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Biomedical subjects

G C Smith

Publications and source records attributed to G C Smith.

At least 109 records · Page 6Linked to original sources

Competitive and noncompetitive inhibition of the DNA-dependent protein kinase.

The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase that is involved in mammalian DNA double-strand break repair. The catalytic subunit of DNA-PK (DNA-PKcs) shares sequence homology in its kinase domain with phosphatidylinositol (PI) 3-kinase. Here, we provide a detailed kinetic analysis of DNA-PK inhibition by the PI 3-kinase inhibitor wortmannin and demonstrate this inhibition to be of a noncompetitive nature, with a Ki of 120 nM. Another inhibitor of PI 3-kinase. LY294002, its parent compound, quercetin, and other derivatives have also been studied. These chemicals are competitive inhibitors of DNA-PK, with LY294002 having a Ki of 6.0 microM. Using an antibody to wortmannin, we found that this compound binds covalently to the kinase domain of DNA-PKcs both in vitro and in vivo. Binding of wortmannin to the active site of DNA-PKcs is inhibited by ATP but not by a peptide substrate. Furthermore, wortmannin is able to bind to DNA-PKcs independently of Ku, and it is not stimulated by the presence of DNA. This suggests that the ATP binding site of DNA-PKcs is open constitutively and that DNA activation of the kinase is mediated via another mechanism.

Androstadienes↗

DNA-PK activation by ionizing radiation-induced DNA single-strand breaks.

PURPOSE: To assess the ability of 60Co gamma-radiation-induced plasmid DNA single-strand breaks (gamma-ssb) to activate the DNA-dependent protein kinase (DNA-PK) in vitro. MATERIALS AND METHODS: Plasmid DNA was gamma-irradiated under aerobic conditions to yield 0-6 gamma-ssb and <0.1 double-strand breaks (dsb) per plasmid molecule. The irradiated DNA was used to stimulate DNA-PK in crude HF19 fibroblast nuclear extracts and/or purified HeLa cell DNA-PK protein, and the activation compared with that obtained with a single enzymatically generated plasmid DNA ssb (GpII endonuclease) or dsb (EcoRI endonuclease). RESULTS: Gamma-Irradiated plasmid DNA activates DNA-PK in both crude and purified preparations and the kinase activity increases linearly with dose. As significant DNA-PK activation was detectable using irradiated plasmids which contain <0.1 dsb/molecule, it was concluded that this activation is due to gamma-ssb. However, using purified DNA-PK, this activation is relatively weak as approximately 3 approximately-ssb is equivalent to one GpII-generated DNA ssb or one end of an EcoRI-generated dsb in DNA-PK assays. CONCLUSIONS: As gamma-ssb are in a approximately 20-fold excess of approximately-dsb in vivo for low LET radiation, gamma-ssb may contribute significantly to DNA-PK signalling of gamma-radiation-induced DNA damage in vivo.

Cells, Cultured↗

Cleavage and inactivation of ATM during apoptosis.

The activation of the cysteine proteases with aspartate specificity, termed caspases, is of fundamental importance for the execution of programmed cell death. These proteases are highly specific in their action and activate or inhibit a variety of key protein molecules in the cell. Here, we study the effect of apoptosis on the integrity of two proteins that have critical roles in DNA damage signalling, cell cycle checkpoint controls, and genome maintenance-the product of the gene defective in ataxia telangiectasia, ATM, and the related protein ATR. We find that ATM but not ATR is specifically cleaved in cells induced to undergo apoptosis by a variety of stimuli. We establish that ATM cleavage in vivo is dependent on caspases, reveal that ATM is an efficient substrate for caspase 3 but not caspase 6 in vitro, and show that the in vitro caspase 3 cleavage pattern mirrors that in cells undergoing apoptosis. Strikingly, apoptotic cleavage of ATM in vivo abrogates its protein kinase activity against p53 but has no apparent effect on the DNA binding properties of ATM. These data suggest that the cleavage of ATM during apoptosis generates a kinase-inactive protein that acts, through its DNA binding ability, in a trans-dominant-negative fashion to prevent DNA repair and DNA damage signalling.

Amino Acid Sequence↗

Dual-component video image analysis system (VIASCAN) as a predictor of beef carcass red meat yield percentage and for augmenting application of USDA yield grades.

An improved ability to quantify differences in the fabrication yields of beef carcasses would facilitate the application of value-based marketing. This study was conducted to evaluate the ability of the Dual-Component Australian VIASCAN to 1) predict fabricated beef subprimal yields as a percentage of carcass weight at each of three fat-trim levels and 2) augment USDA yield grading, thereby improving accuracy of grade placement. Steer and heifer carcasses (n = 240) were evaluated using VIASCAN, as well as by USDA expert and online graders, before fabrication of carcasses to each of three fat-trim levels. Expert yield grade (YG), online YG, VIASCAN estimates, and VIASCAN estimated ribeye area used to augment actual and expert grader estimates of the remaining YG factors (adjusted fat thickness, percentage of kidney-pelvic-heart fat, and hot carcass weight), respectively, 1) accounted for 51, 37, 46, and 55% of the variation in fabricated yields of commodity-trimmed subprimals, 2) accounted for 74, 54, 66, and 75% of the variation in fabricated yields of closely trimmed subprimals, and 3) accounted for 74, 54, 71, and 75% of the variation in fabricated yields of very closely trimmed subprimals. The VIASCAN system predicted fabrication yields more accurately than current online yield grading and, when certain VIASCAN-measured traits were combined with some USDA yield grade factors in an augmentation system, the accuracy of cutability prediction was improved, at packing plant line speeds, to a level matching that of expert graders applying grades at a comfortable rate.

Animals↗

An audit of retail beef loin steak tenderness conducted in eight U.S. cities.

An audit of supermarkets in eight U.S. cities was conducted to characterize retail beef loin steaks with respect to grade, postfabrication aging, and tenderness and to provide an interim measure of progress in industry efforts to improve retail beef tenderness. Top sirloin steaks (n = 819) and strip loin steaks (n = 827 paired steaks) were purchased at retail markets in Atlanta, Chicago, Denver, Houston, Philadelphia, Phoenix, San Francisco, and Seattle and shipped to Colorado State University for measurement of shear force (both cut types) and evaluation by a trained sensory panel (strip loins only). Approximately 80% of the steaks originated from beef plants in Kansas, Texas, Nebraska, and Colorado. Postfabrication aging periods ranged from 2 to 87 d (mean = 20.8 d) for top sirloin steaks and from 2 to 91 d (mean = 19.2 d) for strip loin steaks, although most top sirloin (85%) and strip loin (91%) steaks were available for purchase 7 to 35 d after fabrication. The mix of quality grades was similar for both cuts: 60% Select, 31% "commodity" Choice, 6 to 7% "Certified" Choice, and 2 to 3% Prime. Shear force averaged 3.46 kg (SD = .74) for top sirloins and 3.05 kg (SD = .95) for strip loins; 75% of both steak types had shear force values between 2 and 4 kg. Postfabrication periods shorter than 7 d were associated with reduced (P < .05) tenderness, especially for top sirloin steaks. Higher quality grades were associated with greater (P < .05) tenderness (Prime > Choice > Select) for both cuts. Based on panel tenderness ratings, the approximate odds of obtaining a "slightly tough" or tougher strip loin steak at a retail supermarket were: 0 for Prime, 1 in 10 for "Certified" Choice, 1 in 5 for "commodity" Choice, and 1 in 4 for Select. Audit results suggest that tenderness characteristics of loin steaks have not changed materially since the National Beef Tenderness Survey was conducted in 1991 and that two primary focal points of the beef industry's efforts to improve tenderness should be 1) to prevent short-aged (< 7 d postfabrication) product from reaching consumers and 2) to identify methods for enhancing tenderness of Select and "commodity" Choice beef.

Animals↗

Identification of quality management practices to reduce the incidence of retail beef tenderness problems: development and evaluation of a prototype quality system to produce tender beef.

A prototype quality system for ensuring beef tenderness was designed and tested. The test population of cattle was genetically diverse, but it was constrained to include youthful (14- to 17-mo-old) steers with no more than 3/8 Bos indicus inheritance. Feeding and preharvest management of the cattle were consistent with procedures recommended for production of grain-finished beef of an acceptable quality level. In addition, the target endpoint for harvest (11-mm external fat thickness over the longissimus at the 12th rib) resulted in production of mostly Select and low Choice beef carcasses; 92% of the resulting carcasses qualified for these two grade levels. Application of the prototype quality system reduced the expected rate of nonconformance to desired tenderness specifications from about one in four loin steaks (23% for top sirloins and 26% for strip loins) to approximately one in eight loin steaks (13% for top sirloins and 12% for strip loins). Tenderness comparisons among sires suggested that the rate of nonconformance for strip loin steaks might be reduced even further by control of genetic inputs into the system. Use of process control in a quality management system was demonstrated to be an effective approach for assurance of beef tenderness.

Animal Feed↗

Case life of seven retail products from beef cattle supplemented with alpha-tocopheryl acetate.

Packages (n = 660) of clod, strip loin, tenderloin, bottom round, eye-of-round, and top round steaks, as well as ground beef, from beef cattle fed diets supplemented with vitamin E (500 IU/d for 100 d preharvest) were compared with packages of products derived from carcasses of beef cattle fed control diets to determine the duration of acceptable muscle color during simulated retail display. In addition, the effect of storage before steak fabrication, simulating distribution time, on the acceptability of each treatment and each cut during retail display was investigated. Dietary supplementation of animals with vitamin E increased muscle alpha-tocopherol concentrations (P < .05) in all cuts. The acceptable retail display time of ground beef from vitamin E-supplemented cattle was longer (P < .05) compared to ground beef from unsupplemented cattle by 10.2, 15.6, and 17.6 h following 7, 14, and 21 d of storage in chub packages prior to final grinding and retail display, respectively. However, length of storage in a vacuum package before retail display did not have a consistent effect on retail case life of steaks. Product drip loss during retail display was not affected by storage time or vitamin E treatment. The trained panel color score during retail display of high vitamin E strip loin, bottom round, inside round, and eye-of-round steaks remained acceptable longer (P < .05) by 11.5, 25.2, 8.4, and 29.4 h, respectively, than that of control steaks. The results demonstrated that vitamin E supplementation of cattle feed can be used as a management tool to reduce the economic losses associated with beef muscle color deterioration during retail display of products.

Animal Feed↗

Comparison of sponging and excising as sampling procedures for microbiological analysis of fresh beef-carcass tissue.

Sponging and excising were evaluated as sampling procedures for microbiological analysis of beef-carcass tissue. Brisket tissue portions (10 x 10 cm) were inoculated with 2 ml of an Escherichia coli ATCC 25922 cell suspension (3 x 10(8)CFU/ ml). After 30 min, the portions were sampled by excising (EX) or swabbing (SP) with a sterile sponge and were analyzed for aerobic plate counts on tryptic soy agar and for total coliform counts and E. coli counts on Petrifilm E. coli count plates. Another set of inoculated samples was analyzed after being spray washed, in sequence, with water (6 s, 35 degrees C, 3.4 bar), acetic acid (2%, 6 s, 35 degrees C, 2.1 bar), water (20 s, 42 degrees C, 20.7 bar), and acetic acid (2%, 6 s, 35 degrees C, 2.1 bar). Additional samples were sampled for analysis after chilling at 7 degrees C for 24 h. Bacterial counts recovered were influenced (P < or = 0.05) by procedure of sampling (EX versus SP), time of sampling (0.5 versus 24 h), and by their interactions. Counts recovered 0.5 h after inoculation from unwashed or spray-washed samples were similar between the two sampling procedures (EX and SP). However, counts recovered after 24 h of sample storage were significantly (P < or = 0.05) lower for the SP compared with the EX procedure. The results indicated that as the carcass tissue was stored, recovery of bacteria by SP was less efficient than was recovery by EX.

Animals↗

Sources and extent of microbiological contamination of beef carcasses in seven United States slaughtering plants.

This study determined microbiological loads of beef carcasses at different stages during the slaughtering to chilling process in seven (four steer/heifer and three cow/bull) plants. Potential sources of contamination (feces, air, lymph nodes) were also tested. Each facility was visited twice, once in November through January (wet season) and again in May through June (dry season). Carcasses were sampled by aseptic excision of surface tissue (100 cm2) from the brisket, flank, and rump (30 samples each) after hide removal (pre-evisceration), after final carcass washing, and after 24-h carcass chilling. The samples were analyzed individually by standard procedures for aerobic plate counts (APC), total coliform counts (TCC), Escherichia coli biotype I counts (ECC), and presence of Salmonella. Incidence of Salmonella was higher on dry feces of older compared to younger animals, fresh feces of younger compared to older animals, and on cow/bull carcasses compared to steer/heifer carcasses. Most factors and their interactions had significant (P < or = 0.05) effects on the bacterial counts obtained. Depending on plant and season, APC, TCC, and ECC were < or =10(4), < or =10(2), and < or =10(1) CFU/cm2 in 46.7 to 93.3, 50.0 to 100.0, and 74.7 to 100.0% of the samples, respectively. TCC exceeded 10(3) CFU/cm2 in 2.5% (wet season) and 1.5% (dry season) of the samples. ECC exceeded 10(2) CFU/cm2 in 8.7%, 0.3%, and 1.5% of the pre-evisceration, final carcass-washing, and 24-h carcass-chilling samples, respectively, during the wet season; the corresponding numbers during the dry season were 3.5%, 2.2%, and 3.0%, respectively. These data should serve as a baseline for future comparisons in measuring the microbiological status of beef carcasses, as the new inspection requirements are implemented.

Abattoirs↗

Extent of beef carcass contamination with Escherichia coli and probabilities of passing U.S. regulatory criteria.

In the 1996 U.S. Meat and Poultry Inspection Regulations, Escherichia coli biotype I counts were included as "performance criteria" of the slaughtering process. The criteria were based on a three-class attributes sampling plan applied in a moving window. The values for m and M and c and n were set at 5 and 100 CFU/cm2, and 3 and 13 samples, respectively, for beef carcasses after overnight chilling following slaughter. In this study, beef carcasses were analyzed for counts of E. coli, and the results were expressed according to the above criteria. Furthermore, probabilities of passing E. coli performance criteria were determined. Carcasses were sampled in seven slaughtering plants (four steer and heifer; three cow and bull), during two seasons, and at three plant locations (pre-evisceration, after final carcass washing, and after 24 h of carcass chilling). Each entire carcass sample (100 cm2 from the brisket, flank, and rump) was analyzed individually for E. coli counts. Compared with the regulation, which set the value of m and the acceptable range based on the 80th percentile of E. coli contamination data from U. S. Food Safety and Inspection Service nationwide baseline studies, our results showed that, on the average and depending on plant and season, 84.2 to 100% of the chilled carcass samples were in the acceptable range. The average percentages of chilled samples in the unacceptable range, set at the 98th percentile, were 0 to 6.7%. Depending on plant and season, the overall probabilities of chilled carcasses passing the regulatory requirement were 0.597 to 1.0 (brisket), 0.471 to 1.0 (flank), and 0.485 to 1.0 (rump). The results indicated substantial variation among plants and between seasons in ability to meet the E. coli performance criteria.

Animals↗

Incidence of Salmonella on beef carcasses relating to the U.S. meat and poultry inspection regulations.

This article is part of a major study designed to collect baseline contamination data by sampling beef carcasses in seven slaughtering plants (four steer-heifer and three cow-bull plants) during both a dry season (November to January) and a wet season (May to June). Samples (n = 30) were excised from each of three carcass anatomical sites (brisket, flank, and rump) at each of three points in the slaughtering chain (pre-evisceration, following final carcass washing, after 24-h carcass chilling). A total of 3,780 samples (100 cm2 each) were analyzed for presence of Salmonella; aerobic plate counts, total coliform counts, and Escherichia coli counts were also made. After 24-h chilling, average incidence (expressed as a percentage) of Salmonella in the brisket, flank, and rump samples, respectively, for steer-heifer carcasses was 0.8+/-1.7, 0, and 2.5+/-5.0 for the wet season and 0.8+/-1.7, 0, and 0 for the dry season; the corresponding percentages for cowbull carcasses were 4.4+/-2.0, 2.2+/-3.9, and 1.1+/-1.9 for the wet season and 2.2+/-3.9, 1.1+/-1.9, and 0 for the dry season. Depending on plant and season, ranges of probabilities of chilled steer-heifer carcasses passing the U.S. regulatory requirements for Salmonella contamination were 0.24 to 1.0 for the brisket, 1.0 for the flank, and 0.002 to 1.0 for the rump; the corresponding ranges for the chilled cow-bull carcasses were 0.25 to 1.0, 0.25 to 1.0, and 0.70 to 1.0. When the number of positive brisket, flank, and rump samples were combined, the probabilities of passing the regulatory requirements were 0.242 to 1.0 and 0.772 to 1.0 for the wet and dry seasons, respectively, in steer-heifer plants and 0.368 to 0.974 and 0.865 to 1.0 in cow-bull plants. Correlation coefficients of aerobic plate counts, total coliform counts, and E. coli counts with Salmonella incidence were higher (P< or =0.05) for cow-bull samples that had increased incidence of the pathogen when compared to steer-heifer samples.

Abattoirs↗

DNA-dependent protein kinase and related proteins.

The DNA-dependent protein kinase (DNA-PK) is a nuclear protein serine/threonine kinase that must bind to DNA double-strand breaks to be active. We and others have shown that it is a multiprotein complex comprising an approx. 465 kDa catalytic subunit (DNA-PKcs) and a DNA-binding component, Ku. Notably, cells defective in DNA-PK are hypersensitive to ionizing radiation. Thus X-ray-sensitive hamster xrs-6 cells are mutated in Ku, and rodent V3 cells and cells of the severe combined immune-deficient (Scid) mouse lack a functional DNA-PKcs. Cloning of the DNA-PKcs cDNA revealed that it falls into the phosphatidylinositol (PI) 3-kinase family of proteins. However, biochemical assays indicate that DNA-PK contains no intrinsic lipid kinase activity, but is instead a serine/threonine kinase. We have also found that DNA-PK activity can be inhibited by the PI 3-kinase inhibitors wortmannin and LY294002. Consistent with its proposed role in genome surveillance and the detection of DNA damage, DNA-PKcs is most similar to a subset of proteins involved in cell-cycle checkpoint control and signalling of DNA damage. Furthermore, the recent cloning of the gene mutated in ataxia-telangiectasia (A-T) patients, named ATM (A-T mutated), has revealed that the product of this gene is also a PI 3-kinase family member and is related to DNA-PKcs. Although much is known about the clinical symptoms and cellular phenotypes that arise from disruption of the A-T gene, little is known about the biochemical action of ATM in response to DNA damage. Given its sequence similarity with DNA-PKcs, we speculate that ATM may function in a manner similar to DNA-PK.

Androstadienes↗

First-trimester growth and the risk of low birth weight.

BACKGROUND: Previous studies have demonstrated a correlation between first-trimester size and birth weight. It is not known, however, whether low birth weight is related to first-trimester growth. We sought to determine whether the risk of low birth weight and birth weight that was low for gestational age is related to the size of the embryo or the fetus in the first trimester. METHODS: From a data base of ultrasound records of more than 30,000 pregnancies, we identified women who had no important medical problems, a normal menstrual history, and a first-trimester ultrasound scan in which the crown-rump length of the embryo or fetus had been measured. We examined the relation between the outcome of 4229 pregnancies and the difference between the measured and the expected crown-rump length in the first trimester, expressed as equivalent days of growth. RESULTS: A first-trimester crown-rump length that was two to six days smaller than expected was associated with an increased risk (as compared with a normal or slightly larger than expected crown-rump length) of a birth weight below 2500 g (relative risk, 1.8; 95 percent confidence interval, 1.3 to 2.4), a birth weight below 2500 g at term (relative risk, 2.3; 95 percent confidence interval, 1.4 to 3.8), a birth weight below the fifth percentile for gestational age (relative risk, 3.0; 95 percent confidence interval, 2.0 to 4.4), and delivery between 24 and 32 weeks of gestation (relative risk, 2.1; 95 percent confidence interval, 1.1 to 4.0), but not with delivery between 33 and 36 weeks (relative risk, 1.0; 95 percent confidence interval, 0.7 to 1.5). CONCLUSIONS: Suboptimal first-trimester growth may be associated with low birth weight, low birth-weight percentile, and premature delivery.

Birth Weight↗

Nonacid meat decontamination technologies: model studies and commercial applications.

Increased consumer awareness and concern about microbial foodborne diseases has resulted in intensified efforts to reduce contamination of raw meat, as evidenced by new meat and poultry inspection regulations being implemented in the United States. In addition to requiring operation of meat and poultry slaughtering and processing plants under the principles of the hazard analysis critical control point (HACCP) system, the new regulations have established microbiological testing criteria for Escherichia coli and Salmonella, as a means of evaluating plant performance. These developments have renewed and intensified interest in the development and commercial application of meat and poultry decontamination procedures. Technologies developed and evaluated for decontamination include live animal cleaning/washing, chemical dehairing, carcass knife-trimming to remove physical contaminants, steam/hot water-vacuuming for spot-cleaning/decontamination of carcasses, spray washing/rinsing of carcasses with water of low or high pressures and temperatures or chemical solutions, and exposure of carcass sides to pressurized steam. Under appropriate conditions, the technologies applied to carcasses may reduce mean microbiological counts by approximately one-three log colony forming units (cfu)/cm2, and some of them have been approved and are employed in commercial applications (i.e., steam-vacuuming; carcass spray-washing with water, chlorine, organic acid or trisodium phosphate solutions; hot water deluging/spraying/rinsing, and pressurized steam). The contribution of these decontamination technologies to the enhancement of food safety will be determined over the long term, as surveillance data on microbial foodborne illness are collected. This review examines carcass decontamination technologies, other than organic acids, with emphasis placed on recent advances and commercial applications.

Animals↗

Psychological morbidity and quality of life in Australian women with early-stage breast cancer: a cross-sectional survey.

OBJECTIVE: To determine the prevalence of psychological morbidity and describe quality of life in women with early-stage breast cancer. DESIGN: Cross-sectional descriptive study (3 months after conservative breast surgery or mastectomy) of patients from nine general hospitals in Melbourne, Victoria, October 1994 to March 1997. PARTICIPANTS: 303 women with early-stage breast cancer entering a randomised trial of adjuvant psychological group therapy; mean age, 46 years (SD, 8). MAIN OUTCOME MEASURES: Diagnostic and Statistical Manual of Mental Health (DSM)-IV psychiatric diagnoses generated by the Monash Interview for Liaison Psychiatry; quality-of-life data based on the the European Organization for Research and Treatment of Cancer quality-of-life questionnaire (QLQ)-C30 (core) and QLQ-BR23 (breast module) instruments. RESULTS: 45% of the women (135/303) had a psychiatric disorder; 42% (127) of the sample had depression or anxiety, or both; there was minor depression in 82 (27.1%), an anxiety disorder in 26 (8.6%), major depression in 29 (9.6%) and a phobic disorder in 21 (6.9%). 20% of women (61) had more than one disorder. On quality-of-life measures nearly one-third of the women felt less attractive and most had lost interest in sexual activity. There was substantial distress about hair loss. Symptoms of lymphoedema were described by 13 women (4.3%). Breast conservation surgery was associated with a better body image (P<0.01). CONCLUSION: Women recently diagnosed with early-stage breast cancer have high rates of psychiatric and psychological disturbance. Quality of life is substantially affected. Clinicians should actively explore their patients' psychological adjustment to enable early recognition and treatment of these disorders.

Adaptation, Psychological↗

Movement of badgers (Meles meles) in a high-density population: individual, population and disease effects.

The movement of 1763 badgers trapped between 36 social groups in Woodchester Park, Gloucestershire, over 18 years was analysed to determine the frequency and duration of moves, the factors associated with a predisposition to move and the spatial pattern of movements. Of those badgers whose capture history could be categorized, nearly half had moved. Of these, 73.1% were classified as 'occasional movers', 22.1% as 'permanent movers' and 4.8% as 'frequent movers'. Most adult badgers that moved made occasional moves (78.8%, n = 67). Cubs made all types of move including permanent moves (29%, n = 10). Seventy per cent of females were non-movers compared with 37% of males. Badgers were significantly more likely to move to smaller groups, whereas male badgers were significantly more likely to move to groups with a greater proportion of females. The spatial pattern of movement differed from the distribution of groups with bovine tuberculosis in the study area. However, temporal changes in movement were significantly related to the incidence of Mycobacterium bovis infection in the following year, indicating that as the movement of badgers between groups varies so does the incidence of bovine tuberculosis in the population. This finding is of central importance in the formulation of badger control policy.

Animals↗