Presence Of IMP Research Articles

Prevalence and Molecular Characteristics of Carbapenem Resistant Acinetobacter baumannii Isolates from A Regional Tertiary Care Hospital

Acinetobacter spp., is an emerging opportunistic nosocomial Gram negative bacterial pathogen with increasing prevalence in particular the species Acinetobacter baumannii. It infects the most vulnerable immunocompromised hospitalized patients who are critically ill. Significant levels of morbidity and mortality have been reported with outbreaks and the carbapenem hydrolyzing beta lactamases that includes MBLs and oxacillinases are recognized as important contributors of carbapenem resistance in Acinetobacter spp., Enzymatic degradation of drugs, target modifications, multidrug efflux pumps and permeability defects are some of the important resistance mechanisms present in A. baumannii. Accumulation of various resistance mechanisms made treatment of A. baumannii infection very difficult. The major objective of the study was to identify the pattern of antibiotic resistance and its regional prevalence. Hence this study was aimed and conducted to isolate, identify and distinguish the antibiogram of A. baumannii from clinical specimens and to study a molecular level identification of resistance mechanisms of the isolates from a tertiary care hospital. Various clinical specimens like blood, urine, abscess, vaginal swab were analyzed and evaluated for the presence of Acinetobacter. Four members of Acinetobacter species; A junii, A lowffii, A ursingii and A baumannii, were isolated from clinical specimens. A. baumannii was the predominant species and 15% of the A. baumannii isolates were confirmed to be resistant to carbapenems. A molecular typing was done to identify the genes conferring antibiotic resistance and five major genes were identified in the isolates. The predominant genes present in the isolates wereOXA-58, OXA-23 and GIM. Presence of IMP & VIM were also identified. , ,

Hypoxanthine-guanine phosphoribosyltransferase is activated via positive cooperativity between guanine and IMP.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme in the purine salvage pathway. Here, the reverse reaction of HGPRT from the thermophilic bacterium Hungateiclostridium thermocellum was studied in the presence of IMP and pyrophosphate. As for the human enzyme, the bacterial HGPRT was activated by guanine. Furthermore, guanine was found to operate as both an activator and an inhibitor. Intriguingly, within the concentration range of guanine where it functions as the activator, the Km value for IMP was not influenced by guanine. Consequently, guanine was found to noncompetitively activate the reverse reaction toward IMP. Here, we propose a reaction scheme that explains the activation mechanism in which the enzyme forms a chimeric oligomer bound to both IMP and guanine.

AMP Deaminase 3 Overexpression Reduces Mitochondrial Content in C2C12 Myotubes by Decreasing PGC‐1α Promotor Activation

Skeletal muscles undergoing atrophy have decreased intracellular [ATP], PGC‐1α expression, and mitochondrial content. This combination of findings is unexpected considering that decreased [ATP] is typically associated with increases in [ADP] and [AMP], activation of AMPK and PGC‐1α, and subsequent mitochondrial biogenesis in order to increase energy production. Therefore, it is unclear why atrophying muscles display deficits in PGC‐1α and mitochondrial content despite having decreased [ATP]. A possible explanation is increased activity of the enzyme AMP Deaminase 3 (AMP→IMP+NH3), which is highly upregulated during muscle atrophy. We tested the hypothesis that increased expression of AMPD3 can reduce mitochondrial content in skeletal muscle cells by decreasing the AMPK→ PGC‐1α→ mitochondrial biogenesis intracellular signaling cascade by decreasing [AMP].MethodsC2C12 myotubes were transduced with adenovirus encoding AMPD3 or GFP (control) and allowed to differentiate further for 5 days. Myotubes were then collected and analyzed for nucleotide concentrations (UPLC), protein expression of AMPK, phosphorylated AMPK(Thr172), AMPKα substrates, mitochondrial OXPHOS proteins (Western Blot), 2 Kb PGC‐1α promotor activity (PGC‐1α‐Luciferase assay), and citrate synthase enzyme activity (marker of mitochondrial content).ResultsIncreased AMPD activity was confirmed by the presence of IMP (0.20 μmol/g) in AMPD3 groups compared to undetectable in GFP controls. Overexpression of AMPD3 decreased [ATP] by 32%, [ADP] by 19%, [AMP] by 28%, and the total adenine nucleotide pool + IMP by 31% (p<0.001 for all). Notably, despite the pronounced decrease in [ATP], the [AMP]/[ATP] did not differ between groups. This was reflected by no differences in protein expression of AMPK, pAMPK(Thr172), and AMPKα substrate. However, PGC‐1α‐luciferase activity was significantly lower after 1 and 5 days of AMPD3 overexpression compared to GFP (p<0.0001, p<0.05), suggesting decreased PGC‐1α promotor region activation despite no measurable difference in pAMPK(Thr172). Furthermore, citrate synthase activity was 17% lower in myotubes overexpressing AMPD3 compared to GFP (p=0.01), yet no differences in mitochondrial OXPHOS proteins were detected.Summary/ConclusionsOur results show that increased expression of AMPD3 can decrease adenine nucleotide concentrations without disrupting the relative ATP:ADP:AMP ratios. Consequently, we do not detect measurable differences in AMPK activation. However, we do find significantly lower PGC‐1α promotor activity and citrate synthase activity. These findings indicate that increased expression of AMPD3, as occurs during muscle atrophy, can reduce PGC‐1α activation and mitochondrial biogenesis, ultimately resulting in reduced mitochondrial content. Interestingly, our data suggests that the reduction in PGC‐1α activity occurs independent of AMPK activation. Future studies should be conducted to determine the specific mechanism of PGC‐1α activation along with direct measures of mitochondrial biogenesis, such as, mitochondrial protein synthesis rate.Support or Funding InformationFunded by NIH R01 AR070200This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Two classes of bacterial IMPDHs according to their quaternary structures and catalytic properties.

Inosine-5′-monophosphate dehydrogenase (IMPDH) occupies a key position in purine nucleotide metabolism. In this study, we have performed the biochemical and physico-chemical characterization of eight bacterial IMPDHs, among which six were totally unexplored. This study led to a classification of bacterial IMPDHs according to the regulation of their catalytic properties and their quaternary structures. Class I IMPDHs are cooperative enzymes for IMP, which are activated by MgATP and are octameric in all tested conditions. On the other hand, class II IMPDHs behave as Michaelis-Menten enzymes for both substrates and are tetramers in their apo state or in the presence of IMP, which are shifted to octamers in the presence of NAD or MgATP. Our work provides new insights into the IMPDH functional regulation and a model for the quaternary structure modulation is proposed.

Virulence variation among isolates of western equine encephalitis virus in an outbred mouse model

Little is known about viral determinants of virulence associated with western equine encephalitis virus (WEEV). Here, we have analysed six North American WEEV isolates in an outbred CD1 mouse model. Full genome sequence analyses showed < or =2.7 % divergence among the six WEEV isolates. However, the percentage mortality and mean time to death (MTD) varied significantly when mice received subcutaneous injections of 10(3) p.f.u. of each virus. Two WEEV strains, McMillan (McM) and Imperial 181 (IMP), were the most divergent of the six in genome sequence; McM caused 100 % mortality by 5 days post-infection, whereas IMP caused no mortality. McM had significantly higher titres in the brain than IMP. Similar differences in virulence were observed when McM and IMP were administered by aerosol, intranasal or intravenous routes. McM was 100 % lethal with an MTD of 1.9 days when 10(3) p.f.u. of each virus was administered by intracerebral inoculation; in contrast, IMP caused no mortality. The presence of IMP in the brains after infection by different routes and the lack of observed mortality confirmed that IMP is neuroinvasive but not neurovirulent. Based on morbidity, mortality, MTD, severity of brain lesions, virus distribution patterns, routes of infection and differences in infection of cultured cells, McM and IMP were identified as high- and low-virulence isolates, respectively.

Cavitation as a Mechanism of Substrate Discrimination by Adenylosuccinate Synthetases,

Adenylosuccinate synthetase catalyzes the first committed step in the de novo biosynthesis of AMP, coupling L-aspartate and IMP to form adenylosuccinate. Km values of IMP and 2'-deoxy-IMP are nearly identical with each substrate supporting comparable maximal velocities. Nonetheless, the Km value for L-aspartate and the Ki value for hadacidin (a competitive inhibitor with respect to L-aspartate) are 29-57-fold lower in the presence of IMP than in the presence of 2'-deoxy-IMP. Crystal structures of the synthetase ligated with hadacidin, GDP, and either 6-phosphoryl-IMP or 2'-deoxy-6-phosphoryl-IMP are identical except for the presence of a cavity normally occupied by the 2'-hydroxyl group of IMP. In the presence of 6-phosphoryl-IMP and GDP (hadacidin absent), the L-aspartate pocket can retain its fully ligated conformation, forming hydrogen bonds between the 2'-hydroxyl group of IMP and sequence-invariant residues. In the presence of 2'-deoxy-6-phosphoryl-IMP and GDP, however, the L-aspartate pocket is poorly ordered. The absence of the 2'-hydroxyl group of the deoxyribonucleotide may destabilize binding of the ligand to the L-aspartate pocket by disrupting hydrogen bonds that maintain a favorable protein conformation and by the introduction of a cavity into the fully ligated active site. At an approximate energy cost of 2.2 kcal/mol, the unfavorable thermodynamics of cavity formation may be the major factor in destabilizing ligands at the L-aspartate pocket.

The Effect of Temperature and pH on the Co-operative Behavior of Mg2+-stimulated Acto-myosin-ATPase and the Inhibition by IMP

We have previously reported our finding that IMP inhibits the Mg2+-stimulated acto-myosin-ATPase activity of isolated actin and myosin. These experiments were undertaken at 35°C and pH 7.0. It was also shown that the binding of actin to myosin was cooperative and that in the presence of IMP the Hill coefficient was decreased. The experiments shown here were carried out with isolated actin and myosin at three temperatures (25°C, 31°C and 37°C) and three pH values (6, 7 and 8). The results show that: (i) the Mg2+-stimulated acto-myosin-ATPase activity decreases with decreasing temperature; (ii) the Mg2+-stimulated acto-myosin-ATPase activity is lower at pH = 6 and 8 compared to pH = 7; (iii) the effect of temperature and pH on the Mg2+-stimulated acto-myosin-ATPase activity can be explained by a decrease in co-operativity between actin and myosin; (iv) IMP inhibits the Mg2+-stimulated acto-myosin-ATPase activity at all temperatures and pH values. The greatest inhibition is found at pH = 7; and (v) the inhibition by pH + IMP is about the same for pH = 6 and pH = 7; at pH = 8 this combined inhibition is slightly higher. This leads to the same decrease in Mg2+-stimulated acto-myosin-ATPase activity. Muscle fatigue can be explained by a combination of non-regulatory factors (for example pH) and regulatory factors (such as IMP) and from our results we conclude that IMP serves as an additional regulatory safety switch to maintain the balance between energy consumption and energy production and thereby preventing an energy crisis during exhaustive exercise of short duration.

Carbapenem-resistant Pseudomonas aeruginosa in malaysia producing IMP-7 beta-lactamase.

We have isolated and identified a carbapenem-resistant Pseudomonas aeruginosa strain from Malaysia that produces an IMP-7 metallo-beta-lactamase. This isolate showed high-level resistance to meropenem and imipenem, the MICs of which were 256 and 128 micro g/ml, respectively. Isoelectric focusing analyses revealed pI values of >9.0, 8.2, and 7.8, which indicated the possible presence of IMP and OXA. DNA sequencing confirmed the identity of the IMP-7 determinant.

IMP Alone Organizes the Active Site of Adenylosuccinate Synthetase from Escherichia coli

A complete set of substrate/substrate analogs of adenylosuccinate synthetase from Escherichia coli induces dimer formation and a transition from a disordered to an ordered active site. The most striking of the ligand-induced effects is the movement of loop 40-53 by up to 9 A. Crystal structures of the partially ligated synthetase, which either combine IMP and hadacidin or IMP, hadacidin, and Mg(2+)-pyrophosphate, have ordered active sites, comparable with the fully ligated enzyme. More significantly, a crystal structure of the synthetase with IMP alone exhibits a largely ordered active site, which includes the 9 A movement of loop 40-53 but does not include conformational adjustments to backbone carbonyl 40 (Mg(2+) interaction element) and loop 298-304 (L-aspartate binding element). Interactions involving the 5'-phosphoryl group of IMP evidently trigger the formation of salt links some 30 A away. The above provides a structural basis for ligand binding synergism, effects on k(cat) due to mutations far from the site of catalysis, and the complete loss of substrate efficacy due to minor alterations of the 5'-phosphoryl group of IMP.

A Model for Regulation of the Mg2+-Stimulated Acto-Myosin-ATPase Activity: Inhibition of the Formation of Actin-Myosin Complex and the Mg2+-Stimulated Acto-Myosin-ATPase Activity by IMP and AMP

Previously, we showed that the decrease in force output during continuous isometric contractions in rat skeletal muscle was related to an increase in the concentration of IMP. In this paper we report on additional experiments in which the effect of IMP on the Mg 2+ -stimulated acto-myosin-ATPase activity of isolated actin and myosin is measured at 35°C. The results show that 1) the binding of actin to myosin is co-operative (Hill coefficient = 3.82); 2) in the presence of IMP or AMP the Mg 2+ -stimulated acto-myosin-ATPase activity is inhibited up to 60% at 10 mM; 3) in the presence of IMP or AMP not only the Mg 2+ -stimulated acto-myosin-ATPase activity decreases, but also K 50. From these results we conclude that IMP and AMP may be considered as uncompetitive inhibitors. Our results suggest that IMP and AMP can prevent an ‘energy crisis’ during exhaustive exercise of short duration by down-regulating the contractile machinery.

Effectors of the Stringent Response Target the Active Site ofEscherichia coli Adenylosuccinate Synthetase

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a pleiotropic effector of the stringent response, potently inhibits adenylosuccinate synthetase from Escherichia coli as an allosteric effector and/or as a competitive inhibitor with respect to GTP. Crystals of the synthetase grown in the presence of IMP, hadacidin, NO3-, and Mg2+, then soaked with ppGpp, reveal electron density at the GTP pocket which is consistent with guanosine 5'-diphosphate 2':3'-cyclic monophosphate. Unlike ligand complexes of the synthetase involving IMP and GDP, the coordination of Mg2+ in this complex is octahedral with the side chain of Asp13 in the inner sphere of the cation. The cyclic phosphoryl group interacts directly with the side chain of Lys49 and indirectly through bridging water molecules with the side chains of Asn295 and Arg305. The synthetase either directly facilitates the formation of the cyclic nucleotide or scavenges trace amounts of the cyclic nucleotide from solution. Regardless of its mode of generation, the cyclic nucleotide binds far more tightly to the active site than does ppGpp. Conceivably, synthetase activity in vivo during the stringent response may be sensitive to the relative concentrations of several effectors, which together exercise precise control over the de novo synthesis of AMP.

Mechanistic implications from crystalline complexes of wild-type and mutant adenylosuccinate synthetases from Escherichia coli.

Asp13 and His41 are essential residues of adenylosuccinate synthetase, putatively catalyzing the formation of adenylosuccinate from an intermediate of 6-phosphoryl-IMP. Wild-type adenylosuccinate synthetase and three mutant synthetases (Arg143 --> Leu, Lys16 --> Gln, and Arg303 --> Leu) from Eschericha coli have been crystallized in the presence of IMP, hadacidin (an analogue of L-aspartate), Mg2+, and GTP. The active site of each complex contains 6-phosphoryl-IMP, Mg2+, GDP, and hadacidin, except for the Arg303 --> Leu mutant, which does not bind hadacidin. In response to the formation of 6-phosphoryl-IMP, Asp13 enters the inner coordination sphere of the active site Mg2+. His41 hydrogen bonds with 6-phosphoryl-IMP, except in the Arg303 --> Leu complex, where it remains bound to the guanine nucleotide. Hence, recognition of the active site Mg2+ by Asp13 evidently occurs after the formation of 6-phosphoryl-IMP, but recognition of the intermediate by His41 may require the association of L-aspartate with the active site. Structures reported here support a mechanism in which Asp13 and His41 act as the catalytic base and acid, respectively, in the formation of 6-phosphoryl-IMP, and then act together as catalytic acids in the subsequent formation of adenylosuccinate.

Effects of adenine nucleotides on low Km 5′ nucleotidase from human seminal plasma

1. 1. Low K m 5' nucleotidase purified from human seminal plasma has been used in this study to investigate the response of the enzyme to adenine nucleoside di- and triphosphates in the presence of AMP and IMP as substrates. 2. 2. In the presence of AMP, the addition of 0.5 mM ATP to the enzyme Mg-free results into the highest V max / K m ratio value and other experimental combinations of effectors tested cause variation of the kinetic parameters of the enzyme, indicating a control of AMP dephosphorylation by adenine nucleotides. 3. 3. In the presence of IMP, ATP and ADP activate the enzyme but the response to various experimental combinations of effectors shows no significant difference in the kinetic properties of the enzyme, indicating a different control of the dephosphorylation of IMP.

Adenine-induced hypoxanthine release from IMP-enriched human erythrocytes

Adenine uptake and hypoxanthine release by IMP-enriched human erythrocytes has been studied. The presence of IMP within the erythrocytes leads to an increase in the rate of adenine incorporation. Adenine is taken up by IMP-enriched erythrocytes as AMP, even when intracellular 5-phoshorobosyl-1-pyrophosphate concentration is undetectable and too low to allow IMP synthesis from hypoxanthine. During adenine uptake and AMP synthesis, hypoxanthine is released by the cells. The possibility that 5-phosphoribosyl-1-pyrophosphate, necessary for AMP synthesis, is formed through the hypoxanthine guanine phosphoribosyltransferese-catalyzed IMP pyrophosphorolysis is considered.

The crystal structure of phosphorylase b at 6 Å resolution

The determination of the crystal structure of phosphorylase b in the presence of IMP at 6 Å resolution is described. The structure determination is based on two heavy-atom isomorphous derivatives and their anomalous contributions. The molecular boundary is clearly distinguishable in the electron density map, except in the region of subunit-subunit contact about the crystallographic dyad axis, which is the symmetry axis of the dimer. The dimer molecule is roughly ellipsoidal in shape with dimensions 63 Å × 63 Å × 116 Å. There is a pronounced cavity on the enzyme surface but it is not yet known if this is a substrate binding site.

Enzymes in facultative anaerobiosis of molluscs-II. Basic catalytic properties of phosphoenolpyruvate carboxykinase in oyster adductor muscle

1. 1. p-Enolpyruvate carboxykinase (E.C. 4.1.1.32) from oyster adductor tissue occurs as a single activity peak with a pI value of 6·64. 2. 2. The enzyme is present in high activities only in adductor tissue. 3. 3. It exhibits a relatively high degree of specificity for IDP and GDP and is inactive in the presence of IMP, GMP and ADP. Oyster enzyme, unlike p-enolpyruvate carboxykinases from other sources, exhibits no activity in the presence of Mg 2+. 4. 4. The pH activity profiles in the presence of Zn 2+ and Mn 2+ are similar in general pattern but the pH optima are 5·1 and 6·0 respectively. 5. 5. Kinetic constants for metal ions, IDP and p-enolpyruvate were determined at different pH values. For metal ions, the apparent enzyme-Mn 2+ affinity is greater at pH 6·0 than at pH 5·1 while the apparent enzyme-Zn 2+ affinity is greater at pH 5·1 than at pH 6·0. S 0·5 values for both metal ions under different conditions vary between 0·15 and 0·52 mM. The absolute values of S 0·5 for IDP are comparable in the presence of Zn 2+ and Mn 2+ (in the range of 0·03–0.06 mM) and these are largely independent of pH. p-Enolpyruvate saturation exhibits aberrant behaviour in the present of Mn 2+ while in the presence of Zn 2+ it follows Michaelis-Menten patterns. Equally significant, the apparent K m values are at least one order of magnitude less than observed for the enzyme in the presence of Mn 2+. 6. 6. Low concentrations of Cu 2+ (10–40 μM) inhibit the enzyme in the presence of either Zn 2+ or Mn 2+. Cu 2+ in the presence of Mn 2+ exhibits a competitive inhibition pattern while in the presence of Zn 2+, a non-competitive inhibition pattern occurs. 7. 7. The physiological role of the enzyme is discussed in relation to its cellular distribution, metal ion requirements and pH properties.