor of (p)ppGpp in Bacillus anthracis. A co-structure with Bacillus subtilis PurR reveals that (p)ppGpp binds to a PurR pocket reminiscent of the active site of phosphoribosyltransferase enzymes that has been repurposed to serve a purely regulatory role, where the effectors (p)ppGpp and PRPP compete to allosterically control transcription. PRPP inhibits PurR DNA binding to induce transcription of purine synthesis genes, whereas (p)ppGpp antagonizes PRPP to enhance PurR DNA binding and repress transcription. A (p)ppGpp-refractory purR mutant in B. subtilis fails to downregulate purine synthesis genes upon amino acid starvation. Our work establishes the precedent of (p)ppGpp as an effector of a classical transcription repressor and reveals the key function of (p)ppGpp in regulating nucleotide synthesis through gene regulation, from soil bacteria to pathogens.

The second messenger c-di-AMP mediates bacterial exopolysaccharide biosynthesis: a review.

Cyclic dimeric adenosine 3'-5'-monophosphate (c-di-AMP) is a recently discovered nucleotide messenger in bacteria. It plays an important role in signaling, transcription, and cell physiology, such as in bacterial growth, potassium transport, fatty acid synthesis, the metabolic balance of cell wall components, and biofilm formation. Exopolysaccharides (EPSs) have distinct physico-chemical properties and diverse bioactivities including antibacterial, hypolipidemic, and antioxidative activities, and they are widely used in the food, pharmaceutical, and cosmetic industries. Although c-di-AMP has been demonstrated to regulate the biosynthesis of bacterial EPSs, only a single c-di-AMP receptor, CabpA, has been identified in EPS synthesis. With the aim of describing current understanding of the regulation of microbial EPSs, this review summarizes c-di-AMP biosynthesis and degradation as well as the mechanism through which c-di-AMP regulates bacterial EPSs.

PpGpp Coordinates Nucleotide and Amino-Acid Synthesis in E. coli During Starvation

(p)ppGpp is a nucleotide messenger universally produced in bacteria following nutrient starvation. In E.coli, ppGpp inhibits purine nucleotide synthesis by targeting several different enzymes, but the physiological significance of their inhibition is unknown. Here, we report the structural basis of inhibition for one target, Gsk, the inosine-guanosine kinase. Gsk creates an unprecedented, allosteric binding pocket for ppGpp by restructuring terminal sequences, which restrains conformational dynamics necessary for catalysis. Guided by this structure, we generated a chromosomal mutation that abolishes Gsk regulation by ppGpp. This mutant strain accumulates abnormally high levels of purine nucleotides following amino-acid starvation, compromising cellular fitness. We demonstrate that this unrestricted increase in purine nucleotides is detrimental because it severely depletes pRpp and essential, pRpp-derived metabolites, including UTP, histidine, and tryptophan. Thus, our results reveal the significance of ppGpp's regulation of purine nucleotide synthesis and a critical mechanism by which E.coli coordinates biosynthetic processes during starvation.

Comparison of spectral FRET microscopy approaches for single-cell analysis.

Förster resonance energy transfer (FRET) is a valuable tool for measuring molecular distances and the effects of biological processes such as cyclic nucleotide messenger signaling and protein localization. Most FRET techniques require two fluorescent proteins with overlapping excitation/emission spectral pairing to maximize detection sensitivity and FRET efficiency. FRET microscopy often utilizes differing peak intensities of the selected fluorophores measured through different optical filter sets to estimate the FRET index or efficiency. Microscopy platforms used to make these measurements include wide-field, laser scanning confocal, and fluorescence lifetime imaging. Each platform has associated advantages and disadvantages, such as speed, sensitivity, specificity, out-of-focus fluorescence, and Z-resolution. In this study, we report comparisons among multiple microscopy and spectral filtering platforms such as standard 2-filter FRET, emission-scanning hyperspectral imaging, and excitation-scanning hyperspectral imaging. Samples of human embryonic kidney (HEK293) cells were grown on laminin-coated 28 mm round gridded glass coverslips (10816, Ibidi, Fitchburg, Wisconsin) and transfected with adenovirus encoding a cAMP-sensing FRET probe composed of a FRET donor (Turquoise) and acceptor (Venus). Additionally, 3 FRET "controls" with fixed linker lengths between Turquoise and Venus proteins were used for inter-platform validation. Grid locations were logged, recorded with light micrographs, and used to ensure that whole-cell FRET was compared on a cell-by-cell basis among the different microscopy platforms. FRET efficiencies were also calculated and compared for each method. Preliminary results indicate that hyperspectral methods increase the signal-to-noise ratio compared to a standard 2-filter approach.

Secondary nucleotide messenger c-di-GMP exerts a global control on natural product biosynthesis in streptomycetes.

Cyclic dimeric 3′-5′ guanosine monophosphate, c-di-GMP, is a ubiquitous second messenger controlling diverse cellular processes in bacteria. In streptomycetes, c-di-GMP plays a crucial role in a complex morphological differentiation by modulating an activity of the pleiotropic regulator BldD. Here we report that c-di-GMP plays a key role in regulating secondary metabolite production in streptomycetes by altering the expression levels of bldD. Deletion of cdgB encoding a diguanylate cyclase in Streptomycesghanaensis reduced c-di-GMP levels and the production of the peptidoglycan glycosyltransferase inhibitor moenomycin A. In contrast to the cdgB mutant, inactivation of rmdB, encoding a phosphodiesterase for the c-di-GMP hydrolysis, positively correlated with the c-di-GMP and moenomycin A accumulation. Deletion of bldD adversely affected the synthesis of secondary metabolites in S. ghanaensis, including the production of moenomycin A. The bldD-deficient phenotype is partly mediated by an increase in expression of the pleiotropic regulatory gene wblA. Genetic and biochemical analyses demonstrate that a complex of c-di-GMP and BldD effectively represses transcription of wblA, thus preventing sporogenesis and sustaining antibiotic synthesis. These results show that manipulation of the expression of genes controlling c-di-GMP pool has the potential to improve antibiotic production as well as activate the expression of silent gene clusters.

RelZ-Mediated Stress Response in Mycobacterium smegmatis: pGpp Synthesis and Its Regulation.

Stringent response is a conserved stress response mechanism in which bacteria employ the second messengers guanosine tetraphosphate and guanosine pentaphosphate [collectively termed (p)ppGpp] to reprogram their cellular processes under stress. In mycobacteria, these alarmones govern a multitude of cellular phenotypes, such as cell division, biofilm formation, antibiotic tolerance, and long-term survival. Mycobacterium smegmatis possesses the bifunctional RelMsm as a (p)ppGpp synthetase and hydrolase. In addition, it contains a short alarmone synthetase MS_RHII-RSD (renamed RelZ), which contains an RNase H domain in tandem with the (p)ppGpp synthetase domain. The physiological functions of RelMsm have been well documented, but there is no clear picture about the cellular functions of RelZ in M. smegmatis RelZ has been implicated in R-loop induced stress response due to its unique domain architecture. In this study, we elucidate the differential substrate utilization pattern of RelZ compared to that of RelMsm We unveil the ability of RelZ to use GMP as a substrate to synthesize pGpp, thereby expanding the repertoire of second messengers known in mycobacteria. We have demonstrated that the pGpp synthesis activity of RelZ is negatively regulated by RNA and pppGpp. Furthermore, we investigated its role in biofilm formation and antibiotic tolerance. Our findings highlight the complex role played by the RelZ in cellular physiology of M. smegmatis and sheds light upon its functions distinct from those of RelMsmIMPORTANCE Bacteria utilize nucleotide messengers to survive the hostile environmental conditions and the onslaught of attacks within the host. The second messengers guanosine tetraphosphate and pentaphosphate [(p)ppGpp] have a profound impact on the long-term survival, biofilm formation, antibiotic tolerance, virulence, and pathogenesis of bacteria. Therefore, understanding the stress response mechanism regulated by (p)ppGpp is essential for discovering inhibitors of stress response and potential drug targets. Mycobacterium smegmatis contains two (p)ppGpp synthetases: RelMsm and RelZ. Our study unravels the novel regulatory mechanisms of RelZ activity and its role in mediating antibiotic tolerance. We further reveal its ability to synthesize novel second messenger pGpp, which may have regulatory roles in mycobacteria.

Essentiality of the Maltase AmlE in Maltose Utilization and Its Transcriptional Regulation by the Repressor AmlR in the Acarbose-Producing Bacterium Actinoplanes sp. SE50/110.

Actinoplanes sp. SE50/110 is the wild type of industrial production strains of the fine-chemical acarbose (acarviosyl-maltose), which is used as α-glucosidase inhibitor in the treatment of type II diabetes. Although maltose is an important building block of acarbose, the maltose/maltodextrin metabolism has not been studied in Actinoplanes sp. SE50/110 yet. Bioinformatic analysis located a putative maltase gene amlE (ACSP50_2474, previously named malL; Wendler et al., 2015a), in an operon with an upstream PurR/LacI-type transcriptional regulator gene, named amlR (ACSP50_2475), and a gene downstream (ACSP50_2473) encoding a GGDEF-EAL-domain-containing protein putatively involved in c-di-GMP signaling. Targeted gene deletion mutants of amlE and amlR were constructed by use of the CRISPR/Cas9 technology. By growth experiments and functional assays of ΔamlE, we could show that AmlE is essential for the maltose utilization in Actinoplanes sp. SE50/110. Neither a gene encoding a maltose phosphorylase (MalP) nor MalP enzyme activity were detected in the wild type. By this, the maltose/maltodextrin system appears to be fundamentally different from other described prokaryotic systems. By sequence similarity analysis and functional assays from the species Streptomyces lividans TK23, S. coelicolor A3(2) and S. glaucescens GLA.O, first hints for a widespread lack of MalP and presence of AmlE in the class Actinobacteria were given. Transcription of the aml operon is significantly repressed in the wild type when growing on glucose and repression is absent in an ΔamlR deletion mutant. Although AmlR apparently is a local transcriptional regulator of the aml operon, the ΔamlR strain shows severe growth inhibitions on glucose and – concomitantly – differential transcription of several genes of various functional classes. We ascribe these effects to ACSP50_2473, which is localized downstream of amlE and presumably involved in the metabolism of the second messenger c-di-GMP. It can be assumed, that maltose does not only represent the most important carbon source of Actinoplanes sp. SE50/110, but that its metabolism is coupled to the nucleotide messenger system of c-di-GMP.

Small Regulatory RNAs in the Control of Motility and Biofilm Formation in E. coli and Salmonella

Biofilm formation in Escherichia coli and other enteric bacteria involves the inverse regulation of the synthesis of flagella and biofilm matrix components such as amyloid curli fibres, cellulose, colanic acid and poly-N-acetylglucosamine (PGA). Physiologically, these processes reflect the transition from growth to stationary phase. At the molecular level, they are tightly controlled by various sigma factors competing for RNA polymerase, a series of transcription factors acting in hierarchical regulatory cascades and several nucleotide messengers, including cyclic-di-GMP. In addition, a surprisingly large number of small regulatory RNAs (sRNAs) have been shown to directly or indirectly modulate motility and/or biofilm formation. This review aims at giving an overview of these sRNA regulators and their impact in biofilm formation in E. coli and Salmonella. Special emphasis will be put on sRNAs, that have known targets such as the mRNAs of the flagellar master regulator FlhDC, the stationary phase sigma factor σS (RpoS) and the key biofilm regulator CsgD that have recently been shown to act as major hubs for regulation by multiple sRNAs.

Structural Insights into the Regulatory Mechanism of the Response Regulator RocR from Pseudomonas aeruginosa in Cyclic Di-GMP Signaling

The nucleotide messenger cyclic di-GMP (c-di-GMP) plays a central role in the regulation of motility, virulence, and biofilm formation in many pathogenic bacteria. EAL domain-containing phosphodiesterases are the major signaling proteins responsible for the degradation of c-di-GMP and maintenance of its cellular level. We determined the crystal structure of a single mutant (R286W) of the response regulator RocR from Pseudomonas aeruginosa to show that RocR exhibits a highly unusual tetrameric structure arranged around a single dyad, with the four subunits adopting two distinctly different conformations. Subunits A and B adopt a conformation with the REC domain located above the c-di-GMP binding pocket, whereas subunits C and D adopt an open conformation with the REC domain swung to the side of the EAL domain. Remarkably, the access to the substrate-binding pockets of the EAL domains of the open subunits C and D are blocked in trans by the REC domains of subunits A and B, indicating that only two of the four active sites are engaged in the degradation of c-di-GMP. In conjunction with biochemical and biophysical data, we propose that the structural changes within the REC domains triggered by the phosphorylation are transmitted to the EAL domain active sites through a pathway that traverses the dimerization interfaces composed of a conserved regulatory loop and the neighboring motifs. This exquisite mechanism reinforces the crucial role of the regulatory loop and suggests that similar regulatory mechanisms may be operational in many EAL domain proteins, considering the preservation of the dimerization interface and the spatial arrangement of the regulatory domains.

Investigating cADPR and NAADP in intact and broken cell preparations

The body of literature characterizing cyclic adenosine diphosphoribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) as Ca2+-mobilizing second messengers is growing apace. However, their unique properties may, for the uninitiated, make them difficult to work with. This article reviews many of the available techniques (and associated pitfalls) for investigating these nucleotide messengers, predominantly focusing upon optical techniques using fluorescent reporters to measure Ca2+ in the cytosol as well as Ca2+ or pH within the lumen of intracellular organelles.

Paracrine stimulation of vascular smooth muscle proliferation by diadenosine polyphosphates released from proximal tubule epithelial cells

The purinergic receptor system plays an important role in the regulation of both vascular and tubular functions within the kidney; however, the release of purinergic agonists other than ATP by renal tissue is not known. In this investigation, we determine if kidney tissue is a source of diadenosine polyphosphates, which have high affinity for the P(2X) and P(2Y) receptors. Both diadenosine pentaphosphate and hexaphosphate were identified by matrix-assisted laser desorption ionization-mass spectrometry in extracts purified from both whole porcine kidney and from cloned cells of the LLC-PK1 cell line. Both polyphosphates in nanomolar concentrations were found to significantly stimulate the proliferation of vascular smooth muscle cells derived from rat thoracic aortas. The purinergic-receptor antagonist, suramin, did not significantly affect the growth-stimulatory properties of the polyphosphates. The growth stimulation of vascular smooth muscle cells by platelet-derived growth factor was potentiated by both diadenosine polyphosphates. We conclude that diadenosine polyphosphates are endogenous purinergic agonists of the kidney that have physiologic and pathophysiologic relevance. These epithelial cell metabolic products have vasoregulatory properties while linking the energy supply and tubular function.

Distinct Structural Determinants of Efficacy and Sensitivity in the Ligand-binding Domain of Cyclic Nucleotide-gated Channels

Cyclic nucleotide-gated (CNG) channels open in response to direct binding of cyclic nucleotide messengers. Every subunit in a tetrameric CNG channel contains a cytoplasmic ligand-binding domain (BD) that includes a beta-roll (flanked by short helices) and a single C-terminal helix called the C-helix that was previously found to control efficacy (maximal open probability) and selectivity for cGMP versus cAMP. We constructed a series of chimeric CNG channel subunits, each containing a distinct BD sequence (chosen from among six phylogenetically divergent isoforms) fused to an invariant non-BD sequence. We assayed these "BD substitution" chimeras as homomeric CNG channels in Xenopus oo-cytes to compare their functions and found that the most efficient activation by both cAMP and cGMP derived from the BD of the catfish CNGA4 olfactory modulatory subunit (fCNGA4). We then tested the effects of replacing subregions of the bovine CNGA1 BD with corresponding fCNGA4 sequence and hence identified parts of the fCNGA4 BD producing efficient activation. For instance, replacing either the "hinge" that connects the roll and C-helix subdomains or the BD sequence N-terminal to the hinge greatly enhanced cAMP efficacy. Replacing the "loop-beta 8" region (the C-terminal end of the beta-roll) improved agonist sensitivity for cGMP selectively over cAMP. Our results thus identify multiple BD elements outside the C-helix that control selective ligand interaction and channel gating steps by distinct mechanisms. This suggests that the purine ring of the cyclic nucleotide may interact with both the beta-roll and the C-helix at different points in the mechanism.

Immunomodulation: an important concept in modern anaesthesia.

This review summarises evidence for immunomodulatory effect of drugs administered peri-operatively. The clinical significance of the balance of pro- and anti-inflammatory cytokines may be seen in certain disease states, for example, meningococcal meningitis and Lyme arthritis. This balance may be altered peri-operatively. Traditionally, these changes are considered to be due to the stress response of surgery, the response to cardiopulmonary bypass, or endotoxaemia. This review presents in vitro evidence suggesting that drugs modulating this cytokine balance include non-steroidal anti-inflammatory agents, phosphodiesterase inhibitors and opioids, acting through effects on intracellular cyclic nucleotide messenger systems. An important consequence of the pro-inflammatory cytokine activity is increased adhesion of neutrophils. Aspects of this process may be inhibited by avoiding low blood flow states, by reducing adhesion molecule expression (for example by use of pentoxifylline), or by use of negatively charged anions such as heparin. Neutrophil activity is generally depressed by intravenous anaesthetic induction agents, but is enhanced by opioids. Natural killer cell activity, which is involved in immunity against tumour cells and virally infected cells is transiently depressed by volatile anaesthetic agents and opioids. In contrast catecholamines enhance natural killer cell activity. Whereas decrease in immunoglobulin levels occur peri-operatively, this is not thought to be as a result of drugs at clinically used concentrations but rather due to haemodilution.

Membrane dynamics and permeability

The surface of cells is a repository of a wide variety of receptors that enable cells to sense their surroundings and to respond appropriately. Some receptors are recognized by enveloped viruses that bind to the cell surface and fuse with it to release the capsid into the cytoplasm of the target cell. Surface components are also involved in immune reactions and in the social behaviour of cells in their organisation into tissues and organs. The plasma membrane represents an important site of attack by pathogens many of which produce toxins that introduce pores into the membrane allowing leakage of the cell contents. The translocation of ions and other solutes through channels is the means by which cell membranes create a selectively permeable barrier allowing compartmentation of cellular processes and coordination of a variety of physiological functions. A gathering of distinguished researchers in the field of membrane dynamics and permeability took place under the auspices of the London Membrane Group and the Cell Surface Research Fund at the Royal Society on September 8, 1995 (Fig. 1) to discuss recent developments in the subject. Another purpose was to mark the occasion of the retirement of Professor Charles Pasternak from St George's Hospital Medical School where he founded the Deparment of Biochemistry more than 20 years ago. One of the remarkable aspects of the meeting was that the contributors were all numbered amongst his former students and colleagues. The topics of the symposium divided into membrane dynamics where the mechanisms of surface receptor and glucose transporter recycling were considered together with the role of cyclic nucleotide messengers in mediating cell movement. Cell surface functions in disease considered the identity of surface features of cells concerned with malignancy and infection. The final section was devoted to a consideration of the mechanisms whereby ions and solutes move through channels and pores in membranes. The treatment ranged from theoretical aspects and modelling to facilitated diffusion processes associated with genetic disorders and infection. As editor of the symposium papers I have been impressed by how well they integrate into the theme of the meeting. That such a broad group of topics can be so encompassed is testimony to the wide ranging interests and expertise of the

Functional organization of the riboflavin biosynthesis operon from Bacillus subtilis SHgw

We have sequenced 6006 bp DNA of a region from the Bacillus subtilis SHgw chromosome known to contain riboflavin biosynthesis genes (rib gene cluster, 210 degrees on the B. subtilis genetic map). Five of the seven open reading frames found within the sequence are shown to represent the genes ribG, ribB, ribA, ribH and ribTD. The calculated molecular masses for the putative translation products are 39,305, 23,481, 44,121, 16,287 and 14,574 daltons respectively. The five rib genes are transcribed as a polycistronic 4277 nucleotide messenger RNA. The steady-state level of the transcript is negatively regulated by riboflavin. A cis-acting element necessary for regulation was mapped by analysis of constitutive mutations within the 5' untranslated region of the operon. The element is at least 48 bp in length and does not bear obvious similarity to well defined prokaryotic regulatory elements. The molecular mechanism of regulation remains unknown, but the data presented argue against regulation by attenuation.

Selective inhibition of cyclic AMP phosphodiesterase from various human tissues by milrinone, a potent cardiac bipyridine

We observed the effects of milrinone, an inotropic agent prescribed to treat congestive heart failure, on cyclic nucleotide messenger systems in various human tissues in vitro. Cyclic nucleotide phosphodiesterases (PDEs) from the human heart were separated into three isoforms, FI, FII and FIII, by DEAE-cellulose chromatography. Milrinone proved to be a potent and selective inhibitor of human cardiac FIII PDE, a “low K m ” enzyme for cyclic AMP (cAMP-PDE). The IC 50 value for the inhibition of FIII PDE was 0.42 μM, while those of FI and FII PDEs, “high K m ” enzymes, were 38 and 19 μM, respectively. Kinetic studies showed that milrinone inhibited the activity of FIII PDE, competitively with respect to cAMP, and the K i , was 0.15 μM. Milrinone in doses to 100 μM had no effect on human cardiac cAMP-dependent protein kinase and adenylate cyclase. The activity of cAMP-PDEs from human platelets and the aorta, as well as that from heart, were potently inhibited by milrinone, with much the same IC 50 values. Cyclic AMP-PDEs from human kidney, liver and lung were not readily inhibited by milrinone, and the IC 50 values of cAMP-PDEs from these tissues were about 7-30-fold higher than that from heart. On the other hand, papaverine had a relatively lesser selectivity for any of the cAMP-PDEs. All these results suggest that milrinone exerts inotropic effects by inhibiting cAMP-PDE selectively in the human heart tissues and that this compound can be used to evaluate different forms of cAMP-PDEs present in human tissues.

Sequence of rat liver alpha 2-macroglobulin and acute phase control of its messenger RNA.

Six alpha 2-macroglobulin cDNA clones were isolated from two liver cDNA libraries produced from rats undergoing acute inflammation. The coding sequence for rat alpha 2-macroglobulin including its 27-residue signal peptide and the 3' - and part of the 5' nontranslated regions were determined. The mature protein consisting of 1445 amino acids is coded for by a 4790 +/- 40 nucleotide messenger RNA. It contains a typical internal thiol ester region and 25 cysteine residues which are conserved between rat and human alpha 2-macroglobulin. Although the amino acid sequences of rat and human alpha 2-macroglobulin share 73% identity, two small divergent areas of 17 and 38 residues were found, corresponding to 29 and 11% identity, respectively. These areas are located in the bait region and, therefore, may confer specific proteinase recognition capabilities on rat alpha 2-macroglobulin. Following an inflammatory stimulation, rat alpha 2-macroglobulin mRNA levels increased 214-fold over control values and reached a maximum at 18 h. By 24 h the levels had decreased to less than 30% of the maximum value. Transcription rates from the alpha 2-macroglobulin gene as measured in nuclear run-on experiments showed a less than 3-fold increase in nuclei from acutely inflamed rats as compared to controls. These results suggest that the accummulation of alpha 2M mRNA is due to the combined effects of increased transcription rates and post-transcriptional processing.