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PubMed2005

Temporal profile of replication of human chromosomes.

Chromosomes in human cancer cells are expected to initiate replication from predictably localized origins, firing reproducibly at discrete times in S phase. Replication products obtained from HeLa cells at different stages of S phase were hybridized to cDNA and genome tiling oligonucleotide microarrays to determine the temporal profile of replication of human chromosomes on a genome-wide scale. About 1,000 genes and chromosomal segments were identified as sites containing efficient origins that fire reproducibly. Early replication was correlated with high gene density. An acute transition of gene density from early to late replicating areas suggests that discrete chromatin states dictate early versus late replication. Surprisingly, at least 60% of the interrogated chromosomal segments replicate equally in all quarters of S phase, suggesting that large stretches of chromosomes are replicated by inefficient, variably located and asynchronous origins and forks, producing a pan-S phase pattern of replication. Thus, at least for aneuploid cancer cells, a typical discrete time of replication in S phase is not seen for large segments of the chromosomes.

Chromosomes, Human↗
PubMed1995

Epithelial differentiation fails to support replication of cloned human papillomavirus type 16 DNA in transfected keratinocytes.

Research on the life cycle of human papillomaviruses (HPVs) has suffered from the lack of a model system that allows the use of molecularly cloned HPV DNA. In this study, we analyzed replication of molecularly cloned HPV-16 genomes after transfection into cells of two human keratinocyte cell lines. Transfected cells were grown in cell culture (in vitro) or in transplantation chambers on the flanks of nude mice (in vivo). When DNA was extracted after different time intervals, replication of HPV-16 DNA could not be detected. Even the formation of a stratified epithelium under in vivo conditions failed to support vegetative replication. In contrast, transfection of molecularly cloned HPV-11 DNA resulted in replication of viral DNA in vitro. It seems likely that besides epithelial cell differentiation, a number of other factors influence HPV-16 replication.

Animals↗
PubMed2004

Assessing stress in cancer patients: a second-order factor analysis model for the Perceived Stress Scale.

Using the Perceived Stress Scale (PSS), perceptions of global stress were assessed in 111 women following breast cancer surgery and at 12 and 24 months later This is the first study to factor analyze the PSS. The PSS data were factor analyzed each time using exploratory factor analysis with oblique direct quartimin rotation. Goodness-of-fit indices (root mean square error of approximation [RMSEA]), magnitude and pattern of factor loadings, and confidence interval data revealed a two-factor solution of positive versus negative stress items. The findings, replicated across time, also indicate factor stability. Hierarchical factor analyses supported a second-order factor of "perceived stress." This alternative factor model of the PSS is presented along with observations regarding the measure's use in cancer research.

Adult↗
PubMed2006

The SU(VAR)3-9/HP1 complex differentially regulates the compaction state and degree of underreplication of X chromosome pericentric heterochromatin in Drosophila melanogaster.

In polytene chromosomes of Drosophila melanogaster, regions of pericentric heterochromatin coalesce to form a compact chromocenter and are highly underreplicated. Focusing on study of X chromosome heterochromatin, we demonstrate that loss of either SU(VAR)3-9 histone methyltransferase activity or HP1 protein differentially affects the compaction of different pericentric regions. Using a set of inversions breaking X chromosome heterochromatin in the background of the Su(var)3-9 mutations, we show that distal heterochromatin (blocks h26-h29) is the only one within the chromocenter to form a big "puff"-like structure. The "puffed" heterochromatin has not only unique morphology but also very special protein composition as well: (i) it does not bind proteins specific for active chromatin and should therefore be referred to as a pseudopuff and (ii) it strongly associates with heterochromatin-specific proteins SU(VAR)3-7 and SUUR, despite the fact that HP1 and HP2 are depleted particularly from this polytene structure. The pseudopuff completes replication earlier than when it is compacted as heterochromatin, and underreplication of some DNA sequences within the pseudopuff is strongly suppressed. So, we show that pericentric heterochromatin is heterogeneous in its requirement for SU(VAR)3-9 with respect to the establishment of the condensed state, time of replication, and DNA polytenization.

Animals↗
PubMed1981

Near-infrared reflectance determination of fat, protein, and moisture in fresh meat.

Near-infrared spectral absorption properties were determined for emulsified and ground meat samples. Six corrected log numbers associated with the optical responses of 6 filters in the InfraAlyzer were used as multiple independent variables in regression equations. Dependent variables for the equations were moisture, determined by oven-drying; fat, determined by Goldfisch extraction; and protein, determined by the Kjeldahl method. InfraAlyzer log values, replicated 4 times on each emulsified beef sample and 3 times on each ground lamb sample, increased as the samples were warmed from the heat generated in the sample drawer. Variation in temperature of the meat samples were partially responsible for differences in constant terms and in regression coefficients for equations developed on data from different replications of a sample. Multiple correlation coefficients for fat ranged from 0.91 to 0.94 in emulsified meat and from 0.83 to 0.85 in ground meat; for moisture, 0.90-0.94 and 0.83-0.85, respectively; and for protein, 0.80-0.85 and 0.72-0.77, respectively. Overall, near-infrared reflectance shows promise as a rapid method for determining composition of meat. Nevertheless, some aspects of near-infrared reflectance require further attention.

Animals↗
PubMed2001

A potential role for mini-chromosome maintenance (MCM) proteins in initiation at the dihydrofolate reductase replication origin.

Mini-chromosome maintenance (MCM) proteins were originally identified in yeast, and homologues have been identified in several other eukaryotic organisms, including mammals. These findings suggest that the mechanisms by which eukaryotic cells initiate and regulate DNA replication have been conserved throughout evolution. However, it is clear that many mammalian origins are much more complex than those of yeast. An example is the Chinese hamster dihydrofolate reductase (DHFR) origin, which resides in the spacer between the DHFR and 2BE2121 genes. This origin consists of a broad zone of potential sites scattered throughout the 55-kb spacer, with several subregions (e.g. ori-beta, ori-beta', and ori-gamma) being preferred. We show here that antibodies to human MCMs 2-7 recognize counterparts in extracts prepared from hamster cells; furthermore, co-immunoprecipitation data demonstrate the presence of an MCM2-3-5 subcomplex as observed in other species. To determine whether MCM proteins play a role in initiation and/or elongation in Chinese hamster cells, we have examined in vivo protein-DNA interactions between the MCMs and chromatin in the DHFR locus using a chromatin immunoprecipitation (ChIP) approach. In synchronized cultures, MCM complexes associate preferentially with DNA in the intergenic initiation zone early in S-phase during the time that replication initiates. However, significant amounts of MCMs were also detected over the two genes, in agreement with recent observations that the MCM complex co-purifies with RNA polymerase II. As cells progress through S-phase, the MCMs redistribute throughout the DHFR domain, suggesting a dynamic interaction with DNA. In asynchronous cultures, in which replication forks should be found at any position in the genome, MCM proteins were distributed relatively evenly throughout the DHFR locus. Altogether, these data are consistent with studies in yeast showing that MCM subunits localize to origins during initiation and then migrate outward with the replication forks. This constitutes the first evidence that mammalian MCM complexes perform a critical role during the initiation and elongation phases of replication at the DHFR origin in hamster cells.

Animals↗
PubMed2003

Measuring plant diversity in the tall threetip sagebrush steppe: influence of previous grazing management practices.

In July 2000, a 490-ha wildfire burned a portion of a long-term grazing study that had been established in 1924 at the US Sheep Experiment Station north of Dubois, Idaho, USA. Earlier vegetation measurements in this tall threetip sagebrush (Artemisia tripartita spp. tripartita) bunchgrass plant community documented significant changes in vegetation due to grazing and the timing of grazing by sheep. A study was initiated in May 2001 using 12 multiscale modified Whittaker plots to determine the consequences of previous grazing practices on postfire vegetation composition. Because there was only one wildfire and it did not burn all of the original plots, the treatments are not replicated in time or space. We reduce the potential effects of psuedoreplication by confining our discussion to the sample area only. There were a total of 84 species in the sampled areas with 69 in the spring-grazed area and 70 each in the fall- and ungrazed areas. Vegetation within plots was equally rich and even with similar numbers of abundant species. The spring-grazed plots, however, had half as much plant cover as the fall- and ungrazed plots and the spring-grazed plots had the largest proportion of plant cover composed of introduced (27%) and annual (34%) plants. The fall-grazed plots had the highest proportion of native perennial grasses (43%) and the lowest proportion of native annual forbs (1%). The ungrazed plots had the lowest proportion of introduced plants (4%) and the highest proportion of native perennial forbs (66%). The vegetation of spring-grazed plots is in a degraded condition for the environment and further degradation may continue, with or without continued grazing or some other disturbance. If ecosystem condition was based solely on plant diversity and only a count of species numbers was used to determine plant diversity, this research would have falsely concluded that grazing and timing of grazing did not impact the condition of the ecosystem.

Agriculture↗
PubMed1998

DNA topoisomerase II sites in the histone H4 gene during the highly synchronous cell cycle of Physarum polycephalum.

The nearly perfect synchrony of nuclear division in a plasmodium of Physarum polycephalum provides a powerful system to analyze topoisomerase II cleavage sites in the course of the cell cycle. The histone H4 locus, whose schedule of replication and transcription is precisely known, was chosen for this analysis. Drug-induced topoisomerase II sites are clustered downstream of the histone H4 gene and appear highly dependent on cell cycle stage. They were only detected in mitosis and at the very beginning of S phase, precisely at the time of replication of the histone H4 region. The sites, which were absent in G2 phase, reappeared at the next mitosis. Remarkably, DNase I hypersensitive sites occurred in nearly the same location, but their schedule was totally different: they were absent in mitosis and present in G2. This schedule follows H4 transcription, which peaks in mid-S phase and in the second part of G2 phase and is off during mitosis. These results suggest that topoisomerase II may not be involved in transcription, but plays a role in remodeling chromatin structure, both during chromosome condensation in prophase/metaphase to allow their decatenation and during chromosome decondensation after metaphase to allow replication fork passage throughout the region.

Animals↗
PubMed1984

Examination of the mode of action of ribavirin against tobacco mosaic virus.

Ribavirin inhibited a specific step of tobacco mosaic virus (TMV) replication. Time-courses of sensitivity of TMV multiplication in asynchronous and synchronous infections demonstrated that ribavirin inhibited an early function that occurs prior to the period of viral RNA and protein synthesis. In the synchronous infection, ribavirin inhibited the synthesis of both single-stranded genomic RNA and double-stranded replicative form RNA when treatment began during the first 4 h, but there was no inhibition when treatment began at 12 h or later, even though most RNA synthesis occurred after 12 h. Viral protein synthesis also was inhibited when treatment began during the first 4 h and not when treatment began later. Unlike the mechanisms proposed against animal viruses, ribavirin appears to inhibit TMV replication by preventing an early function that is necessary to initiate viral RNA synthesis.

Kinetics↗
PubMedDate not supplied

Nitrate losses in subsurface drainage from a corn-soybean rotation as affected by time of nitrogen application and use of nitrapyrin.

Subsurface drainage, a water management practice used to remove excess water from poorly drained soils, can transport substantial amounts of NO3 from agricultural crop production systems to surface waters. A field study was conducted from the fall of 1986 through 1994 on a tile-drained Canisteo clay loam soil (fine-loamy, mixed, superactive, calcareous, mesic Typic Endoaquoll) to determine the influence of time of N application and use of nitrapyrin [NP; 2-chloro-6-(trichloromethyl) pyridine] on NO3 losses from a corn (Zea mays L.)-soybean [Glycine max (L.) Merr.] rotation. Four anhydrous ammonia treatments [fall N, fall N + NP, spring preplant N, and split N (40% preplant and 60% sidedress)] were replicated four times and applied at 150 kg N ha(-1) for corn on individual drainage plots. Sixty-two percent of the annual drainage and 69% of the annual NO3 loss occurred in April, May, and June. Flow-weighted NO3-N concentrations in the drainage water were two to three times greater in the two years following the three-year dry period compared with preceding and succeeding years. Nitrate N concentrations and losses in the drainage from corn were greatest for fall N with little difference among the other three N treatments. Nitrate losses from soybean were affected more by residual soil NO3 following corn than by the N treatments per se. Averaged across the four rotation cycles, flow-normalized NO3-N losses ranked in the order: fall N > split N > spring N = fall N + NP. Under these conditions NO3 losses from a corn-soybean rotation into subsurface drainage can be reduced by 13 to 18% by either applying N in the spring or using NP with late fall-applied ammonia.

Agriculture↗
PubMed2005

Influence of sward height, daily timing of concentrate supplementation, and restricted time for grazing on forage utilization by lactating beef cows.

To establish the effect of sward height, concentrate feeding time, and restricted time for grazing on forage utilization by grazing cattle, 32 crossbred beef (24 Angus and eight Hereford) cows (632 kg BW) and calves (104 kg BW) were grouped by weight and calving date. They were assigned randomly to two sward height treatments (4 to 8 or 8 to 12 cm), replicated four times. The herbage comprised mainly Kentucky bluegrass, orchardgrass, some forbs, and white clover. The cows were restricted to 12 h/d grazing (0700 to 1900) or unrestricted to 24 h/d grazing and fed a concentrate supplement (4.1 kg DM.cow(-1).d(-1), approximately 0.65% of BW or 33% of total DMI) either at 0700 or 1800. The experiment was repeated over three 15-d periods in May, June/July, and August 2000. The herbage on high sward height pasture was higher (P = 0.06) in NDF and ADF and lower (P < 0.01) in CP than low sward height herbage. For cows restricted to 12 h/d grazing, supplementing at 0700 as opposed 1800 resulted in greater (P = 0.04) forage DMI (8.6 vs. 8.1 kg/d), whereas cows that were unrestricted showed little change (8.2 kg/d at 0700 vs. 8.4 kg/d at 1800). Supplementing at 1800 as opposed to 0700 resulted in greater (P = 0.03) herbage DM digestibility (67.7 vs. 64.5%) for cows on high sward height, whereas cows on low sward height exhibited minimal differences (65.4% at 1800 vs. 66.3% at 0700). Cows restricted to 12 h/d grazing and supplemented at 0700 as opposed to 1800 resulted in greater (P = 0.06) digestible DMI (5.0 vs. 4.7 kg/d), whereas unrestricted cows exhibited the opposite response (4.6 kg/d digestible DMI at 0700 vs. 4.9 kg/d at 1800). Supplementing at 1800 as opposed to 0700 increased the time spent grazing to a greater (P = 0.09) extent for restricted than for unrestricted cows. When forage availability or grazing time was limiting (due to a low forage allowance and restricted access to forage, respectively) supplementing concentrates at 0700 resulted in greater forage utilization and intake rate because of increased forage DMI, DM digestibility, and digestible DMI. However, when forage or grazing time was not limiting, supplementing concentrates at 1800 resulted in greater forage utilization because of increased forage DM digestibility.

Animals↗
PubMed2005

Time of feed delivery affects the feeding and lying patterns of dairy cows.

The objective of this experiment was to determine whether it is the return from milking or delivery of fresh feed that has the greater effect on the daily patterns of feeding and lying behavior of dairy cattle. Forty-eight lactating Holstein cows were subjected to each of 2 treatments in a 2 x 2 cross-over design replicated over time. The treatments were 1) milking and feed delivery times coinciding and 2) feed delivery 6 h after milking. Cows were milked twice daily at 0500 and 1700 h. An electronic monitoring system was used to measure the time spent at the feed alley. Time-lapse video was used to quantify the lying time and incidence of aggressive displacements of the cows at the feed alley. Cows increased their total daily feeding time by 12.5% when fed 6 h after milking. This change was driven by an 82% increase in feeding time during the first hour immediately following the delivery of fresh feed and a 26% decrease in feeding time during the first hour after milking. The delivery of feed 6 h after milking did not change the daily lying time of the cows, but did decrease the latency to lie down after milking by 20 min. The reduction in feeding time after milking and decreased latency to lie down resulted in a tendency for less aggressive interactions at the feed alley after the cows returned from milking. These results indicate that the delivery of fresh feed has a greater impact on stimulating feeding behavior than does the return from milking and that changes in feeding management can affect both the feeding and lying behavior of dairy cows.

Aggression↗
PubMed2000

Transportation of goats: effects on physiological stress responses and live weight loss.

The management of food animals prior to slaughter influences both profitability and animal well-being. This experiment was conducted as a split-unit design to determine live weight shrink and stress responses in goats due to differences in stocking density during transportation and holding. A total of 150 Spanish does were transported on two different days (replicate) and held overnight (18 h) without feed in low- (LD) or high-density (HD) groups. On each day, 75 does were transported 2.5 h with floor spaces of .18 m2 and .37 m2/animal in LD (25 does) and HD (50 does) groups, respectively. The average temperatures in the trailer during transportation were 34.6 and 35 degrees C, respectively, on d 1 and 2. All animals were blood-sampled before loading (PRELOAD) and four does from each treatment were sampled immediately after loading (POSTLOAD). Animals were blood-sampled in holding pens either at 0, 1, 2, 3, 4, or 18 h after transportation (time) to assess the time course (n = 8 does per time per replicate) of stress responses. Individual animals were weighed just before loading onto a trailer and after overnight holding to assess shrinkage. Treatment or treatment x time did not have a significant effect on any of the dependent variables studied. There were significant effects of time (P < .01) on plasma cortisol, glucose, and urea nitrogen (PUN) concentrations. Time also had significant effects (P < .01) on plasma creatine kinase (CK) activity, differential leukocyte counts (neutrophils, lymphocytes, monocytes, and eosinophils), and ratio of neutrophils to lymphocytes (N:L). However, plasma leptin concentrations were not influenced by time. Cortisol concentrations increased at POSTLOAD sampling, peaked at 0 h, and decreased thereafter before spiking again at 18 h of holding. The PUN was higher at 18 h than at other time periods studied. Plasma glucose concentrations increased and remained at higher levels at 0, 1, and 2 h and began decreasing at 3 h, reaching PRELOAD levels at 18 h. Plasma CK kinase activity peaked at approximately 2 h after transportation. The N:L ratio was higher at all time periods after transportation than prior to starting the journey, indicating a prolonged effect of transportation stress on the immune system. The mean (+/- SE) shrinkage losses were 10.2 +/- .68 and 9.8 +/- .68 in HD and LD treatment groups, respectively. The results indicate that the stress responses of goats due to transportation begin decreasing within 3 h after transportation. However, prolonged holding periods without feed may increase stress responses and bring about metabolic changes.

Animals↗
PubMed2004

Comparing the solid phase and saline extract Microtox assays for two polycyclic aromatic hydrocarbon-contaminated soils.

The performance of remedial treatments is typically evaluated by measuring the concentration of specific chemicals. By adding toxicity bioassays to treatment evaluations, a fuller understanding of treatment performance is obtained. The solid phase Microtox assay is a useful tool in characterizing the toxicity of contaminated soils and sediments. This study compares the performance of the solid phase and saline extract Microtox assays in two experiments using two soils contaminated with polycyclic aromatic hydrocarbons (PAHs). The first experiment, conducted to refine the solid phase assay procedures, evaluated sample holding times, sample replication, and reference toxicant controls. The effective concentration reducing light emission by 50% (EC50) of four samples was measured with eight replicates of each sample. Samples were stored for as long as two weeks without showing substantial changes in toxicity. For future studies, three replicates of each sample are recommended because that degree of replication yielded a statistical power of more than 95% in most samples. Phenol was a reliable reference toxicant with a mean EC50 of 21.76 and a 95% confidence interval of 15.6 to 27.9 mg/L. In a second experiment, the solid phase Microtox assay was compared to saline extract Microtox assays with mixing times ranging from 5 min to 16 h. The solid phase assay was more sensitive yielding EC50s 7 to 50 times lower than the extract EC50s. In addition, the saline extract assays displayed results that varied for mixing times of less than 2 h. Based on these two experiments, the solid phase Microtox test has proved to be a useful assay for measuring the toxicity of PAH-contaminated soils.

Biological Assay↗
PubMed1981

Replication of alpha and beta globin DNA sequences occurs during early S phase in murine erythroleukemia cells.

Murine erythroleukemia cells (MELC) can be induced to express the characteristics of erythroid differentiation by a variety of agents. Previous studies indicate that an action of inducer, occurring during early S phase, may be critical to the expression of differentiated characteristics such as initiation of accumulation of newly synthesized alpha and beta globin mRNAs. In this investigation, the time of replication of globin genes in MELC was studied. DNA was isolated from synchronous populations of cells obtained by centrifugal elutriation. Newly replicated DNA sequences were prepared from synchronized cells cultured for 1 1/2 hr with 5-bromodeoxyuridine; bromodeoxyuridine-containing DNA was isolated by CsCl gradient centrifugation. By employing cloned probes for hybridization to newly synthesized DNA, it was found that alpha and beta globin gene sequences are replicated early in S phase, while ribosomal RNA gene sequences are replicated to about the same extent in early, middle, and late S phases.

Animals↗
PubMed1989

The influence of the timing of preparation on the anxiety of preschool children experiencing surgery.

A quasi-experimental design with two groups of preschool children was used to determine if surgical preparation offered at two different time periods would result in different levels of state anxiety and posthospital behavioral upset. Twenty-three children (3 to 6 years of age) were studied. The children in one group were prepared the afternoon before surgery, and the children in the other group were prepared the morning of surgery. The same surgical preparation program was given to all the children by the principal investigator. The state anxiety level of each child was assessed at six time points during the ambulatory surgical experience. The posthospital behavioral upset of each child was also measured. The parent of each child responded to a 27-item questionnaire one week after discharge. Analysis of the observation and questionnaire data indicated that the children who were prepared the afternoon before surgery did not differ significantly in state anxiety or posthospital behavioral upset from the children who were prepared the morning of surgery. Also, the levels of anxiety of the children in the two preparation groups did not vary significantly with the type of surgery. The state anxiety mean score of the children in the morning preparation group at each time point was lower than the mean score of the children in the afternoon preparation group at each time point. Replication of the study with a larger sample size is recommended.

Affect↗
PubMed1992

Involvement of membrane traffic in the replication of poliovirus genomes: effects of brefeldin A.

Brefeldin A (BFA) is a macrolide antibiotic that has multiple targets in vesicular transport and blocks membrane traffic between the cis- and trans-Golgi compartments, leading to the disruption of the trans-Golgi apparatus (for a review see Pelham, 1991, Cell 67, 449-451). Consequently, BFA interferes with the maturation of viral glycoproteins and suppresses the formation of infectious viruses that contain a lipid envelope. We report that this antibiotic strongly inhibits poliovirus replication even though this virus lacks a lipid envelope and does not encode any glycoproteins. Addition of BFA from the beginning of poliovirus infection blocks the synthesis of late proteins but has no effect on p220 cleavage, indicating that the input viral RNA is translated to produce active 2Apro. The presence of BFA at later times has no effect on poliovirus protein synthesis, indicating that this step is not a direct target for the antibiotic. Indeed, the target of BFA is viral RNA synthesis, because addition of the antibiotic at any time after poliovirus infection drastically reduces the incorporation of labeled uridine into poliovirus RNA. Both plus- and minus-stranded RNA syntheses are diminished when BFA is present from the beginning of infection, but plus-stranded RNA synthesis is more affected when the inhibitor is added at later times. The replication of poliovirus RNA takes place in close association with membrane vesicles that fill the cytoplasm of the infected cells. Little is known about the origin and function of these vesicles that form part of the viral replication complexes. Our findings suggest that the replication of poliovirus genomes may require the maturation of membranous vesicles from a vesicular compartment that is affected by BFA. The effects of BFA on late protein synthesis by other animal viruses varies according to the virus species examined. Among picornaviruses, rhinoviruses are sensitive to the antibiotic, whereas encephalomyocarditis virus is resistant. A negative-stranded RNA virus such as vesicular stomatitis is blocked by BFA, whereas vaccinia virus, a cytoplasmic DNA virus, is resistant.

Antiviral Agents↗