酵母酶系合成三磷酸鸟苷的研究

研究了利用啤酒酵母细胞酶系催化鸟苷酸(GMP)合成三磷酸鸟苷(GTP)的工艺过程。采用分式析因及响应面实验设计对其工艺条件进行优化,分析了温度、pH、酵母、无机盐和表面活性剂等因素对GTP积累的影响,得到了一个可以较好预测实际反应的二次方模型以及优化条件即：GMP 7．16g/L,葡萄糖55g/L,酵母270g/L,硫酸氨1．5g/L,硫酸镁2g/L,磷酸二氢钾27．2g/L,表面活性剂8川L,氯化十六烷基吡啶1．5g/L,pH6．74,温度37．4℃。经过优化,反应得率从71．3％增至92．7％,较国内外报道的水平(80％)提高了12．7％.     

Optimizing the MALDI-TOF-MS Observation of Peptides Containing Disulfide Bonds

The observation of peaks corresponding to both disulfide-bonded and reduced peptides in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of disulfides could suggest that the samples are either mixtures prior to analysis or that the measurement process has converted single compounds into mixtures. This is an important distinction when characterizing potentially disulfide-bonded peptides obtained from proteolyzed proteins or from oxidized synthetic peptides. It is well documented that disulfides can undergo in-source decay (ISD) when using a 337-nm laser. However, the mixed matrix 2-(4-hydroxyphenylazo)benzoic acid:α-cyano-4-hydroxycinnamic acid (1:10) not only suppresses the ISD reduction of disulfides to thiols but allows the same low threshold laser power generally used with α-cyano-4-hydroxycinnamic acid to be applied.     

Limitation in the detection of Listeria monocytogenes in food in the presence of competing Listeria innocua

Aims: Detectability of Listeria monocytogenes at 10⁰ CFU per food sample in the presence of Listeria innocua using standard microbiological detection was evaluated and compared with the real‐time PCR‐based method. Methods and Results: Enrichment in half‐Fraser broth followed by subculture in Fraser broth according to EN ISO 11290‐1 was used. False‐negative detection of 10⁰ CFU L. monocytogenes was obtained in the presence of 10¹ CFU L. innocua per sample using the standard detection method in contrast to more than 10⁵ CFU L. innocua per sample using real‐time PCR. Identification of L. monocytogenes on the chromogenic medium by the standard procedure was impossible if L. innocua was able to overgrow L. monocytogenes by more than three orders of magnitude after the enrichment in model samples. These results were confirmed using naturally contaminated food samples. Conclusions: Standard microbiological method was insufficient for the reliable detection of 10⁰ CFU L. monocytogenes in the presence of more than 10⁰ CFU of L. innocua per sample. On the other hand, if the growth of L. monocytogenes was sufficient to reach the concentration equal to the detection limit of PCR, the amount of the other microflora present in the food sample including L. innocua was not relevant for success of the PCR detection of L. monocytogenes. Significance and Impact of the Study: After the enrichment, the PCR detection is more convenient than the standard one as PCR detection is not compromised by other present microflora.     

Protein thiophosphorylation associated with secretory inhibition in permeabilized chromaffin cells

Permeabilized cells treated with the adenosine triphosphate analog, [35S]adenosine-5'-0-(3-thiotriphosphate) ([gamma-35S]ATP), showed thiophosphorylation of a small number of cellular proteins. A 54 kilodalton (kDa) protein was heavily thiophosphorylated in unstimulated control cells and a 43 kilodalton protein was more heavily thiophosphorylated in calcium stimulated cells. Intact cells incorporated 35S into a series of higher molecular weight proteins. Stimulation of prelabelled, permeabilized cells resulted in a loss of 35S from the cells over a 20 min period. Treatment of permeabilized cells with ATP gamma S inhibited secretion and 35S incorporation into the cells. Pretreatment with ATP gamma S resulted in subsequent inhibition of both secretion and the ability of the cells to incorporate 35S from [gamma-35S]ATP. These results indicate that the sites normally available for phosphorylation were inactivated by thiophosphorylation and were unavailable to participate in the secretory process. The inhibition of secretion associated with thiophosphorylation of these proteins suggests that they may play a role in the control of secretion by chromaffin cells.     

DNA-polycation complexation and polyplex stability in the presence of competing polyanions

Polyelectrolyte complex (polyplex) formation was studied by employing tapping mode atomic force microscopy (AFM) and an ethidium bromide fluorescence assay. The polycations chitosan and poly-L-lysine were used to compact DNA and the stability of the polyplexes was evaluated upon exposure to competing polyanions (alginate and xanthan). Furthermore, the relative preference of these polycations for DNA and the competing polyanion was investigated. The results showed that neither poly-L-lysine nor chitosan displayed any selectivity in binding to DNA relative to the competing polyanions, demonstrating the importance of electrostatics in the binding of a polycation to a polyanion. However, the ability of the polyanions to destabilize the DNA-polycation complexes depended on both the polyanion and the polycation employed, indicating that polymer-specific properties are also important for the complexation behavior and polyplex stability. Destabilization experiments further showed that annealing yielded complexes that were less prone to disruption upon subsequent exposure to alginate. Annealing experiments of plasmid DNA-chitosan complexes showed an increased fraction of rods following temperature treatment, indicating that the rods most likely are the more stable morphology for this system.     

Validation of discrete time-to-event prediction models in the presence of competing risks

Clinical prediction models play a key role in risk stratification, therapy assignment and many other fields of medical decision making. Before they can enter clinical practice, their usefulness has to be demonstrated using systematic validation. Methods to assess their predictive performance have been proposed for continuous, binary, and time-to-event outcomes, but the literature on validation methods for discrete time-to-event models with competing risks is sparse. The present paper tries to fill this gap and proposes new methodology to quantify discrimination, calibration, and prediction error (PE) for discrete time-to-event outcomes in the presence of competing risks. In our case study, the goal was to predict the risk of ventilator-associated pneumonia (VAP) attributed to Pseudomonas aeruginosa in intensive care units (ICUs). Competing events are extubation, death, and VAP due to other bacteria. The aim of this application is to validate complex prediction models developed in previous work on more recently available validation data.     

The analysis of failure times in the presence of competing risks.

Distinct problems in the analysis of failure times with competing causes of failure include the estimation of treatment or exposure effects on specific failure types, the study of interrelations among failure types, and the estimation of failure rates for some causes given the removal of certain other failure types. The usual formation of these problems is in terms of conceptual or latent failure times for each failure type. This approach is criticized on the basis of unwarranted assumptions, lack of physical interpretation and identifiability problems. An alternative approach utilizing cause-specific hazard functions for observable quantities, including time-dependent covariates, is proposed. Cause-specific hazard functions are shown to be the basic estimable quantities in the competing risks framework. A method, involving the estimation of parameters that relate time-dependent risk indicators for some causes to cause-specific hazard functions for other causes, is proposed for the study of interrelations among failure types. Further, it is argued that the problem of estimation of failure rates under the removal of certain causes is not well posed until a mechanism for cause removal is specified. Following such a specification, one will sometimes be in a position to make sensible extrapolations from available data to situations involving cause removal. A clinical program in bone marrow transplantation for leukemia provides a setting for discussion and illustration of each of these ideas. Failure due to censoring in a survivorship study leads to further discussion.     

Quantifying the predictive accuracy of time-to-event models in the presence of competing risks

Prognostic models for time-to-event data play a prominent role in therapy assignment, risk stratification and inter-hospital quality assurance. The assessment of their prognostic value is vital not only for responsible resource allocation, but also for their widespread acceptance. The additional presence of competing risks to the event of interest requires proper handling not only on the model building side, but also during assessment. Research into methods for the evaluation of the prognostic potential of models accounting for competing risks is still needed, as most proposed methods measure either their discrimination or calibration, but do not examine both simultaneously. We adapt the prediction error proposal of Graf et al. (Statistics in Medicine 1999, 18, 2529–2545) and Gerds and Schumacher (Biometrical Journal 2006, 48, 1029–1040) to handle models with competing risks, i.e. more than one possible event type, and introduce a consistent estimator. A simulation study investigating the behaviour of the estimator in small sample size situations and for different levels of censoring together with a real data application follows.     

Case-control methods in the presence of multiple failure times and competing risks

The link between cohort and incident case-control studies has been considered by many authors. In particular, under the Cox proportional hazards model, follow-up data (implicit or explicit) can be analyzed as a case-control study by randomly selecting controls from the risk sets of each incident case, thereby obviating the necessity of working with the entire cohort when interest is primarily on exposure effects. This paper extends this linkage to competing risks and to diseases with multiple incidence or recurrence times by matching to each event (case) a sample of controls from the appropriate risk set. Illustrations are given.     

Evolution of opinions on social networks in the presence of competing committed groups

Public opinion is often affected by the presence of committed groups of individuals dedicated to competing points of view. Using a model of pairwise social influence, we study how the presence of such groups within social networks affects the outcome and the speed of evolution of the overall opinion on the network. Earlier work indicated that a single committed group within a dense social network can cause the entire network to quickly adopt the group''s opinion (in times scaling logarithmically with the network size), so long as the committed group constitutes more than about of the population (with the findings being qualitatively similar for sparse networks as well). Here we study the more general case of opinion evolution when two groups committed to distinct, competing opinions and , and constituting fractions and of the total population respectively, are present in the network. We show for stylized social networks (including Erdös-Rényi random graphs and Barabási-Albert scale-free networks) that the phase diagram of this system in parameter space consists of two regions, one where two stable steady-states coexist, and the remaining where only a single stable steady-state exists. These two regions are separated by two fold-bifurcation (spinodal) lines which meet tangentially and terminate at a cusp (critical point). We provide further insights to the phase diagram and to the nature of the underlying phase transitions by investigating the model on infinite (mean-field limit), finite complete graphs and finite sparse networks. For the latter case, we also derive the scaling exponent associated with the exponential growth of switching times as a function of the distance from the critical point.     

Nonparametric estimation of bivariate failure time associations in the presence of a competing risk

Most research on the study of associations among paired failuretimes has either assumed time invariance or been based on complexmeasures or estimators. Little has accommodated competing risks.This paper targets the conditional cause-specific hazard ratio,henceforth called the cause-specific cross ratio, a recent modificationof the conditional hazard ratio designed to accommodate competingrisks data. Estimation is accomplished by an intuitive, nonparametricmethod that localizes Kendall's tau. Time variance is accommodatedthrough a partitioning of space into ‘bins’ betweenwhich the strength of association may differ. Inferential proceduresare developed, small-sample performance is evaluated, and themethods are applied to the investigation of familial associationin dementia onset.     

铜绿假单胞菌的MALDI-TOF-MS检测方法的建立

目的 建立利用基质辅助激光解吸电离飞行时间质谱仪( MALDI-TOF-MS)对铜绿假单胞菌的快速检测方法.方法 通过MALDI-TOF-MS法对铜绿假单胞菌进行检测分析,并与生化鉴定方法相比较.结果 MALDI-TOF-MS对铜绿假单胞菌的检测后得到肽指纹图片及相关质谱数据,建立MALDI-TOF-MS对铜绿假单胞菌的快速检测方法.结论 MALDI-TOF-MS方法检测铜绿假单胞菌准确快速、操作简单等特点,可发展成为食品检验铜绿假单胞菌的重要(辅助)工具.     

Measurements of cysteine reactivity during protein unfolding suggest the presence of competing pathways

Evidence that proteins may unfold utilizing complex competing pathways comes from a new pulse-labeling protocol in which the change in reactivity of a single cysteine residue in a protein during unfolding is measured, making use of its easily monitored reaction with the Ellman reagent, dithionitrobenzoic acid. The kinetics of unfolding of two single cysteine-containing mutant forms of the small protein barstar, C82A, which contains only Cys40, and C40A, which contains only Cys82, have been studied. The data suggest that unfolding occurs via two parallel pathways, each forming competing intermediates. In one of these early intermediates, Cys40 and Cys82 are already as reactive as they are in the fully unfolded protein, while in the other intermediate, the Cys thiol groups are unreactive. One more long-lived intermediate also needs to be included on the pathway defined by the early intermediate with unreactive Cys thiol groups to account for the difference in the rates of fluorescence change and of change in Cys40 reactivity. The demonstration of multiple intermediates and pathways for unfolding indicates that protein unfolding reactions can be as complex as protein folding reactions.     

Drug modification of protein thiophosphorylation in permeabilized chromaffin cells

Treatment of permeabilized chromaffin cells with low concentrations of the ATP analog adenosine-5′-O-(3-thiotriphosphate)[³⁵S] results in ³⁵S incorporation into a small number of cellular proteins. Of these proteins, a 47 kilodalton protein is most heavily thiophosphorylated. Permeabilized cells were treated with various drugs known to influence cell functions, more specifically chromaffin granule function, to determine the kinase responsible for thiophosphorylation of the 47 kilodalton protein and if its thiophosphorylation is associated with a specific cell function.

Several drugs which influence the activity of cell kinases were examined for their effect on secretion and thiophosphorylation of the 47 kilodalton protein. There was no qualitative effect of cAMP, cGMP or trifluoperazine on thiophosphorylation of the protein. Both cyclic nucleotides slightly enhanced secretion, while trifluoperazine enhanced only unstimulated catecholamine release. Phorbol 12-myristate 13-acetate had no effect on secretion or ³⁵S incorporation into cell proteins. Only the free calcium concentration of the medium influenced thiophosphorylation of the 47 kilodalton protein, with increased calcium producing increased thiophosphorylation.

Drugs affecting chromaffin vesicle functions were used to assess the relationship between specific functions and thiophosphorylation of the protein. Inhibition of nucleotide translocation with atractyloside or 4,4′diisothiocyanatostilbene-2,2′disulfonic acid or inhibition of the proton translocating ATPase by N-ethylmaleimide inhibited thiophosphorylation of the 47 kilodalton protein, with little effect on secretion. Treatment with rotenone markedly enhanced secretion and thiophosphorylation of the protein. Calcium ionophores had no effect on thiophosphorylation of the protein. Dichloroacetic acid, which inhibits phosphorylation of mitochondrial pyruvate dehydrogenase, had no effect on secretion and a variable effect on thiophosphorylation of the 47 kilodalton protein. The data suggest that thiophosphorylation of the protein may be associated with nucleotide translocation across the vesicle membrane.     

Multipoint linkage detection in the presence of heterogeneity

Linkage heterogeneity is common for complex diseases. It is well known that loss of statistical power for detecting linkage will result if one assumes complete homogeneity in the presence of linkage heterogeneity. To this end, Smith (1963, Annals of Human Genetics 27, 175-182) proposed an admixture model to account for linkage heterogeneity. It is well known that for this model, the conventional chi-squared approximation to the likelihood ratio test for no linkage does not apply even when the sample size is large. By dealing with nuclear families and one marker at a time for genetic diseases with simple modes of inheritance, score-based test statistics (Liang and Rathouz, 1999, Biometrics 55, 65-74) and likelihood-ratio-based test statistics (Lemdani and Pons, 1995, Biometrics 51, 1033-1041) have been proposed which have a simple large-sample distribution under the null hypothesis of linkage. In this paper, we extend their work to more practical situations that include information from multiple markers and multi-generational pedigrees while allowing for a class of general genetic models. Three different approaches are proposed to eliminate the nuisance parameters in these test statistics. We show that all three approaches lead to the same asymptotic distribution under the null hypothesis of no linkage. Simulation results show that the proposed test statistics have adequate power to detect linkage and that the performances of these two classes of test statistics are quite comparable. We have applied the proposed method to a family study of asthma (Barnes et al., 1996), in which the score-based test shows evidence of linkage with p-value <0.0001 in the region of interest on chromosome 12. Additionally, we have implemented this score-based test within the frequently used computer package GENEHUNTER.     

The relationship between ATPase activity, isometric force, and myosin light-chain phosphorylation and thiophosphorylation in skinned smooth muscle fiber bundles from chicken gizzard

Isometric force developed by skinned gizzard muscle fiber bundles and levels of phosphorylation and thiophosphorylation of the 20,000-dalton myosin light chain were determined. These data showed a highly non-linear relationship between isometric force and myosin light-chain phosphorylation. Maximum force was developed at approximately 0.2 mol of phosphate/mol of light chain as reported previously (Hoar, P. E., Kerrick, W. G. L., and Cassidy, P. S. (1979) Science 204, 503-506). In contrast, the relationship between isometric force and myosin light-chain thiophosphorylation was linear, with maximum force occurring at 1.0 mol of thiophosphate/mol of myosin light chain. These observations are consistent with the latch-bridge hypothesis for conditions of varying myosin light-chain phosphatase/myosin light-chain kinase activity ratios as discussed by Hai and Murphy [1988) Am. J. Physiol. 254, C99-C106). To further test the latch-bridge hypothesis, ATPase activity was also measured during isometric force development in these fiber bundles. The relationship between isometric force and ATPase activity was linear whether the myosin light chains were phosphorylated or thiophosphorylated. Thus the number of cycling myosin cross-bridges, as measured by ATPase activity, was directly proportional to the force the muscle developed, not to the level of myosin light-chain phosphorylation. This finding that high levels of tension generated at low levels of light-chain phosphorylation are associated with high levels of ATPase activity is inconsistent with the latch-bridge model (Hai and Murphy, 1988).