Assessing bioavailability of ruminally protected methionine and lysine prototypes.

Met and Lys are essential AA that can limit lactational performance in dairy cattle fed protein-sufficient diets. Thus, there is industry demand for ruminally protected (RP) sources of Met and Lys. One method of providing ruminal protection for Met and Lys is lipid encapsulation. The objective of this work was to assess 3 lipid-encapsulated Met prototypes (P1, P2, and P3) and 1 Lys prototype (P4) to determine ruminal protection, small intestine absorption (experiment 1), and animal production responses (experiment 2). Ruminal protection was estimated from 8-h in situ retention during ruminal incubation and intestinal absorption from plasma appearance after an abomasal bolus of the in situ retentate. Blood samples were collected over time to determine plasma Met and Lys concentration responses compared with unprotected Lys and Met infused abomasally. The prototypes were not exposed to the total diet or subjected to typical feed handling methods before evaluation. The bioavailability of P1, P2, and P3 Met prototypes was found to be 14, 21, and 18% of the initial AA material, respectively. The RP-Lys prototype had a bioavailability of 45%. To evaluate production responses, 20 Holstein cows were randomly assigned to 2 trials (n = 10 each) in a replicated Latin square design with 14-d periods. The base diet was predicted to be deficient in metabolizable Met (-14.8 g/d) and Lys (-16.1 g/d) per the Cornell Net Carbohydrate and Protein System (version 6.55). In the Met trial, the base diet was supplemented with RP-Lys to meet Lys requirements, and treatments were as follows: no added RP-Met (NCM), NCM plus Smartamine M (SM; Adisseo, Alpharetta, GA), and NCM plus P1, P2, or P3 at 148% of the Met content of SM. In the Lys trial, the base diet was supplemented with RP-Met to meet the Met requirement, and treatments were as follows: no added Lys (NCL), NCL plus AjiProL (AL; Ajinomoto Heartland Inc., Chicago, IL), and NCL plus P4 at 55, 78, or 102% of the reported absorbed Lys in AL. All products were top dressed on the diet without prior mixing or extended exposure to the rest of the diet. Milk protein concentration significantly increased when diets were supplemented with P2, P3, or SM (3.12, 3.12, and 3.11%, respectively) compared with NCM (3.02%). Only P1 (3.04%) was significantly lower than SM. Prototype P2 had the greatest numerical milk protein output response among the 3 RP-Met prototypes, suggesting that it may have had the greatest efficacy when supplemented into these rations. There was a numerical milk protein concentration response to AL and a linear increase in milk protein concentration for P4. The P4 and AL treatments resulted in comparable milk protein production regardless of P4 dose.

Intracoronary stenting compared with conventional therapy for abrupt vessel closure complicating coronary angioplasty: a matched case-control study.

OBJECTIVES: A case-control analysis was performed to compare clinical outcome after intracoronary stenting with that after conventional therapy for abrupt vessel closure. BACKGROUND: Previous studies have demonstrated that stenting after abrupt vessel closure results in marked angiographic improvement and preservation of coronary flow, leading to the anticipation of similar improvement in clinical outcome. METHODS: Sixty-one of 92 consecutive patients treated at two clinical sites by intracoronary stenting for abrupt vessel closure were matched, according to angiographic features of closure and estimated left ventricular mass threatened by ischemia, with patients treated conventionally during the 18 months before stent availability. In 33 pairs of matched patients, vessel closure was established; in 28 pairs, it was threatened (coronary dissection or worsening stenosis with preservation of normal anterograde flow). Baseline clinical and angiographic characteristics were comparable in the two matched groups. Patients with indeterminate mechanisms of total occlusion (31%) or dissections < 15 mm long (43%) predominated; patients with visible thrombus (8%) or dissections > 15 mm long (18%) were infrequently represented. Stents were successfully deployed in 60 of 61 patients at a median of 52 min (range 3 to 269) after the onset of closure. RESULTS: When compared with conventional treatment, stenting resulted in less residual stenosis (26% vs. 49% diameter stenosis, p < 0.001), a greater likelihood of restoration of Thrombolysis in Myocardial Infarction (TIMI) grade 3 blood flow (97% vs. 72%, p < 0.001) and a reduction in the need for emergency bypass surgery (4.9% vs. 18%, p = 0.02). However, the incidence of Q wave myocardial infarction was nearly the same in the two groups (32% vs. 20%, respectively, p = NS). In the group with stenting, peak creatine kinase level and the frequency of Q wave infarction after established vessel closure increased with the time to stent placement (p = 0.001 and 0.054, respectively); the incidence of procedure-related Q wave infarction in patients who underwent stenting within 45 min of closure was very low (3.9%). In-hospital death occurred in 3.3% of patients in each treatment group. At a mean of 6.3 months of follow-up after hospital discharge, survival free from late cardiac death, myocardial infarction, bypass surgery or coronary angioplasty was 74.9% and 81.3% in the stent and the control treatment group, respectively (p = NS). CONCLUSIONS: Although early treatment of established vessel closure by intracoronary stenting was associated with a low incidence of both myocardial infarction and emergency bypass surgery, the likelihood or severity of infarction was not reduced among those in whom stents were implanted later. Patients with threatened vessel closure could not be shown to benefit from stent treatment. These data provide preliminary indications for stent placement in the acute period to be validated in larger randomized studies.

Large scale synthesis of recombinant human thyrotropin using methotrexate amplification: chromatographic, immunological, and biological characterization.

Studies of human TSH (hTSH) structure and function have been limited by difficulties in producing large quantities of recombinant hormone. We describe a system for the stable expression of high levels of recombinant human TSH (rec hTSH) using a mutant form of dihydrofolate reductase (dhfr) as an amplifiable dominant selectable marker. A vector expressing both the hTSH alpha-subunit and the mutant dhfr was cotransfected with a hTSH beta-subunit expression vector into dhfr-deficient cells. Amplification of the transfected sequences by methotrexate selection, followed by cell culture in a hollow fiber perfusion system, yielded rec hTSH production as high as 100,000 microU/ mL. Immunoradiometric assays using five different antibodies revealed no differences in the immunological activities of rec hTSH and pituitary hTSH. Bioactivity was measured in a novel TSH bioassay coupling the generation of cAMP by a transfected hTSH receptor to the cAMP-dependent regulation of a luciferase reporter gene. The ED50 for bovine TSH in this bioassay was 1.4 ng/mL (3.5 x 10(-11) mol/L). The ratio of the ED50 values for rec hTSH and pituitary hTSH was 1.0:1.1 (P = NS), indicating that the two TSHs were of equivalent potency. In conclusion, we have developed techniques for the high level production of rec hTSH that is immunologically and biologically equivalent to pituitary hTSH. The ability to produce large quantities of rec hTSH using standard laboratory techniques should facilitate future studies, such as the development of clinically useful TSH analogs.

Strategies one hospital group has developed for successful change.

Evangelical Health Systems has developed its own patient care software program and is marketing it to other hospitals under the name FasTrack Systems, Inc. The hospital communications and software package was installed in three EHS hospitals in 18 months.

Effect of total and rumen undegradable protein on the performance of cows fed low fiber diets.

Twelve multiparous and 18 primiparous Holstein cows were fed a 17.3% CP, 21.0% ADF diet during wk 2 through 6 postpartum. Cows then were assigned from wk 7 through 14 to one of three low fiber (10.7% ADF) dietary treatments containing either 14.4 or 18.7% CP, the latter with or without a soybean meal enhanced with rumen undegradable protein. Treatments had no effect on milk yield or composition in multiparous cows, although milk fat percentage was not depressed in multiparous cows receiving the low fiber diets. The soybean meal diet enhanced with rumen undegradable protein increased yields of milk, 4% FCM, fat, protein, and DMI compared with the 14.4% CP diet in primiparous cows; it also increased yields of 4% FCM and fat versus the 18.7% CP, untreated diet in primiparous cows. Blood urea N concentrations were greater for high CP diets than for the low CP diet in both parity groups. Rumen acetate: propionate ratios were higher for both high CP diets than for the low CP diet in multiparous cows. Soybean meal enhanced with rumen undegradable protein improved yields of milk and its components in primiparous cows fed low fiber diets, even when high protein diets were fed.

Influence of dietary protein and supplemental niacin on lactational performance of cows fed normal and low fiber diets.

Forty-seven cows (24 primiparous) were assigned to one of four normal (20.5%) ADF diets for wk 2 to 5 postpartum. Dietary treatments in a 2 x 2 factorial design were diets of 13.8 versus 18.8% CP and 0 versus 12 g/d of niacin per cow. During wk 6 to 13 postpartum, cows were fed low (11.8%) ADF diets while maintaining CP and niacin treatments. Low CP diets contained solvent-extracted soybean meal; rumen soybean meal with enhanced undegradable protein was used in high CP diets. High CP diets increased milk protein percentage in multiparous cows and yields of milk, 4% FCM, fat, protein, and SNF in primiparous cows during the normal fiber period. High dietary CP also increased yields of 4% FCM, fat, protein, and SNF in primiparous cows fed normal fiber diets. When switched to low fiber diets, primiparous cows fed high CP diets decreased more in 4% FCM and fat yields than those fed low CP. Primiparous cows fed niacin decreased more in 4% FCM than controls. High dietary CP increased DMI in primiparous cows fed normal fiber diets, but those fed low CP diets increased more in DMI when switched to low fiber diets. Supplemental niacin appeared to interact with dietary CP in multiparous cows, increasing blood glucose and decreasing blood beta-hydroxybutyrate and NEFA concentrations with the high CP, normal fiber diet. Increased dietary CP improved yields of milk and milk components in primiparous cows.

Effects of protein level and forage source on milk production and composition in early lactation dairy cows.

Twenty multiparous and 12 primiparous Holstein cows were assigned at calving to one of three grass hay-based diets containing either 14, 18, or 22% CP or an alfalfa hay-based diet containing 22% CP to examine the effect of protein level and forage source on milk yield and composition. The diets contained 23% ADF during wk 1 to 4 postpartum, which was lowered to 11% for wk 5 to 12 postpartum. Cows fed the 18 and 22% CP grass-based diets produced higher yields of milk, 4% FCM, fat, protein, and SNF than those fed the 14% CP diet during the high fiber period. In addition, cows fed the 22% CP grass-based diet had higher milk fat tests than those fed the 14% CP diet during the high fiber period, due primarily to an increase in short-chain fatty acid synthesis. Milk fat depression was more severe when cows were changed to low fiber diets while fed the 22% CP alfalfa-based diet than when fed the 22% CP grass-based diet. Depression in milk fat content was 15.0, 17.0, 15.6, and 27.0% for 14, 18, and 22% CP grass-based and 22% CP alfalfa-based diets, respectively. Cows receiving the 18 and 22% CP grass-based diets exhibited higher blood NEFA during the high fiber feeding period than those fed the 14% CP diet. After fiber was lowered, changes in rumen acetate:propionate ratios were unaffected by treatment. Lowering fiber level resulted in an increased milk CP percentage regardless of treatment. Grass hay appeared to be more effective than alfalfa hay in preventing depression in milk fat test upon the change to a low fiber diet.

Effects of intraperitoneal L-leucine, L-isoleucine, L-valine, and L-arginine on milk fat depression in early lactation cows.

Eight Holstein cows were assigned following calving to two groups, balanced for parity, using a continuous completely randomized block design. Cows were fed a diet with 13.5% CP and 22.4% ADF from 35 to 55 DIM and then 13.8% CP and 15% ADF from 56 to 92 DIM. Alfalfa grass hay was the forage source, and concentrate mixtures contained primarily corn and soybean meal. Cows were given daily intraperitoneal infusions of a solution of L-Leu (46.1 g, 84.2 mM), L-Ile (31.4 g, 57.3 mM), L-Val (38.3 g, 78.2 mM), and L-Arg (25.0 g, 34.4 mM) or physiological saline following the a.m. milking from 42 through 84 DIM. Infusion of AA significantly increased plasma concentrations of Leu, Ile, Val, and Arg. Effects of AA infusion occurred during the low fiber period. Cows receiving AA maintained daily milk fat yield, increased p.m. milk fat yield, decreased less in p.m. milk fat percentage, and increased daily and p.m. yields of C16 fatty acids in milk. During the posttreatment period, cows previously receiving AA declined in daily milk fat yield, milk fat percentage, and total daily C4 to C16 milk fatty acid yield. Results suggest that the infused AA may have increased de novo synthesis of C16 milk fatty acids.

Mutations of the human thyrotropin-beta subunit glycosylation site reduce thyrotropin synthesis independent of changes in glycosylation status.

In recent studies, site-directed mutagenesis has been used to alter the tripeptide glycosylation recognition sequences of glycoprotein hormone subunits, thereby affecting their structure and function. However, it is not known whether these effects result from changes in glycosylation status, amino acid sequence, or both. We therefore studied the synthesis of wild-type and mutant recombinant human thyrotropins produced by transient transfection of a human cell line. Mutating the TSH-beta subunit glycosylation recognition sequence, Asn-Thr-Thr (codons 23-25), to either Gln-Thr-Thr or Asn-Thr-Tyr abolished subunit glycosylation, as demonstrated by the inability to incorporate 3H-carbohydrates. However, a third mutation (Asn-Thr-Ser) contained an intact glycosylation recognition sequence site, and was shown to retain glycosylation. The mutations that abolished TSH-beta subunit glycosylation resulted in greater than 90% decreases in TSH synthesis. However, the glycosylation recognition sequence mutant that retained beta subunit glycosylation exhibited a 70% decrease in TSH production. These decreases were not attributable to the intracellular accumulation of TSH or its free beta subunit. We also engineered two TSH-beta subunit mutations that did not alter the glycosylation recognition sequence. A glycine to arginine mutation adjacent to the glycosylation recognition sequence, in a region thought to be critical for heterodimer formation, abolished TSH production. In contrast, shortening the TSH-beta subunit carboxyterminus by six amino acids increased TSH synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)