Biochemical characterization of the Pseudomonas aeruginosa 101/1477 metallo-beta-lactamase IMP-1 produced by Escherichia coli.
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The blaIMP gene coding for the IMP-1 metallo-beta-lactamase produced by a Pseudomonas aeruginosa clinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in Escherichia coli BL21 (DE3), and its product was purified to homogeneity with a final yield of 35 mg/liter of culture. The structural and functional properties of the enzyme purified from E. coli were identical to those of the enzyme produced by P. aeruginosa. The IMP-1 metallo-beta-lactamase exhibits a broad-spectrum activity profile that includes activity against penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems. Only monobactams escape its action. The enzyme activity was inhibited by metal chelators, of which 1,10-o-phenanthroline and dipicolinic acid were the most efficient. Two zinc-binding sites were found. The zinc content of the P. aeruginosa 101/1477 metallo-beta-lactamase was not pH dependent. (+info)
Efficacy of chromium picolinate on performance and tissue accretion in pigs with different lean gain potential.
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We conducted two experiments to determine whether the efficacy of chromium picolinate (CrP) on growth performance, carcass composition, and tissue accretion rates is dependent on the lean gain potential of the pigs. In Exp. 1, 40 barrows (20 from each of two genetic backgrounds; two pigs per pen, five pens per treatment) were fed a fortified, corn-soybean meal basal diet (.95% lysine from 19 to 55 kg BW; .80% lysine from 55 to 109 kg BW) without or with 200 microg/kg of Cr from CrP. The addition of Cr had no effect on performance, carcass measurements, or accretion rates of carcass protein or lipid, regardless of the lean gain potential of the pigs. In Exp. 2, 60 group-penned pigs (three pigs per pen; five pens per treatment) were fed a fortified, corn-soybean meal basal diet without or with 200 microg/kg of Cr from CrP from 21 to 104 kg BW. Within the dietary Cr treatments, half of the pigs received daily injections of 3 mg of porcine somatotropin (pST) from 54 to 104 kg BW. The pST administration resulted in faster growth rates (P < .007), improved feed efficiency (P < .001), increased longissimus area (P < .001), and decreased 10th-rib backfat (P < .001). Administration of pST also increased the percentage and accretion rate of carcass protein (P < .001) and decreased the percentage and accretion rate of carcass lipid (P < .001). The addition of CrP to the diet had no effect on any variable measured in either the untreated or pST-treated pigs. In these studies, Cr was ineffective at altering the composition of the carcass and its effects were not dependent on the pig's potential for lean gain. (+info)
Dietary protein and chromium tripicolinate in Suffolk wether lambs: effects on production characteristics, metabolic and hormonal responses, and immune status.
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Thirty-two Suffolk wether lambs were fed for 84 d in a 2 x 2 factorial experiment using two levels of dietary protein (9.0 to 12.1% CP, low protein, LP; or 12.8 to 14.4% CP, high protein, HP) and supplemental Cr (none, C; or 400 ppb Cr as chromium tripicolinate, Cr). At 14- to 21-d intervals, lambs were weighed, and jugular blood samples were collected. Mean ADG and carcass weight (P > .10) did not differ. In lambs fed HP, Cr reduced liver weight and increased kidney weight (P < .01). Lambs fed HP had elevated plasma urea N (PUN; P < .01) and albumin (P < .04). During an i.v. epinephrine challenge on d 43, plasma cortisol declined in lambs fed Cr (Cr x time, P < .03) and in lambs fed LP (CP x time, P < .001). An i.v. glucose tolerance test conducted 3 h later showed that supplemental Cr decreased glucose clearance rate in lambs fed HP (CP x Cr, P < .10) but not in lambs fed LP. On d 62, PUN was increased in lambs fed HP (P < .001) between 0 and 3 h postprandial, and there was a Cr x CP interaction (P < .04). Postprandial plasma NEFA declined with Cr vs C (Cr x time, P < .07) and with HP vs LP (CP x time, P < .10). By d 66, lambs fed Cr had an elevated (P < .03) blood platelet and fibrinogen content. Chromium increased erythrocyte count in lambs fed HP (Cr x CP, P < .08), and isolated peripheral lymphocytes had greater blastogenic response to 4 microg/mL of phytohemagglutinin (Cr x CP, P < .001). The lymphocyte response to pokeweed mitogen (.2 microg/mL) was reduced in lambs fed Cr (P < .10). In the present experiment, Cr supplementation had minimal and inconsistent effects on production and metabolic criteria of lambs. (+info)
Calcium accumulation during sporulation of Bacillus megaterium KM.
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Accumulation of Ca2+ in Bacilli occurs during stages IV to VI of sporulation. Ca2+ uptake into the sporangium was investigated in Bacillus megaterium KM in protoplasts prepared in stage III of sporulation and cultured to continue sporulation. These protoplasts and whole cells exhibit essentially identical Ca2+ uptake, which is compared with that of forespores isolated in stage V of sporulation. Ca2+, uptake into both sporangial protoplasts and isolated forespores occurs by Ca2+-specific carrier-mediated processes. However, protoplasts exhibit a Km value of 31 micrometer, and forespores have a Km value of 2.1 mM. Sporangial protoplasts accumulate Ca2+ against a concentration gradient. In contrast, Ca2+ uptake into isolated forespores is consistent with downhill transfer in which both rate and extent of uptake are affected by the external Ca2+ concontration. Dipicolinic acid has no effect on Ca2+ uptake by isolated forespores, apart from decreasing the external Ca2+ concentration by chelation. A model for sporulation-specific Ca2+ accumulation is proposed, in which Ca2+ is transported into the sporangium, resulting in a concentration of 3--9 mM in the mother-cell cytoplasm. This high concentration of Ca2+ enables carrier-mediated transfer down a concentration gradient into the forespore compartment, where a low free Ca2+ concentration is maintained by complexing with dipicolinic acid. (+info)
An intracellular iron chelator pleiotropically suppresses enzymatic and growth defects of superoxide dismutase-deficient Escherichia coli.
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Mutants of Escherichia coli that lack cytoplasmic superoxide dismutase (SOD) exhibit auxotrophies for sulfur-containing, branched-chain, and aromatic amino acids and cannot catabolize nonfermentable carbon sources. A secondary-site mutation substantially relieved all of these growth defects. The requirement for fermentable carbon and the branched-chain auxotrophy occur because superoxide (O2-) leaches iron from the [4Fe-4S] clusters of a family of dehydratases, thereby inactivating them; the suppression of these phenotypes was mediated by the restoration of activity to these dehydratases, evidently without changing the intracellular concentration of O2-. Cloning, complementation, and sequence analysis identified the suppressor mutation to be in dapD, which encodes tetrahydrodipicolinate succinylase, an enzyme involved in diaminopimelate and lysine biosynthesis. A block in dapB, which encodes dihydrodipicolinate reductase in the same pathway, conferred similar protection. Genetic analysis indicated that the protection stems from the intracellular accumulation of tetrahydro- or dihydrodipicolinate. Heterologous expression in the SOD mutants of the dipicolinate synthase of Bacillus subtilis generated dipicolinate and similarly protected them. Dipicolinates are excellent iron chelators, and their accumulation in the cell triggered derepression of the Fur regulon and a large increase in the intracellular pool of free iron, presumably as a dipicolinate chelate. A fur mutation only partially relieved the auxotrophies, indicating that Fur derepression assists but is not sufficient for suppression. It seems plausible that the abundant internal iron permits efficient reactivation of superoxide-damaged iron-sulfur clusters. This result provides circumstantial evidence that the sulfur and aromatic auxotrophies of SOD mutants are also directly or indirectly linked to iron metabolism. (+info)
Real-time monitoring of Bacillus subtilis endospore components by attenuated total reflection Fourier-transform infrared spectroscopy during germination.
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Chemical changes of particular Bacillus subtilis spore components were monitored by attenuated total reflection Fourier-transform infrared spectroscopy (ATR/FTIR) during spore germination on a ZnSe internal reflection element. Within minutes of the initiation of spore germination, significant changes in the amount of calcium dipicolinate (DPA-Ca) and proteins were noted in the wild-type strain. The changes in a germination mutant (strain 1G9, gerD) were similar to those in the wild-type strain, but the rates of change were slower. The changes in another germination mutant (strain 1G7, gerA) were very different from those in the first two strains: germination was slow and incomplete, and proteins and DPA-Ca remained unaltered throughout the course of the germination study. This technique thus offers a sensitive and non-destructive method for real-time monitoring of various cellular components during spore germination. (+info)
Influence of chromium tripicolinate on growth and glucose metabolism in yearling horses.
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Thoroughbred and Quarter Horse yearlings (n = 24; 335+/-7 d of age) were used in a 112-d feeding trial to determine whether chromium (Cr) supplementation would alter growth, development, and energy metabolism of growing horses on high-concentrate diets. The horses were assigned at random within breed and gender subgroups to one of four treatment groups: A) basal concentrate; B) basal plus 175 microg of Cr/kg concentrate; C) basal plus 350 microg of Cr/kg concentrate; and D) basal plus 700 microg of Cr/kg concentrate. Chromium was provided via Cr tripicolinate (Prince Agri Products, Quincy, IL). The horses were weighed, measured for withers and hip height, heart girth, and body length and underwent ultrasound evaluation for croup fat thickness. The concentrate was fed for ad libitum consumption for two, 1.5-hr feeding periods daily. Coastal bermudagrass (Cynodon dactylon) hay was group-fed (six animals/group) at 1% of BW daily. Feed intake was 60% concentrate and 40% hay, resulting in a supplemental Cr intake of 0, 105, 210, and 420 microg/kg diet for groups A, B, C, and D, respectively. Colts consumed more concentrate and total feed than did fillies (P < .05), but no dietary effect on feed intake was detected. Colts weighed more than fillies at the completion of the experiment (P = .0754), but no dietary effects on weight, body measurements, or croup fat were detected. An i.v. glucose tolerance test (.2 g of glucose/kg BW) and an i.v. insulin sensitivity test (.1 IU of insulin/kg BW) were conducted on each animal during the third 28-d period of the experiment. Plasma glucose peaked immediately following injection and decreased more rapidly in animals consuming the high-Cr diet than in those consuming the control diet (P < .01). Mean glucose fractional turnover rate values increased (P = .0369) and mean half-life of glucose decreased (P = .0634) in response to the high Cr supplementation. Plasma glucose depletions in animals fed the other two diets were between and not different from (P > .10) the depletions in control animals or in those fed high-Cr diets. No difference in insulin sensitivity was detected (P > .10). Results indicate that Cr tripicolinate supplementation of yearling horses increases the rate at which glucose is metabolized and may lower the plasma glucose concentration. No effect of Cr supplementation on development of the animals was detected. (+info)
Macrophage control of mycobacterial growth induced by picolinic acid is dependent on host cell apoptosis.
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The effects of picolinic acid (PA) on the intramacrophagic growth of Mycobacterium avium were studied. PA reduced M. avium growth inside mouse macrophages and led to a complete control of mycobacterial growth when added together with IFN-gamma. The mechanism involved did not require TNF-alpha, NO, or the respiratory burst, and was not dependent on either iron or zinc withholding. The mycobacteriostatic activity of the macrophages was associated with the induction of morphological changes that culminated in apoptosis at day 4 of treatment. PA alone induced apoptosis in macrophages, and this effect was increased by IFN-gamma treatment. Apoptosis at day 4 of infection was reduced by inhibiting macrophage activation with the prostaglandin 15 deoxy-prostaglandin J2 or by treating the cells with the antioxidant N-acetylcysteine. Mycobacterial growth was partially restored in macrophages treated with PA and IFN-gamma when 15 deoxy-prostaglandin J2 was added, concomitant with a delay in apoptosis. N-Acetylcysteine or glutathione could also completely revert the mycobacteriostatic effects of PA or PA plus IFN-gamma. (+info)