An encyclopedia of mouse genes.

The laboratory mouse is the premier model system for studies of mammalian development due to the powerful classical genetic analysis possible (see also the Jackson Laboratory web site, http://www.jax.org/) and the ever-expanding collection of molecular tools. To enhance the utility of the mouse system, we initiated a program to generate a large database of expressed sequence tags (ESTs) that can provide rapid access to genes. Of particular significance was the possibility that cDNA libraries could be prepared from very early stages of development, a situation unrealized in human EST projects. We report here the development of a comprehensive database of ESTs for the mouse. The project, initiated in March 1996, has focused on 5' end sequences from directionally cloned, oligo-dT primed cDNA libraries. As of 23 October 1998, 352,040 sequences had been generated, annotated and deposited in dbEST, where they comprised 93% of the total ESTs available for mouse. EST data are versatile and have been applied to gene identification, comparative sequence analysis, comparative gene mapping and candidate disease gene identification, genome sequence annotation, microarray development and the development of gene-based map resources.

Hydrogen storage based on physisorption.

Physisorption of molecular hydrogen based on neutral and negatively charged aromatic molecular systems has been evaluated using ab initio calculations to estimate the binding energy, DeltaH, and DeltaG at 298 ( approximately 77 bar) and 77 K (45 bar) in order to compare calculated results with experimental measurements of hydrogen adsorption. The molecular systems used in this study were corannulene (C(20)H(10)), dicyclopenta[def,jkl]triphenylene (C(20)H(10)), 5,8-dioxo-5,8-dihydroindeno[2,1-c]fluorene (C(20)H(10)O(2)), 6-hexyl-5,8-dioxo-5,8-dihydroindeno[2,1-c]fluorene (C(26)H(22)O(2)), coronene (C(24)H(12)), dilithium phthalocyanine (Li(2)Pc, C(32)H(16)Li(2)N(8)), tetrabutylammonium lithium phthalocyanine (TBA-LiPc, C(48)H(52)LiN(9)), and tetramethylammonium lithium phthalocyanine (TMA-LiPc, C(36)H(28)LiN(9)). It was found (a) that the calculated term that corrects 0 K electronic energies to give Gibbs energies (thermal correction to Gibbs energy, TCGE) serves as a good approximation of the adsorbent binding energy required in order for a physisorption process to be thermodynamically allowed and (b) that the binding energy for neutral aromatic molecules varies as a function of curvature (e.g., corannulene versus coronene) or if electron-withdrawing or -donating groups are part of the adsorbent. A negatively charged aromatic ring, the lithium phthalocyanine complex anion, [LiPc](-), introduces charge-induced dipole interactions into the adsorption process, resulting in a doubling of the binding energy of Li(2)Pc relative to corannulene. Experimental hydrogen adsorption results for Li(2)Pc, which are consistent with MD simulation results using chi-Li(2)Pc to simulate the adsorbent, suggest that only one side of the phthalocyanine ring is used in the adsorption process. The introduction of a tetrabutylammonium cation as a replacement for one lithium ion in Li(2)Pc has the effect of increasing the number of hydrogen molecules adsorbed from 10 (3.80 wt %) for Li(2)Pc to 24 (5.93 wt %) at 77 K and 45 bar, suggesting that both sides of the phthalocyanine ring are available for hydrogen adsorption. MD simulations of layered tetramethylammonium lithium phthalocyanine molecular systems illustrate that doubling the wt % H(2) adsorbed is possible via such a system. Ab initio calculations also suggest that layered or sandwich structures can result in significant reductions in the pressure required for hydrogen adsorption.

Influence of maternal protein restriction in primiparous heifers during mid- and/or late-gestation on meat quality and fatty acid profile of progeny.

The objective of this study was to evaluate the influence of metabolizable protein (MP) restriction in mid- and/or late-gestation on meat quality characteristics of progeny. Heifers were assigned to 2 levels of dietary protein (control [CON], 102% of MP requirements; or restricted [RES], 80% of MP requirements) at 2 stages of gestation (mid-gestation [MID] and late-gestation [LATE]) in a Balaam's Design crossover treatment structure resulting in 4 treatment combinations (CON-CON, CON-RES, RES-CON, RES-RES). A carryover effect of MID MP treatment on LATE CON indicated CON-CON steaks were more tender (P < .001) than RES CON. Mid-gestation restriction resulted in progeny with increased (P < .05) carcass water, soft tissue moisture, and decreased soft tissue fat percentage compared with progeny from dams receiving MID CON. Reduced maternal MP also differentially influenced the fatty acid profiles of progeny. Results suggest it is possible for progeny to overcome a moderate gestational MP restriction with minimal impacts on carcass composition or meat characteristics.

Weyl-like points from band inversions of spin-polarised surface states in NbGeSb.

Band inversions are key to stabilising a variety of novel electronic states in solids, from topological surface states to the formation of symmetry-protected three-dimensional Dirac and Weyl points and nodal-line semimetals. Here, we create a band inversion not of bulk states, but rather between manifolds of surface states. We realise this by aliovalent substitution of Nb for Zr and Sb for S in the ZrSiS family of nonsymmorphic semimetals. Using angle-resolved photoemission and density-functional theory, we show how two pairs of surface states, known from ZrSiS, are driven to intersect each other near the Fermi level in NbGeSb, and to develop pronounced spin splittings. We demonstrate how mirror symmetry leads to protected crossing points in the resulting spin-orbital entangled surface band structure, thereby stabilising surface state analogues of three-dimensional Weyl points. More generally, our observations suggest new opportunities for engineering topologically and symmetry-protected states via band inversions of surface states.

AMP-activated protein kinase is negatively associated with intramuscular fat content in longissimus dorsi muscle of beef cattle.

Marbling, or intramuscular fat, is an important factor in meat quality. As a key regulator of lipid metabolism, AMP activated protein kinase (AMPK) may be associated with intramuscular fat accumulation. Our objective was to evaluate the relationship among AMPK and its associated signaling mediators, with marbling and lean growth in beef cattle. Steers with high intramuscular fat content (High IMF, 5.71±0.36%, n=5) and low intramuscular fat content (Low IMF, 2.09±0.19%, n=5) were selected. High IMF was associated with increased tenderness (P<0.05) and backfat thickness (P<0.01). Muscle weights were higher in Low compared to High IMF (P<0.05). High IMF steers had a reduced AMPK activity (P<0.01), reduced acetyl-CoA carboxylase phosphorylation (P<0.05), and reduced total mTOR (P=0.02) content. Data provide evidence that AMPK is involved in IMF deposition in beef cattle.

Effect of nursing-calf implant timing on growth performance and carcass characteristics.

The objective of this study was to compare pre- and postweaning growth performance, carcass characteristics, and meat quality attributes of calves that did not receive an implant or were implanted early or late in the nursing period. Crossbred steer calves ( = 135) were stratified by birth date and birth weight and randomly assigned to the following implant treatments: control (CON; no preweaning implant), 58 d (EARLY; 36 mg zeranol, administered at an average of 58 ± 13 d of age), and 121 d (LATE; 36 mg zeranol, administered at an average 121 ± 13 d of age). After weaning, steers were blocked by initial feed yard BW to 15 pens (5 pens/treatment and 9 steers/pen). All steers were implanted on d 21 after arrival at the feed yard and again on d 108 of finishing. Steer BW and ultrasound assessment of rib eye area (uREA), rib fat thickness (uRFT), and percent intramuscular fat (uIMF) were collected when implants were administered, at weaning, and on harvest day. Carcass measurements included HCW, rib eye area (REA), 12th-rib fat thickness (FT), and marbling score. Objective color (L*, a*, and b*) was recorded, and a 3.8-cm strip loin section was removed from both sides of each carcass and portioned into 2.54-cm steaks that were aged for 3 or 14 d for analysis of cook loss and Warner-Bratzler shear force (WBSF). The remaining portion of each sample was used for analysis of moisture and crude fat. Steer BW, ADG, and G:F did not differ among treatments ( > 0.05). Steers implanted in the EARLY treatment had a greater ( < 0.05) cumulative DMI than CON but were not different from steers implanted in the LATE treatment. Ultrasound REA and uRFT (averaged across all collection days) did not differ ( > 0.05); however, steers on the CON treatment had a greater ( ≤ 0.05) percent uIMF than EARLY implanted steers, whereas steers receiving the LATE implant were intermediate and not different from the other treatments. Hot carcass weight, REA, FT, USDA yield grade, marbling score, and objective color did not differ ( > 0.05) among treatments. The proportion of steers in each USDA yield and quality grade was similar ( > 0.05) among treatments, and no differences were detected for total carcass value or price per 45.4 kg (hundredweight; > 0.05). Treatment did not influence ( > 0.05) percent cook loss, crude fat, moisture, or WBSF. In conclusion, administering a nursing implant, regardless of timing, did not influence live performance, carcass characteristics, or meat quality of steers fed in this study.

Pre-slaughter transport, AMP-activated protein kinase, glycolysis, and quality of pork loin.

Numerous studies have revealed that pre-slaughter stress, like transport, increases the occurrence of pale, soft, and exudative (PSE) pork meat. The molecular mechanism underlying this phenomenon, however, is poorly defined. In this study, we investigated the effects of pre-slaughter transport and subsequent rest on energy metabolism, AMP-activated protein kinase (AMPK) activation, and glycolysis in postmortem pork loin. Results indicated that pre-slaughter transport accelerated ATP depletion, which led to lower energy status in postmortem muscle immediately post-exsanguination when compared with control. The lower energy status led to AMPK activation within 1h postmortem, subsequently increasing glycolysis, leading to rapid glycolysis and high incidence of PSE meat. Allowing pigs to rest after transport restored energy status in muscle ante-mortem. Higher energy status then prevented premature and rapid AMPK activation in postmortem muscle and lessened the negative effects of pre-slaughter transport on meat quality. AMPK regulated glycolysis in postmortem muscle, at least partially, through phosphorylation and activation of phosphofructose kinase-2, since fructose-2,6-diphosphate content, an allosteric activator of phosphofructose kinase-1, was well correlated with AMPK activation and glycolytic rate. This suggests that AMPK is a potential molecular target for the control of PSE incidence in pork.

The influence of maternal nutrition on expression of genes responsible for adipogenesis and myogenesis in the bovine fetus.

The objective of this study was to determine whether altered maternal energy supply during mid-gestation results in differences in muscle histology or genes regulating fetal adipose and muscle development. In total, 22 Angus cross-bred heifers (BW=527.73±8.3 kg) were assigned randomly to the three dietary treatments providing 146% (HIGH; n=7), 87% (INT; n=7) or 72% (LOW; n=8) of the energy requirements for heifers from day 85 to day 180 of gestation. Fetuses were removed via cesarean section at day 180 of gestation and longissimus muscle (LM) and subcutaneous fat were collected and prepared for analysis of gene expression. Samples from the LM and semitendinosus (ST) were evaluated for muscle fiber diameter, area and number. The right hind limb was dissected and analyzed to determine compositional analysis. Fetal growth and muscle histology characteristics of the LM and ST were similar among treatments. Preadipocyte factor-1 expression was up-regulated in fetal LM (P<0.05) of HIGH fetuses as compared with INT, whereas LOW fetuses showed increased CCAAT/enhancer-binding protein-ß (C/EBP-ß) expression in LM as compared with INT (P<0.05). Peroxisome proliferator-activated receptor γand C/EBP-α did not differ as a result of dietary treatment in LM or subcutaneous fat samples. There was a tendency for increased expression of fatty acid synthase in LM of LOW fetuses as compared with INT (P<0.10). Myogenin was more highly expressed (P<0.05) in LM of the LOW fetuses, whereas µ-calpain expression was increased in the HIGH treatment compared with INT. A tendency for increased expression of IGF-II was observed for both LOW and HIGH fetuses compared with INT (P<0.10). Expression of stearoyl-CoA desaturase, myoblast determination protein 1, myogenic factor 5, myogenic regulatory factor-4, m-calpain, calpastatin, IGF-I and myostatin was similar between treatments. Collectively, these results suggest that fetal growth characteristics are not affected by the level of maternal nutritional manipulation imposed in this study during mid-gestation. However, differences in expression of fetal genes regulating adipose and muscle tissue growth and development could lead to differences in postnatal composition and warrants further investigation.

The influence of maternal energy status during mid-gestation on beef offspring tenderness, muscle characteristics, and gene expression.

The objective of this study was to determine if maternal energy status during mid-gestation influences the expression of genes regulating muscle and fat development, and muscle characteristics that may impact meat tenderness. Cows grazed dormant, native range (Positive Energy Status [PES]) or were fed at 80% of maintenance energy requirements (Negative Energy Status [NES]) during mid-gestation. Steer offspring were harvested after 21 d in the feedlot (weaning subsample) or after 208 d in the feedlot (final subsample). Greater 21-d tenderness was observed in NES steers, resulting from reduced collagen content in longissimus lumborum steaks. In the semitendinosus, NES steers had greater soluble collagen, and down-regulated expression of MHC-IIA and TIMP-3 at weaning, while MHC-IIA expression was up-regulated in NES steers in the final harvest. Data show mid-gestational maternal energy status may impact offspring tenderness and collagen, but differences were not detected in expression of genes important in myogenesis and adipogenesis in muscle samples obtained from steers at weaning or slaughter.

The influence of maternal energy status during midgestation on beef offspring carcass characteristics and meat quality.

Research has suggested that maternal undernutrition may cause the development of a thrifty phenotype in the offspring, potentially resulting in greater adiposity and reduced muscle mass. These alterations in adipose and muscle development could have lasting impacts on offspring growth, carcass characteristics, and meat quality. However, limited research exists evaluating the impact of maternal energy status on these economically important traits of the offspring. Therefore, the objective of this study was to determine the influence of maternal energy status during midgestation on offspring carcass characteristics and meat quality. To alter maternal energy status, cows either grazed dormant, winter range (positive energy status [PES]) or were fed in a drylot at 80% of the energy requirements for BW maintenance (negative energy status [NES]) during a mean period of 102 ± 10.9 to 193 ± 10.9 d of gestation. Changes in BCS, BW, LM area (LMA), and 12th rib backfat were measured throughout midgestation. At the end of midgestation, cows in the NES group had a reduction (P ≤ 0.05) in BCS, BW, LMA, and 12th rib backfat when compared with PES dams. Cows and calves were managed similarly after midgestation through weaning and calves were managed and fed a common diet through the receiving, backgrounding, and finishing phases in the feedlot. Calves were harvested after 208 d in the feedlot, carcass characteristics were recorded, and strip loins were recovered for analysis of objective color and Warner-Bratzler shear force (WBSF). Maternal energy status had no influence on offspring HCW, dressing percent, LMA, percent KPH, marbling score, percent intramuscular fat, objective color, or WBSF (P > 0.10). Progeny of NES cows tended to have improvements in 12th rib backfat and USDA yield grade (P < 0.10). Greater ratio of marbling score to 12th rib fat thickness and ratio of percent intramuscular fat to 12th rib fat thickness (P < 0.05) were discovered in progeny from cows experiencing a NES during midgestation. These results suggest that maternal energy status during midgestation may impact fat deposition in intramuscular and subcutaneous fat depots without impacting muscle mass.

Association of N-acetyl-beta-glucosaminidase levels with seriousness of suicide attempts.

We hypothesized that levels of N-acetyl-beta-glucosaminidase (NAG) would differ between patients making serious versus nonserious suicide attempts. The levels of NAG from 7 patients hospitalized for serious suicide attempts were compared to 3 patients whose suicide attempts were not rated serious. There was a significant difference in the mean NAG levels of the serious suicide attempters and the nonserious attempters, 25 versus 61; p = .036. Implications of this finding are discussed.

Risk factors in Alzheimer's disease: a clinicopathologic study.

We investigated potential risk factors for Alzheimer's disease (AD) in a clinicopathologic study of 407 patients with definite AD, 100 non-Alzheimer dementia patients, and 50 normal subjects. The AD patients had more first-degree relatives with dementia than the non-AD dementia group (odds ratio of 1.85, 95% confidence interval of 1.07-3.20) or the normal elderly (odds ratio of 3.60, 95% confidence interval of 1.50-8.64) but did not have significantly more head injuries, medical and psychiatric illnesses, or relatives with Down's syndrome. The AD patients with a family history of dementia had their dementia at a later age than those without an affected relative. These findings indicate a familial risk for AD that is greater than for other dementing illnesses and has age-related penetrance. This study does not support other putative risk factors for AD such as head trauma and familial Down's syndrome.

Development of scoring criteria for the clock drawing task in Alzheimer's disease.

OBJECTIVE: To investigate the reliability and validity of free-hand clock drawings, a frequently used measure of constructional apraxia, in patients with Alzheimer's disease. DESIGN: Survey for the purpose of testing reliability and validity of a new scale. SETTING: Memory Disorder Clinic at a university-affiliated hospital in the Upper Midwest. PATIENTS: Forty-six patients were diagnosed with clinically probable dementia of the Alzheimer type after a dementia evaluation, and 26 normal elderly controls were research volunteers without a history of cognitive dysfunction. MEASUREMENTS: Neuropsychological tests, dementia-related scales, and clock drawings rated by a new 20-item Clock Drawing Interpretation Scale. Reliability measures, correlations, and clustering of items in the CDIS. RESULTS: The CDIS had inter-rater reliability (r = .94), internal consistence (rtt = .95), and reproducibility over a 6-month interval. CDIS scores were significantly correlated with two dementia-related scales and all neuropsychological tests and had the highest correlations with other measures of constructional apraxia. All but four Alzheimer patients (91%) and none of the controls had CDIS scores of 18 or less. CONCLUSION: Clinicians may reliably screen patients with Alzheimer's disease with the clock-drawing task, a measure sensitive to deficits in constructional apraxia.

The effects of fludrocortisone acetate and deoxycorticosterone acetate on salt appetite in mice.

Three genotypes of mice were shown to increase their intake of 3% sodium chloride (NaCl) following administration of fludrocortisone acetate (25 mg/kg), a synthetic steroid having strong mineralocorticoid/glucocorticoid activity. However, treatment with deoxycorticosterone acetate (DOCA) (10 mg/kg), a corticosteroid with predominantly mineralocorticoid activity, did not increase 3% NaCl consumption by the stock tested. In contrast, both fludrocortisone and DOCA have been shown to be effective activators of sodium appetite in rats. The essential importance of glucocorticoids in potentiating mineralocorticoid activation of sodium hunger in mice is suggested.

Genetic influences on locomotor activating effects of ethanol and sodium pentobarbital.

The paradoxical capability of sedative-hypnotics to produce behavioral disinhibition varies among genotypes. In DBA/2 mice ethanol (ETOH) produced strong locomotor stimulation with the peak of the biphasic curve at 1.5 g/kg IP. C57BL/6 mice showed no activation, and F1S were intermediate. These characterizations held for a variety of behavioral indices derived from 15 min tests, such as distance, speed, and rest time, at doses in the 0-2.0 g/kg range. Analogous studies with sodium pentobarbital (0-40 mg/kg) yielded a similar pattern of strain differences in locomotor stimulation. In contrast, loss of righting reflex durations (60 mg/kg PENTO, IP) were similar in the two strains, indicating dissociation of activating and sedative effects. In complementary studies, long- and short-sleep mice, which were bred for differences in soporific effects of ETOH, showed similar activation profiles at ETOH doses up to 1.5 g/kg and PENTO doses up to 30 mg/kg. These studies provide support for an hypothesis of common genetic control of the activation effect for ETOH and PENTO.

Bully/victim problems among middle school children.

Bully/victim problems among six classes of 8-9 year-old and six classes of 11-12 year-old children, attending three middle schools, were investigated by means of Olweus's self-report Bullying Inventory. About 21 per cent of the children reported being bullied, and about 17 per cent reported bullying others, "sometimes" or more often. Reports of both bullying and being bullied were more prevalent among boys than among girls, and among the younger group than among the older group. The two most common forms of bullying were reported to be teasing and hitting/kicking. Fewer of the younger group than the older group reported being bullied by same-age pupils, and more of the former reported being bullied by older pupils. Most boys were bullied by other boys only, whereas girls were more likely to be bullied by children of either sex. Besides being bullied in school, children also reported that this happened on the journey to/from school and in other places such as in the street near where they live. Many children expressed negative attitudes towards bullying, although nearly a third said that they could understand why it happens. The majority of the children who reported being bullied/bullying others had not been spoken to about this by teachers or by someone at home. Victims of bullying, but not bullies, were found to be most likely to report feeling unhappy and lonely at school, and to report having fewer good friends. In Study 2, children identified as bullies, victims and not involved in this type of problem were interviewed to find out why certain children and/or themselves bully/get bullied by others, and the feelings of the children involved. The pattern of responses by the three groups differed in some important ways.

The influence of growth stage on carcass composition and factors associated with marbling development in beef cattle.

Subcutaneous fat and marbling both increase in beef cattle during the feeding phase but are antagonistic in regard to their contribution to beef carcass value. The objective of this study was to determine whether cellular factors associated with marbling development change with growth stage throughout the feeding period and whether they are correlated to marbling relative to carcass composition. Twenty-four steers of known origin with the cytosine and thymine (CT) leptin genotype were allotted to 3 harvest groups. Six steers per harvest group were harvested at the following predetermined points: 35 d on feed (early feeding period, EF), average live weight of 464 kg (middle feeding period, MF), and 1.17-cm 12th-rib subcutaneous fat thickness (late feeding period, LF). Longissmus muscle samples were collected within 30 min postmortem and snap frozen for real-time PCR and Western blot analysis of lipoprotein lipase, adenosine monophosphate-activated protein kinase α (AMPKα), stearoyl-coenzyme A desaturase (SCD), PPARγ, C/EBP-ß, and myostatin. Carcass data were recorded, and LM samples were collected and aged 2, 7, 14, and 21 d postmortem for Warner-Bratzler shear force determination. Carcass composition was estimated by dissection of the 9-10-11 rib section and subsequent proximate analysis of the soft tissue. Intramuscular fat content of the LM increased linearly throughout the feeding period, giving additional support to marbling as an early developing tissue. Expression of AMPKα was found to be downregulated, whereas SCD expression was upregulated in the LF group relative to the first 2 harvest groups. Additionally, SCD and PPARγ were downregulated in the EF group relative to the latter 2 harvest groups. These changes in gene expression resulted in a linear increase in only PPARγ protein abundance, whereas myostatin tended to increase quadratically. A correlation was found between intramuscular fat and PPARγ abundance. This gives further evidence of the importance of adipocyte hyperplasia in increasing marbling. Targeting and increasing PPARγ expression may serve as a mechanism to increase marbling deposition. Last, LF steaks were more tender than MF or EF steaks, indicating improved tenderness with increased days on feed.

Calpain-1 activity in bovine muscle is primarily influenced by temperature, not pH decline.

The objectives of this study were to 1) determine the conditions (temperature and pH) that exist in early postmortem muscle of normally chilled and delay chilled beef carcasses to provide a model for in vitro work and 2) determine the mechanism by which early postmortem temperature/pH conditions found in beef muscle influence the enzymes that regulate the aging process in vitro. For objective 1, 7 finished beef animals (HCW 385 ± 8 kg) were harvested with the right sides subjected to normal chilling (2.3°C) approximately 1.25 h postmortem and the left sides subjected to ambient temperature (delay chilling; 22.6°C) for an additional 4.75 h postmortem and then allowed to chill at 2.3°C. Delay chilled carcasses had a more rapid pH decline (P < 0.05) and a slower rate of carcass cooling (P < 0.05). No differences were observed between normally chilled and delay chilled samples for sarcomere length or postmortem proteolysis of troponin T (TnT; P > 0.10). Warner-Bratzler shear force (WBSF) was reduced in steaks from normally chilled carcasses at 14 d (P < 0.05), while results indicated a strong, positive correlation between 14-d WBSF and 3-h longissimus dorsi muscle (LM) temperature (r = 0.67, P < 0.01) as well as a strong, negative correlation between 14-d WBSF and 6-h LM pH (r = -0.65, P < 0.02). These results were used to design the methodology for objective 2, where isolated myofibrils were subjected to µ-calpain digestion at 4 or 22°C with either a fast or slow initial pH decline. As expected, digestions with a fast initial pH decline had lower pH values in the early time points of the incubation (P < 0.05). No differences were detected in µ-calpain activity or in the degradation of intact TnT between the fast and slow pH decline treatments (P > 0.10); however, warmer digestions resulted in a tendency for increased activation of µ-calpain (P < 0.10) and a significant reduction in intact TnT (P < 0.05). Additionally, a temperature × time interaction was revealed in µ-calpain activity and in the degradation of intact TnT (P < 0.05). Specifically, assayed calpain activity was lower at 0.17, 0.33, 1, and 3 h and greater at 72 h in warmer digestions, while intact TnT disappearance was greater as both time and digestion temperature increased. Meat aging and µ-calpain activity are influenced by both temperature and pH, but more research is necessary to fully realize their relationships.

Caspase-3 does not enhance in vitro bovine myofibril degradation by µ-calpain.

Tenderness is a key component of palatability, which influences consumers' perception of meat quality. There are a variety of factors that contribute to the tenderness of beef carcasses, including postmortem proteolysis. A more complete understanding of this biological mechanism regulating tenderness is needed to ensure consistently tender beef. Numerous reports indicate µ-calpain is primarily responsible for the degradation of proteins postmortem. Meanwhile, it has been shown that caspase-3 can cleave calpastatin, the inhibitor of µ-calpain. Therefore, the objective of this study was to determine if in vitro degradation of calpastatin by caspase-3 can enhance the postmortem breakdown of myofibrillar proteins by µ-calpain. Bovine semitendinosus muscles were excised from two carcasses 20 min postmortem. Muscle strips were dissected from the semitendinosus, restrained to maintain length, and placed in a neutral buffer containing protease inhibitors. Upon rigor completion, myofibrils were isolated from each strip, and sarcomere length was determined. Samples with similar sarcomere lengths were selected to minimize the effect of sarcomere length on proteolysis. Myofibrils were then incubated at 22°C with either µ-calpain, µ-calpain+calpastatin, µ-calpain+caspase-3+calpastatin, or caspase-3+calpastatin for 0.25, 1, 3, 24, 48, or 72 h at a pH of 6.8. Proteolysis of troponin T (TnT) and calpastatin was evaluated using SDS-PAGE and Western blotting techniques. Analysis of Western blots confirmed significant degradation of calpastatin by caspase-3 (P<0.05). Additionally, Western blots revealed intact calpastatin disappeared rapidly as a result of digestion by µ-calpain. Although caspase-3 did not significantly degrade TnT (P>0.05), all µ-calpain digestion treatments resulted in substantial TnT breakdown (P<0.05). Degradation of TnT did not differ between the µ-calpain+calpastatin and µ-calpain+caspase-3+calpastatin digestions (P>0.05). Results of this study indicate caspase-3 cleavage of calpastatin does not enhance in vitro degradation of TnT by µ-calpain.

Effects of early- to mid-gestational undernutrition with or without protein supplementation on offspring growth, carcass characteristics, and adipocyte size in beef cattle.

Angus × Gelbvieh cows with 2 to 3 previous pregnancies were used to evaluate effects of maternal nutrient restriction on offspring adipose tissue morphology at standard production endpoints. At 45 d after AI to a single sire, pregnancy was confirmed and cows randomly allotted into groups and fed a control (Con, 100% of NRC recommendations), nutrient-restricted (NR, 70% of Con diet), or nutrient-restricted + protein-supplemented (NRP, 70% of Con + essential AA supply to the small intestine equal to Con) diet. At d 185 of gestation, cows were commingled and received the Con diet thereafter. Bull calves were castrated at 2 mo of age. Calves were weaned at 210 d, backgrounded for 28 d, and then placed in the feedlot for 195 d. Steers and heifers were slaughtered at an average 12th-rib fat thickness of 7.6 mm. Adipose tissue from selected depots was collected for adipocyte size analysis. There was no significant difference in BW or BCS between Con, NRP, and NR cows at d 45 of gestation, which averaged 489.7 ± 17.7 kg and 5.35 ± 0.13, respectively. At d 185 of gestation, Con and NRP groups had similar BW (566.1 ± 14.8 and 550.2 ± 14.8 kg) and BCS (6.34 ± 0.27 and 5.59 ± 0.27), but NR cows exhibited reduced (P < 0.05) BW (517.9 ± 14.8 kg) and BCS (4.81 ± 0.27). Among offspring (steers and heifers) at slaughter, there were no significant differences in BW or organ weights among treatment groups. Yield grade was reduced (P < 0.05) and semitendinosus weight/HCW tended (P = 0.09) to be reduced in NR offspring compared with Con and NRP offspring. Average adipocyte diameter was increased (P < 0.05) in subcutaneous, mesenteric, and omental adipose tissue and tended (P = 0.09) to increase in perirenal adipose tissue in NR compared with Con offspring with NRP offspring adipocyte diameter being either intermediate or similar to Con calves. The adipocyte size alterations observed in NR offspring were confirmed by DNA concentration of the adipose tissue depots. There also was an increased mRNA expression (P < 0.05) of fatty acid transporter 1 in subcutaneous adipose tissue from NR offspring compared with Con and NRP offspring. Nutritional restriction during early and mid gestation increased or tended to increase (P < 0.09) adipocyte diameter in all adipose tissue depots in finished steer and heifer calves.