Model of severe malaria in young mice suggests unique response of CD4 T cells.

Severe malaria occurs most in young children but is poorly understood due to the absence of a developmentally-equivalent rodent model to study the pathogenesis of the disease. Though functional and quantitative deficiencies in innate response and a biased T helper 1 (Th1) response are reported in newborn pups, there is little information available about this intermediate stage of the adaptive immune system in murine neonates. To fill this gap in knowledge, we have developed a mouse model of severe malaria in young mice using 15-day old mice (pups) infected with Plasmodium chabaudi. We observe similar parasite growth pattern in pups and adults, with a 60% mortality and a decrease in the growth rate of the surviving young mice. Using a battery of behavioral assays, we observed neurological symptoms in pups that do not occur in infected wildtype adults. CD4+ T cells were activated and differentiated to an effector T cell (Teff) phenotype in both adult and pups. However, there were relatively fewer and less terminally differentiated pup CD4+ Teff than adult Teff. Interestingly, despite less activation, the pup Teff expressed higher T-bet than adults' cells. These data suggest that Th1 cells are functional in pups during Plasmodium infection but develop slowly.

On the kinetic mechanism of dimethylarginine dimethylaminohydrolase.

Dimethylarginine dimethylaminohydrolase (DDAH, EC 3.5.3.18) catalyzes the hydrolysis of asymmetric Nω,Nω-dimethyl-l-arginine (ADMA), an endogenous inhibitor of human nitric oxide synthases. The active-site cysteine residue has been proposed to serve as the catalytic nucleophile, forming an S-alkylthiourea reaction intermediate, and serving as a target for covalent inhibitors. Inhibition can lead to ADMA accumulation and downstream inhibition of nitric oxide production. Prior studies have provided experimental evidence for formation of this covalent adduct but have not characterized it kinetically. Here, rapid quench-flow is used with ADMA and the DDAH from Pseudomonas aeruginosa to determine the rate constants for formation (k2 = 17 ± 2 s-1) and decay (k3 = 1.5 ± 0.1 s-1) of the covalent S-alkylthiourea adduct. A minimal kinetic mechanism for DDAH is proposed that supports the kinetic competence of this species as a covalent reaction intermediate and assigns the rate-limiting step in substrate turnover as hydrolysis of this intermediate. This work helps elucidate the different reactivities of S-alkylthiourea intermediates found among the mechanistically diverse pentein superfamily of guanidine-modifying enzymes and provides information useful for inhibitor development.

Discovery of halopyridines as quiescent affinity labels: inactivation of dimethylarginine dimethylaminohydrolase.

In an effort to develop novel covalent modifiers of dimethylarginine dimethylaminohydrolase (DDAH) that are useful for biological applications, a set of "fragment"-sized inhibitors that were identified using a high-throughput screen are tested for time-dependent inhibition. One structural class of inactivators, 4-halopyridines, show time- and concentration-dependent inactivation of DDAH, and the inactivation mechanism of one example, 4-bromo-2-methylpyridine (1), is characterized in detail. The neutral form of halopyridines is not very reactive with excess glutathione. However, 1 readily reacts, with loss of its halide, in a selective, covalent, and irreversible manner with the active-site Cys249 of DDAH. This active-site Cys is not particularly reactive (pK(a) ca. 8.8), and 1 does not inactivate papain (Cys pK(a) ca. ≤4), suggesting that, unlike many reagents, Cys nucleophilicity is not a predominating factor in selectivity. Rather, binding and stabilization of the more reactive pyridinium form of the inactivator by a second moiety, Asp66, is required for facile reaction. This constraint imparts a unique selectivity profile to these inactivators. To our knowledge, halopyridines have not previously been reported as protein modifiers, and therefore they represent a first-in-class example of a novel type of quiescent affinity label.

On the mechanism of dimethylarginine dimethylaminohydrolase inactivation by 4-halopyridines.

Small molecules capable of selective covalent protein modification are of significant interest for the development of biological probes and therapeutics. We recently reported that 2-methyl-4-bromopyridine is a quiescent affinity label for the nitric oxide controlling enzyme dimethylarginine dimethylaminohydrolase (DDAH) (Johnson, C. M.; Linsky, T. W.; Yoon, D. W.; Person, M. D.; Fast, W. J. Am. Chem. Soc. 2011, 133, 1553-1562). Discovery of this novel protein modifier raised the possibility that the 4-halopyridine motif may be suitable for wider application. Therefore, the inactivation mechanism of the related compound 2-hydroxymethyl-4-chloropyridine is probed here in more detail. Solution studies support an inactivation mechanism in which the active site Asp66 residue stabilizes the pyridinium form of the inactivator, which has enhanced reactivity toward the active site Cys, resulting in covalent bond formation, loss of the halide, and irreversible inactivation. A 2.18 Å resolution X-ray crystal structure of the inactivated complex elucidates the orientation of the inactivator and its covalent attachment to the active site Cys, but the structural model does not show an interaction between the inactivator and Asp66. Molecular modeling is used to investigate inactivator binding, reaction, and also a final pyridinium deprotonation step that accounts for the apparent differences between the solution-based and structural studies with respect to the role of Asp66. This work integrates multiple approaches to elucidate the inactivation mechanism of a novel 4-halopyridine "warhead," emphasizing the strategy of using pyridinium formation as a "switch" to enhance reactivity when bound to the target protein.

Expert training with standardized operative technique helps establish a successful penile prosthetics program for urologic resident education.

INTRODUCTION: The challenge of resident education in urologic surgery programs is to overcome disparity imparted by diverse patient populations, limited training times, and inequalities in the availability of expert surgical educators. Specifically, in the area of prosthetic urology, only a small proportion of programs have full-time faculty available to train residents in this discipline. AIM: To examine whether a new model using yearly training sessions from a recognized expert can establish a successful penile prosthetics program and result in better outcomes, higher case volumes, and willingness to perform more complex surgeries. METHODS: A recognized expert conducted one to two operative training sessions yearly to teach standardized technique for penile prosthetics to residents. Each session consisted of three to four operative cases performed under the direct supervision of the expert. Retrospective data were collected from all penile prosthetic operations before (February, 2000 to June, 2004: N = 44) and after (July, 2004 to October, 2007: N = 79) implementation of these sessions. MAIN OUTCOME MEASURES: Outcomes reviewed included patient age, race, medical comorbidities, operative time, estimated blood loss, type of prosthesis, operative approach, drain usage, length of stay, and complications including revision/explantation rates. Statistical analysis was performed using Student's t-tests, Fisher's tests, and survival curves using the Kaplan-Meier technique (P value ≤ 0.05 to define statistical significance). RESULTS: Patient characteristics were not significantly different pre- vs. post-training. Operative time and estimated blood loss significantly decreased. Inflatable implants increased from 19/44 (43.2%, pre-training) to 69/79 (87.3%, post-training) (P < 0.01). Operations per year increased from 9.96 (pre-training) to 24 (post-training) (P < 0.01). Revision/explantation occurred in 11/44 patients (25%, pre-training) vs. 7/79 (8.9%, post-training) (P < 0.05). CONCLUSIONS: These data demonstrate that yearly sessions with a recognized expert can improve surgical outcomes, type, and volume of implants and can reduce explantation/revision rates. This represents an excellent model for improved training of urologic residents in penile prosthetics surgery.

Prostaglandin E2mediates spontaneous rhythmic contraction in rabbit detrusor muscle.

INTRODUCTION: The purpose of this investigation was to determine if prostaglandin E2(PGE2) is produced by rabbit detrusor free of urothelium and demonstrate that PGE2 is responsible for the generation of spontaneous rhythmic contraction (SRC). METHODS: A bioassay was performed in which contraction frequency in strips of rabbit detrusor was compared before and after addition of superfusate from incubating sections of rabbit detrusor. Specificity was determined by testing the effects of SC-51089, a PGE2(EP1) antagonist. Effects on development of tension were determined in artery segments after treatment with increasing doses of PGE2, PGF2α, and TXA2, and a section of femoral artery was used as a negative control. Confirmation of PGE2 production was then determined using EIA kits. RESULTS: Increased rhythmic frequency was identified after superfusate from a section of rabbit detrusor free of urothelium was added to strips of detrusor from the same animal. Additional experiments demonstrated that rhythmic frequency generated after treatment with PGE2 was significantly reduced after treatment with SC-51089. In artery smooth muscle, prostaglandin dose response experiments demonstrated that only TXA2 induced contraction at physiologic doses (<10⁻7M). As a negative control, subsequent treatment of a section of femoral artery with detrusor superfusate failed to increase tension, confirming a lack of TXA2 production. EIA confirmed that PGE2 production increased by 4.8-fold in strips of detrusor free of urothelium after 15 minutes of incubation and that this production was blocked by ibuprofen and a COX-1 inhibitor. CONCLUSIONS: Rabbit detrusor produces PGE2 which is the most likely mediator of SRC.

Spatial Segregation of Phosphatidylinositol 4,5-Bisphosphate (PIP(2)) Signaling in Immune Cell Functions.

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is a prevalent phosphoinositide in the inner leaflet of the plasma membrane. PIP(2) associates with an ever-growing list of proteins, and participates in a variety of cellular processes. PIP(2) signaling to the actin cytoskeleton transduces specific signals necessary for changes in morphology, motility, endocytosis, exocytosis, phagocytosis, and cell activation. The mechanism(s) by which PIP(2) signaling pathways are specific is a topic of intense investigation. One working model is the compartmentalization of PIP(2)-mediated signaling by concentrating PIP(2) in cholesterol-dependent membrane rafts, therefore providing spatial and temporal regulation. Here we discuss properties of PIP(2) signaling to the actin cytoskeleton in immune cell functioning, the association of PIP(2) cellular pools with membrane rafts, and recent work investigating models for compartmentalization of PIP(2)-mediated signaling in membrane rafts to the actin cytoskeleton.

Compartmentalization of phosphatidylinositol 4,5-bisphosphate signaling evidenced using targeted phosphatases.

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is a prevalent phosphoinositide in cell membranes, with important functions in cell signaling and activation. A large fraction of PIP(2) associates with the detergent-resistant membrane "raft" fraction, but the functional significance of this association remains controversial. To measure the properties of raft and nonraft PIP(2) in cell signaling, we targeted the PIP(2)-specific phosphatase Inp54p to either the raft or nonraft membrane fraction using minimal membrane anchors. Interestingly, we observed that targeting Inp54p to the nonraft fraction resulted in an enrichment of raft-associated PIP(2) and striking changes in cell morphology, including a wortmannin-sensitive increase in cell filopodia and cell spreading. In contrast, raft-targeted Inp54p depleted the raft pool of PIP(2) and produced smooth T cells void of membrane ruffling and filopodia. Furthermore, raft-targeted Inp54p inhibited capping in T cells stimulated by cross-linking the T cell receptor, but without affecting the T cell receptor-dependent Ca(2+) flux. Altogether, these results provide evidence of compartmentalization of PIP(2)-dependent signaling in cell membranes such as predicted by the membrane raft model.

The serine acetyltransferase reaction: acetyl transfer from an acylpantothenyl donor to an alcohol.

Serine acetyltransferase is a member of the left-handed parallel beta-helix family of enzymes that catalyzes the committed step in the de novo synthesis of l-cysteine in bacteria and plants. The enzyme has an ordered kinetic mechanism with acetyl CoA bound prior to l-serine and O-acetyl-l-serine released prior to CoA. The rate-limiting step along the reaction pathway is the nucleophilic attack of the serine hydroxyl on the thioester of acetyl CoA. Product release contributes to rate-limitation at saturating concentrations of reactants. The reaction is catalyzed by an active site general base with a pK of 7, which accepts a proton from the serine hydroxyl as a tetrahedral intermediate is formed between the reactants, and donates it to the thiol of CoA as the intermediate collapses to give products. This mechanism is likely the same for all O-acyltransferases that catalyze their reaction by direct attack of the alcohol on the acyl donor, using an active-site histidine as the general base. Serine acetyltransferase is regulated by feedback inhibition by the end product l-cysteine, which acts by binding to the serine site in the active site and inducing a conformational change that prevents reactant binding. The enzyme also associates with O-acetylserine sulfhydrylase, the final enzyme in the biosynthetic pathway, which contributes to stabilizing the acetyltransferase.

Chemical mechanism of the serine acetyltransferase from Haemophilus influenzae.

The pH dependence of kinetic parameters was determined in both reaction directions to obtain information about the acid-base chemical mechanism of serine acetyltransferase from Haemophilus influenzae (HiSAT). The maximum rates in both reaction directions, as well as the V/K(serine) and V/K(OAS), decrease at low pH, exhibiting a pK of approximately 7 for a single enzyme residue that must be unprotonated for optimum activity. The pH-independent values of V(1)/E(t), V(1)/K(serine)E(t), V/K(AcCoA)E(t), V(2)/E(t), V(2)/K(OAS)E(t), and V/K(CoA)E(t) are 3300 +/- 180 s(-1), (9.6 +/- 0.4) x 10(5) M(-1) s(-1), 3.3 x 10(6) M(-1) s(-1), 420 +/- 50 s(-1), (2.1 +/- 0.5) x 10(4) M(-1) s(-1), and (4.2 +/- 0.7) x 10(5) M(-1) s(-1), respectively. The K(i) values for the competitive inhibitors glycine and l-cysteine are pH-independent. The solvent deuterium kinetic isotope effects on V and V/K in the direction of serine acetylation are 1.9 +/- 0.2 and 2.5 +/- 0.4, respectively, and the proton inventories are linear for both parameters. Data are consistent with a single proton in flight in the rate-limiting transition state. A general base catalytic mechanism is proposed for the serine acetyltransferase. Once acetyl-CoA and l-serine are bound, an enzymic general base accepts a proton from the l-serine side chain hydroxyl as it undergoes a nucleophilic attack on the carbonyl of acetyl-CoA. The same enzyme residue then functions as a general acid, donating a proton to the sulfur atom of CoASH as the tetrahedral intermediate collapses, generating the products OAS and CoASH. The rate-limiting step in the reaction at limiting l-serine levels is likely formation of the tetrahedral intermediate between serine and acetyl-CoA.

Kinetic mechanism of the serine acetyltransferase from Haemophilus influenzae.

The kinetic mechanism of serine acetyltransferase from Haemophilus influenzae was studied in both reaction directions. The enzyme catalyzes the conversion of acetyl CoA and L-serine to O-acetyl-L-serine (OAS) and coenzyme A (CoASH). In the direction of L-serine acetylation, an equilibrium ordered mechanism is assigned at pH 6.5. The initial velocity pattern in the absence of added inhibitors is best described by a series of lines converging on the ordinate when L-serine is varied at different fixed levels of acetyl CoA. The initial velocity pattern at pH 7.5 is also intersecting, but the lines are nearly parallel. Product inhibition by OAS is noncompetitive against acetyl CoA, while it is uncompetitive against L-serine. Product inhibition by L-serine in the reverse reaction direction is noncompetitive with respect to both OAS and CoASH. Glycine and S-methyl-L-cysteine (SMC) were used as dead-end analogs of L-serine and OAS, respectively. Glycine is competitive versus L-serine and uncompetitive versus acetyl CoA, while SMC is competitive against OAS and uncompetitive against CoASH. Desulfo-CoA was used as a dead-end analog of both acetyl CoA and CoASH, and is competitive versus both substrates in the direction of L-serine acetylation; while it is competitive against CoASH and noncompetitive against OAS in the direction of CoASH acetylation. All of the above kinetic parameters are consistent with those predicted for an ordered mechanism at pH 6.5 with the exception of the uncompetitive inhibition by OAS vs. serine. The latter inhibition pattern suggests combination of OAS with the central E:acetyl CoA:serine complex. Cysteine is known to regulate its own biosynthesis at the level of SAT. As a dead-end inhibitor, L-cysteine is competitive against both substrates in both reaction directions. These results are discussed in terms of the mechanism of regulation.