Effect of methionine, norleucine, and lysine derivatives on cephalosporin C formation in chemically defined media.
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Demain, A. L. (Merck Sharp & Dohme Research Laboratories, Rahway, N.J.), Joanne F. Newkirk, and D. Hendlin. Effect of methionine, norleucine, and lysine derivatives on cephalosporin C formation in chemically defined media. J. Bacteriol. 85: 339-344. 1963.-Chemically defined media were developed for production of cephalosporin C by Cephalosporium sp. In such media, the requirement for methionine can be satisfied by norleucine. Further stimulation of antibiotic production was obtained with the lysine derivatives epsilon-N-acetyl-l-lysine and epsilon-aminocaproic acid but not with lysine itself. Also inactive were alpha-aminoadipic and ketoadipic acids. Other lysine derivatives were found to inhibit cephalosporin C production at 0.01 m. The final medium supported the production of approximately 0.5 g of cephalosporin C per liter of medium. (+info)
Sleep time following anesthesia in mouse lines selected for resistance or susceptibility to fescue toxicosis.
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In previous work, a mouse line selected for resistance (R) to fescue toxicosis had higher activities of two hepatic Phase II detoxification enzymes than a mouse line selected for fescue toxicosis susceptibility (S). The primary objective of the present study was to determine whether those same lines also differed in hepatic Phase I enzyme activity, estimated from sleep time (ST) following sodium pentobarbital anesthesia. Additional objectives were to determine whether ST differences between lines were modulated by endophyte-infected fescue in the diet (with or without an enzyme inducer) and whether ST of individual mice was correlated with the effect of a toxin-containing diet on the postweaning growth of those mice. In Exp. I, 24 males from each line were randomly assigned to each of five diets: control (commercial rodent food meal); E+ (50% endophyte-infected fescue seed, 50% control); E+P (the E+ diet supplemented with 1,000 ppm phenobarbital); E- (50% endophyte-free fescue seed, 50% control); and E-P (the E- diet supplemented with 1,000 ppm phenobarbital). After 4 wk on these diets, ST was measured on all the mice. A second ST was recorded on each mouse by randomly sampling one-fourth of the population after 1, 2, 3, or 4 wk on a pelleted rodent food diet. Regardless of diet, R mice had shorter first and second ST than S mice (P < 0.01), suggesting higher hepatic Phase I microsomal enzyme activity. Mice on both phenobarbital-supplemented diets had shorter first ST than mice whose diets did not include that microsomal enzyme inducer (P < 0.01). In Exp. II, ST was measured on male and female R and S mice (n = 280) after they had been fed the E- diet for 2 wk, then the E+ diet for 2 wk, and then a pelleted rodent food diet for 2 wk. Growth response to the E+ diet was the percentage of reduction in gain on the E+ diet compared to gain on the E- diet the previous 2 wk. As in Exp. I, S mice slept longer than R mice (P < 0.01). The residual correlation between ST and gain reduction associated with the E+ diet equaled 0.04. Thus, an animal's apparent Phase I enzyme activity did not predict its growth rate depression on the toxin-containing diet. Based on these and previous studies, divergent selection for toxicosis response in mice was successful partially by causing divergence in activities of hepatic Phase I and II detoxification enzymes. (+info)
Domperidone can ameliorate deleterious reproductive effects and reduced weight gain associated with fescue toxicosis in heifers.
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The aim of this study was to evaluate the effect of a dopamine antagonist, domperidone, in nonpregnant, reproductively cycling heifers consuming endophyte-infected (EI) fescue diets. Thirty crossbred heifers (Angus x Holstein or Hereford x Holstein) were assigned to one of three treatment groups (n = 10); endophyte-free (EF) fescue diet, EI fescue diet, or endophyte-infected diet and treated with domperidone (EID). Heifers fed EI diets had decreased weight gains compared with heifers fed EF or EID (P < 0.05) during a 21-d treatment period. Ovarian structures were monitored via transrectal ultrasound to determine follicle size and day of ovulation. Blood plasma samples were collected daily and analyzed for progesterone concentration to determine luteal function. Heifers ingesting EI diets had estrous cycles of shorter duration and lower mid-cycle progesterone concentrations than heifers in the EF or EID treatments (P < 0.05). Ovaries from a subset of heifers in each group (n = 3 per group) were harvested and in vitro secretion of progesterone from luteal tissue extracts was determined. No differences in progesterone concentrations were detected among luteal tissue incubates (P > 0.05). These results suggest that domperidone supplementation of heifers consuming EI fescue may ameliorate certain symptoms of fescue toxicosis. (+info)
Structural elucidation of novel phosphocholine-containing glycosylinositol-phosphoceramides in filamentous fungi and their induction of cell death of cultured rice cells.
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Novel ZGLs (zwitterionic glycosphingolipids) have been found in and extracted from the mycelia of filamentous fungi ( Acremonium sp.) isolated from soil. Five ZGLs (ZGL1-ZGL5) were structurally elucidated by sugar compositional analysis, methylation analysis, periodate oxidation, matrix-assisted laser-desorption ionization-time-of-flight MS, (1)H-NMR spectroscopy and fast-atom bombardment MS. Their chemical structures were as follows: GlcN(alpha1-2)Ins1-P-1Cer (ZGL1), Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL2), Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL3), PC-->6Man(alpha1-6)GlcN(alpha1-2)Ins1- P -1Cer (ZGL4), and PC-->6Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL5) (where Cer is ceramide and PC is phosphocholine). In addition, one acidic glycosphingolipid, which was the precursor of ZGLs, was also characterized as inositol-phosphoceramide. The core structure of the ZGLs, GlcN(alpha1-2)Ins1- P, is rather different from those found in other fungi, such as Man(alpha1-2)Ins1- P and Man(alpha1-6)Ins1- P. Interestingly, the terminal mannose residue of ZGL4 and ZGL5 was modified further with a PC group. The presence of PC-containing glycosylinositol-phosphoceramides has not been reported previously in any organism. The ceramide constituents of both ZGLs and acidic glycosphingolipid were essentially the same, and consisted of a 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid base and 2-hydroxytetracosanoic acid (>90%) as the major fatty acid. ZGLs were found to cause cell death in suspensions of cultured rice cells. The cell death-inducing activity of ZGLs is probably due to the characteristic glycan moiety of Man(alpha1-6)GlcN, and PC-containing ZGLs had high activity. This study is the first to demonstrate that fungal glycosylinositol-phosphoceramides induce cell death in cultured rice cells. (+info)
Effects of feeding diets containing endophyte-infected fescue seed on luteinizing hormone secretion in postpartum beef cows and in cyclic heifers and cows.
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Two experiments were conducted to investigate the effect of feeding endophyte (Acremonium coenophialum)-infected fescue (Festuca arundinacea Shreb.) seed on LH secretion in postpartum beef cows and in cycling heifers and cows. In Exp. 1, spring-calving primiparous Angus cows (n = 16) were pair-fed for 75 d diets that contained endophyte-free or endophyte-infected (95%) fescue seed that contained 1.3 micrograms/g of ergovaline and 5.2 mg/g of saturated pyrrolizidines. Serial blood samples for basal and GnRH-stimulated serum LH analysis were obtained on d 7, 28, 42, and 56 of the study. The endophyte had no effect on LH secretion (basal, pulse frequency, and amplitude) or milk production. Average daily gain was decreased (P < .05) in cows that consumed infected fescue seed compared with controls (-.20 vs -.01 kg, respectively). Basal serum prolactin concentrations were reduced (P < .01) in treated compared with control cows (8.9 vs 25.4 ng/mL, respectively) on d 70. In Exp. 2, cycling Angus heifers (n = 8; age = 2 yr) and cows (n = 8; age = 4 yr) stratified by age were pair-fed for 40 d diets that contained the noninfected or the highly infected fescue seed. Estrus was synchronized by prostaglandin F2 alpha (d 18 and 28). Serial blood samples for serum LH analysis were obtained on d 28 (luteal phase) and d 30 (follicular phase). The endophyte did not affect LH (P > .28) or prolactin (P > .16) secretion, whereas ADG was decreased (P < .05) in treated compared with control animals (.32 vs .70 kg/d, respectively). (+info)
Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase.
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Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutarate-dependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with site-directed mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (Delta310 mutant) enhanced ring-expansion of penicillin G by approximately 2-fold. A double mutant, Delta310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The Delta310/N305L/M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K(m) = 2.00 +/- 0.47 compared with 6.02 +/- 0.97 mm for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305. (+info)
Winged helix transcription factor CPCR1 is involved in regulation of beta-lactam biosynthesis in the fungus Acremonium chrysogenum.
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Winged helix transcription factors, including members of the forkhead and the RFX subclasses, are characteristic for the eukaryotic domains in animals and fungi but seem to be missing in plants. In this study, in vitro and in vivo approaches were used to determine the functional role of the RFX transcription factor CPCR1 from the filamentous fungus Acremonium chrysogenum in cephalosporin C biosynthesis. Gel retardation analyses were applied to identify new binding sites of the transcription factor in an intergenic promoter region of cephalosporin C biosynthesis genes. Here, we illustrate that CPCR1 recognizes and binds at least two sequences in the intergenic region between the pcbAB and pcbC genes. The in vivo relevance of the two sequences for gene activation was demonstrated by using pcbC promoter-lacZ fusions in A. chrysogenum. The deletion of both CPCR1 binding sites resulted in an extensive reduction of reporter gene activity in transgenic strains (to 12% of the activity level of the control). Furthermore, Acremonium transformants with multiple copies of the cpcR1 gene and knockout strains support the idea of CPCR1 being a regulator of cephalosporin C biosynthesis gene expression. Significant differences in pcbC gene transcript levels were obtained with the knockout transformants. More-than-twofold increases in the pcbC transcript level at 24 and 36 h of cultivation were followed by a reduction to approximately 80% from 48 to 96 h in the knockout strain. The overall levels of the production of cephalosporin C were identical in transformed and nontransformed strains; however, the knockout strains showed a striking reduction in the level of the biosynthesis of intermediate penicillin N to less than 20% of that of the recipient strain. We were able to show that the complementation of the cpcR1 gene in the knockout strains reverses pcbC transcript and penicillin N amounts to levels comparable to those in the control. These results clearly indicate the involvement of CPCR1 in the regulation of cephalosporin C biosynthesis. However, the complexity of the data points to a well-controlled or even functional redundant network of transcription factors, with CPCR1 being only one player within this process. (+info)
Effects of endophyte-infected tall fescue on indicators of thermal status and growth in Hereford and Senepol steers.
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Poor growth often occurs in cattle consuming ergot alkaloids associated with endophyte-infected (EI) tall fescue. Hyperthermia may contribute significantly to poor growth resulting from fescue toxicosis. This study examined indicators of thermal status and growth in Hereford (n = 30; heat-sensitive Bos taurus; H) and Senepol (n = 28; heat-tolerant Bos taurus; S) steers fed EI tall fescue (TF) or orchardgrass (OG) in 2 x 2 factorial experiments. Respiration rates, daytime shade use, tail skin temperatures, and body weights were measured during the summer and fall of 2000 (Exp. 1) and 2001 (Exp. 2). Experimental diets consisted of hay and seed for 12 wk in 2000, hay for 6 wk during the summer of 2001, and hay plus seed for 6 wk during the fall of 2001. In Exp. 1, EI tall fescue increased (P < 0.01) respiration rates, shade use, and skin temperatures in both breeds. Breed x diet affected (P < 0.01) 12-wk ADG in Exp. 1. Growth rate was lower for H-TF (262 g/d) than for S-TF, S-OG, and H-OG (475, 497, and 524 g/d, respectively). In Exp. 2, Senepol had lower (P < 0.01) respiration rates, shade use, and skin temperatures compared with Hereford, but diet did not alter (P > 0.14) these indicator traits in either breed. Breed x diet affected (P < 0.01) summer growth rates. Growth rate was lower for H-TF (88 g/d) than for H-OG, S-TF, and S-OG (508, 555, and 566 g/d, respectively). Adding seed to the diets in Exp. 2 decreased (P < 0.01) ADG for both breeds on TF during the fall. Thermal status indicator traits in Senepol and Hereford steers were similarly altered by TF; however, only Hereford showed consistently poor growth. Senepol showed resilience in their capacity for growth under conditions of fescue toxicosis. Senepol influence may enhance cattle performance in production systems that use EI tall fescue as the base forage. (+info)