ATP bound to the origin recognition complex is important for preRC formation.

The origin recognition complex (ORC) binds origins of replication and directs the assembly of a higher order protein complex at these sites. ORC binds and hydrolyzes ATP in vitro. ATP binding to the largest subunit of ORC, Orc1p, stimulates specific binding to origin DNA; however, the function of ATP hydrolysis by ORC is unknown. To address the role of ATP hydrolysis, we have generated mutants within Orc1p that are dominant lethal. At physiological ATP concentrations, these mutants are defective for ATP hydrolysis but not ATP binding in the absence of DNA. These mutants inhibit formation of the prereplicative complex when overexpressed. The dominant lethal phenotype of these mutant ORC complexes is suppressed by simultaneous overexpression of wild-type, but not mutant, Cdc6p. Our findings suggest that these hydrolysis-defective mutants inhibit growth by titrating Cdc6p away from the origin. Based on these observations, we propose that Cdc6p specifically recognizes the ATP-bound state of Orc1p and that ATP hydrolysis is coupled to preRC disassembly.

Efficacy of a vitamin A-fortified wheat-flour bun on the vitamin A status of Filipino schoolchildren.

BACKGROUND: Wheat flour is a possible food vehicle for vitamin A fortification. OBJECTIVE: This study assessed the efficacy of consumption of a vitamin A-fortified wheat-flour bun (pandesal) on the vitamin A status of school-age children. DESIGN: This was a double-masked clinical trial conducted in 396 and 439 children aged 6-13 y attending 4 rural schools in the Philippines. The children were randomly assigned to a vitamin A-fortified (experimental) or nonfortified (control) group. A 60-g vitamin A-fortified pandesal (containing approximately 133 microg retinol equivalents) or a nonfortified pandesal was consumed by the children 5 d/wk for 30 wk. Vitamin A status, hemoglobin concentration, anthropometric status, morbidity, and dietary intake were assessed at baseline and 30 wk later. A modified relative dose response (MRDR) was assessed in a subsample of 20% of the children ( approximately 75/group) with the lowest initial serum retinol concentration at the 30-wk follow-up. RESULTS: Baseline serum retinol significantly modified the effect of the intervention. The fortified group, whose initial serum retinol concentrations were below the median, had a 0.07 +/- 0.03-micromol/L greater improvement in serum retinol at the 30-wk follow-up than did the control group (P: = 0.02). Improved vitamin A status was also evident in the MRDR subsample. End-of-study differences in the MRDR showed that vitamin A- fortified pandesal intake decreased the percentage of children with inadequate liver vitamin A stores by 50% (15.3% compared with 28.6%; P: = 0.05). CONCLUSIONS: Daily consumption of vitamin A-fortified pandesal significantly improved the vitamin A status of Filipino school-age children with marginal-to-low initial serum retinol concentrations.

Regulation of origin recognition complex conformation and ATPase activity: differential effects of single-stranded and double-stranded DNA binding.

The Saccharomyces cerevisiae origin recognition complex (ORC) is bound to origins of DNA replication throughout the cell cycle and directs the assembly of higher-order protein-DNA complexes during G(1). To examine the fate of ORC when origin DNA is unwound during replication initiation, we determined the effect of single-stranded DNA (ssDNA) on ORC. We show that ORC can bind ssDNA and that ORC bound to ssDNA is distinct from that bound to double-stranded origin DNA. ssDNA stimulated ORC ATPase activity, whereas double-stranded origin DNA inhibited the same activity. Electron microscopy studies revealed two alternative conformations of ORC: an extended conformation stabilized by origin DNA and a bent conformation stabilized by ssDNA. Therefore, ORC appears to exist in two distinct states with respect to its conformation and ATPase activity. Interestingly, the effect of ssDNA on these properties of ORC is correlated with ssDNA length. Since double-stranded origin DNA and ssDNA differentially stabilize these two forms of ORC, we propose that origin unwinding triggers a transition between these alternative states.

Effects of form of the diet on anatomical, microbial, and fermentative development of the rumen of neonatal calves.

Eight neonatal, Holstein bull calves were paired by birth date and birth weight and randomly assigned to either a finely ground or unground control diet (chopped hay and rolled grain) to study the effects of the physical form of the diet on anatomical, microbial, and fermentative development of the rumen. The diets varied in particle size but were identical in composition (25% alfalfa hay and 75% grain mix). Calves were fed milk at 8% of birth weight daily until weaning. Feed intake was equalized for each pair of calves. Ruminal fluid samples were collected from ruminal cannulas to determine pH, fermentation products, and buffering capacity and to enumerate bacteria. Calves were slaughtered at 10 wk of age, and weights of the full and empty reticulorumen, abomasum, and omasum were recorded. Ruminal tissue samples were taken to assess papillary development by morphometric measurements. Calves had similar body weights at wk 10. Ruminal pH was affected by age and was lower for calves fed the ground diet. Total anaerobic bacterial counts were not affected by the physical form of the diet; however, calves fed the ground diet had lower numbers of cellulolytic bacteria and higher numbers of amylolytic bacteria than did calves fed the unground diet. Physical form of the diet did not affect the weights of the reticulorumen whether full or empty. However, calves fed the ground diet had heavier omasum weights, both full and empty. Physical form of the diet affected papillary size and shape but did not influence the muscle thickness of rumen. Results indicated that the physical form of the diet had a significant influence on the anatomical and microbial development of the forestomac and, therefore, might influence future performance.

Coordinate binding of ATP and origin DNA regulates the ATPase activity of the origin recognition complex.

The Origin Recognition Complex (ORC) is a six-protein assembly that specifies the sites of DNA replication initiation in S. cerevisiae. Origin recognition by ORC requires ATP. Here, we demonstrate that two subunits, Orc1p and Orc5p, bind ATP and that Orc1p also hydrolyzes ATP. ATP binding and hydrolysis by Orc1p are both regulated by origin DNA in a sequence-specific manner. ATP binding to Orc1p, but not ATP hydrolysis, is responsible for the ATP dependence of the ORC-origin interaction, indicating that ATP is a cofactor that locks ORC on origin DNA. These data demonstrate that occupancy of the Orc1p ATP-binding site has a profound effect on ORC function and that ATP hydrolysis by Orc1p has the potential to drive transitions between different functional states of ORC.

Molecular cloning and expression of the 32-kDa subunit of human TFIID reveals interactions with VP16 and TFIIB that mediate transcriptional activation.

Transcription factor TFIID consists of TATA binding protein (TBP) and at least eight TBP-associated factors (TAFs). As TAFs are required for activated but not basal transcription, we have proposed that TAFs act as coactivators to mediate signals between activators and the basal transcription machinery. Here we report the cloning, expression, and biochemical characterization of the 32-kDa subunit of human (h) TFIID, termed hTAFII32. We find that hTAFII32 is the human homologue of Drosophila TAFII40. In vitro protein-protein interaction assays reveal that as observed with Drosophila TAFII40, hTAFII32 interacts with the C-terminal 39-amino acid activation domain of the acidic transactivator viral protein 16 (VP16) as well as with the general transcription factor TFIIB. Moreover, a partial recombinant TFIID complex containing hTAFII32 was capable of mediating in vitro transcriptional activation by the VP16 activation domain. These findings indicate that specific activator-coactivator interactions have been conserved between human and Drosophila and provide additional support for the function of these interactions in mediating transcriptional activation.

Stress protein induction in skeletal muscle: comparison of laboratory models to naturally occurring hypertrophy.

The purpose of the study was to compare stress protein [heat shock protein (HSP) 72] response in laboratory models of hypertrophy to naturally occurring work-induced hypertrophy. Two laboratory models of hypertrophy inducement, namely, compensatory hypertrophy and stretch hypertrophy, were compared with hypertrophy resulting from migratory flight in the blue-winged teal. We hypothesized that HSP 72 would be expressed more strongly in hypertrophied muscle than in control muscle. Furthermore, we hypothesized that changes occurring in laboratory models would also occur in work-induced enlargement. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses were used to assess HSP 72 levels in control and hypertrophied muscle. Laboratory models elicited similar responses, with increased HSP 72 content in hypertrophied muscle. Work-induced hypertrophy or disuse atrophy did not change the degree of HSP 72 expression in the blue-winged teal. The presence of HSP 72 in these conditions may indicate that stressors eliciting changes in muscle protein expression, including the loss of muscle mass, may elicit HSP 72 synthesis.

A prevalence study of xerophthalmia in the Philippines: implications for supplementation strategies.

In three provinces of the Philippines (Quezon, Northern Samar and Zamboanga del Sur), 11,378 children between 6 and 83 months of age were examined for signs of xerophthalmia and weighed to determine weight-for-age status. Xerophthalmia prevalence ranged from 1.6% to 4.4% for nightblindness and 0.6% to 2.7% for Bilot's spots in the three provinces indicating a serious vitamin A deficiency problem. These levels contrast sharply with the recent national xerophthalmia prevalence (0.7% for night blindness and 0.2% for Bitot's spots). There was a preponderance of mild xerophthalmia among males, and among children 4-6 years of age. No consistent association between weight-for-age status and xerophthalmia was found. The study recommends the universal distribution of vitamin A to children in high-prevalence regions, rather than the current practice of providing vitamin A supplements to moderately and severely underweight children identified through the annual village-based child weighing sessions, to ensure reaching children most at risk for xerophthalmia. The study also suggests the need for regional vitamin A assessments to identify areas endemic for vitamin A deficiency because the aggregate national prevalence may mask a more serious localized problem.

Comparison of stainless steel fascial staples and polypropylene suture material for closure of the linea alba in dogs.

Five centimeter segments of the linea alba in mature, female, mixed breed dogs were incised and then approximated with either stainless steel fascial staples or size 0 polypropylene suture material. Breaking strength of the linea alba closures was determined at 0 (Phase I, 18 dogs) and at 7, 14, and 31 days (Phase II, 18 dogs). Histology and videointeractive planar morphometry were used to evaluate healing of the linea alba. Breaking strength and histologic and morphometric variables were analyzed statistically using analysis of variance in a split plot design. In a clinical trial, 20 cm linea alba incisions were approximated with stainless steel fascial staples after elective ovariohysterectomy in mature, female, mixed breed dogs (Phase III, eight dogs). The dogs were evaluated at 1, 2, 7, 14, 31, 90, and 180 days. Linea alba incisions approximated with polypropylene suture material were significantly stronger than linea alba incisions approximated with stainless steel fascial staples on day 0. There was no significant difference between breaking strength values, histologic, and morphometric appearance of linea alba incisions closed with stainless steel fascial staples or polypropylene suture material when compared 7, 14, and 31 days after surgery. All of the dogs in the clinical trial appeared to be normal at all evaluation times. Approximation of linea alba incisions with stainless steel fascial staples compares favorably to closure with a simple continuous pattern of polypropylene suture material with regard to breaking strength, clinical, histologic, and morphometric appearance.

Measuring suitability of soybean products for early-weaned pigs with immunological criteria.

Two trials were conducted to determine the suitability of soybean products for baby pigs. Weanling pigs (n = 40 and 48 in Trials 1 and 2, respectively) were infused orally (6 g/d) with dried skim milk, soybean meal (SBM, 48% CP); soy protein concentrate, moist extruded soy protein concentrate, or soy protein isolate from d 7 to 12 of age. Pigs were then fed a diet containing the same protein source for 1 (Trial 1) or 2 (Trial 2) wk after weaning (d 21 of age). To avoid exposure of pigs to soybean proteins, the dams of pigs were fed a corn-corn gluten meal-based diet supplemented with lysine and tryptophan from d 109 of gestation. All pigs in Trial 1 were killed at 28 d of age, and samples of ileal digesta and small intestine were obtained. In Trial 2, the soy protein isolate was not included, and all pigs were fed a diet containing 4% soybean oil and 1.25% lysine for the last 3 wk of the trial. Growth performance, skin-fold thickness, after intradermal injection of extracts of the corresponding proteins, and anti-soy immunoglobulin G (IgG) titers were measured. Results indicated that pigs fed diets containing SBM had lower (P less than .05) villus height and xylose absorption but higher (P less than .05) serum anti-soy IgG titers and increased skin-fold thickness compared with the mean of pigs given milk and all other soy treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of stressful exercise on leukocytes in cattle with experimental pneumonic pasteurellosis.

Seven yearling bulls were treated with stressful exercise and intrabronchial Pasteurella haemolytica A1. Group 1 bulls (nos. 1-4) underwent treadmill exercise and, 24 days later, intrabronchial instillation of P. haemolytica A1. Group 2 bulls (nos. 5-7) underwent treadmill exercise, followed 30 min later by intrabronchial P. haemolytica A1. Blood lactic acid values were raised (p less than 0.05) by treadmill exercise only, but plasma cortisol was raised (p less than 0.05) by treadmill exercise and by P. haemolytica A1 infection. Neutrophils in bronchoalveolar lavage (BAL) differed from control values 24 h after treadmill exercise, and 1 h and 4 h after P. haemolytica A1 infection. Respiratory disease was more severe and the gross lung lesions were larger in group 2 bulls than in group 1 bulls. P. haemolytica A1 was recovered from the livers, spleens and mesenteric lymph nodes of group 2 but not group 1 bulls, suggesting that group 2 bulls had experienced bacteraemia. Decreased neutrophils in BAL fluid from group 2 bulls at 1 h and 4 h after infection suggests that exercise transiently inhibited neutrophil egress from the blood to the alveoli; BAL neutrophils peaked at 1 h and 4 h after infection in group 1 bulls but declined at 24 h. We conclude that group 2 bulls were made more susceptible to experimental pneumonic pasteurellosis by stressful exercise.

Pneumonic pasteurellosis induced experimentally in gnotobiotic and conventional calves inoculated with Pasteurella haemolytica.

Experimental pneumonia caused by Pasteurella haemolytica was induced in 2-week-old gnotobiotic (n = 4) and conventional (n = 6) calves by endobronchial inoculation into the right caudal lung lobe of 7.9 x 10(10) +/- 0.6 x 10(10) (mean +/- SD) colony-forming units of P haemolytica in the 6-hour log phase of growth. The calves were studied for 24 hours or less. Regression lines for the relationship between clinical index and time for the gnotobiotic group and conventional group of calves were compared, and the clinical index was found to be significantly (P less than or equal to 0.005) more rapid in the gnotobiotic group. There was also a significant difference in the preinoculation, absolute segmented neutrophil count (P less than or equal to 0.05), and in the total serum protein, albumin, and globulin values (P less than or equal to 0.05). Comparison of the preinoculation and post inoculation blood cell and blood chemical values revealed a significant increase (P less than or equal to 0.05) in the numbers of band neutrophils and fibrinogen in conventional calves, and a significant decrease (P less than or equal to 0.05) in the total WBC count in gnotobiotic calves. Necropsy of both groups of calves revealed a circular to oblong lesion that was congested, edematous, and firm, and which occupied 20% to 100% of the right caudal lung lobe and involved the remaining lung lobes to a more minor degree. When mean lesion scores of the 2 groups of calves were compared, no significant difference (P less than or equal to 0.05) was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical and pathologic studies of experimentally induced Pasteurella haemolytica pneumonia in calves.

Pasteurella haemolytica pneumonia of the right caudal lung lobe was experimentally induced in 2-week-old Holstein calves (n = 11) by endobronchial inoculation of 7.9 x 10(10) colony-forming units of 6-hour log-phase bacteria. Calves were studied for 72 hours after inoculation. The challenge procedure consistently induced a lesion in the right caudal lung lobe, which was consistent radiographically with results of pathologic examination and a similar volume of bronchography contrast medium. Clinically, the calves developed a significant increase in rectal temperature within 24 hours after inoculation. Seventy-two hours after inoculation, the total WBC counts, absolute band neutrophil counts, monocyte counts, and blood fibrinogen concentrations were significantly higher than normal and albumin concentration was significantly decreased. Necropsy revealed a circular to oblong lesion that was congested, edematous, and firm and occupied 20 to 40% of the right caudal lung lobe. Histologic examination revealed a severe acute inflammatory reaction characterized by cellular exudate and proteinaceous fluid in the alveoli, interlobular septa, and pleura.

Evaluation of attributes that affect longissimus muscle tenderness in Bos taurus and Bos indicus cattle.

Biological tenderness differences between longissimus muscles (LM) from Bos indicus and Bos taurus breeds were evaluated. Steers and heifers of Hereford x Angus (H x A, n = 10), 3/8 Sahiwal x H, A or H x A (3/8 SAH, n = 6) and 5/8 Sahiwal x H, A or H x A (5/8 SAH, n = 11) crosses were utilized. Muscle temperature and pH were monitored every 3 h for the first 12 h and at 24 h. Samples were obtained within 1 h and at 24 h postmortem from the LM for determination of calcium-dependent protease (CDP) -I and -II and CDP inhibitor (INH) activities. At 1 and 14 d postmortem, LM samples were removed for determining cathepsin B and B + L activity, soluble and total collagen, sarcomere length, muscle-fiber histochemistry, shear force and sensory-panel traits. Data were analyzed using least squares procedures with fixed effects of breed cross, sex and their interaction. No significant breed cross effects were observed for carcass traits or rates of pH and temperature decline. Steaks from H x A had lower (P less than .05) shear-force values and higher (P less than .05) sensory scores for tenderness at 1 and 14 d postmortem than steaks from 3/8 and 5/8 SAH. Correspondingly, 5/8 SAH had lower (P less than .05) myofibril fragmentation indices than H x A at 1, 3, 7 and 14 d postmortem. Breed cross effects were not significant for sarcomere length, fiber types, soluble and total collagen, cathepsin B and B + L specific activity, CDP-I and -II activity and INH activity within 1 h postmortem. However, INH total activity/100 g of muscle was greater (P less than .01) at 24 h postmortem for 5/8 SAH (208.8 +/- 14.8) and 3/8 SAH (195.6 +/- 19.3) than for H x A (136.3 +/- 14.9). For H x A, SDS-PAGE revealed that by d 1 desmin had been subjected to proteolysis, and by d 14 desmin could not be detected, but a 30,000-dalton component was clearly evident. However, in 5/8 SAH, desmin remained visible at d 14 without a 30,000-dalton component appearing. This reduced protein hydrolysis may account for less tender meat in SAH; INH apparently influences this process.

Transient hypersensitivity to soybean meal in the early-weaned pig.

An experiment was conducted to determine whether baby pigs develop hypersensitivity to dietary soybean proteins. Thirty-two pigs were orally infused with either dried skim milk (5 g/d; control) or soybean meal (48% CP; 5 g/d) from d 7 to 14 after birth. Sows were fed a corn-corn gluten meal-based diet supplemented with lysine and tryptophan to avoid exposure of pigs to soybean proteins. Pigs were weaned at 21 d of age and fed diets containing either soybean meal or milk proteins until d 56. One half of the pigs were killed at 28 d of age and the rest at 56 d of age. Segments of small intestine were collected, and intraepithelial lymphocytes were isolated. At 28 d of age, pigs fed diets containing soybean meal had lower (P less than .05) villus height (221 vs 298 microns) and rate of gain (86 vs 204 g/d) than control pigs did. Pigs fed a diet containing soybean meal had higher (P less than .05) immunoglobulin G (IgG) titers to soybean protein than did pigs fed a milk protein-based diet. Blood and intestinal lymphocytes collected on d 28 and 56 did not exhibit any proliferative response when cultured with purified soy proteins (2.5 or 5 microns/ml). Phytohemagglutinin- and pokeweed mitogen-induced lymphocyte proliferations were higher (P less than .05) at d 56 than at d 28, but there were no differences attributable to protein source. There were no differences (P greater than .05) in skin-fold thickness measurements following intradermal injection with soy or milk proteins. Decreased villus height and increased serum IgG titers to soybean proteins coinciding with inferior performance of early weaned pigs fed diets containing soybean meal indicate that conventionally processed, commercial soybean meal may retain some antigens that can cause transient hypersensitivity in piglets.

New host and locality record for Trypanosoma peromysci.

Trypanosoma peromysci Watson, 1912 (Sarcomastigophora: Kinetoplastida), is described from a new host and locality. One of 20 (5.0%) Peromyscus leucopus collected from Pottawatomie and Riley counties in Kansas was found to harbor the parasite. Morphometric and statistical analysis confirmed the trypanosome to be indistinguishable from T. peromysci, the only difference being a greater mean flagellar length than reported previously. This is the first reported occurrence of T. peromysci in the white-footed mouse (Peromyscus leucopus noveboracensis Fischer, 1829) and also the first record of its occurrence in Kansas.

Time of appearance of esterase-positive alveolar macrophages in bovine fetal lung.

Bovine fetal lung tissue was examined histochemically, using alpha-naphthyl butyrate (nonspecific method), to detect the presence of esterase-positive pulmonary alveolar macrophages (PAM). Positively stained PAM were first seen in the alveolar septa and lumina of the lungs of 4 of 8 fetuses 240 days old. Macrophages (n = 100) in 240-day-old fetuses were 6.74 +/- 0.07 microm, and in most cells, esterase-positive granules were sparsely distributed in the cytoplasm. In 10 fetuses 250 days old, the alveolar septa and lumina of lungs contained positively stained macrophages, as did those of all older fetal lungs examined. Compared with macrophages of the 240-day-old fetuses, those (n = 50) of 250-day-old fetuses were significantly larger, 7.32 +/- 0.10 micron (P less than 0.0001), and esterase-positive granules were heavily distributed throughout the cytoplasm. In all fetal lungs, T lymphocytes contained a single, small, esterase-positive granule, compared with the numerous diffuse esterase-positive granules in PAM. Type-II pneumonocytes with well-developed lamellar bodies had no evidence of positive nonspecific esterase reaction in lungs.

The embryonic development of theproctodeal gland of Coturnix coturnix japonica (Aves).

The embryogenesis of the proctodeal gland and development of the connective tissue of the associated lamina propria in the dorsal wall of the proctodeum of Common Coturnix (Coturnix c. japonica) were studied on embryos collected at 12-hour intervals from day 7 of incubation through hatching. Gland development began at 9.5 days from ectodermal-derived epithelial buds developing in the craniolateral region of the dorsal proctodeal epithelium. At 11 days, the epithelial buds had become solid convoluted epithelial cords which, by 11.5 days branced to form solid individual glandular units. Lumina of the unit were formed by degeneration and vacuolization of the central cells of these solid units. Squamous epithelial caps, several layers thick, separated the vacuolizing units from the proctodeal cavity at day 13; these caps subsequently thinned, bulged into the proctodeal cavity and, by day 16, began to disappear opening the lumen of the units to the proctodeal cavity. Sequential formation of epithelial buds, by region, was: craniolateral at 9.5 days, craniomedial expansion at 10.5 days followed, beginning at 11 days, by caudal expansion along entire dorsal proctodeal wall. Sequence of appearance of gland-associated connective tissue fibers was: 9.5 days, reticular fibers already present; 11 days, collagen fibers; 11.5 days elastic fibers. Serially reconstructed models of an individual glandular unit at hatching enabled classification of the unit as a simple branched alveolar gland.