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At least 631 records · Page 35Linked to original sources
PubMed1997

Sampling and analytical quality control of the determination of aluminium in soybean leaves.

A pot trial was set up with nine soil types, each replicated five times, in randomized blocks, to investigate the effect of soil properties on the uptake of Al by soybean plants. The variance of the measurements was estimated using five replicates of each soil type. This then enabled the statistical significance of the difference between results for each soil type to be established. The analytical variance was determined by two analyses per pot. Systematic errors during the analytical procedure were estimated by the analysis of reagent blanks and CRMs. Analysis of variance (ANOVA) was used to estimate the proportions of total variance contributed by analysis, sampling and geochemistry. Robust statistics were required because of the high sensitivity of classical ANOVA to a small number of outlying values, due to either technical or geochemical causes. In the case study described here, traditional analytical quality control apparently gave a satisfactory result. However, the results from robust ANOVA showed that the sampling error was the limiting factor for this application. It is suggested that the environmental analyst must consider errors from procedures of both sampling and analysis of environmental samples.

Aluminum↗
PubMedDate not supplied

Analysis of the dynamics of Pneumocystis carinii in vitro.

Three culture systems employing mammalian lung cell monolayers were evaluated in terms of their ability to support the replication of primary isolates of rat-derived Pneumocystis carinii. For each system, analysis of variance was used to identify changes in the numbers of P. carinii over time. Significant replication was consistently demonstrated over lung fibroblasts (MRC-5), however, increases perceived in the culture supernatant were clearly influenced by the density of the underlying monolayer.

Animals↗
PubMed1995

[Viral hepatitis and liver transplantation].

Chronic liver diseases due to hepatitis viruses are the main indication for liver transplantation. Recurrence of viral infection on the liver graft is responsible for the occurrence of chronic hepatitis, liver cirrhosis, or liver failure. The risk of viral recurrence may influence the indications for liver transplantation. The risk of viral B recurrence is higher in patients transplanted for viral B liver cirrhosis than for viral B-delta liver cirrhosis, and for fulminant hepatitis B. This risk is higher in patients with active viral B replication at time of transplantation. Long-term administration of anti-HBs immunoglobulins reduces significantly the HBV recurrence rate and the related mortality, especially in patients without active HBV replication at transplantation. Middle-term results for patients transplanted for viral C related liver cirrhosis are good, but the recurrence rate of HCV infection is high. Protocols of prevention of HCV recurrence after transplantation are mandatory.

Hepatic Encephalopathy↗
PubMed2004

Real-time monitoring of intracellular Staphylococcus aureus replication.

A high-throughput system to rapidly assess the intracellular replication of Staphylococcus aureus has been developed utilizing S. aureus transformed with a dual gfp-luxABCDE reporter operon under the control of a growth-dependent promoter. Replication of tagged bacteria internalized into bovine mammary epithelial cells (MAC-T) could be measured by monitoring fluorescence and bioluminescence from the reporter operon following removal of extracellular bacteria from the plates. Bacterial replication inside cells was confirmed by a novel ex vivo time-lapse confocal microscopic method. This assay of bacterial replication was used to evaluate the efficacy of antibiotics which are commonly used to treat staphylococcal infections. Not all antibiotics tested were able to prevent intracellular replication of S. aureus and some were ineffective at preventing replication of intracellular bacteria at concentrations above the MIC determined for bacteria in broth culture. Comparison of the fluorescence and bioluminescence signals from the bacteria enabled effects on protein synthesis and metabolism to be discriminated and gave information on the entry of compounds into the eukaryotic cell, even if bacterial replication was not prevented. Elevated resistance of S. aureus to antibiotics inside host cells increases the likelihood of selecting S. aureus strains which are resistant to commonly used antimicrobial agents within the intracellular niche. The approach presented directly assesses intracellular efficacy of antibiotics and provides an evidence-based approach to antibiotic selection for prescribing physicians and medical microbiologists.

Animals↗
PubMedDate not supplied

Analysis of the bacterial cell cycle using strains in which chromosome replication is controlled by plasmid R1.

As an alternative approach in the study of the Escherichia coli cell cycle, we have constructed strains in which chromosome replication is under the control of various plasmid R1 derivatives, IntR1 strains. The physiological properties of such strains are described. In intR1 strains, chromosome replication can be manipulated independently of cell-cycle-related control mechanisms, and the effects on cell division can be analysed. Using this approach, we have found that the timing of replication during the cell cycle is random in intR1 strains, that overreplication of the chromosome is lethal, and that chromosome replication does not trigger cell division. Current investigations include the study of the E. coli cell cycle in the absence of the DnaA protein, the effect on cell division of a specific inhibition of the initiation of chromosome replication, and the molecular basis of uni- and bidirectional replication.

Cell Cycle↗
PubMed2004

Role of the proximal enhancer of the major immediate-early promoter in human cytomegalovirus replication.

The human cytomegalovirus (CMV) enhancer has a distal component (positions -550 to -300) and a proximal component (-300 to -39) relative to the transcription start site (+1) of the major immediate-early (MIE) promoter. Without the distal enhancer, human CMV replicates slower and has a small-plaque phenotype. We determined the sequence requirements of the proximal enhancer by making 5'-end deletions to positions -223, -173, -116, -67, and -39. Even though recombinant virus with the proximal enhancer deleted to -39 has the minimal TATA box-containing MIE promoter element, it cannot replicate independently in human fibroblast cells. Recombinant virus with a deletion to -67 has an Sp-1 transcription factor binding site which may represent a minimal enhancer element for recombinant virus replication in human fibroblast cells. Although recombinant virus with a deletion to -223 replicates to titers at least 100-fold less than that of the wild-type virus, it replicates to titers 8-fold higher than that of recombinant virus with a deletion to -173 and 20-fold higher than that of virus with a deletion to -67. Recombinant virus with a deletion to -173 replicates more efficiently than that with a deletion to -116. There was a direct correlation between the level of infectious virus replication and time after infection, amount of MIE gene transcription, MIE and early viral protein synthesis, and viral DNA synthesis. The extent of the proximal enhancer determines the efficiency of viral replication.

Activating Transcription Factors↗
PubMed2001

Granulosa cells of the cumulus oophorus are different from mural granulosa cells in their response to gonadotrophins and IGF-I.

There are two types of granulosa cells: those which surround the oocyte are cumulus cells (CC) and those which surround the antrum are mural granulosa cells (MGC). These cells are under the influence of several hormones and growth factors, the most important of which are gonadotrophins and IGF-I. In this article, we report novel observations on the differences between these two types of granulosa cells and their interaction with gonadotrophins and IGF-I. We were able to conduct physiological studies on the role of IGF-I by using an analogue of IGF-I which does not bind to IGF-I-binding proteins (LR3-IGF-I). Immature rats received saline, equine chorionic gonadotrophin (eCG), LR3-IGF-I or eCG plus LR3-IGF-I by infusion using a pump from 24-29 days of age. The rats were killed and the ovaries removed. Surface follicles were punctured and MGC and oocyte cumulus complexes were removed. These were cultured in saline (control) and in three different doses of FSH. Cell replication was assessed by 3H-thymidine incorporation and differentiation was evaluated by the measurement of progesterone secretion. It was noted that CC replicated ten times more than MGC. Similarly, progesterone secretion by CC was six times more than by MGC. In vivo exposure to gonadotrophins (eCG) positively influenced in vitro treatment with FSH in both cell types. This phenomenon was observed in both cell replication and progesterone secretion. The IGF-I analogue had a positive effect on cell replication of MGC but a negative effect on the cell replication of CC. With respect to progesterone secretion, the IGF-I analogue had a negative effect on CC but a positive effect on MGC. In conclusion, CC behaved differently from MGC in response to gonadotrophins and the IGF-I analogue. IGF-I and FSH acted additively, synergistically or antagonistically in different circumstances.

Analysis of Variance↗
PubMed1995

Design and analysis of in vitro antitumor pharmacodynamic studies.

The relationship between drug concentration (C), exposure time (t), and the resulting effect (h) for a chemotherapeutic agent is expressed as Cn x t = h. The value of n, derived from curve fitting of the C versus t plot, indicates the relative importance of concentration and exposure time. The selection of concentrations and exposure times in a pharmacodynamic experiment may affect the precision and accuracy of parameter estimation. The use of optimal designs is even more critical when the numbers of experimental conditions are limited by tumor availability (e.g., small size of surgical specimens from patients). The present study used computer-simulated data to define the most efficient in vitro pharmacodynamic experimental designs and the optimal method of pharmacodynamic data analysis. All studies used Monte Carlo simulations to compare designs with varying numbers of drug concentrations, exposure times, and replications. For each selected design, 50-100 error-containing data sets were created by addition of experience-based random errors to expected concentration-response profiles. To compare methods of data analysis, the same 1250 simulated data sets were analyzed by two methods (i.e., surface response method and traditional method). The results showed that simultaneous fitting of drug effect at all concentrations and all exposure times by the surface response method yielded n estimates that had greater precision and accuracy than a traditional method that required sequential determination of the effective inhibitory concentration (e.g., IC50) and then the n value using the IC50 at different exposure times. Subsequent studies were analyzed using the surface response method. To evaluate the effect of selection of concentrations and exposure times on the precision of n estimation, between 1100 and 2200 simulated data sets, with 400 observations per data set, were generated using different exposure times and drug concentrations. Because the number of observations was limited to 400, the number of replications at each condition varied depending on the total number of selected conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Antineoplastic Agents↗
PubMed2005

Detection and quantitation of HPV DNA replication by Southern blotting and real-time PCR.

This provides a brief introduction into the mechanism of DNA replication by the E1 and E2 proteins and describes the traditional Southern blotting technique that is used to monitor E1- and E2-mediated DNA replication. It also includes a novel real-time polymerase chain reaction (PCR) approach for monitoring E1- and E2-mediated DNA replication that has enhanced sensitivity and quantitation compared with Southern blotting, and a discussion of when to use the Southern blotting and real-time PCR techniques.

Base Sequence↗
PubMed2005

Stimulation of poliovirus synthesis in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro.

The plus-strand RNA genome of poliovirus serves three distinct functions in the life cycle of the virus. The RNA is translated and then replicated, and finally the progeny RNAs are encapsidated. These processes can be faithfully reproduced in a HeLa cell-free in vitro translation-RNA replication system that produces viable poliovirus. We have previously observed a stimulation of virus synthesis when an mRNA, encoding protein 3CD(pro), is added to the translation-RNA replication reactions of poliovirus RNA. Our aim in these experiments was to further define the factors that affect the stimulatory activity of 3CD(pro) in virus synthesis. We observed that purified 3CD(pro) protein also enhances virus synthesis by about 100-fold but has no effect on the translation of the polyprotein. Optimal stimulation is observed only when 3CD(pro) is present early in the incubation period. The stimulation, however, is abolished by a mutation either in the RNA binding domain of 3CD(pro), 3C(pro)R84S/I86A, or by each of two groups of complementary mutations R455A/R456A and D339A/S341A/D349A at interface I in the 3D(pol) domain of 3CD(pro). Surprisingly, virus synthesis is strongly inhibited by the addition of both 3C(pro) and 3CD(pro) at the beginning of incubation. We also examined the effect of other viral or cellular proteins on virus synthesis in the in vitro system. No enhancement of virus synthesis occurred with viral proteins 3BC, 3ABC, 3BCD, 3D(pol), and 3C(pro) or with cellular protein PCBP2. These results suggest that 3CD(pro) has to be present in the reaction at the time the replication complexes are assembled and that both the 3C(pro) and 3D(pol) domains of the protein are required for its activity that stimulates virus production.

3C Viral Proteases↗
PubMedDate not supplied

Ultrastructural study of avian synovium infected with an arthrotropic reovirus.

Three-week-old chicks were inoculated via the foot pad with the avian reovirus (strain WVU 2937) that has been proved to be the causative agent in avian viral arthritis. The initial stages of the avian reovirus infection in the synovium of the hock joint were studied by transmission electron microscopy to shed light on the early virus-host relationship of an arthrotropic virus and synovial tissue. At 48 hours postinoculation (PI), coated, partially coated, and uncoated virus particles were seen in a dense lysosome-like inclusion in the cytoplasm of subsynovial fibroblasts. Virus replication was demonstrated in fibroblasts of the subsynovium 4 days PI. Infiltration of leukocytes into the area of the virus replication was noted at the time of virus replication (4 days PI). By the second week of the infection (11 days PI), the subsynovium was heavily infiltrated with lymphocytes and plasma cells, and proliferative changes had occurred in the synovial lining cells.

Animals↗
PubMed2004

Set1 is required for meiotic S-phase onset, double-strand break formation and middle gene expression.

The Set1 protein of Saccharomyces cerevisiae is a histone methyltransferase (HMTase) acting on lysine 4 of histone H3. Inactivation of the SET1 gene in a diploid leads to a sporulation defect. We have studied various processes that take place during meiotic differentiation in set1delta diploid cells. The absence of Set1 leads to a delay of meiotic S-phase onset, which reflects a defect in DNA replication initiation. The timely induction of meiotic DNA replication does not require the Set1 HMTase activity, but depends on the SET domain. In addition, set1delta displays a severe impairment of the DNA double-strand break formation, which is not only the consequence of the replication delay. Transcriptional profiling experiments show that the induction of middle meiotic genes, but not of early meiotic genes, is affected by the loss of Set1. In contrast to meiotic replication, the transcriptional induction of the middle meiotic genes appears to depend on the methylation of H3-K4. Our results unveil multiple roles of Set1 in meiotic differentiation and distinguish between HMTase-dependent and -independent Set1 functions.

DNA Damage↗
PubMed2005

Leishmania (Viannia) braziliensis growth in vitro culture relies more on folic acid availability than Leihsmania (Leishmania) amazonensis.

We compared the in vitro growth of promastigotes from two Leishmania species in TC-100 and Schneider media. Leishmania (Leishmania) amazonensis replication rates were similar in both tissue culture media and reached maximum rates by 48 h. In contrast Leishmania (Viannia) braziliensis growth was significantly greater in TC-100 but maximum rates were achieved by 96 h. Folic acid appears to be the limiting factor and supplementation of Schneider media with this nutrient improved L. (V.) braziliensis replication rates and decreased the time of maximum replication to 48 h.

Animals↗
PubMed1986

Individual differences in auditory electric responses: comparisons of between-subject and within-subject variability. I. Absolute latencies of brainstem vertex-positive peaks.

Seven subjects were tested, each on eight separate sessions, for brainstem auditory evoked responses to monaural right, monaural left, and binaural stimulus presentations. Comparisons of between-subject vs. within-subject variability of the absolute latencies of vertex-positive peaks expressed in terms of the coefficient of variation indicate that: within-subject stability is greater than between-subject stability for the five brainstem peaks; between-subject variability shows significant differences due to peak but not to ear of presentation; within-subject variability shows significant differences due to both peak and ear; comparisons of within-subject variability over time show significant differences due to peak but not to time; patterns of individual variation within the brainstem series are characterized by increases in stability of peak latencies over time, and by replicability of stability profiles over time. Other measures of latency and amplitude based on this series of responses are planned for a subsequent report.

Brain Stem↗
PubMed2005

A polymeric master replication technology for mass fabrication of poly(dimethylsiloxane) microfluidic devices.

A protocol of producing multiple polymeric masters from an original glass master mold has been developed, which enables the production of multiple poly(dimethylsiloxane) (PDMS)-based microfluidic devices in a low-cost and efficient manner. Standard wet-etching techniques were used to fabricate an original glass master with negative features, from which more than 50 polymethylmethacrylate (PMMA) positive replica masters were rapidly created using the thermal printing technique. The time to replicate each PMMA master was as short as 20 min. The PMMA replica masters have excellent structural features and could be used to cast PDMS devices for many times. An integration geometry designed for laser-induced fluorescence (LIF) detection, which contains normal deep microfluidic channels and a much deeper optical fiber channel, was successfully transferred into PDMS devices. The positive relief on seven PMMA replica masters is replicated with regard to the negative original glass master, with a depth average variation of 0.89% for 26-microm deep microfluidic channels and 1.16% for the 90 mum deep fiber channel. The imprinted positive relief in PMMA from master-to-master is reproducible with relative standard deviations (RSDs) of 1.06% for the maximum width and 0.46% for depth in terms of the separation channel. The PDMS devices fabricated from the PMMA replica masters were characterized and applied to the separation of a fluorescein isothiocyanate (FITC)-labeled epinephrine sample.

Dimethylpolysiloxanes↗
PubMed1973

Replication of deoxyribonucleic acid during the division cycle of Salmonella typhimurium.

The rate of thymidine incorporation into cells of Salmonella typhimurium growing in different media has been measured. In glucose-minimal medium, deoxyribonucleic acid (DNA) replication occurs during the first two-thirds of the division cycle; the final one-third of the division cycle was devoid of DNA replication. The measured doubling time of S. typhimurium in this medium is approximately 48 min, indicating that C (the time for a round of replication) and D (the time between termination and cell division) are approximately 32 and 16 min, respectively. At slower growth rates the pattern of replication is the same as glucose minimal medium. At faster growth rates the "gap" in DNA synthesis disappears. At rapid growth rates evidence for multiple forks is obtained.

Carbon Isotopes↗
PubMed1987

[Genetic transformation of somatic cells. XII. Mutations in human satellite DNA introduced into Chinese hamster cells].

Data are presented about detailed restriction mapping, hybridization and sequence analysis of plasmid pRT301 rescued into E. coli 25-30 cell divisions after transfection of Chinese hamster cells. This plasmid contains an insert of human satellite III fragment (HS3), which was replicated many times in mammalian cells, and we found multiple point mutations in this non-selectable fragment, mainly single inserts of GC base pair. Specificity of mutagenesis of HS3 induced by transfection and replication in Chinese hamster cells differs from mutagenesis specificity found by other authors for selectable genes.

Animals↗
PubMed1986

Cell cycle-specific replication of Escherichia coli minichromosomes.

The timing of Escherichia coli minichromosome replication in the cell division cycle was examined using an improved procedure for studying plasmid replication frequency. Cultures growing exponentially in glucose/Casamino acids minimal medium were pulse-labeled with [3H]thymidine, and the radioactivity incorporated into plasmid DNA in cells of different ages was analyzed. At the end of the labeling period the bacteria were bound to the surface of a nitrocellulose membrane filter, and the radioactivity in new daughter cells, which eluted continuously from the membrane, was quantitated following agarose gel electrophoresis. The minichromosomes replicated during a discrete interval in the cell division cycle that appeared to coincide with initiation of chromosome replication. In contrast, plasmid pBR322 replicated throughout the division cycle at a rate that increased gradually as a function of cell age. The difference in minichromosome and pBR322 replication was clearly discernible in cells harboring both plasmids. It was also found that the 16 kD gene adjacent to oriC was not a determinant of the timing of minichromosome replication during the division cycle. The results are consistent with the conclusion that minichromosome replication frequency is governed by the same mechanism that controls chromosome replication.

Cell Cycle↗