Biomedical subjects

T Yan

Publications and source records attributed to T Yan.

At least 19 recordsLinked to original sources

PubMed2006

Stability in a denitrifying fluidized bed reactor.

This study evaluates changes in the microbial community structure and function of a pilot-scale denitrifying fluidized bed reactor during periods of constant operating conditions and periods of perturbation. The perturbations consisted of a shutdown period without feed, two disturbances in which biofilms were mechanically sheared from carrier particles, and a twofold step increase in feed nitrate concentration. In the absence of perturbations, nitrate removal was stable and consistently greater than 99%. The structure and dynamics of the microbial community were studied using cloning and sequencing techniques and terminal restriction fragment length polymorphism (T-RFLP) of the SSU rRNA gene. Under unperturbed operating conditions, stable function was accompanied by high constancy and low variability of community structure with the majority of terminal restriction fragments (T-RFs) appearing throughout operation at consistent relative abundances. Several of the consistently present T-RFs correlated with clone sequences closely related to Acidovorax (98% similarity), Dechloromonas (99% similarity), and Zoogloea (98% similarity), genera recently identified by molecular analyses of similar systems. Significant changes in community structure and function were not observed after the shutdown period. In contrast, following the increase in loading rate and the mechanical disturbances, new T-RFs appeared. After both mechanical disturbances, function and community structure recovered. However, function was much more resilient than community structure. The similarity of response to the mechanical disturbances despite differences in community structure and operating conditions suggests that flexible community structure and potentially the activity of minor members under nonperturbation conditions promotes system recovery.

Biodegradation, Environmental↗

PubMed2006

Phylogenetic and functional biomakers as indicators of bacterial community responses to mixed-waste contamination.

Few studies have demonstrated changes in community structure along a contaminant plume in terms of phylogenetic, functional, and geochemical changes, and such studies are essential to understand how a microbial ecosystem responds to perturbations. Clonal libraries of multiple genes (SSU rDNA, nirK, nirS, amoA, pmoA, and dsrAB) were analyzed from groundwater samples (n = 6) that varied in contaminant levels, and 107 geochemical parameters were measured. Principal components analyses (PCA) were used to compare the relationships among the sites with respect to the biomarker (n = 785 for all sequences) distributions and the geochemical variables. A major portion of the geochemical variance measured among the samples could be accounted for by tetrachloroethene, 99Tc, No3, SO4, Al, and Th. The PCA based on the distribution of unique biomarkers resulted in different groupings compared to the geochemical analysis, but when the SSU rRNA gene libraries were directly compared (deltaC(xy) values) the sites were clustered in a similar fashion compared to geochemical measures. The PCA based upon functional gene distributions each predicted different relationships among the sites, and comparisons of Euclidean distances based upon diversity indices for all functional genes (n = 432) grouped the sites by extreme or intermediate contaminant levels. The data suggested that the sites with low and high perturbations were functionally more similar than sites with intermediate conditions, and perhaps captured the overall community structure better than a single phylogenetic biomarker. Moreover, even though the background site was phylogenetically and geochemically distinct from the acidic sites, the extreme conditions of the acidic samples might be more analogous to the limiting nutrient conditions of the background site. An understanding of microbial community-level responses within an ecological framework would provide better insight for restoration strategies at contaminated field sites.

Bacteria↗

PubMed2006

Small intestinal submucosa improves islet survival and function in vitro culture.

INTRODUCTION: Most centers maintain isolated human islet preparations in tissue culture to improve the safety as well as the practicality of islet transplantation. However, maintaining viability and recovery of islets remains a challenge. Extracellular matrix (ECM) is one of the most important components of the islet microenvironment. Reconstruction of the cell-matrix relationship seems to be necessary to sustain the structure and function of differentiated islets. Small intestinal submucosa (SIS), a natural ECM, is well known to promote wound healing, tissue remodeling, and cell growth. The purpose of this study was to evaluate recovery and function of isolated rat pancreatic islets during in vitro culture with SIS. METHODS: Pancreatic islets isolated from Wistar rats following intraductal collagenase distension, mechanical dissociation, and EuroFicoll purification were cultured in plates coated with multilayer SIS (SIS-treated group) or without (standard cultured group) for 7 or 14 days in an islet culture media of RPMI 1640 (Gibco). The islets from both experimental groups were stained and counted with dithizone. Islet recovery following culture was determined by the ratio of counts after culture to the yield of islets immediately following islet isolation. The viability of the islets was assessed by a glucose challenge test with low glucose (2.7 mmol/L), high glucose (16.7 mmol/L), and high glucose solution supplemented with 50 micromol/L 3-isobutyl-1-methylxanthine solution. The apoptosis of islet cells was measured by relative quantification of histone-complexed DNA fragments by using enzyme-linked immunosorbent assay. RESULTS: After 7 or 14 days of in vitro tissue culture, the recovery in SIS-treated islets group was about double of that cultured in the plates without SIS coating. In the SIS-treated group, there was no significant difference between the short- and the long-term periods of culture (95.8%+/-1.0% vs 90.8%+/-1.5%, P>.05). Following incubation with high glucose (16.7 mmol/L) solution, the insulin secretion in the SIS-treated group showed a greater increase than the control group after 14 days of culture (20.7+/-1.1 mU/L vs 11.8+/-1.1 mU/L, P<.05). When islets were placed in the high glucose solution containing IBMX, the stimulated insulin secretion was more increased in the SIS-treated than in the control group despite the duration of the culture. The calculated stimulation index of SIS-treated group was about two to three times greater than the control group. In addition, the stimulation index of the SIS-treated group remained constant regardless of short-term versus long-term culture (9.5+/-0.2 vs 10.2+/-1.2, P>.05). Much less apoptosis of islet cells occurred in the SIS-treated than in the control group. CONCLUSION: Coculture of isolated rat islets with native sheetlike small intestinal submucosa seemed to build an ECM for islets providing possible biotrophic and growth factors that promote the recovery and subsequent function of islets.

Animals↗

PubMed2006

Effects of dairy cow genotype with two planes of nutrition on energy partitioning between milk and body tissue.

The data used in the present study were derived from a 2 (genotype) x 2 (plane of nutrition) factorial design production study using Holstein-Friesian (n = 32) and Norwegian (n = 32) first-lactation dairy cattle offered grass silage-based diets from 1 to 44 wk of lactation. The high nutrition diet had concentrate inclusions (g/kg of dry matter) of 600, 500, and 400 for lactation days of < 101, 101 to 200, and > 200, respectively, and the low nutrition diet included concentrates at 300, 200, and 100 for the same periods. Dietary metabolizable energy (ME) concentrations were measured in calorimetric chambers at lactation d 80, 160, and 240 respectively, and then applied to production data to calculate ME intake. From wk 1 to 44 of lactation, Holstein-Friesian cows had a consistently lower accumulated live weight gain and body condition score, and a consistently higher ME intake and milk energy output than Norwegian cows, irrespective of the plane of nutrition. Compared with Norwegian cows using mean data derived from the 2 planes of nutrition, Holstein-Friesian cows produced a significantly higher proportion of milk energy output over ME intake in early and mid lactation, although this increase was not significant in late lactation. In contrast, Holstein-Friesian cows partitioned a significantly lower proportion of ME intake into body tissue than Norwegian cows in early lactation, although the differences were not significant in mid or late lactation. When ME intake and energy used for maintenance, milk, and body tissue were taken into account, the efficiency of ME use for lactation was similar between the 2 genotypes offered the high or low concentrate diet during the whole lactation. It is concluded that Holstein-Friesian cows can produce more milk energy than Norwegian cows, mainly as a result of higher ME intake and because of a greater ability to partition more energy into milk and less into body tissue. The effect on energy partitioning mainly occurs in early and midlactation and is particularly evident with high concentrate diets.

Animal Nutritional Physiological Phenomena↗

PubMed2006

Relationships among manure nitrogen output and dietary and animal factors in lactating dairy cows.

A large data set derived from total diet digestibility assessments on lactating dairy cows (535 Holstein-Friesian and 29 Norwegian) was used to examine effects of dietary and animal factors on manure (feces and urine) nitrogen (N) output and to develop mitigation strategies and prediction equations for manure N output in lactating dairy cows. Manure N output was positively and significantly related to live weight, milk yield, dietary crude protein (CP) concentration, dry matter intake, and N intake. Reducing the dietary CP concentration or increasing the milk yield decreased manure N output per kilogram of milk yield. Prediction equations for manure N output using live weight and milk yield, either alone or combined, had relatively low R2 (0.227 to 0.474) and large standard error (70.6 to 85.6) values. Addition of dietary CP concentration to these relationships considerably increased R2 to 0.754 and reduced the standard error to 48.2. Relating manure N output to N intake produced a very high r2 (0.901) and a very low standard error (30.6). The addition of live weight and milk yield to this relationship as supporting predictors only marginally increased R2 to 0.910 and reduced the standard error to 29.3. The internal validation of these equations revealed that use of N intake as the primary predictor produced a very accurate prediction of manure N output. In situations in which data on N intake are not available, prediction equations based on dietary CP concentration, live weight, and milk yield together can produce a relatively accurate assessment of manure N output.

Animal Feed↗

PubMed2005

Confidence intervals of similarity values determined for cloned SSU rRNA genes from environmental samples.

The goal of this research was to investigate the influence of the error rate of sequence determination on the differentiation of cloned SSU rRNA gene sequences for assessment of community structure. SSU rRNA cloned sequences from groundwater samples that represent different bacterial divisions were sequenced multiple times with the same sequencing primer. From comparison of sequence alignments with unedited data, confidence intervals were obtained from both a 'double binomial' model of sequence comparison and by non-parametric methods. The results indicated that similarity values below 0.9946 are likely derived from dissimilar sequences at a confidence level of 0.95, and not sequencing errors. The results confirmed that screening by direct sequence determination could be reliably used to differentiate at the species level. However, given sequencing errors comparable to those seen in this study, sequences with similarities above 0.9946 should be treated as the same sequence if a 95% confidence is desired.

Base Sequence↗

PubMed2005

Relationships between urea dilution measurements and body weight and composition of lactating dairy cows.

The objective of the present study was to investigate the potential of the urea dilution technique, coupled with live animal measures to predict the body components of dairy cattle. The study involved 104 lactating Holstein-Friesian cows offered grass silage-based diets. Urea space volume (USV) was calculated from 2 collection periods of blood samples following infusion of urea at 12 (USV12, kg) and 30 (USV30, kg) min after infusion, and then as a proportion of live weight (LW) or empty body weight (EBW). All cows were slaughtered within 2 d of the USV trials. Large ranges existed in EBW and empty body concentrations of water, crude protein (CP), lipid, ash, and gross energy (GE). The USV12 and USV30 were both positively related to LW, EBW, and empty body component weights. The r2 values for USV12 were greater than USV30. The r2 values in the relationships of EBW and empty body composition with USV, however, were smaller than those with LW. Nevertheless, the relationships were improved when both USV and LW were used as predictors, rather than using either alone. Adding milk yield and body condition score as supporting predictors to prediction equations using USV and LW data for EBW, lipid, and GE contents further improved the relationships (r2 = 0.93, 0.66, and 0.77, respectively). Internal evaluation of one-third of the present data using equations developed from two-thirds of the present data indicated that using USV, live weight, and other live animal variables as predictors, rather than using USV alone, considerably improved the prediction accuracy. It was concluded that USV can be used to predict body composition, but the relationships with USV were poorer than those with LW. The USV can only be used as a supporting variable to live weight for prediction of body components in lactating dairy cows.

Animals↗

PubMed2004

Experimental and simulation study of neon collision dynamics with a 1-decanethiol monolayer.

A study of the energy accommodation of neon colliding with a crystalline self-assembled 1-decanethiol monolayer adsorbed on Au(111) is presented. The intensity and velocity dependencies of the scattered neon as a function of incident angle and energy were experimentally measured. Scattering calculations show good agreement with these results, which allows us to examine the detailed dynamics of the energy and momentum exchange at the surface. Simulation results show that interaction times are, at most, a few picoseconds. Even for these short times, energy exchange with the surface, both normal and in-plane, is very rapid. An important factor in determining the efficiency of energy exchange is the location at which the neon collides with the highly corrugated and structurally dynamic unit cell. Moreover, our combined experimental and theoretical results confirm that these are truly surface collisions in that neon penetration into the organic boundary layer does not occur, even for the highest incident energies explored, 560 meV.

Journal Article↗

PubMed2004

A conical piezoelectric transducer with integral sensor as a self-calibrating acoustic emission energy source.

The experimental results of a conical piezoelectric transducer with integral backing sensor as a self-calibrating simulated acoustic emission (SAE) energy source are presented. It has been shown that there is a negative linear relationship between the energy of SAE signal detected by the backing sensor and the relative strength (signal energy) of SAE source in the structure detected by a reference sensor under different transducer-to-structure coupling efficiencies, with AC drives of the same wave packet, frequency and peak amplitude to excite the conical transducer as a SAE source at all the investigated frequencies over the frequency range of interest in AE measurements (nominally from 50 kHz to around 1 MHz). This should enable the relative strength of the SAE source in a structure to be determined using the SAE measurement from the backing sensor for the selected electrical inputs to the conical transducer, and hence to remove the concerns about inconsistent transducer-to-structure coupling affecting the relative strength of the SAE source for calibration.

Journal Article↗

PubMed2004

Coupling of functional gene diversity and geochemical data from environmental samples.

Genomic techniques commonly used for assessing distributions of microorganisms in the environment often produce small sample sizes. We investigated artificial neural networks for analyzing the distributions of nitrite reductase genes (nirS and nirK) and two sets of dissimilatory sulfite reductase genes (dsrAB1 and dsrAB2) in small sample sets. Data reduction (to reduce the number of input parameters), cross-validation (to measure the generalization error), weight decay (to adjust model parameters to reduce generalization error), and importance analysis (to determine which variables had the most influence) were useful in developing and interpreting neural network models that could be used to infer relationships between geochemistry and gene distributions. A robust relationship was observed between geochemistry and the frequencies of genes that were not closely related to known dissimilatory sulfite reductase genes (dsrAB2). Uranium and sulfate appeared to be the most related to distribution of two groups of these unusual dsrAB-related genes. For the other three groups, the distributions appeared to be related to pH, nickel, nonpurgeable organic carbon, and total organic carbon. The models relating the geochemical parameters to the distributions of the nirS, nirK, and dsrAB1 genes did not generalize as well as the models for dsrAB2. The data also illustrate the danger (generating a model that has a high generalization error) of not using a validation approach in evaluating the meaningfulness of the fit of linear or nonlinear models to such small sample sizes.

Bacteria↗

PubMed2004

Prediction of nutritive values in grass silages: I. Nutrient digestibility and energy concentrations using nutrient compositions and fermentation characteristics.

Grass silages (n = 136) were selected from commercial farms across Northern Ireland according to their pH, ammonia nitrogen, DM, and predicted ME concentration. Each silage was offered to four sheep as a sole feed at maintenance feeding level to determine nutrient digestibility and urinary energy output. Dry matter concentration was determined as alcohol-corrected toluene DM and was subsequently used as the basis for all nutrient concentrations. The objectives were to use these data to examine relationships between nutritive value and nutrient concentration or fermentation characteristics in silages and then develop prediction equations for silage nutritive values using stepwise multiple regression techniques. The silages had a large range in quality (DM = 15.5 to 41.3%, ME = 7.7 to 12.9 MJ/kg of DM, pH = 3.5 to 5.5) and a relatively even distribution over the range. There was a positive relationship (P < 0.001) between silage GE and DE or ME concentration. Digestible OM in total DM (DOMD); ME/GE; and digestibility of DM, OM, and GE were positively related (P < 0.05) to CP, soluble CP, ether extract, lactic acid concentration, and lactic acid/ total VFA, whereas they were negatively related (P < 0.05) to ADF, NDF, lignin, individual VFA concentration, pH, and ammonia N/total N. Concentrations of DE and ME and digestibility of CP and NDF had similar relationships with those variables, although some relationships were not significant. Three sets of multiple prediction equations for DE and ME concentration; ME/ GE; DOMD; and digestibility of DM, OM, GE, CP, and NDF were therefore developed using three sets of predictors. The first set included GE, CP, soluble N/total N, DM, ash, NDF, lignin, lactic acid/total VFA, and ammonia N/total N; the second set excluded soluble N/ total N and lignin because they are not typically measured; the third set further excluded the fermentation data. The R2 values generally decreased with exclusion of predictors. The second and third sets of equations, except for NDF digestibility, were validated using the mean-square-prediction-error model and an independent grass silage data set published since 1977 (n = 17 [DM digestibility] to 28 [DOMD and OM digestibility]). The validation indicated that the equations developed in the present experiment could accurately predict DE and ME concentrations and DE/GE and ME/GE in grass silages.

Animal Feed↗

PubMed2004

Prediction of nutritive values in grass silages: II. Degradability of nitrogen and dry matter using digestibility, chemical composition, and fermentation data.

One hundred thirty-six perennial rye-grass silages with wide variations in quality were evaluated for N and DM degradability in three beef steers offered grass silage of medium quality ad libitum. The silages were incubated in the rumen of each animal in triplicate for 0, 6, 12, 24, 48, and 72 h, respectively. The disappearance rates of N or DM were used to calculate the readily soluble fraction ("a" value), potentially degradable fraction ("b" value), and the fractional degradation rate of"b" ("c" value). The effective degradability (P) of N or DM was then estimated assuming a ruminal outflow rate of 0.02, 0.05, or 0.08/h (P0.02, P0.05, or P0.08). The objective was to use these data to develop prediction equations for N and DM degradability in grass silages. There were considerable variations in "a," "b," and "c" values and the P0.02, P0.05, or P0.08 of N and DM (e.g., the P0.02 of N ranged from 75.0 to 93.4% and the P0.02 of DM from 51.5 to 82.5%). The P0.02, P0.05, or P0.08 of N and DM were negatively related (P < 0.001) to ADF, NDF, and lignin concentrations but positively related (P < 0.001) to protein fractions (CP, soluble CP, and true protein concentrations) and digestibility of DM, OM, GE, CP, and NDF and digestible OM in the total DM (measured with sheep). The N and DM degradability data were also positively related to silage lactic acid concentration, but the relationships between DM degradability data and pH, ammonia N/total N, and VFA concentration in silages were negative (P < 0.05). Several sets of prediction equations (linear and multiple) were thus developed for N and DM degradability using CP or NDF concentration, "a" value or digestibility data as primary predictors, together with or without other nutrient concentration and silage fermentation variables. All these relationships were highly significant (P < 0.001), and each predictor had a significant effect on the relationship (P < 0.05). The R2 values in multiple regression for N and DM degradability were generally over 0.70 and higher than in linear regression equations. Four equations were also developed to convert N and DM degradability at a given ruminal outflow rate, predicted using the above-mentioned equations, to their counterparts at any ruminal outflow rate (0.02 to 0.10/h), respectively.

Animals↗

PubMed2004

Short communication: Effects of feeding level on energy concentration in grass silage-based diets offered to dairy cattle.

Twelve grass silages were offered to sheep as a sole diet at maintenance and to lactating dairy cows ad libitum as mixed silage and concentrates diets (n = 13 diets). Fecal and urinary energy outputs were measured for silages and mixed diets. Digestible energy (DE) and metabolizable energy (ME) concentrations for mixed diets with sheep at maintenance were estimated based on the silage dry matter (DM) proportion obtained in the cattle trials, the silage energy utilization values (methane energy-predicted) determined using sheep, and tabulated concentrate values. A comparison of dietary mean data (n = 13) indicated that concentrations of ME (P < 0.01) and DE (P < 0.001) in mixed diets were significantly lower for cows at production feeding level than for sheep at maintenance. The reductions were proportionately 0.015 and 0.020 with each unit increase in feeding level above maintenance, respectively. These ME and DE data were also used to evaluate the feeding level correction factors previously proposed by Van Es (1975) (ME, 0.018) and Yan et al. (2002) (ME, 0.016; DE, 0.025) using the mean square prediction error technique. The ME correction factor proposed by Yan et al. (2002) had a greater prediction accuracy than that proposed by Van Es (1975) for the prediction of ME concentration in mixed diets offered to dairy cattle at production feeding level.

Animal Nutritional Physiological Phenomena↗

PubMed2003

Evaluation of different energy feeding systems with production data from lactating dairy cows offered grass silage-based diets.

A set of data from 838 lactating dairy cows, drawn from 12 long-term feeding studies (at least 8 wk/period), was used to evaluate the energy feeding systems for dairy cows currently adopted in Australia, France, The Netherlands, the United Kingdom, and the United States. The animals were offered mixed diets of concentrates, forage [grass silages (n = 33) and corn silages (n = 5)] ad libitum. Data used in the present evaluation were either measured [dry matter (DM) intake, milk production and live weight], measured/estimated [dietary metabolizable energy (ME) concentration] or estimated [milk energy output and live weight change (LWC)]. The mean-square prediction error (MSPE) was used for the evaluation. Total ME intake, milk yields, and LWC varied from 91 to 338 MJ/d, 7.7 to 48.9, and -1.23 to 1.73 kg/d, respectively. Australian and French systems predicted total energy requirement and milk yield relatively well, while British, Dutch and American systems underpredicted total energy requirement by proportionately 0.06, 0.04, and 0.03, respectively; and overpredicted milk yield by 0.09, 0.06, and 0.04. The Agricultural and Food Research Council (AFRC) each produced a relatively larger error of the bias (predicted - actual data) over the total MSPE for ME requirement and milk yield and a relatively smaller error of random than other systems. However, an addition of proportionately 0.05 to the total predicted ME requirement of AFRC, as suggested in this system and currently used in the UK, indicated the prediction accuracy of ME requirement and milk yield is similar to Australian and French systems. Nevertheless, all the systems had a poor prediction of LWC. For each system, the total prediction error (total MSPE) was mainly derived from the line (slope; 0.49 to 0.64 of total MSPE), while less derived from the random (0.20 to 0.48 of total MSPE), indicating a large variation between the predicted and actual LWC existed among individual cows. The residual plots of the residual differences in LWC against predicted LWC revealed that the prediction error was greater with increasing LWC. It is concluded that Australian and French systems have a better prediction of total energy requirement and milk yield than other systems, and LWC is an inappropriate indicator of energy balance in lactating dairy cows.

Animal Nutritional Physiological Phenomena↗

PubMed2003

Alternatives to linear analysis of energy balance data from lactating dairy cows.

The current energy requirements system used in the United Kingdom for lactating dairy cows utilizes key parameters such as metabolizable energy intake (MEI) at maintenance (MEm), the efficiency of utilization of MEI for 1) maintenance, 2) milk production (kl), 3) growth (kg), and the efficiency of utilization of body stores for milk production (kt). Traditionally, these have been determined using linear regression methods to analyze energy balance data from calorimetry experiments. Many studies have highlighted a number of concerns over current energy feeding systems particularly in relation to these key parameters, and the linear models used for analyzing. Therefore, a database containing 652 dairy cow observations was assembled from calorimetry studies in the United Kingdom. Five functions for analyzing energy balance data were considered: straight line, two diminishing returns functions, (the Mitscherlich and the rectangular hyperbola), and two sigmoidal functions (the logistic and the Gompertz). Meta-analysis of the data was conducted to estimate kg and kt. Values of 0.83 to 0.86 and 0.66 to 0.69 were obtained for kg and kt using all the functions (with standard errors of 0.028 and 0.027), respectively, which were considerably different from previous reports of 0.60 to 0.75 for kg and 0.82 to 0.84 for kt. Using the estimated values of kg and kt, the data were corrected to allow for body tissue changes. Based on the definition of kl as the derivative of the ratio of milk energy derived from MEI to MEI directed towards milk production, MEm and kl were determined. Meta-analysis of the pooled data showed that the average kl ranged from 0.50 to 0.58 and MEm ranged between 0.34 and 0.64 MJ/kg of BW0.75 per day. Although the constrained Mitscherlich fitted the data as good as the straight line, more observations at high energy intakes (above 2.4 MJ/kg of BW0.75 per day) are required to determine conclusively whether milk energy is related to MEI linearly or not.

Animal Nutritional Physiological Phenomena↗

PubMed2001

Loss of DNA mismatch repair imparts defective cdc2 signaling and G(2) arrest responses without altering survival after ionizing radiation.

Our previous data demonstrated that cells deficient in MutL homologue-1 (MLH1) expression had a reduced and shorter G(2) arrest after high-dose-rate ionizing radiation (IR), suggesting that the mismatch re pair (MMR) system mediates this cell cycle checkpoint. We confirmed this observation using two additional isogenetically matched human MLH1 (hMLH1)-deficient and -proficient human tumor cell systems: human ovarian cancer cells, A2780/CP70, with or without ectopically expressed hMLH1, and human colorectal carcinoma cells, RKO, with or without azacytidine treatment to reexpress hMLH1. We also examined matched MutS homologue-2 (hMSH2)-deficient and -proficient human endometrial carcinoma HEC59 cell lines to determine whether hMSH2, and MMR in general, is involved in IR-related G(2) arrest responses. As in MLH1-deficient cells, cells lacking hMSH2 demonstrated a similarly altered G(2) arrest in response to IR (6 Gy). These differences in IR-induced G(2) arrest between MMR-proficient and -deficient cells were found regardless of whether synchronized cells were irradiated in G(0)/G(1) or S phase, indicating that MMR indeed dramatically affects the G(2)-M checkpoint arrest. However, unlike the MMR-dependent damage tolerance response to 6-thioguanine exposures, no significant difference in the clonogenic survival of MMR-deficient cells compared with MMR-proficient cells was noted after high-dose-rate IR. In an attempt to define the signal transduction mechanisms responsible for MMR-mediated G(2) arrest, we examined the levels of tyrosine 15 phosphorylation of cdc2 (phospho-Tyr15-cdc2), a key regulator of the G(2)-M transition. Increased phospho-Tyr15-cdc2 levels were observed in both MMR-proficient and -deficient cell lines after IR. However, the levels of the phospho-Tyr15-cdc2 rapidly decreased in MMR (hMLH1 or hMSH2)-deficient cell lines at times coincident with progress from the IR-induced G(2) arrest through M phase. Thus, differences in the levels of phospho-Tyr15-cdc2 after high-dose-rate IR correspond temporally with the observed differences in the IR-induced G(2) arrest, suggesting that MMR proteins may exert their effect on IR-induced G(2) arrest by signaling the cdc2 pathway. Although MMR status does not significantly affect the survival of cells after high-dose-rate IR, it seems to regulate the G(2)-M checkpoint and might affect overall mutation rates.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Inhibition of egg hatching success and larvae survival of the scallop, Chlamys farreri, associated with exposure to cells and cell fragments of the dinoflagellate Alexandrium tamarense.

We report an apparently novel toxic effect of the dinoflagellate Alexandrium tamarense, manifested by inhibition of the egg hatching success of the scallop, Chlamys farreri. The hatching rate of C. farreri approached only 30% of controls when its fertilised eggs were exposed for 36h to A. tamarense cells or cellular fragments at a concentration of 100 cells/ml, and the hatching rate was just 5% after exposure to A. tamarense of 500 cells/ml. Similar exposures of the fertilised scallop eggs to two other algal species, the diatom Phaeodactylum tricornutum and the raphidophyte Heterosigma carterae, resulted in no such toxicity or inhibitory effects. Likewise, exposure of eggs to standard STX toxin, as well as to A. tamarense cell contents (supernant of re-suspended algal cells following ultrasonication and centrifugation), did not elicit this inhibitory response. However, exposure of the scallop eggs to cell cultures, intact algal cells, or cell fragments of A. tamarense produced marked toxicity. The alga also influenced larvae at early D-shape stage of scallop. The survival rates began to decrease significantly after exposed for 6 days at concentration of 3000 cells/ml and above; no larvae could survive after 14-day exposure to A. tamarense at 10,000 cells/ml or 20-day at 5000 cells/ml. The results indicated the production of novel substances from A. tamarense which can cause adverse effects on egg hatching and survival of the scallop larvae. The experiment also found that the developmental stages before blastula was the developmental period most sensitive to the A. tamarense toxin(s) and the alga at early exponential stage had the strongest effect on egg hatching comparing with other growth phases. The adverse effect of A. tamarense on early development of scallops may cause decline of shellfish population and may have further impact on marine ecosystem.

Animals↗

PubMed2001

Inhibition of human osteoblast marker gene expression by retinoids is mediated in part by insulin-like growth factor binding protein-6.

All-trans -retinoic acid (atRA) inhibits osteoblast marker gene expression and markedly increases expression of insulin-like growth factor binding protein-6 (IGFBP-6) in human osteoblasts. The possibility that IGFBP-6 inhibits the osteoblast phenotype and also mediates the inhibitory effect of atRA on osteoblast marker gene expression was explored using an antisense approach. Stable human osteoblast-like osteosarcoma SaOS-2 cells were prepared that expressed antisense IGFBP-6 RNA under basal and atRA-stimulated conditions. The functional expression of IGFBP-6 antisense RNA was confirmed by measuring IGFBP-6 mRNA by Northern analysis or by measuring IGFBP-6 protein in the conditioned media (CM) by radioimmunoassay. Antisense clones produced less mRNA and had less IGFBP-6 protein in the CM than controls. IGFBP-6 protein levels in the CM were inversely correlated with alkaline phosphatase (ALP) activity, whereas IGFBP-3 and IGFBP-4 protein levels were not. We reasoned that atRA would have little or no effect on ALP activity in IGFBP-6 antisense clones if atRA mediated its inhibitory effects by recruiting IGFBP-6. In the majority of IGFBP-6 antisense clones with the lowest IGFBP-6 mRNA and CM protein levels and only modest changes in other IGF system components, atRA did not significantly decrease ALP activity. These findings provide evidence that atRA recruits IGFBP-6 to inhibit the human osteoblast phenotype.

Alkaline Phosphatase↗