Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes.

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial signalling molecule produced by diguanylate cyclases of the GGDEF-domain family. Elevated c-di-GMP levels or increased GGDEF protein expression is frequently associated with the onset of sessility and biofilm formation in numerous bacterial species. Conversely, phosphodiesterase-dependent diminution of c-di-GMP levels by EAL- and HD-GYP-domain proteins is often accompanied by increased motility and virulence. In this study, we individually overexpressed 23 predicted GGDEF, EAL or HD-GYP-domain proteins encoded by the phytopathogen Pectobacterium atrosepticum strain SCRI1043. MS-based detection of c-di-GMP and 5'-phosphoguanylyl-(3'-5')-guanosine in these strains revealed that overexpression of most genes promoted modest 1-10-fold changes in cellular levels of c-di-GMP, with the exception of the GGDEF-domain proteins ECA0659 and ECA3374, which induced 1290- and 7660-fold increases, respectively. Overexpression of most EAL domain proteins increased motility, while overexpression of most GGDEF domain proteins reduced motility and increased poly-ß-1,6-N-acetyl-glucosamine-dependent flocculation. In contrast to domain-based predictions, overexpression of the EAL protein ECA3549 or the HD-GYP protein ECA3548 increased c-di-GMP concentrations and reduced motility. Most overexpression constructs altered the levels of secreted cellulases, pectinases and proteases, confirming c-di-GMP regulation of virulence in Pe. atrosepticum. However, there was no apparent correlation between virulence-factor induction and the domain class expressed or cellular c-di-GMP levels, suggesting that regulation was in response to specific effectors within the network, rather than total c-di-GMP concentration. Finally, we demonstrated that the cellular localization patterns vary considerably for GGDEF/EAL/HD-GYP proteins, indicating it is a likely factor restricting specific interactions within the c-di-GMP network.

Effects of feeding different amounts of supplemental glycerol on ruminal environment and digestibility of lactating dairy cows.

A replicated 3×3 Latin square study was conducted to evaluate the effects of replacing a portion of ground corn in the diet with dietary glycerol on rumen environment, blood metabolites, and nutrient digestibility. Six rumen-cannulated Holstein cows, averaging 56±18 DIM and 38.0±8.2 kg of milk/d, were used in the study. Experimental periods included 3 wk for treatment adjustment period followed by 1 wk for data collection. Diets were corn silage based and balanced to be isocaloric and isonitrogenous. Treatments were 0 g of glycerol/d (control, CON), 200 g of glycerol/d (G2), and 400 g of glycerol/d (G4). Dry matter intake (DMI) decreased as the amount of glycerol fed increased. Milk yield was higher for CON and G2 cows in comparison with G4 cows. Milk fat percentage and yield were reduced when glycerol was fed compared with CON cows but increased the milk protein percentage at the highest concentration of dietary glycerol. These changes resulted in decreased energy-corrected milk yield and efficiency (milk/DMI) in diets supplemented with G4 compared with CON. No differences were observed in ruminal pH and ammonia concentrations. Molar proportions of acetate, valerate, and acetate:propionate ratio decreased, whereas propionate increased as the amount glycerol fed increased. Molar proportions of butyrate were greatest when glycerol was included in the diet compared with CON. Nutrient intake and digestion were not different among treatments. Results of this trial suggest that feeding increasing amounts of glycerol may decrease DMI and alters ruminal fermentation, resulting in reduced yield of milk, fat, and energy-corrected milk.

The impact of ingested potato type II inhibitors on the production of the major serine proteases in the gut of Helicoverpa armigera.

The flowers of the ornamental tobacco produce high levels of a series of 6 kDa serine protease inhibitors (NaPIs) that are effective inhibitors of trypsins and chymotrypsins from lepidopteran species. These inhibitors have a negative impact on the growth and development of lepidopteran larvae and have a potential role in plant protection. Here we investigate the effect of NaPIs on the activity and levels of serine proteases in the gut of Helicoverpa armigera larvae and explore the adaptive mechanisms larvae employ to overcome the negative effects of NaPIs in the diet. Polyclonal antibodies were raised against a Helicoverpa punctigera trypsin that is a target for NaPIs and two H. punctigera chymotrypsins; one that is resistant and one that is susceptible to inhibition by NaPIs. The antibodies were used to optimize procedures for extraction of proteases for immunoblot analysis and to assess the effect of NaPIs on the relative levels of the proteases in the gut and frass. We discovered that consumption of NaPIs did not lead to over-production of trypsins or chymotrypsins but did result in excessive loss of proteases to the frass.

Effects of the addition of direct-fed microbials and glycerol to the diet of lactating dairy cows on milk yield and apparent efficiency of yield.

A study was conducted to evaluate the effects of a direct-fed microbial (M) and dietary glycerol (G) on milk yield, efficiency of yield, and nutrient digestibility during hot weather. Sixty Holstein cows averaging 120 d in milk (DIM) and 36.2 kg/d of milk were used in a 12-wk 2×2 factorial design trial from June through September 2008. Cows were fed a common diet during the 2-wk standardization period and were blocked by milk yield, DIM, parity, and dry matter intake. Diets were based on corn and ryegrass silages and balanced to be isocaloric and isonitrogenous. Treatments included a negative control (M- or G-), 4 × 10(9) cfu/head of a combination of Lactobacillus acidophilus NP51 and Propionibacterium freudenreichii NP24 (M+), control plus 400 g/h per day of 99% pure food-grade glycerol (G+), and 4×10(9) cfu/h per day of a combination of Lactobacillus acidophilus NP51 and Propionibacterium freudenreichii NP24 plus 400 g/h per day of 99% pure food-grade glycerol (MG++). No interactions were observed between direct-fed microbials and dietary glycerol in the study except on apparent nutrient digestibility. No differences were observed in dry matter intake, which averaged 22.7, 23.1, 23.4, and 22.9 for M-, G-, M+, and G+, respectively. Milk yield was increased for M+ compared with M- at 34.1 and 31.7 kg/d, but G+ had no effect on yield. No treatment effect was noted for milk fat percentage or milk protein percentage among diets. Milk protein yield was higher for M+ compared with M- at 0.93 versus 0.87 kg/d. Energy-corrected milk was improved for the M+ versus M- groups at 33.5 and 31.6 kg/d, respectively. No differences in respiratory rate, skin temperature, body temperature, or concentrations of serum glucose or urea N were observed among treatments. Improvement in apparent digestibility was observed with M+ and G+ compared with M-/G- in this experiment. The addition of a direct-fed microbial alone improved milk and protein yield, energy-corrected milk, and apparent digestibility of crude protein, neutral detergent fiber, and acid detergent fiber, and the inclusion of glycerol (G+) had a positive effect on apparent dry matter and acid detergent fiber digestibility compared with M-/G-. The addition of a direct-fed microbial and dietary glycerol may improve yield and digestibility for cows subject to heat stress.

Effect of feeding alfalfa hay or Tifton 85 bermudagrass haylage with or without a cellulase enzyme on performance of Holstein cows.

Forty-four lactating Holstein cows (173±30 DIM, 42.5±6.8 kg of milk, 4.03±0.69% fat, 674±78 kg of body weight) were used in an 8-wk, completely randomized trial with a 2 × 2 factorial arrangement of treatments to determine the effect of forage source and supplemental cellulase enzyme on performance. Treatments included 2 forage combinations (corn silage plus 12.2% dry matter, DM, from either alfalfa hay or Tifton 85 bermudagrass haylage) with or without a commercial cellulase enzyme applied to the total mixed ration at the rate of 4 g/head per day (Promote N.E.T.-L, Cargill Animal Nutrition, Minneapolis, MN). Experimental diets were formulated to provide similar concentrations of protein (16.5% of DM), energy (1.63 Mcal of net energy for lactation/kg of DM), and neutral detergent fiber (41.7% of DM) and were fed once daily as a total mixed ration behind Calan doors for ad libitum intake. The cellulase enzyme provided 1,200 cellulase units of activity/g of product and was applied to the total mixed ration and allowed to mix for 5min before feeding. Before beginning the trial, all cows were trained to use Calan (American Calan, Northwood, NH) doors and then fed the alfalfa hay-based diet for 2 wk. Data collected during wk 2 were used as a covariate in the statistical analysis. At the beginning of the 6-wk experimental period, cows were assigned randomly to 1 of the 4 experimental diets. No interactions were observed between forage and enzyme for any measures. Daily DM intake; milk yield; concentrations of milk fat, true protein, lactose, and solids not fat; energy-corrected milk yield; and dairy efficiency were not different among alfalfa or Tifton 85 bermudagrass rations with or without cellulase enzyme supplementation. The results of this trial indicate that Tifton 85 bermudagrass haylage can replace alfalfa hay in diets fed to high-producing, lactating dairy cows without depressing DM intake or milk yield when rations are balanced for NDF. Although supplemental cellulase enzymes have been shown to improve ration digestibility and animal performance in previous trials, no advantages were observed in the current trial.

Pigeon peas as a supplement for lactating dairy cows fed corn silage-based diets.

Holstein rumen-cannulated cows [n=7; initial body weight (BW) 640.56±71.43 kg] were fed a corn silage basal diet with 1 of 3 concentrates (C=control; P10=10% pigeon peas; P20=20% pigeon peas). Cows were randomly assigned to treatments in a replicated 3×3 Latin square and individually fed using Calan gates. Each experimental period was 21 d with 7 d for adaption and 14 d for sample collection. Ruminal fluid samples were taken the last day of each experimental period and analyzed for pH, ammonia, long-chain fatty acids, and volatile fatty acids (VFA). Consecutive a.m. and p.m. milk samples were taken during the last 2 wk of the 21-d period and analyzed for fat, protein, long-chain fatty acids, and somatic cell count. Dry matter intake (kg/d) was reduced during the second period and was greater for P10 diets. Milk protein was greater for cows fed P20 compared with P10. Energy-corrected milk was greater for cows fed the control diet compared with P10. Treatment had no effect on milk yield. Ruminal fluid pH decreased over sampling times; however, pH remained at or above 5.5. Diets did not affect ruminal fluid pH; however, pH was different for sampling periods. Ruminal ammonia decreased until 8h postfeeding at which time it peaked consistent with changes in ammonia concentrations that usually peak 3 to 5h postfeeding on diets high in plant proteins. Dietary treatments altered ruminal fluid VFA with reduced concentrations of acetate and greater concentrations of propionate for control diet, resulting in reduced acetate:propionate ratio. Isobutyrate exhibited an hour by treatment interaction, in which isobutyrate decreased until 8h postfeeding and then tended to be greater for P10 than for other treatments. Animals fed the P10 diet had greater concentrations of ruminal isovalerate. Ruminal cis-9,trans-11 and trans-10,cis-12 conjugated linoleic acid (CLA) isomers were not affected by dietary treatments. The P10 diet had greatest ruminal synthesis of cis-9,trans-11, but control cows had greatest ruminal synthesis of trans-10,cis-12. Milk CLA isomers were similar among treatments. Trends were observed for greater cis-9,trans-11 and trans-10,cis-12 for the P10 diet. Pigeon peas may be used as a protein supplement in dairy diets without affecting milk production, dry matter intake, or ruminal environment when they replace corn and soybean meal.

Performance of lactating dairy cows fed ryegrass silage and corn silage with ground corn, steam-flaked corn, or hominy feed.

Forty-eight mid-lactation Holstein cows were used in a 6-wk completely randomized block design trial with a 4 x 3 factorial arrangement of treatments to determine the effects of feeding different proportions of corn silage and ryegrass silage with supplemental ground corn (GC), steam-flaked corn (SFC), and hominy feed (HF) on the performance of lactating dairy cows. Forage provided 49% of the dietary dry matter in the experimental diets, which were formulated to meet National Research Council requirements. Ryegrass silage provided 100, 75, 50, or 25% of the total forage dry matter, with corn silage supplying the remainder. There were no interactions between the proportion of forage provided by ryegrass silage and energy supplement. Dry matter intake and milk protein percentage decreased linearly with increasing proportions of ryegrass silage, but milk protein yield was similar among forage treatments. There were no differences among forage treatments in milk yield, milk fat percentage and yield, and energy-corrected milk yield. Dry matter intake was higher and there was a tendency for increased milk fat percentage for GC compared with SFC or HF. No other differences were observed in milk yield or composition among energy supplements. Plasma urea nitrogen and glucose concentrations were similar among treatments. Under the conditions of this trial, our results indicate that feeding a combination of corn silage and ryegrass silage is more desirable than feeding ryegrass silage alone, whereas supplementation with GC, SFC, or HF supports similar levels of milk production.

Performance of dairy cows fed annual ryegrass silage and corn silage with steam-flaked or ground corn.

Twenty-four lactating Holstein cows were used in a 6-wk randomized block design trial with a 2 x 2 factorial arrangement of treatments to determine the effects of feeding ground corn (GC) or steam-flaked corn (SFC) in diets based on either annual ryegrass silage (RS) or a 50:50 blend of annual ryegrass and corn silages (BLEND). Experimental diets contained 49.6% forage and were fed as a total mixed ration once daily for 4 wk after a 2-wk preliminary period. No interactions were observed among treatments. Cows fed BLEND consumed more dry matter (DM), organic matter (OM), neutral detergent fiber (NDF), and acid detergent fiber (ADF) than those fed RS, but total-tract digestibility of OM, NDF, and ADF was greater for RS than for BLEND. No differences in nutrient intake were observed among treatments during wk 4 when nutrient digestibility was measured, but digestibility of DM and OM was greater for SFC than for GC. Cows fed BLEND tended to produce more energy-corrected milk than those fed RS, resulting in improved efficiency (kg of milk per kg of DM intake). When diets were supplemented with SFC, cows consumed less DM and produced more milk that tended to have lower milk fat percentage. Yield of milk protein and efficiency was greatest with SFC compared with GC. Blood glucose and milk urea nitrogen concentrations were similar among treatments, but blood urea nitrogen was greater for cows fed GC compared with those fed SFC. Results of this trial indicate that feeding a blend of annual ryegrass and corn silage is more desirable than feeding diets based on RS as the sole forage. Supplementing diets with SFC improved performance and efficiency compared with GC across forage sources.

New insights into the role of vitamin D and calcium in osteoporosis management: an expert roundtable discussion.

BACKGROUND: Adequate vitamin D and calcium nutrition play a critical role in the maintenance of musculoskeletal health and are considered the first step in osteoporosis treatment. ROUNDTABLE DISCUSSION: In February 2008 Merck Sharp & Dohme sponsored a 2-day, evidence-based expert panel on the benefits of vitamin D for the patient with osteoporosis and the role of vitamin D in combination with antiresorptive therapy for the management of osteoporosis. One of the primary objectives of the meeting was to review new data on the optimal serum 25-hydroxy vitamin D [25(OH)D] levels. The symposium was attended by 29 researchers and clinicians from Europe and the Middle East. The discussion focused on optimizing vitamin D and calcium nutrition and reducing falls and fractures in osteoporotic patients. CONCLUSIONS: Current evidence and expert opinion suggests that optimal serum 25(OH)D concentrations should be at least 50 nmol/L (20 ng/mL) in all individuals. This implies a population mean close to 75 nmol/L (30 ng/mL). In order to achieve this level, vitamin D intake of at least 20 microg daily is required. There is a wider therapeutic window for vitamin D than previously believed, and doses of 800 IU per day, regardless of sun exposure, season or additional multivitamin use, appear to present little risk of toxicity. Apart from fracture and fall prevention, optimization of vitamin D status may also have additional general health benefits. Based on newly emerging data regarding calcium supplementation, and recommendations for increased vitamin D intake, the current recommendations for calcium intake in postmenopausal women may be unnecessarily high. In addition to vitamin D and calcium, treatment of patients with osteoporosis at high risk of fractures should also include pharmacologic agents with proven vertebral and non-vertebral fracture efficacy.

Performance and ruminal fermentation of dairy cows fed whole cottonseed with elevated concentrations of free fatty acids in the oil.

Twenty-four lactating Holstein cows were used in an 8-wk completely randomized design trial to examine the effects of feeding whole cottonseed (WCS) with elevated concentrations of free fatty acids (FFA) in the oil on intake and performance. Treatments included WCS with normal concentrations of FFA (6.8%, control) and 2 sources of WCS with elevated FFA [HFFA1 (24.1%) or HFFA2 (22.3%)]. The 2 sources of WCS with elevated FFA differed in that HFFA2 were discolored from being initially stored with excess moisture, which led to heating and deterioration during storage, whereas HFFA1 were normal in appearance and the increase in FFA occurred without heating and visible damage to the WCS. Nutrient concentrations were similar among WCS treatments, which provided 14% of the total dietary dry matter. Dry matter intake tended to be higher for cows fed HFFA2 compared with control and HFFA1. Yield of milk and components was similar among treatments, but milk fat percentage was lower for HFFA1 and HFFA2 compared with control. In a concurrent 3 x 3 Latin square trial with 6 ruminally cannulated Holstein cows, molar proportions of isobutyrate were higher for HFFA2 than control and HFFA1, but no differences were observed in acetate or propionate. Results of these trials indicate that feeding WCS with high concentrations of FFA decreases milk fat percentage but does not alter dry matter intake, milk yield, or concentrations of other components. The minor changes in ruminal fermentation that were observed do not account for the decrease in milk fat percentage.

Performance of lactating dairy cows fed whole cottonseed coated with gelatinized starch plus urea or yeast culture.

Thirty lactating Holstein cows were used in an 8-wk randomized design trial to test the viability of select additives included in the gelatinized corn starch coating applied to whole cottonseed (WCS) on nutrient intake and digestibility and milk yield and composition. Treatments included WCS coated with 2.5% gelatinized corn starch (control); control plus 0.5% urea; or control plus 2.0% yeast culture. The treated WCS represented 12.6% of the dietary dry matter. Cellulose intake was lower for the control coating compared with either the urea or yeast coating because of slightly lower cellulose concentrations in the control treatment. Intake of all other nutrients was similar for all treatments. Whole-tract nutrient apparent digestibility was not altered by treatment. Dry matter intake and milk yield were similar among treatments. Percentage solids-not-fat was lower for the yeast treatment compared with control, but no other differences were observed in milk composition among treatments. Efficiency of milk production (energy-corrected milk yield per unit of dry matter intake) was higher for the urea and yeast treatments compared with control because of slightly higher yield of milk fat and energy-corrected milk. No differences were observed in body weight change during the trial between treatments. Results of this trial indicate that including urea or yeast culture in the gelatinized starch coating does not change whole tract digestibility, but does improve milk production efficiency.

Effect of prepartum dietary calcium on intake and serum and urinary mineral concentrations of cows.

Nine multiparous and 12 primiparous cows were fed diets containing an anionic salt supplement and moderate Ca (0.99%) or high Ca (1.50%) concentrations for 21 d prepartum to determine the effects of dietary Ca concentration on serum and urine electrolytes and on postpartum intake and milk yield. Blood samples were collected during 21 to 1 d prepartum, 0 to 2 d postpartum, and 3 to 21 d postpartum. Dietary cation-anion difference (DCAD) for prepartum diets was approximately -6 mEq/100 g of dry matter (Na + K - Cl - S). Immediately postpartum, cows were fed diets with positive DCAD with greater than 1.00% Ca concentration. Mean serum Ca concentrations 21 to 1 d prepartum, 0 to 2 d postpartum, and 3 to 21 d postpartum were 9.62, 8.41, and 9.38 mg/dL. There were no treatment effects on serum Ca concentration. Mean serum Ca concentration was higher for primiparous than multiparous cows (9.34 vs. 8.93 mg/dL) for the trial and at calving (8.77 vs. 8.13 mg/dL). Mean serum HCO(3)(-) and urinary pH, respectively, were 20.32 mEq/L and 5.67 prepartum, 25.82 mEq/L and 7.62 at calving, and 26.08 mEq/L and 8.25 postpartum. No differences due to treatment were observed for serum and urinary concentrations of HCO(3)(-), pH, Mg, Na, K, and Cl. Milk yield was similar for 0.99 and 1.50% Ca treatments (22.8 and 20.7 kg/d). Diets containing 0.99 or 1.5% Ca maintained serum Ca at adequate levels around parturition and resulted in similar dry matter intake and postpartum milk yield.

Effect of supplemental L-lysine-HCL and corn source on rumen fermentation and amino acid flow to the small intestine.

Four lactating Jersey cows fitted with ruminal and duodenal cannulae were used in a 4 x 4 Latin square design trial to determine the effect of supplemental lysine in diets containing dry ground (GC) or steam-flaked (SFC, 360 g/L) corn on ruminal fermentation and amino acid (AA) flow to the duodenum. Supplemental L-lysine-HCL provided 10 g/d of additional Lys to the total mixed rations. There were no interactions between supplemental Lys and corn source. Supplemental Lys increased Lys intake, but did not alter nutrient intake and digestibility or N flow to the duodenum. Intake of dry matter (DM), organic matter (OM), and neutral detergent fiber (NDF) and ruminal digestibility of starch tended to be higher, whereas ruminal digestibility of DM, OM, acid detergent fiber, and NDF was lower for diets supplemented with SFC compared with GC. Whole-tract digestibility was similar for both corn supplements. Ruminal pH and molar proportions of volatile fatty acids were not affected by supplemental Lys or corn source; however, ruminal NH(3) concentrations were lowest when SFC was fed. Intake of N tended to be higher and the flow of total N and individual AA to the duodenum was higher for diets supplemented with SFC. There was a trend for increased flow of microbial N for diets supplemented with SFC. Supplemental L-lysine-HCL did not alter ruminal fermentation, flow of amino acid to the small intestine, or nutrient digestibility, but feeding SFC reduced ruminal fiber digestion and increased microbial protein synthesis and flow of amino acid to the duodenum.

Ruminal fermentation and bacterial protein synthesis of whole cottonseed coated with combinations of gelatinized corn starch and urea.

Four ruminally and duodenally cannulated Jersey cows were used in a 4 x 5 incomplete Latin square study to determine the effects of including urea in the gelatinized corn starch coating applied to whole cottonseed (WCS) on ruminal fermentation, fiber digestion, and bacterial protein synthesis. Treatments included uncoated WCS (control) and four coated WCS treatments. The coatings provided two concentrations each of gelatinized corn starch (2.5 [2S] or 5% [5S]) and feed grade urea (0.25 [2U] or 0.5% [5U]). Treated WCS comprised 15% of the ration dry matter that was fed as a total mixed ration once daily. Ruminal pH and molar proportions of isobutyrate was higher and NH3-N concentrations lower for control compared with coated WCS. Molar proportions of propionate tended to be higher and valerate was lower with 2S compared with 5S. Molar proportions of acetate tended to be lower, whereas butyrate was higher for 5U than 2U. Nutrient intake was lower for WCS coated with 5S5U compared with 2S5U. Ruminal NDF digestibility of NDF tended to be higher with 5U compared with 2U, but no differences were observed in ruminal or total tract apparent digestibility of nutrients. No differences were observed in the flow of total N or bacterial N to the duodenum, but the flow of nonbacterial N tended to be higher for WCS coated with 5U. Coating WCS appears to slightly alter ruminal metabolism while providing similar amounts of N flowing to the duodenum without altering fiber digestion.

Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance.

Metal nanoshells are a class of nanoparticles with tunable optical resonances. In this article, an application of this technology to thermal ablative therapy for cancer is described. By tuning the nanoshells to strongly absorb light in the near infrared, where optical transmission through tissue is optimal, a distribution of nanoshells at depth in tissue can be used to deliver a therapeutic dose of heat by using moderately low exposures of extracorporeally applied near-infrared (NIR) light. Human breast carcinoma cells incubated with nanoshells in vitro were found to have undergone photothermally induced morbidity on exposure to NIR light (820 nm, 35 W/cm2), as determined by using a fluorescent viability stain. Cells without nanoshells displayed no loss in viability after the same periods and conditions of NIR illumination. Likewise, in vivo studies under magnetic resonance guidance revealed that exposure to low doses of NIR light (820 nm, 4 W/cm2) in solid tumors treated with metal nanoshells reached average maximum temperatures capable of inducing irreversible tissue damage (DeltaT = 37.4 +/- 6.6 degrees C) within 4-6 min. Controls treated without nanoshells demonstrated significantly lower average temperatures on exposure to NIR light (DeltaT < 10 degrees C). These findings demonstrated good correlation with histological findings. Tissues heated above the thermal damage threshold displayed coagulation, cell shrinkage, and loss of nuclear staining, which are indicators of irreversible thermal damage. Control tissues appeared undamaged.

Blockade of phencyclidine-induced cortical apoptosis and deficits in prepulse inhibition by M40403, a superoxide dismutase mimetic.

Repetitive administration of phencyclidine (PCP) in the perinatal period results in cortical apoptosis and a long-lasting deficit in sensorimotor gating. Because these changes are olanzapine-sensitive, we have suggested that the effects of perinatal PCP could be used to model certain aspects of schizophrenia. Studies of PCP and N-methyl-D-aspartate-induced cell death suggested that superoxide could play a role in the pathway leading to death after PCP administration. The purpose of the current study was to determine whether the in vivo administration of M40403, a superoxide dismutase mimetic, could prevent PCP-induced cortical apoptosis and/or deficits in prepulse inhibition. Perinatal rat pups were administered 10 mg/kg PCP on postnatal (PN) days 7, 9, and 11 with or without treatment with 10 mg/kg M40403. Pups were either killed on PN 12 for analysis of various apoptotic markers or they were assessed for prepulse inhibition on PN 24 to 26. Treatment with M40403 2 and 24 h after each PCP treatment prevented PCP-induced increases in two measures of apoptosis in the dorsolateral frontal cortex and in the olfactory cortex. PCP-induced proapoptotic changes in Bax and Bcl-X(L) were also prevented by M40403 treatment. This regimen did not prevent the deficit in prepulse inhibition caused by PCP treatment, but when the treatment regimen was extended through PN 23, M40403 completely prevented the PCP-induced deficit in prepulse inhibition. These data suggest that perinatal PCP treatment leads to long-lasting changes in the pathway(s), leading to cell death and behavioral deficits, and that the superoxide radical plays a critical role in the underlying mechanism.

Ingestive responses to administration of stress hormones in baboons.

Experimental stress and the administration of the stress hormone ACTH have been reported to stimulate sodium appetite in many nonprimate species. Experiments were conducted to determine whether prolonged intracerebroventricular infusions of the neuropeptides corticotropin-releasing factor (CRF) and urocortin (Ucn), or systemic administration of ACTH, affected ingestive behaviors in a nonhuman primate, the baboon. Intracerebroventricular infusions of CRF or Ucn significantly decreased daily food intake. The decrease with Ucn continued into the postinfusion period. These infusions did not alter daily water intake. Daily voluntary intake of 300 mM NaCl solution was not increased, and there was evidence of reductions on days 2-4 of the infusions. Intramuscular injections of porcine ACTH or synthetic ACTH (Synacthen) for 5 days did not affect daily NaCl intake, although the doses were sufficient to increase cortisol secretion and arterial blood pressure. Sodium depletion by 3 days of furosemide injections did induce a characteristic sodium appetite in the same baboons. These results demonstrate the anorexigenic action of CRF and Ucn in this primate. Also, CRF, Ucn, and ACTH did not stimulate sodium appetite at the doses used.

Zinc supplementation does not attenuate alcohol-induced cerebellar Purkinje cell loss during the brain growth spurt period.

BACKGROUND: Alcohol-induced zinc deficiency is one of the mechanisms proposed as a cause of developmental brain damage associated with fetal alcohol syndrome. It is known that alcohol exposure during the brain growth spurt period leads to cerebellar Purkinje cell loss. Therefore, this study examined whether zinc supplementation was capable of preventing alcohol-induced Purkinje cell loss in the cerebellar vermis in a neonatal rat model system. METHODS: Sprague-Dawley rat pups were given alcohol (EtOH; 4.5 g/kg/day), zinc (Zn; 0.54 mg/ml diet; [10 times the regular diet Zn concentration]), or both from postnatal days (PD) 4 through 9 using the artificial-rearing paradigm. A gastrostomy control (GC) and a suckle control group (SC) also were included. All pups were killed on PD 10. Following perfusion, the cerebellar vermis was dissected and processed for stereological cell counting. The total number of Purkinje cells and the volume of the cerebellar vermis were determined. RESULTS: Alcohol produced a significant loss of Purkinje cells compared with that in the GC group (no EtOH and no Zn supplement). The zinc supplementation had no effect in attenuating alcohol-induced Purkinje cell loss in the cerebellar vermis. In fact, the serum zinc concentration data indicated higher zinc concentrations following either EtOH or Zn treatment. Interestingly, the GC group showed a significantly lower zinc concentration compared with the SC group, even though no significant difference in Purkinje cell numbers was observed between these two control groups. CONCLUSION: These findings indicate that alcohol exposure during the third trimester equivalent did not result in zinc deficiency in this neonatal rat model system, nor did zinc supplementation rescue the alcohol-induced Purkinje cell loss in the cerebellar vermis. These findings showed clearly that the serum zinc concentration was not correlated with Purkinje cell loss, suggesting that alcohol-induced loss of cerebellar Purkinje cells in this neonatal rat model system is independent of the availability of serum zinc.

Adjuncts to preparing wounds for closure: hyperbaric oxygen, growth factors, skin substitutes, negative pressure wound therapy (vacuum-assisted closure).

Achieving closure in a chronic wound requires provision of adequate oxygen delivery to the tissue, adequate protein and other nutritional factors, a moist environment, an appropriate inflammatory milieu, dèbridement, and correction of contributing medical diagnoses. In some patients, these conditions are achieved easily, whereas in others, greater effort is required. Adjunctive treatments, including HBO2, growth factors, skin substitutes, and negative-pressure wound therapy (e.g., V.A.C.) can provide the proper conditions for healing in appropriately selected patients.