Novel differential elimination method for determining kinetic coefficients under substrate self-inhibition

A differential elimination method (DEM) is developed to determine the kinetic coefficients for substrate self-inhibition. Finite differentiation of the equation eliminates either K_I or K_S, which enables the equation to be linearized so that [^(\textq)] {\hat{\text{q}}} , K_S, and K_I can be estimated without using nonlinear least square regression (NLSR). The DEM options that eliminate K_I or K_S computed the parameter values exactly when the data did not contain any errors. If one-point or random errors were not too large, both DEM options worked as well as NLSR when data were acquired with geometric intervals for substrate concentration. The DEM was more accurate for fitting the data for the smallest and largest values of S, but relatively weaker in estimating the observed maximum substrate utilization rate, q_max. The estimates for S_max, the concentration at which the maximum specific substrate utilization rate is observed, were relatively invariant among the methods, even when K_S and K_I differed. When the intervals were arithmetic (i.e., equal intervals of substrate concentration) and the data contained errors, the DEM and NLSR estimated the parameters poorly, indicating that collecting data with an arithmetic interval greatly increases the risk of poor parameter estimation. Parameter estimates by DEM fit very well experimental data from nitrification or photosynthesis, which were taken with geometric intervals of substrate concentration or light intensity, but fit poorly phenol-degradation data, which were obtained with arithmetic substrate intervals. Besides providing a reasonable substitute for NLSR, the DEM also can be used as a tool to diagnose the quality of experimental data by comparing its estimates between the DEM options, or, more rigorously, to those from NLSR.     

Standardization of the nutrient medium for determining the biological activity of a number of antibiotics by the diffusion-in-agar method

The possibility of using 2 variants of a unified nutrient medium containing no Hottinger's broth for determination of the biological activity of a large number of antibacterial antibiotics was shown. The medium provided satisfactory reproducibility of the results. It contained yeast extract as a source of nitrogen. The medium was selected with the method of the fractional factor experiment. Clear inhibition growth zones and the slope of the dose-response curve were used as the criteria for estimation of the optimal medium. The described nutrient medium allowed a 2-time increase in the sensitivity of the method used for determination of the biological activity of neomycin, monomycin, streptomycin, oxytetracycline, chlortetracycline and erythromycin.     

The application of a sequential procedure with elimination as a method for the screening for metabolic diseases

A sequential classification procedure with early elimination, for the screening for metabolic diseases, is presented. Asymptotic properties of the procedure are derived in the Appendix and it is shown that the procedure is asymptotically distribution-free under certain assumptions, and asymptotically at least as efficient as a comparable fixed-sample procedure. With the use of data obtained from 36 mentally retarded patients, the procedure was evaluated by means of a bootstrap simulation. The procedure was then applied to this set of data, with satisfactory results and a considerable economy in observations.     

An efficient and reliable method for determining the number of transgenes inserted into transgenic plants

A simple and reliable method is described for determining the number of transgenes inserted into transgenic plants. Analysis of tomato plants transformed with an antisense polygalacturonase gene is used to demonstrate the method. The use of the method to analyze other plants transformed with endogenous plant gene coding regions or promoters is discussed.     

A modified method of determining amylase activity in grain by falling number

The standard method for determination of amylase activity by the falling number was modified by us to study the carbohydrate-amylase complex and time course of starch hydrolysis by amylases from rye grain. The method is based on the use of potato starch as a standard substrate and aqueous extract of grain amylases as an enzyme source.     

Stereospecific synthesis of a GS 4104 metabolite: determination of absolute stereochemistry and influenza neuraminidase inhibitory activity.

The total synthesis for the determination of the absolute stereochemistry of a GS 4104 metabolite 3 is described. In addition, the influenza neuraminidase inhibitory activity of 3 and related intermediates are reported.     

A new method for determining ribosomal DNA copy number shows differences between Saccharomyces cerevisiae populations

Ribosomal DNA genes (rDNA) encode the major ribosomal RNAs and in eukaryotes typically form tandem repeat arrays. Species have characteristic rDNA copy numbers, but there is substantial intra-species variation in copy number that results from frequent rDNA recombination. Copy number differences can have phenotypic consequences, however difficulties in quantifying copy number mean we lack a comprehensive understanding of how copy number evolves and the consequences. Here we present a genomic sequence read approach to estimate rDNA copy number based on modal coverage to help overcome limitations with existing mean coverage-based approaches. We validated our method using Saccharomyces cerevisiae strains with known rDNA copy numbers. Application of our pipeline to a global sample of S. cerevisiae isolates showed that different populations have different rDNA copy numbers. Our results demonstrate the utility of the modal coverage method, and highlight the high level of rDNA copy number variation within and between populations.     

A general method for determining the functional role of a muscle

This paper presents a general classification method for determining the functional role of any muscle, and a procedure for determining the sensitivity of that role to small changes in system parameters. The classification method is based on the premise that the system model, when acted upon solely by the muscle of interest, will depart from any initial rest configuration in such a way as to decrease the muscle's length. This method is particularly useful for multiple-joint muscles, and is illustrated by examining a slider-crank mechanism and straight line muscle model to determine the functional role of the hamstrings during a constrained leg flexion motion.     

Quantitative disk diffusion as a convenient method for determining minimum inhibitory concentrations of oxacillin for staphylococci strains

In this study the susceptibility of 58 coagulase-negative staphylococci (CoNS) strains and 58 Staphylococcus aureus strains to oxacillin was evaluated by a novel method called quantitative disk diffusion (DD) method. The results obtained were compared to phenotypic methods as agar dilution (AD) for oxacillin, disk diffusion (DD) for cefoxitin, and related to the presence of the mecA gene detected by PCR. Minimum inhibitory concentrations (MIC) determined by the quantitative DD method were equivalent to MICs determined in the AD method for S. aureus (Student's t test, p=0.99) and CoNS (Student's t test, p=0.97). Incongruent results between PCR mecA gene determinations and the quantitative DD method were obtained in 8 strains (5 S. aureus and 3 CoNS) where the mecA gene expression was blocked. However, oxacillin resistance was detected by the proposed method even in staphylococci strains showing low-level or heterogeneous resistance to the antibiotic while other phenotypic methods failed. The single quantitative DD method is not expensive, it can be performed in any laboratory and permits accurate identification of oxacillin resistant staphylococci.     

Use of a polarographic method for determining trichothecin]

The polarographic behaviour of trichothecin was studied. It was shown that the antibiotic could be detected in solutions at concentrations of 7.10(-7) moles with the help of the polarographic method. Conditions for the polarographic determination of trichothecin in fermentation broth were developed. The error was not more than 3 per cent. The reliability of the results was shown by statistical treatment of data performed in accordance with the requirement of the USSR State Pharmacopeia, X ed., prescribing that the precision of the assay is such that the fiducial limits at p = 95 per cent deviate from the average value by not more than 5 per cent. Comparison of the results of trichothecin determination in the fermentation broth with the polarographic and biological methods showed no significant difference. Therefore, the polarographic method may be recommended for trichothecin determination in the fermentation broth.     

A new method for determining the stereochemistry of DNA cleavage reactions: application to the SfiI and HpaII restriction endonucleases and to the MuA transposase

A new method was developed for tracking the stereochemical path of enzymatic cleavage of DNA. DNA with a phosphorothioate of known chirality at the scissile bond is cleaved by the enzyme in H218O. The cleavage produces a DNA molecule with the 5'-[16O,18O, S]-thiophosphoryl group, whose chirality depends on whether the cleavage reaction proceeds by a single-step hydrolysis mechanism or by a two-step mechanism involving a protein-DNA covalent intermediate. To determine this chirality, the cleaved DNA is joined to an oligonucleotide by DNA ligase. Given the strict stereochemistry of the DNA ligase reaction, determined here, the original chirality of the phosphorothioate dictates whether the 18O is retained or lost in the ligation product, which can be determined by mass spectrometry. This method has advantages over previous methods in that it is not restricted to particular DNA sequences, requires substantially less material, and avoids purification of the products at intermediate stages in the procedure. The method was validated by confirming that DNA cleavage by the EcoRI restriction endonuclease causes inversion of configuration at the scissile phosphate. It was then applied to the reactions of the SfiI and HpaII endonucleases and the MuA transposase. In all three cases, DNA cleavage proceeded with inversion of configuration, indicating direct hydrolysis of the phosphodiester bond by water as opposed to a reaction involving a covalent enzyme-DNA intermediate.     

The water-sweet aftertaste of neohesperidin dihydrochalcone and thaumatin as a method for determining their sweet persistence

The water–sweet aftertaste produced in humans in responseto tasting intensive sweeteners such as neohesperidin dihydrochalconeand thaumatin was studied. This water–sweet aftertasteincreased with sweetener concentration and diminished with time.The decay in the sweet intensity–time relationships fitteda negative exponential function in a pattern similar to thatwhich occurs when other methods for determining persistenceare employed. Persistence time contants (T) were dependent uponthe maximal perceived sweet intensity (Ip-max) observed at theinitial time of tasting. The use of this procedure is proposedfor determining persistence of intensive sweeteners under circumstanceswhere controlled pH and temperature are desired.