Effect of Lactobacillus acidophilus strain NP51 on Escherichia coil O157:H7 fecal shedding and finishing performance in beef feedlot cattle.

A 2-year study was conducted during the summer months (May to September) to test the effectiveness of feeding Lactobacillus acidophilus strain NP51 on the proportion of cattle shedding Escherichia coli O157:H7 in the feces and evaluate the effect of the treatment on finishing performance. Steers (n = 448) were assigned randomly to pens, and pens of cattle were assigned randomly to NP51 supplementation or no supplementation (control). NP51 products were mixed with water and applied as the feed was mixed daily in treatment-designated trucks at the rate of 10(9) CFU per steer. Fecal samples were collected (n = 3,360) from the rectum from each animal every 3 weeks, and E. coli O157:H7 was isolated by standard procedures, using selective enrichment, immunomagnetic separation, and PCR confirmation. The outcome variable was the recovery of E. coli O157:H7 from feces, and was modeled using logistic regression accounting for year, repeated measures of pens of cattle, and block. No significant differences were detected for gain, intakes, or feed efficiency of control or NP51-fed steers. The probability for cattle to shed E. coli O157:H7 varied significantly between 2002 and 2003 (P = 0.004). In 2002 and 2003, the probability for NP51-treated steers to shed E. coli O157:H7 over the test periods was 13 and 21%, respectively, compared with 21 and 28% among controls. Over the 2 years, NP51-treated steers were 35% less likely to shed E. coli O157: H7 than were steers in untreated pens (odds ratio = 0.58, P = 0.008). This study is consistent with previous reports that feeding NP51 is effective in reducing E. coli O157:H7 fecal shedding in feedlot cattle.

Use of rope devices to describe and explain the feedlot ecology of Escherichia coli O157:H7 by time and place.

Escherichia coli O157:H7 is an important pathogen of humans, and cattle populations serve as an important reservoir for human exposure. The organism is ubiquitous to feedlot cattle populations, although the nature of its occurrence is quite dynamic. Why E. coli O157:H7 varies by time and place in fed cattle is poorly understood. This study was designed to describe and explain the occurrence of E. coli O157:H7 by pen-level factors of time and place. From each pen, we cultured seven ropes placed within pens for cattle to rub and chew (ROPES), in order to classify the pens as high or low prevalence in longitudinal studies conducted during the summer and winter feeding periods of 2 full years. We observed differences in occurrence of ROPES-positive pens by season, weeks within season, and feedyard. ROPES-positive pens clustered temporally. Factors associated with ROPES-positive pen-weeks during both the summer and winter feeding periods were feedyard, prior 7-day mean air temperature, recovery of E. coli O157:H7 from the composite fecal sample, and recovery of E. coli O157:H7 from the water tank. Pens of summer-fed cattle were less likely to be ROPES-positive for E. coli O157:H7 if the ROPES were positive for Salmonella spp. The condition of the pen surface was associated with the likelihood for winter-fed pens of cattle to be ROPES-positive. We were able to monitor these pens of cattle using ROPES at minimal cost and without disturbing individual cattle. These observations improve our understanding of the ecology of E. coli O157:H7 in fed cattle, and also illustrate the importance of designing and analyzing observational studies and clinical trials to account for time- and place-dependent variables that affect the probability of detecting E. coli O157:H7.

Use of rope devices to describe and explain the feedlot ecology of Salmonella by time and place.

Salmonella spp. are important zoonotic pathogens, and cattle can serve as an important source of this organism for human exposure through food. This study was designed to describe and explain the occurrence of Salmonella spp. by pen-level factors of time and place using a pen-level test-method previously validated for E. coli O157:H7. From each pen, we cultured seven ropes placed within the pen for cattle to rub and chew (ROPES), in order to classify the pens as ROPES-positive or ROPES-negative each week in longitudinal studies conducted during the summer and winter feeding periods of 2 full years. We observed differences in occurrence of ROPES-positive pens by week within each season such that, at times, a high proportion of pens were of the same ROPES-status even though feedyards were separated by 50-200 km. Factors associated with ROPES-positive pen-weeks during both the summer and winter feeding periods were the condition of the pen surface and recovery of Salmonella spp. from the water tank. The probability for pens of summer-fed cattle to be ROPES-positive for Salmonella spp. increased as the number of cattle in the pen increased and decreased when the pen was ROPES-positive for E. coli O157:H7. Pens of winter-fed cattle differed by feedyard in the probability of their being ROPES-positive for Salmonella spp. We were able to monitor these pens using ROPES at minimal expense, without disturbing individual cattle, and observe important time and place relationships of Salmonella spp. in fed cattle. These observations illustrate the importance of accounting for time- and place-dependent variables that affect the probability of detecting Salmonella spp. when designing and analyzing observational studies and clinical trials.

Utilization of Bt corn residues by grazing beef steers and Bt corn silage and grain by growing beef cattle and lactating dairy cows.

Three experiments were conducted to evaluate the impact of the Bacillus thuringiensis (Bt)-11 transformation event in two parental corn hybrids differing in date of maturity on beef and dairy cattle performance. Sixteen lactating Holstein dairy cows in replicated 4 x 4 Latin squares were assigned to four diets in a 2 x 2 factorial arrangement: Bt vs non-Bt trait and early- vs late-maturing corn hybrids. The diets contained 40% of the test corn silage plus 28% corn grain from the same corn hybrid (DM basis). There was no effect of the Bt trait on efficiency of milk production, ruminal pH, acetate:propionate ratio, or in situ digestion kinetics of NDF. The early-maturing corn hybrids resulted in greater total VFA concentrations in the rumen and efficiency of 4% fat-corrected milk production than the later-maturing hybrids (P < 0.05). Sixty-seven steer calves were used in a 70-d corn residue grazing trial for the late-maturing corn hybrids only. Daily BW gain of steers was similar for those grazing Bt and non-Bt corn residues, and the steers exhibited no grazing preference between Bt and non-Bt corn residue. One hundred twenty-eight steer calves were assigned to four silage-based growing diets in a 2 x 2 factorial arrangement: Bt vs non-Bt trait and early- vs late-maturing corn hybrids. The diets contained 90% corn silage and 10% supplement (DM basis). The DMI was higher for steers fed Bt compared with non-Bt hybrids (P < 0.02). An interaction (P < 0.03) was observed for feed efficiency between hybrid genotype and incorporation of the Bt trait. Feed efficiency was greater (P < 0.05) for steers fed the later-maturing non-Bt hybrid compared with the later-maturing Bt hybrid; however, feed efficiency was similar between steers fed early-maturing Bt and non-Bt corn silages. Steers fed the early-maturing hybrid gained 11% faster and were 7% more efficient compared with those fed the late-maturing hybrid. These latter results agree with the dairy experiment in which the early-maturing hybrid resulted in 5% greater efficiency of milk production than the later-maturing corn hybrid. In all experiments, incorporation of the Bt trait into corn had no consistent effect on cattle performance. In addition, background genetics of the corn hybrids appeared to have a more consistent impact on performance than did presence of the Bt trait.

Age-related increase in mitochondrial superoxide generation in the testosterone-producing cells of Brown Norway rat testes: relationship to reduced steroidogenic function?

Aging in Brown Norway rats is accompanied by the reduced production of testosterone by the Leydig cells, the testicular cells responsible for synthesizing and secreting this essential steroid. As yet, the mechanism by which Leydig cell steroidogenesis is reduced is unknown. Herein we assess the production of mitochondrial reactive oxygen species by intact Leydig cells isolated from the testes of young and old rats. To this end, Leydig cells were incubated with lucigenin (bis-N-methylacridinium nitrate), a probe that enters cells, localizes to mitochondria, and yields a significant chemiluminescent response following its reaction with intramitochondrial superoxide. Leydig cells from old rats elicited significantly greater lucigenin-derived chemiluminescence (LDCL) than those from young rats. Electron microscopic stereological analysis revealed that the absolute volume of mitochondria in the old cells was reduced from that in the young. These results, taken together, suggest that there are age-related changes in the production of reactive oxygen species by the mitochondria of Leydig cells, with those of old Leydig cells producing significantly greater levels than those of young Leydig cells. The results are consistent with the proposal that mitochondrial-derived reactive oxygen may play a role in the irreversible decline in the ability of old Leydig cells to produce testosterone.

Total syntheses of the Securinega alkaloids (+)-14,15-dihydronorsecurinine, (-)-norsecurinine, and phyllanthine.

A new strategy for enantiospecific construction of the Securinega alkaloids has been developed and applied in total syntheses of (+)-14,15-dihydronorsecurinine (8), (-)-norsecurinine (6), and phyllanthine (2). The B-ring and C7 absolute stereochemistry of these biologically active alkaloids originated from trans-4-hydroxy-L-proline (10), which was converted to ketonitrile 13 via a high-yielding eight-step sequence. Treatment of this ketonitrile with SmI2 afforded the 6-azabicyclo[3.2.1]octane B/C-ring system 14, which is a key advanced intermediate for all three synthetic targets. Annulation of the A-ring of (-)-norsecurinine (6) with the required C2 configuration via an N-acyliminium ion alkylation was accomplished using radical-based amide oxidation methodology developed in these laboratories as a key step, providing tricycle 33. Annulation of the D-ring onto alpha-hydroxyketone 33 with the Bestmann ylide 45 at 12 kbar gave (+)-14,15-dihydronorsecurinine (8). In the securinine series, the D-ring was incorporated using an intramolecular Wadsworth-Horner-Emmons olefination of phenylselenylated alpha-hydroxyketone 47. The C14,15 unsaturation was installed late in the synthesis by an oxidative elimination of the selenoxide derived from tetracyclic butenolide 50 to give (-)-norsecurinine (6). The A-ring of phyllanthine (2) was formed from hydroxyketone 14 using a stereoselective Yb(OTf)3-promoted hetero Diels-Alder reaction of the derived imine 34 with Danishefsky's diene, affording adduct 35. Conjugate reduction and stereoselective equatorial ketone reduction of vinylogous amide 35 provided tricyclic intermediate 36, which could then be elaborated in a few steps to stable hydroxyenone 53 via alpha-selenophenylenone intermediate 52. The D-ring was then constructed, again using an intramolecular Wadsworth-Horner-Emmons olefination reaction to give phyllanthine (2).

Oxidative stress and iron are implicated in fragmenting vacuoles of Saccharomyces cerevisiae lacking Cu,Zn-superoxide dismutase.

The absence of the antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1) is shown here to cause vacuolar fragmentation in Saccharomyces cerevisiae. Wild-type yeast have 1-3 large vacuoles whereas the sod1Delta yeast have as many as 50 smaller vacuoles. Evidence that this fragmentation is oxygen-mediated includes the findings that aerobically (but not anaerobically) grown sod1Delta yeast exhibit aberrant vacuoles and genetic suppressors of other oxygen-dependent sod1 null phenotypes rescue the vacuole defect. Surprisingly, iron also is implicated in the fragmentation process as iron addition exacerbates the sod1Delta vacuole defect while iron starvation ameliorates it. Because the vacuole is reported to be a site of iron storage and iron reacts avidly with reactive oxygen species to generate toxic side products, we propose that vacuole damage in sod1Delta cells arises from an elevation of iron-mediated oxidation within the vacuole or from elevated pools of "free" iron that may bind nonproductively to vacuolar ligands. Furthermore, additional pleiotropic phenotypes of sod1Delta cells (including increased sensitivity to pH, nutrient deprivation, and metals) may be secondary to vacuolar compromise. Our findings support the hypothesis that oxidative stress alters cellular iron homeostasis which in turn increases oxidative damage. Thus, our findings may have medical relevance as both oxidative stress and alterations in iron homeostasis have been implicated in diverse human disease processes. Our findings suggest that strategies to decrease intracellular iron may significantly reduce oxidatively induced cellular damage.

Neurofilament-dependent radial growth of motor axons and axonal organization of neurofilaments does not require the neurofilament heavy subunit (NF-H) or its phosphorylation.

Neurofilaments are essential for establishment and maintenance of axonal diameter of large myelinated axons, a property that determines the velocity of electrical signal conduction. One prominent model for how neurofilaments specify axonal growth is that the 660-amino acid, heavily phosphorylated tail domain of neurofilament heavy subunit (NF-H) is responsible for neurofilament-dependent structuring of axoplasm through intra-axonal crossbridging between adjacent neurofilaments or to other axonal structures. To test such a role, homologous recombination was used to generate NF-H-null mice. In peripheral motor and sensory axons, absence of NF-H does not significantly affect the number of neurofilaments or axonal elongation or targeting, but it does affect the efficiency of survival of motor and sensory axons. Loss of NF-H caused only a slight reduction in nearest neighbor spacing of neurofilaments and did not affect neurofilament distribution in either large- or small-diameter motor axons. Since postnatal growth of motor axon caliber continues largely unabated in the absence of NF-H, neither interactions mediated by NF-H nor the extensive phosphorylation of it within myelinated axonal segments are essential features of this growth.

Subunit composition of neurofilaments specifies axonal diameter.

Neurofilaments (NFs), which are composed of NF-L, NF-M, and NF-H, are required for the development of normal axonal caliber, a property that in turn is a critical determinant of axonal conduction velocity. To investigate how each subunit contributes to the radial growth of axons, we used transgenic mice to alter the subunit composition of NFs. Increasing each NF subunit individually inhibits radial axonal growth, while increasing both NF-M and NF-H reduces growth even more severely. An increase in NF-L results in an increased filament number but reduced interfilament distance. Conversely, increasing NF-M, NF-H, or both reduces filament number, but does not alter nearest neighbor interfilament distance. Only a combined increase of NF-L with either NF-M or NF-H promotes radial axonal growth. These results demonstrate that both NF-M and NF-H play complementary roles with NF-L in determining normal axonal calibers.

Increased expression of keratin 16 causes anomalies in cytoarchitecture and keratinization in transgenic mouse skin.

Injury to epidermis and other stratified epithelia triggers profound but transient changes in the pattern of keratin expression. In postmitotic cells located at the wound edge, a strong induction of K6, K16, and K17 synthesis occurs at the expense of the keratins produced under the normal situation. The functional significance of these alterations in keratin expression is not known. Here, we report that overexpression of a wild-type human K16 gene in a tissue-specific fashion in transgenic mice causes aberrant keratinization of the hair follicle outer root sheath and proximal epidermis, and it leads to hyperproliferation and increased thickness of the living layers (acanthosis), as well as cornified layers (hyperkeratosis). The pathogenesis of lesions in transgenic mouse skin begins with a reorganization of keratin filaments in postmitotic keratinocytes, and it progresses in a transgene level-dependent fashion to include disruption of keratinocyte cytoarchitecture and structural alterations in desmosomes at the cell surface. No evidence of cell lysis could be found at the ultrastructural level. These results demonstrate that the disruption of the normal keratin profile caused by increased K16 expression interferes with the program of terminal differentiation in outer root sheath and epidermis. They further suggest that when present at sufficiently high intracellular levels, K16, along with K6 and K17, appear capable of inducing a reorganization of keratin filaments in the cytoplasm of skin epithelial cells.

Effects of microgravity or simulated launch on testicular function in rats.

Testes from flight rats on COSMOS 2044 and simulated-launch, vivarium, or caudal-elevation control rats (5/group) were analyzed by subjective and quantitative methods. On the basis of observations of fixed tissue, it was evident that some rats had testicular abnormalities unassociated with treatment and probably existing when they were assigned randomly to the four treatment groups. Considering rats without preexisting abnormalities, diameter of seminiferous tubules and numbers of germ cells per tubule cross section were lower (P less than 0.05) in flight than in simulated-launch or vivarium rats. However, ratios of germ cells to each other or to Sertoli cells and number of homogenization-resistant spermatids did not differ from values for simulated-launch or vivarium controls. Expression of testis-specific gene products was not greatly altered by flight. Furthermore, there was no evidence for production of stress-inducible transcripts of the hsp70 or hsp90 genes. Concentration of receptors for rat luteinizing hormone in testicular tissue and surface density of smooth endoplasmic reticulum in Leydig cells were similar in flight and simulated-launch rats. However, concentrations of testosterone in testicular tissue or peripheral blood plasma were reduced (P less than 0.05) in flight rats to less than 20% of values for simulated-launch or vivarium controls. Thus spermatogenesis was essentially normal in flight rats, but production of testosterone was severely depressed. Exposure to microgravity for greater than 2 wk might result in additional changes. Sequelae of reduced androgen production associated with microgravity on turnover of muscle and bone should be considered.