Carbamoyl phosphate synthetase: a tunnel runs through it

BACKGROUND: Sensitivity to specific allergens and increased sensitivity to common spasmogens are characteristic features of allergic asthma and are also features demonstrated by tissues passively sensitized with serum from atopic donors, displaying high levels of IgE. It is evident that the specific response to allergen is related to circulating levels of allergen-specific IgE, but it is unclear whether the histamine hypersensitivity is also related to this immunoglobulin. OBJECTIVE: The objective was to deplete IgE in the serum of a donor with high levels of total and allergen-specific IgE and compare specific-allergen sensitivity and sensitivity to histamine in tissues passively sensitized with either the whole serum or the IgE-depleted serum. METHODS: Serum from a Dermatophagoides farinae-sensitive asthmatic (total IgE = 1047 U/mL, D. farinae-specific IgE > 17.5 U/mL) was subjected to an immunomagnetic separation technique to reduce the levels of IgE (total and specific) to below 10 U/mL. Bronchial tissue from six non-atopic donors was then passively sensitized overnight with either the whole serum or IgE-depleted serum and D. farinae and histamine sensitivity were evaluated the next day using standard organ bath techniques. RESULTS: Passive sensitization with the whole serum resulted in the development of sensitivity to D. farinae and increased sensitivity to histamine (750+/-169 mg contraction to 10 U/mL D. farinae, histamine pEC50 5.64+/-0.16 and max. 813+/-109 mg in sensitized vs 37+/-34 mg contraction to 10 U/mL D. farinae histamine pEC50 5.05+/-0.23 and max. 490+/-84 in non-sensitized tissues, P>0.05). Incubation with IgE-depleted serum still produced histamine hypersensitivity (histamine pEC50 5.57+/-0.16 and max. 737+/-70 mg P>0.05), but no significant response to allergen was detected (20+/-13 mg contraction to 10 U/mL D. farinae). CONCLUSION: These results demonstrate, that increased reactivity to histamine and airway contraction to allergen induced by passive sensitization, occur through independent mechanisms and that, unlike allergen-sensitivity, histamine hypersensitivity is caused by a serum factor other than IgE.     

Carbamoyl phosphate synthetase: caught in the act of glutamine hydrolysis

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the production of carbamoyl phosphate from two molecules of Mg2+ATP, one molecule of bicarbonate, and one molecule of glutamine. The enzyme consists of two polypeptide chains referred to as the large and small subunits. While the large subunit provides the active sites responsible for the binding of nucleotides and other effector ligands, the small subunit contains those amino acid residues that catalyze the hydrolysis of glutamine to glutamate and ammonia. From both amino acid sequence analyses and structural studies it is now known that the small subunit belongs to the class I amidotransferase family of enzymes. Numerous biochemical studies have suggested that the reaction mechanism of the small subunit proceeds through the formation of the glutamyl thioester intermediate and that both Cys 269 and His 353 are critical for catalysis. Here we describe the X-ray crystallographic structure of carbamoyl phosphate synthetase from E. coli in which His 353 has been replaced with an asparagine residue. Crystals employed in the investigation were grown in the presence of glutamine, and the model has been refined to a crystallographic R-factor of 19.1% for all measured X-ray data from 30 to 1.8 A resolution. The active site of the small subunit clearly contains a covalently bound thioester intermediate at Cys 269, and indeed, this investigation provides the first direct structural observation of an enzyme intermediate in the amidotransferase family.     

Orientation specificity in spatial memory: What makes a path different from a map of the path?

Investigated factors that lead spatial information (SI) to be stored in an orientation-specific vs orientation-free manner. Exp 1 showed that learning paths from a small map vs learning paths directly from viewing a world lead to different functional characteristics of spatial memory. Whether the route display was presented as the path itself or as a large map of the path did not affect how the information was stored. Exp 2 examined effects of size of stimulus display, size of world, and scale transformations on how SI in maps is stored and available for use in later judgments. Exp 3 examined the effect of size on orientation specificity of the spatial coding of paths that are viewed directly. The main determinant of whether SI was stored and used in an orientation-specific manner or orientation-free manner was size of the display. Data support the view that there are distinct spatial representations (1 more perceptual and episodic and 1 more integrated and model-like) that have developed to meet different demands faced by mobile organisms. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Energy conservation behavior: The difficult path from information to action.

Presents a social-psychological model of energy-use behavior that draws on behavioral and social research to explain influence processes and behavioral change related to energy conservation behavior. The model consists of 2 interacting sets of factors: psychological factors that refer to how information is processed by individual decision makers and positional factors that relate to characteristics of the decision makers' situations that support or constrain action. Suggestions for maximizing the effectiveness of informational appeals to conserve energy by convincing the consumer that a pay-off will result from the use of energy conserving devices are discussed. It is suggested that the adoption of a conservatory attitude is influenced by the vividness of the argument to conserve energy, the credibility of the source, the understanding and retention of the message, and the degree to which an individual is able and willing to install conservation devices in his/her home. Alternatives to informational appeals through mass media to encourage energy conservation are proposed. (47 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus, a missing link in the evolution of carbamoyl phosphate biosynthesis

Microbial carbamoyl phosphate synthetases (CPS) use glutamine as nitrogen donor and are composed of two subunits (or domains), one exhibiting glutaminase activity, the other able to synthesize carbamoyl phosphate (CP) from bicarbonate, ATP, and ammonia. The pseudodimeric organization of this synthetase suggested that it has evolved by duplication of a smaller kinase, possibly a carbamate kinase (CK). In contrast to other prokaryotes the hyperthermophilic archaeon Pyrococcus furiosus was found to synthesize CP by using ammonia and not glutamine. We have purified the cognate enzyme and found it to be a dimer of two identical subunits of Mr 32,000. Its thermostability is considerable, 50% activity being retained after 1 h at 100 degrees C or 3 h at 95 degrees C. The corresponding gene was cloned by PCR and found to present about 50% amino acid identity with known CKs. The stoichiometry of the reaction (two ATP consumed per CP synthesized) and the ability of the enzyme to catalyze at high rate a bicarbonate-dependent ATPase reaction however clearly distinguish P. furiosus CPS from ordinary CKs. Thus the CPS of P. furiosus could represent a primeval step in the evolution of CPS from CK. Our results suggest that the first event in this evolution was the emergence of a primeval synthetase composed of subunits able to synthesize both carboxyphosphate and CP; this step would have preceded the duplication assumed to have generated the two subdomains of modern CPSs. The gene coding for this CK-like CPS was called cpkA.     

Links from emotional distress to adolescent drug use: A path model.

Administered anonymous surveys asking about drug use, emotional distress, and peer drug associations to 11th and 12th grade high school students (N?=?563). Emotional distress variables accounted for only 4.8% of the variance in drug use. The addition of peer drug associations as a predictor variable increased the variance accounted for to 43.4%. A path model of adolescent drug use based on peer cluster theory was tested using LISREL, and this provided a good fit with the data. As predicted, peer drug associations dominated the prediction of drug use and mediated the effect of emotional distress on drug use, with the exception of a small residual path directly from anger to drug use. The hypothesis that young people take drugs to alleviate emotional distress does not hold up well; emotional distress variables, with the exception of anger, produced only very small and indirect links to drug use. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

PIPkins1, their substrates and their products: new functions for old enzymes

The phosphatidylinositolphosphate kinases (PIPkins) are a unique family of enzymes that catalyse the production of phosphorylated inositol lipids. Recent advances have revealed that, due to their ability to utilise a number of different lipid substrates (at least in vitro), this family is potentially able to generate several distinct, physiologically important inositol lipids. Despite their importance, however, our understanding of the regulation of the PIPkins and of their physiological role in cellular signalling and regulation is still poor. Here we describe in turn the diverse physiological functions of the known substrates and major products of the PIPkins. We then examine what is known about the members of the PIPkin family themselves, and their characteristics and regulation.     

beta-Lactam synthetase: a new biosynthetic enzyme

The principal cause of bacterial resistance to penicillin and other beta-lactam antibiotics is the acquisition of plasmid-encoded beta-lactamases, enzymes that catalyze hydrolysis of the beta-lactam bond and render these antibiotics inactive. Clavulanic acid is a potent inhibitor of beta-lactamases and has proven clinically effective in combating resistant infections. Although clavulanic acid and penicillin share marked structural similarities, the biosyntheses of their bicyclic nuclei are wholly dissimilar. In contrast to the efficient iron-mediated oxidative cyclization of a tripeptide to isopenicillin N, the critical beta-lactam ring of clavulanic acid is demonstrated to form by intramolecular closure catalyzed by a new type of ATP/Mg2+-dependent enzyme, a beta-lactam synthetase (beta-LS). Insertional inactivation of its encoding gene in wild-type Streptomyces clavuligerus resulted in complete loss of clavulanic acid production and the accumulation of N2-(carboxyethyl)-L-arginine (CEA). Chemical complementation of this blocked mutant with authentic deoxyguanidinoproclavaminic acid (DGPC), the expected product of the beta-LS, restored clavulanic acid synthesis. Finally, overexpression of this gene gave the beta-LS, which was shown to mediate the conversion of CEA to DGPC in the presence of ATP/Mg2+. Primary amino acid sequence comparisons suggest that this mode of beta-lactam formation could be more widely spread in nature and mechanistically related to asparagine synthesis.     

Mechanism of carbamoyl phosphate synthetase from Escherichia coli--binding of the ATP molecules used in the reaction and sequestration by the enzyme of the ATP molecule that yields carbamoyl phosphate

A directly energized vacuolar pump for glutathione (GS) conjugates has been described for several plant species. Since glucuronate conjugates also occur in plants, we addressed the question whether plant vacuoles take up the abiotic glucuronate conjugate estradiol 17-(beta-glucuronide) (E217G) via a GS conjugate pump, which in some cases has been reported to accept various organic anions as substrates, or via a distinct glucuronate transporter. Uptake studies into vacuoles from rye and barley were performed with E217G and metolachlor-GS (MOC-GS), a substrate of the GS conjugate ATPase, to compare glucuronate conjugate transport into vacuoles containing endogenous flavone glucuronides with those lacking specific glucuronate conjugates, respectively. Our results indicate that E217G and MOC-GS are taken up into vacuoles of both plants via a directly energized mechanism since transport was (i) strictly ATP-dependent; (ii) inhibited by vanadate but not by bafilomycin A1, azide, verapamil, nor by dissipation of the vacuolar DeltapH or DeltaPsi; (iii) E217G uptake into rye vacuoles was partially driven by other nucleotides in the following order of efficiency: ATP > GTP > UTP congruent with CTP, whereas the non-hydrolyzable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate, ADP, or PPi did not energize uptake. E217G transport into rye vacuoles was saturable (Km approximately 0.2 mM). The rye-specific luteolin glucuronides decreased uptake rates of E217G and MOC-GS into rye and barley vacuoles to comparable degrees with the mono- and diglucuronidated derivatives (40-60% inhibition) being more effective than the triglucuronide. Inhibition of E217G uptake by luteolin 7-O-diglucuronide was competitive (Ki = 120 microM). Taurocholate had no effect on E217G transport, and uptake of MOC-GS was not inhibited by E217G. Although GS conjugates and oxidized GS decreased MOC-GS transport, E217G uptake into rye and barley vacuoles was stimulated up to 7-fold in a concentration-dependent manner by these substances, with dinitrobenzene-GS being most effective. The stimulation of the GS conjugates was not due to detergent or redox effects and was specific for the E217G pump. GS conjugate stimulation of glucuronate uptake was unique for plants as E217G uptake into yeast microsomal vesicles was not affected. By comparison with a DeltaYCF1 yeast mutant, defective in vacuolar transport of GS conjugates mediated by YCF1, it was shown that E217G was taken up into yeast vesicles via a YCF1-independent directly energized pump. These results indicate that E217G as a glucuronate conjugate is transported across the vacuolar membranes of plants and yeast by a carrier distinct from the GS conjugate ATPase.     

Cloning and sequencing of a gene from the archaeon Pyrococcus furiosus with high homology to a gene encoding phosphoenolpyruvate synthetase from Escherichia coli

A gene from the hyperthermophilic archaeon Pyrococcus furiosus, strain Vc1 (DSM 3638), contains an 817-amino-acid open reading frame which shows 42% identity to the phosphoenolpyruvate (PEP) synthetase of Escherichia coli. This putative P. furiosus PEP synthetase is slightly larger than the E. coli enzyme, the region between residues 58 and 89 being absent from the latter.     

AIR synthetase in cowpea nodules: a single gene product targeted to two organelles?

A cDNA (VUpur5) encoding phosphoribosyl aminoimidazole (AIR) synthetase, the fifth enzyme of the de novo purine biosynthesis pathway has been isolated from a cowpea nodule cDNA library. It encodes a 388 amino acid protein with a predicted molecular mass of 40.4 kDa. The deduced amino acid sequence has significant homology with AIR synthetase from other organisms. AIR synthetase is present in both mitochondria and plastids of cowpea nodules. A signal sequence encoded by the VUpur5 cDNA has properties associated with plastid transit sequences but there is no consensus cleavage site as would be expected for a plastid targeted protein. Although the signal sequence does not have the structural features of a mitochondrial targeted protein, it has a mitochondrial cleavage site motif (RX/XS) close to the predicted N-terminus of the mature protein. Southern analysis suggests that AIR synthetase is encoded by a single gene raising questions as to how the product of this gene is targeted to the two organelles. VUpur5 is expressed at much higher levels in nodules compared to other cowpea tissues and the gene is active before nitrogen fixation begins. These results suggest that products of nitrogen fixation do not play a role in the initial induction of gene expression. VUpur5 was expressed in Escherichia coli and the recombinant protein used to raise antibodies. These antibodies recognize two forms of AIR synthetase which differ in molecular size. Both forms are present in mitochondria, although the larger protein is more abundant. Only the smaller protein was detected in plastids.     

Microbial dehalogenases: enzymes recruited to convert xenobiotic substrates

Ocular torsion was measured in five subjects during sinusoidal lateral tilt (amplitude 25 degrees, 0.2 Hz). The cervical contribution to ocular torsion was best visible as the difference between the signals obtained in conditions with only head tilt and conditions with whole body tilt. Contribution of the neck did not affect the slow component, but produced an anticompensatory modulation of the beating field offset by means of saccades (analogous to gaze shift). Static tilt conditions (25 degrees tilt) of the trunk only, the head only or the whole body showed similar data, although of smaller amplitude. The results from patients suffering from post-whiplash syndrome were similar to those of healthy subjects, showing large intersubject variability. The reduced tolerance to head tilt of whiplash patients restricts useful implementation of this sort of test in the clinic.     

The path to a clinical pathway: collaborative care for the patient with an ostomy

A colostomy and ileostomy clinical pathway was developed at a southeastern teaching hospital in 1990 in response to excessive lengths of stay and costs at our hospital compared with national data for this patient group. A multidisciplinary clinical pathway team was formed and charged with the development, implementation, and ongoing monitoring of the clinical pathway tool and its effect on the outcomes of the population of patients with colostomies and ileostomies. Through this multidisciplinary collaboration, length of stay and cost have been reduced while quality care indicators have been maintained. This article presents the sample pathway we developed and describes the pathway development process, documentation, the variance analysis process, and the outcomes achieved with implementation. A urostomy/urinary diversion pathway that was developed after variance analysis review of the colostomy and ileostomy clinical pathway is also presented.