Use of Trinitrophenylation for Quantification of Protease and Peptidase Activities

A sensitive and precise method for quantifying protease and peptidase activities is suggested. N-Terminal amino groups of peptides which are formed during hydrolysis of the substrates react with trinitrobenzenesulfonic acid (TNBS), and the trinitrophenyl (TNP) derivatives are determined spectrophotometrically. Spontaneous hydrolysis of TNBS is considerably diminished on trinitrophenylation at pH 7.4 rather than at pH 9-10 as is usually used. The trinitrophenylation method can be used to determine the initial rate of hydrolysis and the kinetics of reactions catalyzed by proteases and peptidases.     

A new method for spectrophotometric assay of activity of cross-linked penicillin acylase aggregates

A new method for monitoring reactions catalyzed by an immobilized enzyme, cross-linked penicillin acylase aggregates (PA CLEA), is suggested. Appropriate chromogenic substrates for spectrophotometric assay of catalytic activity of immobilized enzyme were chosen and their kinetic parameters determined. Active sites in PA CLEA preparations were titrated by the suggested method; it is shown that almost all active sites are retained during immobilization. This method is characterized as highly expressive, simple, and precise and may be used for control of PA immobilization efficiency as well as for study of operational, thermal, and pH stability of immobilized enzyme preparations.     

A new graphic method for determining the rate constant for affinity of a ligand for its receptor]

A new method of determination of the equilibrium constant for a ligand binding to acceptor and evaluation of the number of binding sites on the acceptor molecules (or cells) is suggested. The method is simpler, more convenient, and more precise than Klotz's or Scatchard's method.     

Developing a low-cost milliliter-scale chemostat array for precise control of cellular growth

Background: Multiplexed milliliter-scale chemostats are useful for measuring cell physiology under various degrees of nutrient limitation and for carrying out evolution experiments. In each chemostat, fresh medium containing a growth rate-limiting metabolite is pumped into the culturing chamber at a constant rate, while culture effluent exits at an equal rate. Although such devices have been developed by various labs, key parameters — the accuracy, precision, and operational range of flow rate — are not explicitly characterized. Methods: Here we re-purpose a published multiplexed culturing device to develop a multiplexed milliliter-scale chemostat. Flow rates for eight chambers can be independently controlled to a wide range, corresponding to population doubling times of 3~13 h, without the use of expensive feedback systems. Results: Flow rates are precise, with the maximal coefficient of variation among eight chambers being less than 3%. Flow rates are accurate, with average flow rates being only slightly below targets, i.e., 3%–6% for 13-h and 0.6%–1.0% for 3-h doubling times. This deficit is largely due to evaporation and should be correctable. We experimentally demonstrate that our device allows accurate and precise quantification of population phenotypes. Conclusions: We achieve precise control of cellular growth in a low-cost milliliter-scale chemostat array, and show that the achieved precision reduces the error when measuring biological processes.     

Determination of the binding parameters for a pre-existing antigen-antibody mixture

A new method, which allows to evaluate parameters of interaction between antibodies (or receptors) and an antigen (or ligand) is suggested. The method is based on the use of so-called coordinates of dilution suggested by the author earlier. Representation of the data of the titration curves for the mixtures of antibodies (or receptors) and antigen (or ligand) in these coordinates allows one to determine the affinity of interaction and the concentration of antigen (or ligand), which can reversibly block antibodies (or receptors). Simple formulas, which allow to estimate which part of paratopes or bivalent antibodies is free and which part is blocked by the antigen, depending on dilution of the considered system, are also suggested. Such a method could be useful for characterization of infection and autoimmune processes when the antigen and antibodies circulate together in the bloodstream.     

森林流域坡面流与壤中流耦合模型的构建与应用

采用饱和入渗理论、Saint-Venant方程和Richards方程构建了以有限差分法求解的坡面流与壤中流耦合模型,并模拟了不同坡度和不同雨强下的坡面产汇流室内实验.结果表明：该模型模拟的坡面流和壤中流过程与实测过程基本一致,峰现时间、径流历时、峰值流量、出流总量模拟值与实测值的相对误差均较小,基本小于10%.模型的模拟精度较高,实用性较强,为深入研究壤中流机制和改进流域降雨-径流模型提供了理论依据.     

Making a robust biomolecular time scale for phylogenetic studies

The further evolution of informational molecular sequences should depend on the number of viable alternatives possible for the sequences as set by selection, the unrepaired mutation rate, and time. Most biomolecular clocks are based on Kimura's nearly neutral mutation random-drift hypothesis. This clock assumes that informational sequences are in equilibrium, i.e., the nucleotides mutate at a uniform rate and the number of nucleotides unconstrained by selection remains constant. Correcting for deviations from these assumptions should produce a more accurate clock. Informational molecules probably formed from polynucleotides having some other function such as nitrogen or nucleotide storage, thus being initially functionally unselected. At any time the rate of development of functionality in a protein may be expected to be proportional to the number of viable alternatives of sequence in its potentially interacting regions. Assuming the rate of unrepaired mutations is constant, these clocks should exponentially slow as they evolve, each with a different rate toward individual equilibria. Also if the degree of selection changes, its clock rate should change. For a more precise clock two approaches are suggested to estimate these time dependent changes in evolutionary rate. An improved clock could improve estimation of phylogeny and put a time scale on that phylogeny.     

Field Evaluation of Alternative Earthen Final Covers

Five methods to assess percolation rate from alternative earthen final covers (AEFCs) are described in the context of the precision with which the percolation rate can be estimated: trend analysis, tracer methods, water balance method, Darcy's Law calculations, and lysimetry. Trend evaluation of water content data is the least precise method because it cannot be used alone to assess the percolation rate. The precision of percolation rates estimated using tracer methods depends on the tracer concentration, percolation rate, and the sensitivity of the chemical extraction and analysis methods. Percolation rates determined using the water balance method have a precision of approximately 100 mm/yr in humid climates and 50 mm/yr in semiarid and drier climates, which is too large to demonstrate that an AEFC is meeting typical equivalency criterion (30 mm/yr or less). In most cases, the precision will be much poorer. Percolation rates computed using Darcy's Law with measured profiles of water content and matric suction typically have a precision that is about two orders of magnitude (or more) greater than the computed percolation rate. The Darcy's Law method can only be used for performance assessment if the estimated percolation rate is much smaller than the equivalency criterion and preferential flow is not present. Lysimetry provides the most precise estimates of percolation rate, but the precision depends on the method used to measure the collected water. The lysimeter used in the Alternative Cover Assessment Program (ACAP), which is described in this paper, can be used to estimate percolation rates with a precision between 0.00004 to 0.5 mm/yr, depending on the measurement method and the flow rates.     

Development and validation of a RP-HPLC method for quantification of isoflavone aglycones in hydrolyzed soy dry extracts

Isoflavones are widely used as an alternative treatment to hormone replacement therapy and also for prevention of several chronic diseases, including cancers. Genistein, daidzein and glycitein are the most abundant isoflavone aglycones found in soy extracts, where they also occur as glycosides. This paper describes the development and validation of an isocratic reversed-phase HPLC (RP-HPLC) method for the analysis of isoflavone aglycones, released after acid hydrolysis of soy dry extracts, used as pharmaceutical raw material. The quantification was carried out in a C(18) endcapped column, using a mobile phase composed of 0.1% acetic acid and methanol (52:48), at a flow rate of 1.0 ml/min and diode array detection (DAD) at 254 nm. The method showed to be linear (r(2)>0.99), precise (R.S.D.<2%), accurate (recovery of 98.88% for daidzein and 98.12% for genistein), robust and specific.     

New carrier for specific delivery of drugs to the brain

1,4-Dihydropyridines were thoroughly investigated as carriers for specific drug delivery to the brain and were found very efficient. The main problem which arises in their application in pharmaceutical preparations, is the short shelf-life time due to hydration and/or oxidation. To overcome these problems, a new carrier system is suggested. Many of 1,4-dihydropyridine-3,5-dicarboxylate derivatives are used as calcium channel blockers which are known to have long shelf-life time, and at the same time, they are safe, with no reported neurotoxicity. Since efficiency of brain specific delivery depends on the rate of oxidation of the dihydropyridine carrier, (the faster the rate, the higher the efficiency), these 3,5-dicarbonyl compounds have almost of no brain-specific delivery properties. N-alkoxycarbonylmethyl derivatives of 1,4-dihydropyridine-3,5-dicarboxylate, a new carrier system is suggested and expected to be stable enough for formulation and storage. The drug moiety will be connected to the 3,5-dicarbonyl groups in the form of esters or amides. The suggested drug-carrier once delivered and distributed in the body, will be subjected to several sequential enzymatic hydrolytic and oxidative processes. The D-carrier is designed so that, the rate of hydrolysis of the ester group at the nitrogen atom should be faster than that of the hydrolysis and release of the drug moiety. The stability and efficiency of brain specific delivery of model drugs were investigated. The in vitro and in vivo studies proved the efficiency of brain specific delivery of the carrier and at the same time its shelf life stability. The results suggest that the designed carrier is promising to be used in application of pharmaceutical formulation.     

Variance estimation for statistics computed from single recurrent event processes

This article is concerned with variance estimation for statistics that are computed from single recurrent event processes. Such statistics are important in diagnosis for each individual recurrent event process. The proposed method only assumes a semiparametric form for the first-order structure of the processes but not for the second-order (i.e., dependence) structure. The new variance estimator is shown to be consistent for the target parameter under very mild conditions. The estimator can be used in many applications in semiparametric rate regression analysis of recurrent event data such as outlier detection, residual diagnosis, as well as robust regression. A simulation study and application to two real data examples are used to demonstrate the use of the proposed method.     

Graft-union development: a delicate process that involves cell-cell communication between scion and stock for local auxin accumulation

Grafting is an ancient cloning method that has been used widely for thousands of years in agricultural practices. Graft-union development is also an intricate process that involves substantial changes such as organ regeneration and genetic material exchange. However, the molecular mechanisms for graft-union development are still largely unknown. Here, a micrografting method that has been used widely in Arabidopsis was improved to adapt it a smooth procedure to facilitate sample analysis and to allow it to easily be applied to various dicotyledonous plants. The developmental stage of the graft union was characterized based on this method. Histological analysis suggested that the transport activities of vasculature were recovered at 3 days after grafting (dag) and that auxin modulated the vascular reconnection at 2 dag. Microarray data revealed a signal-exchange process between cells of the scion and stock at 1 dag, which re-established the communication network in the graft union. This process was concomitant with the clearing of cell debris, and both processes were initiated by a wound-induced programme. The results demonstrate the feasibility and potential power of investigating various plant developmental processes by this method, and represent a primary and significant step in interpretation of the molecular mechanisms underlying graft-union development.     

Efficient Methods for Estimating Amino Acid Replacement Rates

Replacement rate matrices describe the process of evolution at one position in a protein and are used in many applications where proteins are studied with an evolutionary perspective. Several general matrices have been suggested and have proved to be good approximations of the real process. However, there are data for which general matrices are inappropriate, for example, special protein families, certain lineages in the tree of life, or particular parts of proteins. Analysis of such data could benefit from adaption of a data-specific rate matrix. This paper suggests two new methods for estimating replacement rate matrices from independent pairwise protein sequence alignments and also carefully studies Müller-Vingron’s resolvent method. Comprehensive tests on synthetic datasets show that both new methods perform better than the resolvent method in a variety of settings. The best method is furthermore demonstrated to be robust on small datasets as well as practical on very large datasets of real data. Neither short nor divergent sequence pairs have to be discarded, making the method economical with data. A generalization to multialignment data is suggested and used in a test on protein-domain family phylogenies, where it is shown that the method offers family-specific rate matrices that often have a significantly better likelihood than a general matrix. [Reviewing Editor: Dr. Nicolas Galtier]     

Measuring the heart rate of the spider, Aphonopelma hentzi: a non-invasive technique

In this work we describe a non‐invasive and precise technique to record the heartbeats of a spider. A linear output Hall effect transducer in conjunction with a small magnet was used to monitor the micromovements on the dorsal surface of the abdomen of the tarantula Aphonopelma hentzi (Girard) (Theraphosidae). The exoskeleton in this region is in direct contact with suspensory ligaments connected to the heart, and the dorsal cuticle of the opisthosoma moves with each heartbeat. The technique allowed the discrimination of the different stages of the spider's cardiac cycle. The method can be also adapted for a smaller spider or other arthropods. We believe that the method proposed in this paper allows investigators to gain insights into a spider's natural heart rate by gathering unbiased data with a non‐invasive and very precise technique. We have found the resting heart rate of A. hentzi to be 5.6 ± 1.47 beats/min, which is lower than previously reported values.     

Assessment of rate constants for isolating a metabolite by a model-independent method]

A model-independent method for estimating an elimination rate constant of a metabolite of exogenous substance is suggested as an alternative to known methods. The new method (named the initial slope method) uses blood (plasma) concentration-time data of both the substance and the metabolite obtained after an extravascular impulse input of the substance. The metabolite input is not needed substantially facilitating the experiment. The method is based upon the assessment of areas under the substance and metabolite concentration-time curves, the initial substance concentration, and the initial slope of the metabolite concentration-time curve. The method was tested using artificial data generated on the basis of a compartment model for the substance and metabolite kinetics. It was shown providing nonbiased estimates of a true metabolite elimination rate constant irrespective of the structure of the model used to generate data. Other methods failed to provide such estimates.     

Microcontact printing of axon guidance molecules for generation of graded patterns

Microcontact printing (microCP) of proteins has been successfully used for patterning surfaces in various contexts. Here we describe a simple 'lift-off' method to print precise patterns of axon guidance molecules, which are used as substrate for growing chick retinal ganglion cell (RGC) axons. Briefly, the etched pattern of a silicon master is transferred to a protein-coated silicone cuboid (made from polydimethylsiloxane, PDMS), which is then used as a stamp on a glass coverslip. RGC explants are placed adjacent to the pattern and cultured overnight. Fluorescent labeling of the printed proteins allows the quantitative analysis of the interaction of axons and growth cones with single protein dots and of the overall outgrowth and guidance rate in variously designed patterns. Patterned substrates can be produced in 3-4 h and are stable for up to one week at 4 degrees C; the entire protocol can be completed in 3 d.