Effects of dry or wet conditions during the preweaning phase on subsequent feedlot performance and carcass composition of beef cattle.

Our objective was to determine the effects of dry and wet conditions during the preweaning on subsequent feedlot performance and carcass characteristics of beef cattle. Steers (n = 7,432) and heifers (n = 2,361) finished in 16 feedlots in southwestern Iowa through the Tri-County Steer Carcass Futurity Cooperative were used for a retrospective analysis. Cattle originated in the Midwest (Iowa, Missouri, Indiana, Illinois, and Minnesota) and were born in February, March, or April of 2002 through 2013. Feedlot performance and carcass composition data were obtained for each animal. Palmer Drought Severity Index (PDSI) values were obtained for each animal's preweaning environment on a monthly basis. Mean PDSI values were used to classify conditions as dry (≤-2.0), normal (>-2.0 and <2.0), or wet (≥2.0) for the cool (April and May), warm (June through August), and combined (April through August) forage growing seasons preweaning. Mixed models were used to evaluate the effects of PDSI class on subsequent performance. Calf sex, date of birth (as day of year), year, and feedlot were also included as fixed effects. When considering PDSI class during the cool season, cattle from normal and wet classes had a greater feedlot delivery BW (P < 0.0001) than dry. Dry and normal classes had greater (P ≤ 0.02) delivery BW than wet during the warm and combined seasons, however. For the cool season, average daily gain was greater (P < 0.0001) for the dry class than normal and wet. Cattle from the normal class for the cool season had greater (P = 0.001) final BW than wet, but the wet class had the greatest (P < 0.04) and dry class had the lowest (P < 0.01) final BW during the warm season. During the cool season, HCW was greater (P < 0.007) for the normal than wet class, although HCW was greater (P ≤ 0.02) for wet compared with dry and normal during the warm season. Calculated yield grade was lower (P ≤ 0.006) for the normal class during the cool season compared with dry and wet. For both the warm and combined seasons, the dry class had lower (P ≤ 0.004) calculated yield grade compared with normal and wet. Carcasses from cattle that experienced normal or wet warm seasons had greater (P ≤ 0.0005) marbling scores than dry, and normal had greater (P = 0.0009) marbling score than dry for the combined seasons. In conclusion, these data indicate that both dry and wet conditions during the preweaning phase may impact ultimate feedlot performance and carcass composition.

An anti-αVß3 antibody inhibits coronary artery atherosclerosis in diabetic pigs.

BACKGROUND AND AIMS: Diabetes is a major risk factor for the development of atherosclerosis. Hyperglycemia stimulates vascular smooth muscle cells (VSMC) to secrete ligands that bind to the αVß3 integrin, a receptor that regulates VSMC proliferation and migration. This study determined whether an antibody that had previously been shown to block αVß3 activation and to inhibit VSMC proliferation and migration in vitro, inhibited the development of atherosclerosis in diabetic pigs. METHODS: Twenty diabetic pigs were maintained on a high fat diet for 22 weeks. Ten received injections of anti-ß3 F(ab)2 and ten received control F(ab)2 for 18 weeks. RESULTS: The active antibody group showed reduction of atherosclerosis of 91 ± 9% in the left main, 71 ± 11%, in left anterior descending, 80 ± 10.2% in circumflex, and 76 ± 25% in right coronary artery, (p < 0.01 compared to lesions areas from corresponding control treated arteries). There were significant reductions in both cell number and extracellular matrix. Histologic analysis showed neointimal hyperplasia with macrophage infiltration, calcifications and cholesterol clefts. Antibody treatment significantly reduced number of macrophages contained within lesions, suggesting that this change contributed to the decrease in lesion cellularity. Analysis of the biochemical changes within the femoral arteries that received the active antibody showed a 46 ± 12% (p < 0.05) reduction in the tyrosine phosphorylation of the ß3 subunit of αVß3 and a 40 ± 14% (p < 0.05) reduction in MAP kinase activation. CONCLUSIONS: Blocking ligand binding to the αVß3 integrin inhibits its activation and attenuates increased VSMC proliferation that is induced by chronic hyperglycemia. These changes result in significant decreases in atherosclerotic lesion size in the coronary arteries. The results suggest that this approach may have efficacy in treating the proliferative phase of atherosclerosis in patients with diabetes.

Impact of oral meloxicam and long-distance transport on cell-mediated and humoral immune responses in feedlot steers receiving modified live BVDV booster vaccination on arrival.

The objective of this study was to investigate the impact of oral meloxicam (MEL) and long-distance transportation on cell-mediated immunity (CMI) in preconditioned steers receiving a booster vaccination on arrival. We hypothesized that steers treated with MEL at 1mg/kg body weight, 6h before night-time transport, would be less immunocompromised on arrival (day 0) and after 7days, and that CMI following vaccination with a modified live bovine viral diarrhea virus (BVDV) recall antigen would be increased. Brahman crossbreed steers, 13-17 months of age (n=87), were randomly assigned to one of four treatment groups: MEL, transported (MTR) (n=22), MEL, non-transported (MNT) (n=22), lactose placebo, transported (CTR) (n=21), and lactose placebo, non-transported (CNT) (n=22). MTR and CTR steers were transported for approximately 16h non-stop on a truck from Mississippi to Iowa (approximately 1300km), whereas steers in the MNT and CNT groups remained in Mississippi as non-transported controls. Body weight was measured and jugular blood was collected at -1, 0, and 7days from all steers at the same time, regardless of location. Multi-parameter flow cytometry (MP-FCM) was used to identify T-cell subsets and detect the expression of three activation markers (CD25 [interleukin (IL)-2 receptor], intracellular interferon-gamma [IFNγ], and IL-4) after in vitro stimulation with BVDV recall antigen. Plasma cortisol concentration was measured on day -1, 0, and 7 as a marker of transport-associated stress. Serum antibody titer to BVDV was assessed on day -1 and day 7 post-booster vaccination. Whole-blood samples were analyzed using MP-FCM on days 0 and 7. Results were log transformed and analyzed using repeated measures of analysis of variance. Compared with non-transported controls, transport led to an increase in BVDV-induced expression of CD25, IFNγ, and IL-4 in CD4(+), CD8(+), and γδ(+) T-cell subsets (P<0.05). MEL treatment mitigated the transportation-associated increase in CD25 expression by peripheral blood mononuclear cells (PBMCs), CD4(+), and γδ(+) T cells. CMI outputs for the MTR group were less than those of the CTR group (P<0.05); however, the MTR and NT groups did not differ (P>0.10). A treatment*transport interaction was noted for the increase in IL-4 expression by CD8(+) T cells after transport, with a significant difference between the CTR and MTR groups at day 7. In conclusion, the use of oral MEL prior to transport appears to have inhibitory or homeostatic effects, but further research is needed to validate the effect of MEL treatment on specific T-cell subsets in transported cattle.

An audit of the current U.S. Department of Agriculture frame size scoring system.

Feedlot and carcass data from steers (n = 16,700) and heifers (n = 6,357) originating from 16 different states and fed in 17 feedlots located in southwest Iowa were used to evaluate the accuracy of the USDA frame score for predicting final BW of fed cattle. Frame score was recorded by USDA or state personnel for cattle either before leaving the state of origin or on arrival at the terminal feedlot. Mixed model procedures were used to investigate relationships between USDA frame score and measures of live performance and carcass traits. Other fixed effects included in the model included USDA muscle score, sex, age classification on feedlot entry (calf: ≤270 d of age, yearling: 271-365 d of age, and long yearling: >365 d of age), BCS on feedlot arrival, number of treatments for respiratory disease, hide color, and site of frame or muscle scoring; the interactions of sex × frame score and hide color × frame score were also included; fat thickness was included as a fixed effect (covariate) in the analysis of ADG, final BW, days on feed, LM area, marbling score, and quality grade. Random effects included in the model were year of feedlot arrival and feedlot in which cattle were fed. The system accurately projects the minimum target final BW for large frame steers and heifers; however, the final BW of the smallest medium frame steers and heifers exceeds the target minimum final BW by 35 and 40 kg, respectively. When frame score was assigned post facto based on actual final BW (adjusted to 1.27 cm fat thickness), it was determined that large frame was over-assigned by graders (62 vs. 35% for steers and 54 vs. 32% for heifers, actual score vs. postharvest score, respectively), medium frame was underassigned (37 vs. 51% and 46 vs. 58% for steers and heifers), and small frame was underassigned (0.7 vs. 15% and 0.6 vs. 10% for steers and heifers; K = 0.01, P < 0.01). Across sexes, of the cattle assigned to small, medium, or large frame score, 40, 59, and 43% actually had final BW (adjusted to 1.27 cm fat thickness) within the guidelines for the target final BW of each of the frame scores (P < 0.01). The present frame score system accurately reflects the modern feedlot cattle population; however, adjustments in the assignment of frame scores to reflect changes in technologies and final weights may be warranted.

Impact of oral meloxicam on circulating physiological biomarkers of stress and inflammation in beef steers after long-distance transportation.

Transportation stress can result in significant economic losses to producers due to decreased animal productivity and increased medication costs associated with sickness such as bovine respiratory disease (BRD). Meloxicam (MEL) provides pain relief and anti-inflammatory effects in cattle for several days after a single oral treatment. Our hypothesis was that MEL administration before shipping would reduce the impact of long-distance transportation on circulating physiological biomarkers of stress and inflammation in beef steers. Ninety-seven beef steers were blood sampled for baseline biomarker determination and then randomly assigned to receive either 1 mg/kg MEL (n = 49) or a placebo (CONT; n = 48) per os before a 1,316-km transportation event lasting approximately 16 h. Calves were then blood sampled on arrival and 5 d later. Changes in the hemogram, circulating plasma proteins, total carbon dioxide (TCO2), fibrinogen, substance P (SP), cortisol, haptoglobin (Hp)-matrix metalloproteinase-9 (MMP-9) complexes, and tumor necrosis factor α (TNFα) between treatments over time were compared using a mixed effects model with statistical significance designated as P < 0.05. Analysis of covariance was conducted to assess the relationship between circulating MEL concentrations and biomarker changes over time. An increase in neutrophil, platelet, monocyte, white blood cell, and red blood cell counts occurred after transportation (P < 0.0001) and a decrease in lymphocyte count were observed (P < 0.0001). Meloxicam treatment reduced the stress-induced neutrophilia (P = 0.0072) and circulating monocyte count (P = 0.013) on arrival. Mean corpuscle hemoglobin (P = 0.05), mean corpuscle volume (P = 0.05), and lymphocyte count (P = 0.05) were also greater in the CONT calves compared with MEL calves after transportation. Furthermore, Hp-MMP-9 complexes, TCO2, TNFα, plasma proteins, and SP increased and cortisol decreased after shipping (P < 0.01). Meloxicam treatment tended to reduce serum cortisol concentrations (P = 0.08) and there was evidence of a time × treatment interaction (P = 0.04). An inverse relationship between plasma MEL concentrations and circulation cortisol concentrations (P = 0.002) and neutrophil (P = 0.04) and basophil counts (P = 0.03) was also observed. The results suggest that MEL administration may reduce the impact of long-distance transportation on circulating physiological biomarkers of stress and inflammation in beef calves.

Comparison of steer behavior when housed in a deep-bedded hoop barn versus an open feedlot with shelter.

The use of hoop barns as an alternative housing system for beef cattle has not been widely researched. The objectives of this study were to determine the main effects of behavior of steers 1) over winter and summer, 2) when housed in either a hoop barn or a conventional feedlot, and 3) interactions between season and housing system. A total of 960 crossbred Bos taurus steers were used [August 2006 to April 2008 (2 winter and 2 summer trials)]. Steers were housed in either 1 deep-bedded hoop barn (n = 12 pens; 4.65 m(2)/steer) or 1 open feedlot with shelter (n = 12 pens; 14.7 m(2)/steer). Steers were ear tagged, implanted, and weighed (414 ± 36 kg) on arrival and allotted to treatments that were balanced for source, BW, and hide color. Behavioral data (3 postures and 2 behaviors) were collected using a 10-min live scan. The experimental unit for behavior was a pen of steers. Behavioral data were arcsine transformed to achieve a normal distribution. There were no (P > 0.05) differences for time spent at bunk or waterer for steers between housing treatments. Steers housed in an open feedlot with shelter spent less time lying and more time standing and walking (P < 0.05) compared with steers housed in a hoop barn. There were no (P = 0.32) differences between seasons for standing. Steers spent more time at the bunk (P < 0.0001) and waterer (P < 0.0001) in the summer compared with the winter. In the winter, steers engaged in more lying (P = 0.0002) and walking (P < 0.0001). Overall, steers stood less (P = 0.006) and spent more time lying (P = 0.024) when housed in a hoop barn than in the open feedlot with shelter regardless of season. Steers housed in the open feedlot with shelter walked more (P < 0.0001) than steers housed in the hoop barn and walked more (P < 0.0001) in winter than in summer months (6 vs. 3%). There were no (P > 0.05) differences in time spent at bunk and waterer between housing systems within season, but time spent at the waterer and bunk decreased (P < 0.05) for both housing systems during the winter. In conclusion, housing 40 steers per pen in a cornstalk-bedded hoop barn at 4.65 m(2)/steer does not result in adverse behavioral alterations and can be considered as a housing alternative for finishing steers in the Midwestern United States when compared with steers fed in an open feedlot with shelter provided.

Performance and carcass characteristics of finishing beef cattle managed in a bedded hoop-barn system.

The use of bedded hoop barns in finishing systems for beef cattle has not been widely researched. In this management system, beef cattle are confined to hoop barns throughout finishing, and bedding is used to absorb animal waste, which results in minimal effluent. The objective of this study was to compare the performance and carcass characteristics of finishing beef steers (n = 1,428) managed in a bedded hoop-barn management system vs. an open-feedlot system with shelter. Six feeding trials were conducted over a 3-yr period. Three trials were conducted during summer-fall and 3 trials were conducted during winter-spring. Crossbred steers were allotted to 3 pens in the hoop-barn system and to 3 pens in the open-lot system (approximately 40 steers per pen in both facility systems). Stocking densities for the steers were 4.65 m(2) per steer in the hoop-barn system and 14.7 m(2) per steer in the open-lot system. The steers were begun on trial weighing 410 and 411 kg (SD = 21), were fed for 102.3 and 103.0 d (SD = 3.8), and were weighed off test at 595 and 602 kg (SD = 21) for the hoop-barn and open-lot systems, respectively. Steer performance measures consisted of ADG, DMI, and G:F. Carcass characteristics were HCW, fat thickness, LM area, KPH percentage, marbling score, USDA yield grade, and USDA quality grade. No year, season, or pen (management system) main effects, or season x management system and year x management system interactions were observed for any of the items measured related to cattle performance or carcass characteristics (P > 0.05). Final mud scores (a subjective evaluation of the amount of soil and manure adhering to the hair coat of the animals) were greater for the steers from the open-lot system compared with those from the hoop-barn system (P < 0.02), suggesting steers in the hoop-barn system carried less mud than steers from the open-lot system. Average daily cornstalk bedding use in the hoop-barn system was 2.3 kg/steer during summer-fall and 2.6 kg/steer during winter-spring. The performance of finishing cattle managed in a hoop-barn system was not different from the performance of cattle managed in an open-feedlot system with shelter during summer and winter. Managing beef cattle in hoop barns required more bedding but resulted in decreased mud scores compared with cattle managed in an open-lot system with shelter. Hoop barns are a viable alternative housing management system for finishing beef cattle.

Evaluation of fixed sources of variation and estimation of genetic parameters for incidence of bovine respiratory disease in preweaned calves and feedlot cattle.

The primary objective of this study was to estimate variance components and heritability of bovine respiratory disease (BRD) incidence in beef calves before weaning and during the finishing phase. The second objective was to investigate the impact of BRD incidence and treatment frequency on performance and carcass traits. Bovine respiratory disease is the biggest and most costly health challenge facing the cattle industry. The 2 populations used consisted of 1,519 preweaned calves and 3,277 head of feedlot cattle. The incidence rate of BRD in preweaned calves was 11.39%, and among treated cattle, 82.1% were treated once, 13.9% were treated twice, and 4.0% were treated 3 times or more. The incidence of BRD (P = 0.35) and the number of treatments (P = 0.77) had no significant effect on weaning BW. Heritability estimates of the entire preweaned population for BRD resistance and number of treatments were 0.11 +/- 0.06 and 0.08 +/- 0.05, respectively. The genetic correlation estimates for BRD incidence with weaning BW and birth BW were low (-0.02 +/- 0.32 and 0.07 +/- 0.27, respectively). The same estimate for the number of BRD treatments with weaning BW and birth BW was 0.25 +/- 0.35 and 0.30 +/- 0.27, respectively. The observed BRD incidence rate for feedlot cattle was observed at 9.43%. Incidence of BRD significantly (P < 0.01) decreased overall and acclimation ADG by 0.06 +/- 0.01 kg/d and 0.28 +/- 0.03 kg/d, respectively. Carcass traits were also significantly (P < 0.05) affected by BRD incidence; untreated cattle had a 9.1 +/- 1.7-kg heavier HCW. Results were similar in the analysis of treatment frequency. The heritability estimate of BRD incidence and the number of treatments were 0.07 +/- 0.04 and 0.02 +/- 0.03, respectively. Estimates of genetic correlations of BRD incidence with production traits were -0.63 +/- 0.22 for acclimation ADG, -0.04 +/- 0.23 for on-test ADG, -0.31 +/- 0.21 for overall ADG, -0.39 +/- 0.21 for final BW, -0.22 +/- 0.22 for HCW, -0.03 +/- 0.22 for LM area, 0.24 +/- 0.25 for fat, and -0.43 +/- 0.20 for marbling score. Similar results for the number of treatments and production traits were -1.00 +/- 0.68 for acclimation ADG, -0.04 +/- 0.39 for on-test ADG, -0.47 +/- 0.41 for overall ADG, -0.66 +/- 0.40 for final BW, -0.58 +/- 0.45 for HCW, -0.12 +/- 0.38 for LM area, 0.42 +/- 0.50 for fat, and -0.32 +/- 0.37 for marbling score. Because of the high economic cost associated with BRD incidence, even these modest estimates for heritability of BRD resistance should be considered for incorporation into beef cattle breeding programs.

Relationship of various incoming cattle traits with feedlot performance and carcass traits.

Steers (n = 15,631) and heifers (n = 5,897) fed at 18 feedlots (total confinement, partial confinement, or open lots) in southwest Iowa between 2002 and 2006 as part of the Tri-County Steer Carcass Futurity sponsored by Iowa State University were used to correlate various phenotypic traits with feedlot performance and carcass traits. Dependent variables [ADG, respiratory morbidity, HCW, fat thickness, calculated yield grade, marbling score, presence or absence of lung damage, loin muscle area (LMA), and LMA x 100/HCW] were evaluated on the basis of various phenotypic traits [initial BW, disposition score (1 = calm, 6 = extremely excitable), muscle score, frame score, BCS, number of treatments for respiratory disease, presence of lung lesions, breed makeup, and percentage Angus genetics]. Subjectively evaluated phenotypic traits were evaluated by Iowa State University or USDA personnel. Cattle with greater disposition score (more excitable) had decreased initial BW, final BW, ADG, HCW, yield grade, quality grade, marbling score, and mortality (P < 0.01). Respiratory morbidity was negatively correlated with initial BW, ADG, yield grade, HCW, and marbling score (P < 0.01). As initial BW increased, final BW and HCW increased and respiratory morbidity decreased (P < 0.01). Cattle with greater BCS on arrival had greater initial BW but were lighter at slaughter (P < 0.01). Increased number of treatments for respiratory disease was associated with decreased ADG, greater mortality rate, and greater incidence of lung lesions (P < 0.01). Body weight gain was similar between English- and Continental-breed cattle (P > 0.05), although final BW and HCW were greater and yield grade and yield grade-adjusted marbling score were less for Continental-breed cattle (P < 0.01). Cattle with a poorer muscling score had reduced HCW and LMA and greater yield grade, marbling score, and quality grade (P < 0.01). Animal disposition, health, breed type, and frame score have dramatic effects on live feedlot performance and carcass traits.

An evaluation of bovine respiratory disease complex in feedlot cattle: Impact on performance and carcass traits using treatment records and lung lesion scores.

The objective of this study was to investigate the effects of bovine respiratory disease (BRD) complex on economically important production traits with the use of health records in combination with lung lesion scores obtained at slaughter. Records from 5,976 animals were used in this study from cattle that were managed in Midwestern feedlots. Average daily gain for 3 different feeding periods (acclimation, on-test, and overall test) along with final BW were evaluated as performance measures. Hot carcass weight, LM area, subcutaneous fat cover, and marbling score were collected at slaughter. All calves were monitored by experienced feedlot personnel and treated according to the specific health protocol of each feedlot. Incidence of BRD was observed at a rate of 8.17%, and lung lesions at slaughter were present in 61.9% of cattle from a subpopulation (n = 1,665). From this group of cattle, the overall incidence of BRD, which was defined as cattle that had lung lesions, that were treated for BRD in the feedlot, or both, was 64.4%. Incidence of BRD in the feedlot decreased ADG during both the acclimation period (0.37 +/- 0.03 kg) and the overall test period (0.07 +/- 0.01 kg). Incidence of BRD also had significant effects on HCW and marbling score with reduction of 8.16 +/- 1.38 kg and 0.13 +/- 0.04, respectively, in treated cattle. The adverse effects on production traits tended to increase as the number of treatments increased. Potential decrease in performance and carcass merit observed in this study were associated with a decline of $23.23, $30.15, and $54.01 in carcass value when comparing cattle never treated with cattle treated once, twice, or 3 or more times, respectively. The presence of lung lesions did not have a significant effect on any of the traits; however, there was an association between the presence of active bronchial lymph nodes and less productivity of feedlot cattle.

Complement 1s is the serine protease that cleaves IGFBP-5 in human osteoarthritic joint fluid.

UNLABELLED: Insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) are trophic factors for cartilage and have been shown to be chondroprotective in animal models of osteoarthritis (OA). IGFBP-5 is degraded in joint fluid and inhibition of IGFBP-5 degradation has been shown to enhance the trophic effects of IGF-I. OBJECTIVE: To determine the identity of IGFBP-5 protease activity in human OA joint fluid. METHOD: OA joint fluid was purified and the purified material was analyzed by IGFBP-5 zymography. RESULTS: Both crude joint fluid and purified material contained a single band of proteolytic activity that cleaved IGFBP-5. Immunoblotting of joint fluid for complement 1s (C1s) showed a band that had the same Mr estimate, e.g., 88 kDa. In gel tryptic digestion and subsequent peptide analysis by LC-MS/MS showed that the band contained human C1s. A panel of protease inhibitors was tested for their ability to inhibit IGFBP-5 cleavage by the purified protease. Three serine protease inhibitors, FUT175 and CP-143217 and CB-349547 had IC50's between 1 and 6 microM. Two other serine protease inhibitors had intermediate activity (e.g., IC50's 20-40 microM) and MMP inhibitors had no detectible activity at concentrations up to 300 microM. CONCLUSION: Human OA fluid contains a serine protease that cleaves IGFBP-5. Zymography, immunoblotting and LC-MS/MS analysis indicate that C1s is the protease that accounts for this activity.

Protease-resistant insulin-like growth factor (IGF)-binding protein-4 inhibits IGF-I actions and neointimal expansion in a porcine model of neointimal hyperplasia.

IGF-I has been shown to play a role in the progression of atherosclerosis in experimental animal models. IGF-binding protein-4 (IGFBP-4) binds to IGF-I and prevents its association with receptors. Overexpression of a protease-resistant form of IGFBP-4 has been shown to inhibit the ability of IGF-I to stimulate normal smooth muscle cell growth in mice. Based on these observations, we prepared a protease-resistant form of IGFBP-4 and infused it into hypercholesterolemic pigs. Infusion of the protease-resistant mutant inhibited lesion development by 53.3 +/- 6.1% (n = 6; P < 0.01). Control vessels that received an equimolar concentration of IGF-I and the protease-resistant IGFBP-4 showed no reduction in lesion size compared with control lesions that were infused with vehicle. Infusion of a nonmutated form of IGFBP-4 did not significantly inhibit lesion development. Proliferating cell nuclear antigen analysis showed that the mutant IGFBP-4 appeared to inhibit cell proliferation. The area occupied by extracellular matrix was also reduced proportionally compared with total lesion area. Immunoblotting revealed that the mutant IGFBP-4 remained intact, whereas the wild-type IGFBP-4 that was infused was proteolytically cleaved. Further analysis of the lesions revealed that a marker protein, IGFBP-5, whose synthesis is stimulated by IGF-I, was decreased in the lesions that received the protease-resistant, IGFBP-4 mutant, whereas there was no change in lesions that received wild-type IGFBP-4 or the mutant protein plus IGF-I. These findings clearly illustrate that infusion of protease-resistant IGFBP-4 into the perilesion environment results in inhibition of cell proliferation and attenuation of the development of neointima. The findings support the hypothesis that inhibiting IGFBP-4 proteolysis in the lesion microenvironment could be an effective means for regulating neointimal expansion.

Recombinant, nonglycosylated human insulin-like growth factor-binding protein-3 (IGFBP-3) is degraded preferentially after administration to type II diabetics, resulting in increased endogenous glycosylated IGFBP-3.

CONTEXT: Administration of IGF-binding protein-3 (IGFBP-3) with IGF-I stabilizes IGF-I concentrations and prolongs its half-life. One determinant of IGFBP-3 stability is proteolysis. Normal subjects have minimal IGFBP-3 protease activity; however, with pregnancy, acute catabolic illness, or diabetes, IGFBP-3 protease activity is increased. OBJECTIVE: This study was conducted to determine the degree of proteolysis that occurs in glycosylated, endogenous serum IGFBP-3 and nonglycosylated IGFBP-3 after administration of an IGF-I/IGFBP-3 combination to patients with diabetes. DESIGN: Thirty-two patients received either 1 (n = 8) or 2 (n = 24) mg/kg x d IGF-I/IGFBP-3 by bolus s.c. injection (n = 16) or continuous s.c. infusion (n = 16). RESULTS: When nonglycosylated IGFBP-3 was given, the abundance of both glycosylated and nonglycosylated forms of IGFBP-3 in serum was increased. Incubation of nonglycosylated IGFBP-3 with diabetic serum in vitro resulted in more rapid degradation compared with glycosylated IGFBP-3. When the serum obtained from subjects who had received nonglycosylated IGFBP-3 was analyzed, significant differences in the stability of glycosylated and nonglycosylated IGFBP-3 were present. The addition of increasing concentrations of nonglycosylated IGFBP-3 to diabetic serum resulted in a dose-dependent increase in the abundance of endogenous, glycosylated IGFBP-3. Administration of IGF-I and nonglycosylated IGFBP-3 for 2 wk to 32 subjects increased glycosylated IGF-I/IGFBP-3 by 20-40%. The increases were the greatest in the groups that received IGFBP-3 by infusion (e.g. 31% and 40%). CONCLUSIONS: After administration to diabetics, nonglycosylated IGFBP-3 is degraded more rapidly than glycosylated IGFBP-3. By acting as a preferential substrate for the IGFBP-3 protease, nonglycosylated IGFBP-3 protects endogenous, glycosylated IGFBP-3 from degradation, allowing total IGFBP-3 concentrations to increase.

Use of ultrasound to predict body composition changes in steers at 100 and 65 days before slaughter.

Steers from research crossbreeding projects (n = 406) were serially scanned using real-time ultrasound at 35-d intervals from reimplant time until slaughter. Cattle were evaluated for rump fat depth, longissimus muscle area (ULMA), 12th-rib fat thickness (UFAT), and percentage of intramuscular fat (IMF) to determine the ability of ultrasound to predict carcass composition at extended periods before slaughter. Additional background information on the cattle, such as live weight, ADG, breed of sire, breed of dam, implant, and frame score was also used. Carcass data were collected by trained personnel at "chain speed," and samples of the 12th-rib LM were taken for ether extract analysis. Simple correlation coefficients showed positive relationships (P < 0.01) between ultrasound measures taken less than 7 d before slaughter and carcass measures: ULMA and carcass LM area (CLMA, r = 0.66); UFAT and carcass 12th-rib fat thickness (CFAT, r = 0.74); and IMF and carcass numeric marbling score (r = 0.61). The same correlation coefficients for ultrasound measures taken 96 to 105 d before slaughter and carcass values (P < 0.01) were 0.52, 0.58, and 0.63, respectively. Steers were divided into source-verified and nonsource-verified groups based on the level of background information for each individual. Regression equations were developed for the carcass measurements; 46% of the variation could be explained for CLMA and 44% of CFAT at reimplant time, 46% of the variation in quality grade and 42% of the variation in yield grade could be explained. Significant predictors of quality grade were IMF (P < 0.001), natural log of 12th-rib fat thickness (LUFAT, P < 0.001), and ADG (P < 0.01), whereas LUFAT (P < 0.001), ULMA (P < 0.01), live weight (P < 0.001), hip height (P < 0.001), and frame score (P < 0.001) were significant predictors of yield grade. Regressions using ultrasound data taken 61 to 69 d before slaughter showed increasing R2. Live ultrasound measures at reimplant time are a viable tool for making decisions regarding future carcass composition.

Emulsion-derived foams (PolyHIPEs) containing poly(epsilon-caprolactone) as matrixes for tissue engineering.

The preparation of PolyHIPE foams containing poly(epsilon-caprolactone) from macromonomers by free radical homo- or copolymerization is described. The macromonomers are synthesized from PCL diols and are polymerized in the continuous phase of high internal phase emulsions (HIPEs). Subsequent drying yields low-density foams with cell diameters of 5-100 microm. Foam morphology, as determined by scanning electron microscopy, depends on the type of diluent (styrene, methyl methacrylate, or toluene) added to the emulsion organic phase and on the PCL content. Increasing the latter increases the continuous phase viscosity to a point where emulsion formation is impeded. Foam swelling in toluene, 2-propanol, and water was investigated by solvent imbibition and increased with increasing solvent hydrophobicity. Furthermore, it was found generally to decrease with increasing PCL content, due to increasing cross-link density. Swelling generally increased when higher molar mass PCL macromonomer was used due to the formation of a less tightly cross-linked network. One type of foam sample was shown to support the growth of human fibroblasts over a period of 2.5 days.

Biochemical and functional analysis of a conserved IGF-binding protein isolated from rainbow trout (Oncorhynchus mykiss) hepatoma cells.

Rainbow trout (Oncorhynchus mykiss) serum contains several IGF-binding proteins (IGFBPs) that specifically bind to IGFs. The structures of these fish IGFBPs have not been determined and their physiological functions are poorly defined. In this study, we identified a 30 kDa IGFBP present in rainbow trout serum and secreted by cultured trout hepatoma cells. This IGFBP binds to IGFs but not to insulin. This IGFBP was purified to homogeneity using a three-step procedure involving Phenyl-Sepharose chromatography, IGF-I affinity chromatography and reverse-phase HPLC. Affinity cross-linking studies indicated that this IGFBP binds to IGF-I with a higher affinity than to IGF-II. N-terminal sequence analysis of the trout IGFBP suggests that it shares high sequence identity with that of human IGFBP-1 in the N-terminal region. When added to cultured fish and human cells, the trout IGFBP inhibited IGF-I-stimulated DNA synthesis and cell proliferation in a concentration-dependent manner. The inhibitory effect of the fish IGFBP was comparable to those of human IGFBP-1 and -4. These results indicate that the IGFBP molecule is structurally and functionally conserved in evolutionarily ancient vertebrate species such as bony fish.

Tissue transglutaminase facilitates the polymerization of insulin-like growth factor-binding protein-1 (IGFBP-1) and leads to loss of IGFBP-1's ability to inhibit insulin-like growth factor-I-stimulated protein synthesis.

Insulin-like growth factor-binding protein-1 (IGFBP-1) binds to insulin-like growth factors (IGFs) and has been shown to inhibit or stimulate cellular responses to IGF-I in vitro. This capacity of IGFBP-1 to inhibit or stimulate IGF-I actions correlates with its ability to form stable high molecular weight multimers. Since the ability of some proteins to polymerize is dependent upon transglutamination, we determined if tissue transglutaminase could catalyze this reaction and the effect of polymerization of IGFBP-1 upon IGF-I action. Following incubation with pure tissue transglutaminase (Tg), IGFBP-1 formed covalently linked multimers that were stable during SDS-polyacrylamide gel electrophoresis using reducing conditions. Dephosphorylated IGFBP-1 polymerized more rapidly and to a greater extent compared with native (phosphorylated) IGFBP-1. Exposure to IGF-I stimulated transglutamination of IGFBP-1 in vitro. An IGFBP-1 mutant in which Gln(66)-Gln(67) had been altered to Ala(66)-Ala(67) (Q66A/Q67A) was relatively resistant to polymerization by Tg compared with native IGFBP-1. Tg localized in fibroblast membranes was also shown to catalyze the formation of native IGFBP-1 multimers, however, Q66A/Q67A IGFBP-1 failed to polymerize. Although the mutant IGFBP-1 potently inhibited IGF-I stimulated protein synthesis in pSMC cultures, the same concentration of native IGFBP-1 had no inhibitory effect. The addition of higher concentrations of native IGFBP-1 did inhibit the protein synthesis response, and this degree of inhibition correlated with the amount of monomeric IGFBP-1 that was present. In conclusion, IGFBP-1 is a substrate for tissue transglutaminase and Tg leads to the formation of high molecular weight covalently linked multimers. Polymerization is an important post-translational modification of IGFBP-1 that regulates cellular responses to IGF-I.

The complement component C1s is the protease that accounts for cleavage of insulin-like growth factor-binding protein-5 in fibroblast medium.

Cultured fibroblasts secrete an 88-kDa serine protease that cleaves insulin-like growth factor binding protein-5 (IGFBP-5). Because IGFBP-5 has been shown to regulate IGF-I actions, understanding the chemical identity and regulation of this protease is important for understanding how IGF-I stimulates anabolic functions. The protease was purified from human fibroblast-conditioned medium by hydrophobic interaction, lectin affinity, and heparin Sepharose affinity chromatography followed by SDS-polyacrylamide gel electrophoresis. An 88-kDa band was excised and digested with lysyl-endopeptidase. Sequencing of the high pressure liquid chromatography-purified peptides yielded the complement components C1r and C1s. To confirm that C1r/C1s accounted for the proteolytic activity in the medium, immunoaffinity chromatography was performed. Most of the protease activity adhered to the column, and the eluant was fully active in cleaving IGFBP-5. SDS-polyacrylamide gel electrophoresis with silver staining showed two bands, and IGFBP-5 zymography showed a single 88-kDa band. Amino acid sequencing confirmed that the 88-kDa band contained only C1r and C1s. C1r in the fibroblast medium underwent autoactivation, and the activated form cleaved C1s. C1s purified from the conditioned medium cleaved C(4), a naturally occurring substrate. The purified protease cleaved IGFBP-5 but had no activity against IGFBP-1 through -4. C1 inhibitor, a protein known to inhibit activated C1s, was shown to inhibit the cleavage of IGFBP-5 by the protease in the conditioned medium. In summary, human fibroblasts secrete C1r and C1s that actively cleave IGFBP-5. The findings define a mechanism for cleaving IGFBP-5 in the culture medium, thus allowing release of IGF-I to cell surface receptors.

Substitutions for hydrophobic amino acids in the N-terminal domains of IGFBP-3 and -5 markedly reduce IGF-I binding and alter their biologic actions.

Insulin-like growth factor-binding protein-3 and -5 (IGFBP-3 and -5) have been shown to bind insulin-like growth factor-I and -II (IGF-I and -II) with high affinity. Previous studies have proposed that the N-terminal region of IGFBP-5 contains a hydrophobic patch between residues 49 and 74 that is required for high affinity binding. These studies were undertaken to determine if mutagenesis of several of these residues resulted in a reduction of the affinity of IGFBP-3 and -5 for IGF-I. Substitutions for residues 68, 69, 70, 73, and 74 in IGFBP-5 (changing one charged residue, Lys(68), to a neutral one and the four hydrophobic residues to nonhydrophobic residues) resulted in an approximately 1000-fold reduction in the affinity of IGFBP-5 for IGF-I. Substitutions for homologous residues in IGFBP-3 also resulted in a >1000-fold reduction in affinity. The physiologic consequence of this reduction was that IGFBP-3 and -5 became very weak inhibitors of IGF-I-stimulated cell migration and DNA synthesis. Likewise, the ability of IGFBP-5 to inhibit IGF-I-stimulated receptor phosphorylation was attenuated. These changes did not appear to be because of alterations in protein folding induced by mutagenesis, because the IGFBP-5 mutant was fully susceptible to proteolytic cleavage by a specific IGFBP-5 protease. In summary, residues 68, 69, 70, 73, and 74 in IGFBP-5 appear to be critical for high affinity binding to IGF-I. Homologous residues in IGFBP-3 are also required, suggesting that they form a similar binding pocket and that for both proteins these residues form an important component of the core binding site. The availability of these mutants will make it possible to determine if there are direct, non-IGF-I-dependent effects of IGFBP-3 and -5 on cellular physiologic processes in cell types that secrete IGF-I.

Thrombospondin and osteopontin bind to insulin-like growth factor (IGF)-binding protein-5 leading to an alteration in IGF-I-stimulated cell growth.

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) has been shown to bind to extracellular matrix (ECM) with relatively high affinity, but the ECM components that mediate this interaction have not been identified. These studies show that radiolabeled IGFBP-5 specifically coprecipitates with two ECM proteins, thrombospondin-1 (TSP-1) and osteopontin (OPN). As TSP-1 binds avidly to heparin, as does IGFBP-5, the effect of glycosaminoglycans on the TSP-1/IGFBP-5 interaction was analyzed. Heparan and dermatan sulfate inhibited binding, whereas heparin increased binding. Chondroitin sulfate A and B had no effect. In contrast, both heparin and heparan sulfate significantly inhibited the OPN-IGFBP-5 interaction and chondroitin sulfate A, B, and C had no effect. To determine the region of IGFBP-5 that was involved in each interaction, synthetic peptides that spanned several regions of IGFBP-5 were tested for their capacity to competitively inhibit coprecipitation. A peptide that contained the amino acids between positions 201 and 218 resulted in 76% and 86% inhibition of binding to TSP-1 and OPN, respectively. Three other synthetic peptides that spanned regions ofIGFBP-5 with several charged residues had no effect. IGFBP-5 mutants that contained substitutions for basic residues in the 201-218 region were tested for their ability to bind to TSP-1 or OPN. A mutant with substitutions for amino acids at positions R201 and K202 and a mutant with substitutions for K211, R214, K217, and R218 had the greatest reduction in binding to TSP-1. Mutants containing substitutions for R214 alone and the combined K217A, R218A mutant had the greatest reductions in OPN binding. When the smooth muscle cell growth response to these components was assessed, IGF-I plus IGFBP-5 or the combination of TSP-1 or OPN with IGF-I potentiated the IGF-I effect. The addition of IGFBP-5 to these combinations resulted in further significant growth stimulation. Both OPN and TSP-1 specifically bind to IGFBP-5 with high affinity. These interactions may be important for concentrating intact IGFBP-5 in extracellular matrix and for modulating the cooperative interaction between the IGF-I receptor and integrin receptor signaling pathways.