Characterization of MB-1. A dimeric helical protein with a compact core.
(33/4907)
MB-1 is a de-novo protein designed to incorporate a large number of the nutritionally important amino acids methionine, lysine, leucine and threonine into a stable four-helix bundle protein. MB-1 has been expressed and purified from Escherichia coli, indicating it was resistant to intracellular proteases [Beauregard, M., Dupont, C., Teather, R.M. & Hefford, M.A. (1995) Bio/Technology 13, 974]. Here we report an analysis of the secondary, tertiary and quaternary structures in MB-1 using circular dichroism, fluorospectroscopy and size-exclusion chromatography. Our data indicate that the MB-1 structure is close to the target structure, an alpha-helical bundle, in many respects and is highly helical in solution. The single tyrosine incorporated into the designed protein as a spectrocopic probe of tertiary structure, is buried in a compact, folded core and becomes accessible on protein denaturation, as per design. Furthermore, MB-1 was found to be native-like in many respects: (a) protein denaturation induced by urea is cooperative and fully reversible; (b) its oligomeric state at moderate concentration is well defined; and (c) MB-1 has very low affinity for 8-anilino-1-naphthalenesulfonic acid (ANSA), leading to enhancement of ANSA fluorescence that resembles that of other native proteins. On the other hand, our analysis revealed two aspects that command further attention. The folding stability of MB-1 as assessed by urea and thermal denaturation is somewhat less than that found for natural globular proteins of similar size. Size-exclusion chromatography experiments and analysis of MB-1 denaturation indicate that MB-1 is dimeric, not monomeric as designed. In light of these results, the utility and the current limitations of our design approach are discussed. (+info)
Interaction of cattle health/immunity and nutrition.
(34/4907)
The usual means of assessing the health of newly received beef cattle susceptible to bovine respiratory disease (BRD) are subjective, typically involving visual evaluation aided by minimal clinical measurements. Recent evidence based on the occurrence of pneumonic lung lesions at slaughter indicates a need for more accurate methods of diagnosing BRD. Inadequate passive immune transfer at birth may be an important risk factor in susceptibility to BRD, suggesting the need for management to improve passive transfer success rates. Preweaning management and vaccination practices offer opportunities for beef cattle producers to improve the immune status of newly weaned calves and decrease postweaning BRD. Feeding diets with higher levels of concentrate typically improves performance by newly weaned or received cattle, as does feeding diets supplemented with protein; however, limited data suggest that increasing concentrate and protein in receiving diets increases the rate and severity of subjectively determined BRD morbidity. Research with receiving diet concentrate/protein level relative to humoral and cell-mediated immune function coupled with indicators of health and performance is needed. Supplemental B vitamins are sometimes useful in receiving diets, but the effects have been variable, presumably reflecting differences in stress and associated feed intake responses. Vitamin E added to receiving diets to supply > or = 400 IU/animal daily seems beneficial for increasing gain and decreasing BRD morbidity; however, further dose titration experiments are needed. Supplemental Zn, Cu, Se, and Cr can alter immune function of newly received calves, and some field trials have shown decreases in BRD morbidity rate with supplementation; however, several experiments have shown no performance or health/immune benefits from supplementation of these trace minerals. Formulation of receiving diets should take into account decreased feed intake by highly stressed, newly received beef cattle and known nutrient deficiencies, but fortification of such diets with trace minerals beyond the levels needed to compensate for these effects is difficult to justify from present data. (+info)
Comparative protein and amino acid digestibilities in growing pigs and sows.
(35/4907)
An experiment was conducted to compare apparent total tract protein digestibilities and apparent ileal digestibilities of protein and amino acids in growing pigs and adult pregnant and lactating sows. Twelve growing pigs and 12 sows were used and surgically fitted with simple T-cannulas at the distal ileum. Six experimental diets based on corn, barley, wheat, soybean meal, canola meal, or meat and bone meal were formulated, and each diet was fed to growing pigs, gestating sows, and lactating sows for 7 d. Chromium oxide was included in all diets as an indigestible marker (.25%) for calculating nutrient digestibilities. Fecal material was collected on d 5 of each feeding period by grab sampling, and ileal samples were collected for 12 h/d during the last 2 d of each feeding period. Apparent fecal protein digestibilities for all feed ingredients were higher (P < .05) in gestating and lactating sows compared to growing pigs, but no differences between the two groups of sows were observed (P > .05). At the distal ileum, no differences (P > .05) in protein digestibilities were detected between sows and growing pigs regardless of feed ingredient. For all feed ingredients tested, lactating sows had apparent ileal digestibilities of most amino acids that were two to six percentage units higher than those obtained in growing pigs, but not all of the differences were significant. Gestating sows had digestibilities of most amino acids that were intermediate between those of growing pigs and lactating sows. The combined results from the six feed ingredients showed that lactating sows had higher (P < .05) digestibilities of all indispensable amino acids except arginine, and gestating sows had higher (P < .05) digestibilities of five of the indispensable amino acids than did growing pigs. The results of this experiment indicate that apparent fecal protein and apparent ileal amino acid digestibilities obtained in growing pigs are not always representative of digestibilities in either gestating or lactating sows. (+info)
The effect of feeding level and physiological status on total flow and amino acid composition of endogenous protein at the distal ileum in swine.
(36/4907)
An experiment was conducted to investigate the effects of BW, feed intake, and the physiological condition of the animal on the loss and amino acid composition of endogenous protein in swine. Ten growing barrows and five multiparous sows were equipped with a T-cannula in the distal ileum for digesta collection. A protein-free diet was fed to all animals. The barrows were given free access to the experimental diet. The sows also were allowed to consume the diet on an ad libitum basis, and digesta were collected during lactation and in the following gestation period. In addition, digesta from the gravid sows were collected after restricting the sows to 2 kg of feed per day. For each animal group, the endogenous losses of protein and amino acids were calculated in relation to DMI, and the amino acid composition of endogenous protein was calculated. The total endogenous gut protein loss at the distal ileum of growing pigs, lactating sows, and gestating sows, given free access to feed, was 12.4, 9.4, and 11.2 g/kg DMI, respectively. These values were not different (P > .10). However, when gestating sows were fed only 2 kg/d, 17.8 g of endogenous protein was lost per kilogram of DMI, which was higher (P < .05) than for any of the other groups. This difference was mainly caused by higher (P < .05) losses of glycine, proline, and serine. There were no differences (P > .05) in amino acid composition of endogenous protein between growing pigs, lactating sows, and gestating sows given free access to feed, but restricted-fed gestating sows had an amino acid composition of endogenous protein that was significantly different from that of the other groups. The results from the experiment showed that age, BW, and the physiological condition of the animal have little or no effect on the amount of endogenous protein and amino acids lost at the distal ileum of hogs if calculated in relation to DMI. Likewise, the amino acid composition was not affected by the BW or physiological condition of the animal. However, DMI had a significant effect on endogenous protein losses in sows as well as on amino acid composition of endogenous protein. (+info)
Dietary protein and chromium tripicolinate in Suffolk wether lambs: effects on production characteristics, metabolic and hormonal responses, and immune status.
(37/4907)
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)
Effects of increasing ruminally degraded nitrogen and abomasal casein infusion on net portal flux of nutrients in yearling heifers consuming a high-grain diet.
(38/4907)
Seven Meat Animal Research Center (MARC) III heifers (410+/-25 kg) fitted with hepatic portal, mesenteric venous, carotid catheters, and an abomasal cannula were used in a 7 x 5 incomplete Latin square design experiment. The objective was to evaluate the effects of increasing levels of ruminally degradable N (RDN) with or without the addition of abomasally infused casein on portal-drained visceral (PDV) flux of nutrients. Treatments consisted of dietary CP percentage levels of 9.5 (control), control plus .72% dietary urea (11.5U), control plus 1.44% dietary urea (13.5U), control plus abomasally infused casein (250 g/d; 11.5C), or control plus .72% dietary urea and abomasally infused casein (250 g/d; 13.5UC). All diets contained (DM basis) 80% ground corn, 15% corn silage, and 5% dry supplement and were provided for ad libitum consumption. Nitrogen intake increased (linear, P < .001) as CP increased from 9.5 to 13.5%. Portal-drained visceral release of ammonia N increased (linear, P < .10) as RDN increased, and was greater (P < .05) when protein was fed compared with heifers fed control (P < .10). Urea N removal by PDV was not affected ( P > . 10) by level of RDN but was greatest when 11.5C was fed and least when 13.5UC was fed. Net alpha-amino N (AAN) release by PDV was greatest when 13.5UC was fed (309 mmol/h), least when 9.5% CP was fed (112 mmol/h), and intermediate for the other groups (205 to 252 mmol/h). These data suggest that removal of N by the PDV may promote microbial protein synthesis when dietary RDN is low. When RDN needs have been met and amino acids are deficient for the host, escape protein should be fed to increase amino acid absorption. (+info)
Dietary soybean protein moderates the deleterious disturbance of lipid metabolism caused by exogenous oxidized cholesterol in rats.
(39/4907)
Effects of dietary protein on oxidized cholesterol-induced disturbance of lipid metabolism were examined in 4 week old male Sprague-Dawley rats, using casein and soybean protein as dietary protein source. The rats were given one of the two proteins in 0. 078% cholesterol (control), 0.25% cholesterol or 0.25% oxidized cholesterol mixture (containing 0.078% cholesterol) diets. Dietary oxidized cholesterol, compared to cholesterol, tended to inhibit hepatic sterol biosynthesis in casein-fed rats, whereas this inhibitory action was slightly moderated by intake of soybean protein. As a result, the hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity was rather higher in the rats fed oxidized cholesterol than in those fed cholesterol in the soybean protein-fed group. The hepatic cholesterol 7alpha-hydroxylase activity tended to be higher in the rats fed oxidized cholesterol than in those fed control diet in the soybean protein-fed group, despite the fact that oxidized cholesterol lowered the hydroxylase activity in the casein-fed group. On the other hand, dietary oxidized cholesterol tended to slightly enhance the hepatic Delta6 desaturase activity in the casein-fed group; however, this observation was not shown in the soybean protein-fed group. Moreover, dietary soybean protein facilitated fecal oxidized cholesterol excretion and simultaneously inhibited the accumulation of oxidized cholesterol in serum and liver. In conclusion, dietary soybean protein alleviated the deleterious actions of exogenous oxidized cholesterol on hepatic cholesterol and linoleic acid metabolism, although these efficacies were not necessarily significant. A great part of these moderations may be exerted by the specific hypocholesterolemic function of soybean protein, such as the stimulation of fecal oxidized cholesterol excretion, the change of hormonal release and modulation of lipoprotein catabolism. (+info)
High dietary folate supplementation affects gestational development and dietary protein utilization in rats.
(40/4907)
There is new evidence that good folate status may play a critical role in the prevention of neural tube defects and in the maintenance of adequate homocysteine levels, an amino acid recently identified as a risk factor for cardiovascular disease. This has led to different folate recommendations, all of them much higher than the present dietary recommendations. Folic acid is a water-soluble vitamin with a low potential toxicity. However, the possible consequences of long-term, high folate intakes are unknown. Therefore, the present study was undertaken to determine the effects of long-term, high dietary folate supplementation on gestational and nutritional markers in pregnant and virgin rats. Four groups of Wistar rats were classified on the basis of physiological status (virgin or pregnant) and the experimental diets administered (folic acid supplemented, 40 mg/kg diet; or control diet, 2 mg folic acid/kg diet). Rats were fed their respective diets for 3 wk. Two critical periods were used for metabolic balance studies (experimental d 1-5 and 17-21), which involved the determination of fat and protein digestibilities as well as metabolic protein utilization (MPU) and net protein utilization (NPU). Gestational development (number of live fetuses) was adequate in both diet groups regardless of folate supplementation. However, body weight and vertex-coccyx length in fetuses from supplemented dams were less than (P < 0.0001) in fetuses of control dams. Fat and nitrogen digestibilities were not affected by supplementation, but MPU and NPU coefficients were significantly lower (P < 0.05) in the folic acid-supplemented groups, irrespective of physiological status, compared to control rats. These new findings of macro-micronutrient interactions caused by high folate supplementation are discussed on the basis that the vitamin may act as a xenobiotic more than as a nutrient. (+info)