Structures of the Mycobacterium tuberculosis GlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition.

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) from Mycobacterium tuberculosis at 2.6 Šresolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase from Synechocystis (strain 6803) as well as the equivalent enzyme from Thermosynechococcus elongatus. This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII from Escherichia coli and is probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII from M. tuberculosis (MtFBPaseII) is conserved and is analogous to that described previously for the E. coli enzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures of MtFBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for the Synechocystis enzyme. The structural and functional insights derived from the structure of MtFBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.

Net efflux of citrate in Penicillium simplicissimum is mediated by a transport protein.

Penicillium simplicissimum excreted citrate, isocitrate, and succinate when grown in a strongly buffered medium [1 M Mes (pH 6) or 1 M Hepes (pH 7.3)]. Growth in a weakly buffered medium did not lead to citrate excretion despite a similar intracellular citrate concentration. When nongrowing, citrate-excreting hyphae were aerated in a glucose solution, the following steady-state intracellular concentrations of organic acids were measured: succinate (25 mM); citrate, isocitrate, malate, and fumarate (all less than 5 mM). After 2 h of incubation, the extracellular concentrations of these acids were [micromol (g dry wt.)-1]: isocitrate [100], citrate [60], succinate [30], and malate, fumarate, and alpha-ketoglutarate [<5]. The excretion of citrate was due neither to an unspecific change in the permeability of the plasma membrane nor to simple diffusion of undissociated citric acid. The involvement of a transport protein in citrate excretion was indicated because N-ethylmaleimide and sodium azide inhibited citrate excretion strongly despite an unchanged outward-directed citrate gradient. Arguments are given why efflux via a citrate uptake carrier is not considered probable. These results indicate that citrate is excreted by P. simplicissimum via a transport protein that probably specifically mediates the efflux of citrate.

Specific inhibition of basic calcium phosphate and calcium pyrophosphate crystal-induction of metalloproteinase synthesis by phosphocitrate.

Calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystal deposition diseases are a group of heterogeneous arthritides which are a significant source of morbidity in the elderly. Both crystals induced mitogenesis and metalloproteinase (MP) synthesis and secretion by fibroblasts and chondrocytes which may promote degradation of intra-articular tissue. We have previously shown that phosphocitrate (PC), an inhibitor of hydroxyapatite crystallization, specifically blocks BCP crystal-induced mitogenesis in 3T3 cells. This led us to examine the effect of PC on BCP and CPPD crystal induction of MP synthesis in human fibroblasts. PC (10(-3) to 10(-4) M) specifically inhibited the crystal-induced collagenase and stromelysin mRNA accumulation while having no effect on epidermal growth factor-induced or basal levels of mRNA for both enzymes. Western blots (collagenase) of conditioned media confirmed that PC blocked crystal-induced proteinase secretion as well. Moreover, PC (10(-3) M) also blocked the crystal induction of c-fos and c-jun. Since FOS and JUN proteins form a transacting activator (AP-1) for expression of collagenase and stromelysin genes, PC may block the synthesis of both enzymes by inhibiting the transcription of c-fos and c-jun.

Capsazepine inhibits cough induced by capsaicin and citric acid but not by hypertonic saline in guinea pigs.

Acidic solutions mimick many of the effects of capsaicin (Cap), including pain, bronchoconstriction, cough, and sensory neuropeptide release. Evidence from the use of the Cap antagonist capsazepine suggests that in some cases protons act at the Cap receptor. In the present study, we have investigated whether cough evoked by Cap and citric acid (CA) is mediated specifically via the Cap receptor on airway sensory nerves. We have examined the effects of capsazepine on Cap-, CA-, and hypertonic saline-induced cough and also on CA-induced nasal irritation in awake guinea pigs. Capsazepine was nebulized for 5 min before cough challenges with Cap for 5 min and CA for 10 min. Control animals were pretreated with vehicle alone. Capsazepine (100 microM) inhibited the cough response to 30 microM Cap from 0.77 +/- 0.14 coughs/min in control animals to 0.23 +/- 0.08 coughs/min (P < 0.05) and to 80 microM Cap from 1.4 +/- 0.23 to 0.3 +/- 0.11 coughs/min (P < 0.01). There was no effect, however, of lower concentrations of capsazepine (5 and 10 microM) against Cap-evoked cough. At a concentration of 100 microM, capsazepine also inhibited the coughing induced by 0.25 M CA from 1.8 +/- 0.26 to 0.93 +/- 0.31 coughs/min (P < 0.05) but not that induced by 0.5 M CA. Nasal irritation induced by 0.25 M CA, but not by 0.5 M CA, was also inhibited by capsazepine from 2.47 +/- 0.37 to 0.75 +/- 0.31 nose wipes/min (P < 0.05). This inhibitory effect of capsazepine did not appear to reflect a nonspecific suppression of the cough reflex, since cough evoked by exposure to hypertonic (7%) saline for 10 min was unaffected by pretreatment with capsazepine (100 microM). These data show that capsazepine is a specific inhibitor of Cap- and CA-induced cough in guinea pigs. Moreover, they suggest that low pH stimuli evoke cough and nasal irritation by an action at the Cap receptor, either directly or through the release of an intermediate agent.

Suppressive effect of clenbuterol on citric acid-induced cough reflex in guinea pigs.

We previously demonstrated that clenbuterol suppressed bronchial hyperresponsiveness in acute bronchitic models. However the effect of clenbuterol on the cough reflex, the main symptom of acute bronchitis, is not clear. The present study was thus undertaken to investigate the influence of clenbuterol on the cough reflex. Oral administration of clenbuterol (3 and 10 micrograms/kg) to guinea pigs markedly inhibited the increase in the respiratory resistance in response to 5-HT in a dose-dependent manner. At doses of 10 micrograms/kg and above, clenbuterol significantly inhibited the cough reflex induced by citric acid in guinea pigs. These doses are comparable with those causing broncho-dilation as described above, suggesting that the suppressive effect of clenbuterol on the cough reflex in guinea pigs may result from mainly its broncho-dilative action via stimulation of beta-2 adrenoceptors in airway smooth muscles however, other mechanisms cannot be ruled out. These results indicate that this agent may be useful for treatment of cough, the main symptom of acute bronchitis.

Differential effect of glucocorticoids on pulmonary responses and eosinophils.

The persistent airway hyperresponsiveness of Basenji-Greyhound (BG) dogs to 10% citric acid (CA) is abolished by chronic administration of methylprednisolone (MP) and is accompanied by the disappearance of eosinophils from the bronchoalveolar lavage (BAL) fluid. To determine whether the disappearance of eosinophils from BAL fluid was temporally related to the loss of airway responsiveness to CA, we investigated the time course of the reduction in airway responsiveness to CA and correlated it with changes in cell profiles in BAL fluid in a group of BG dogs treated with MP for 1 to 7 days. Six dogs in separate studies were pretreated with MP (2 mg/kg/day) subcutaneously for either 1, 3 or 7 days. Each dog served as its own control for each set of studies. Under thiopental anesthesia, lung resistance (RL) was calculated from transpulmonary pressure and flow measurements prior to and 5 minutes following the completion of a 10% CA aerosol. BAL was performed on a separate occasion with the animals pretreated with MP for either 1, 7, 10 or 14 days. Baseline RL was not significantly different in each control and treatment group. The pulmonary response to CA challenge was diminished following 1, 3 and 7 days of MP pretreatment. Although eosinophils disappeared from the peripheral blood following 1 day of MP treatment, eosinophils in BAL did not begin decreasing until 10 days of MP pretreatment. This temporal dissociation between CA hyperresponsiveness and eosinophils in the BAL fluid suggests that epithelial damage by toxic products of eosinophils in the airway lumen does not play a direct role in citric acid induced airway hyperresponsiveness in BG dogs.

Biological properties of pancreatic elastase in relation to atherosclerotic disease.

The inhibitory effect of elastase on experimental atherosclerosis has been reported in numerous studies. In our investigation, performed in the rat, a pancreatic extract provided with elastolytic activity has been shown to possess an anti-aggregative effect in vitro and ex vivo and anti-thrombotic properties. In addition, the elastase was capable of inhibiting endothelial exfoliation induced by the desquamatory agent sodium citrate. This agent was tested for its microhaemorrhoeological activity in acute and subacute experiments. In both these conditions, elastase was able to increase the flexibility of red blood cells and their resistance to lysis provoked by hypotonic solutions. In animals fed on an atherogenic diet, this substance limited the lipoprotein accumulation in the aorta wall. Moreover, it reduced the enhanced calcium content, induced by vitamin D administration, in the tissue of arteries. These data indicate that elastase can counteract some pathobiological aspects that characterize atherosclerotic events.