An efficient method for calculation of dynamic logarithmic gains in biochemical systems theory

Biochemical systems theory (BST) characterizes a given biochemical system based on the logarithmic gains, rate-constant sensitivities and kinetic-order sensitivities defined at a steady state. This paper describes an efficient method for calculation of the time courses of logarithmic gains, i.e. dynamic logarithmic gains L(Xi, Xj; t), which expresses the percentage change in the value of a dependent variable Xi at a time t in response to an infinitesimal percentage change in the value of an independent variable Xj at t=0. In this method, one first recasts the ordinary differential equations for the dependent variables into an exact canonical nonlinear representation (GMA system) through appropriate transformations of variables. Owing to the structured mathematical form of this representation, the recast system can be fully described by a set of numeric parameters, and the differential equations for the dynamic logarithmic gains can be set up automatically without resource to computer algebra. A simple general-purpose computer program can thus be written that requires only the relevant numeric parameters as input to calculate the time courses of the variables and of the dynamic logarithmic gains for both concentrations and fluxes. Unlike other methods, the proposed method does not require to derive any expression for the partial differentiation of flux expressions with respect to each independent variable. The proposed method has been applied to two kinds of reaction models to elucidate its usefulness.     

An effective method for plasma lipoprotein separation: studies of various animal species

1. Plasma lipoprotein separation by density gradient ultracentrifugation largely depends on visual examination based on the natural yellow pigments of lipoproteins. 2. In non-human species and in humans with dyslipoproteinemia, some lipoproteins are not well visualized due to the lack of pigments. 3. Using a fluorescent probe in minute quantity (1,6 diphenyl-1,3,5-hexatriene) we were able to demonstrate an effective plasma lipoprotein separation using a discontinuous density gradient ultracentrifugation technique. 4. Plasma lipoproteins of human, chicken, rat and carp were compared showing the unique character of carp HDL.     

An efficient method for introducing defined lipids into the plasma membrane of mammalian cells

An efficient method has been devised to introduce lipid molecules into the plasma membrane of mammalian cells. This method has been applied to fuse lipid vesicles with the apical plasma membrane of Madin-Darby canine kidney cells. The cells were infected with fowl plague or influenza N virus. 4 h after infection, the hemagglutinin (HA) spike glycoprotein of the virus was present in the apical plasma membrane of the cells. Lipid vesicles containing egg phosphatidylcholine, cholesterol, and an HA receptor (ganglioside) were then bound to the cells at 0 degrees C. More than 85% of the vesicles were released by external neuraminidase at 0 degrees C or by simply warming the cells to 37 degrees C for 10 s, probably because of the action of the viral neuraminidase at the cell surface. However, when the cells were warmed to 37 degrees C in a pH 5.3 medium for 30 s, 50% of the bound vesicles could no longer be released by external neuraminidase. This only occurred when the HA protein had been cleaved into its HA1 and HA2 subunits. When we used influenza N virus, whose HA is not cleaved in Madin-Darby canine kidney cells, cleavage with external trypsin was required. The fact that the HA protein has fusogenic properties at low pH only in its cleaved form suggests that fusion of the vesicles with the plasma membrane had taken place. Further confirmation for fusion was obtained using an assay based on the decrease of energy transfer between two fluorescent phospholipids in a vesicle upon fusion of the vesicle with the plasma membrane (Struck, D. K., D. Hoekstra, and R. E. Pagano. 1981. Biochemistry, 20:4093-4099).     

An efficient method for multi-locus molecular haplotyping

Many methods exist for genotyping—revealing which alleles an individual carries at different genetic loci. A harder problem is haplotyping—determining which alleles lie on each of the two homologous chromosomes in a diploid individual. Conventional approaches to haplotyping require the use of several generations to reconstruct haplotypes within a pedigree, or use statistical methods to estimate the prevalence of different haplotypes in a population. Several molecular haplotyping methods have been proposed, but have been limited to small numbers of loci, usually over short distances. Here we demonstrate a method which allows rapid molecular haplotyping of many loci over long distances. The method requires no more genotypings than pedigree methods, but requires no family material. It relies on a procedure to identify and genotype single DNA molecules, and reconstruction of long haplotypes by a ‘tiling’ approach. We demonstrate this by resolving haplotypes in two regions of the human genome, harbouring 20 and 105 single-nucleotide polymorphisms, respectively. The method can be extended to reconstruct haplotypes of arbitrary complexity and length, and can make use of a variety of genotyping platforms. We also argue that this method is applicable in situations which are intractable to conventional approaches.     

An efficient mutational method for photosynthetic bacteria

The pigment and auxotrophic mutants of Rhodobacter sphaeroides Y6 were obtained by treatment with ethyl methanesulfonate (EMS) followed by lithium chloride (LiCl). Treatment with 0.081 MEMS and subsequent treatment with 0.071 M LiCl resulted in 12% higher frequency og than that by 0.081 mol/L EMS alone, and the same frequency of pigment mutations than application of 0.081 M EMS alone; the frequency of auxotrophic mutations increased 2.5-fold when treatment with lithium chloride was applied. A blue shift by 10 nm was recorded in the absorption spectrum of carotenoids form YM5-3 green mutant; considerable accumulation of neurosporine was revealed by HPLC and mass spectrometry. The method is efficient for isolating the mutants of photosynthetic bacteria. Published in Russian in Mikrobiologiya, 2006, Vol. 75, No. 6, pp. 758–764. The text was submitted by the authors in English.     

An efficient mutational method for photosynthetic bacteria

The pigment and auxotrophic mutants of Rhodobacter sphaeroides Y6 were obtained by treatment with ethyl methanesulfonate (EMS) followed by lithium chloride (LiCI). Treatment with 0.081 M EPS and subsequent treatment with 0.071 M LiCI resulted in 12% higher frequency of pigment mutations than application of 0.081 M EMS alone; the frequency of auxotrophic mutations increased 2.5-fold when treatment with lithium chloride was applied. A blue shift 10 nm was recorded in the absorption spectrum of carotenoids form YM5-3 green mutant; considerable accumulation of neurosporine was revealed by HPLC and mass spectrometry. The method is efficient for isolating mutants of photosynthetic bacteria.     

An efficient method for in vitro fertilization in rabbits

Abstract

This experiment was carried out to study a simple and efficient method for in vitro production of rabbit embryos. Newly ejaculated rabbit spermatozoa were used to fertilize superovulated oocytes after capacitation in vitro with four different media: (A) isotonic defined medium (DM)+heparin, (B) DM only,(C) DM+ high ionic strength defined medium (HIS), and (D) DM supplemented with 10mM NaHCO₃ (mDM) +HIS supplemented with 10mM NaHCO₃ (mHIS). The presumptive zygotes were cultured in M199 supplemented with 10% FCS, 1.25mM Na Pyruvate and 0.1mM EDTA (mM199). The cleavage rates after 24h of incubation were 29.3%, 32.1%, 64.9%, and 91.6% respectively, and the rates of blastocyst formation after 72h were 0, 27.3%, 58.4% and 85.2%, respectively. The results in the (D) treatment were significantly better than the other three treatments (p<0.01). Developmental potential of in vivo and in vitro derived zygotes was also compared using the mM199. The percentages of blastocyst and hatching blastocyst in the two groups were 92.5% and 87.2% after 84h, and 84.9% and 83.7% after 108h, respectively, and the two groups were not significantly different (p>0.05). The developmental progress of the two groups was nearly synchronous towards the end of culture. When IVF embryos from 2‐ to 4‐cell stage were transferred into recipients, the pregnancy rate did not differ from in vivo fertilization, but the rate of live young from IVF was significantly lower than from in vivo. The results of this experiment showed that ejaculated rabbit sperm could be capacitated efficiently after treatment of mDM and mHIS, and rabbit IVF embryos achieved great development in mM199 in vitro.     

An efficient method for in vitro fertilization in rabbits

This experiment was carried out to study a simple and efficient method for in vitro production of rabbit embryos. Newly ejaculated rabbit spermatozoa were used to fertilize superovulated oocytes after capacitation in vitro with four different media: (A) isotonic defined medium (DM)+heparin, (B) DM only,(C) DM+ high ionic strength defined medium (HIS), and (D) DM supplemented with 10mM NaHCO3 (mDM) +HIS supplemented with 10mM NaHCO3 (mHIS). The presumptive zygotes were cultured in M199 supplemented with 10% FCS, 1.25mM Na Pyruvate and 0.1mM EDTA (mM199). The cleavage rates after 24h of incubation were 29.3%, 32.1%, 64.9%, and 91.6% respectively, and the rates of blastocyst formation after 72h were 0, 27.3%, 58.4% and 85.2%, respectively. The results in the (D) treatment were significantly better than the other three treatments (p<0.01). Developmental potential of in vivo and in vitro derived zygotes was also compared using the mM199. The percentages of blastocyst and hatching blastocyst in the two groups were 92.5% and 87.2% after 84h, and 84.9% and 83.7% after 108h, respectively, and the two groups were not significantly different (p>0.05). The developmental progress of the two groups was nearly synchronous towards the end of culture. When IVF embryos from 2- to 4-cell stage were transferred into recipients, the pregnancy rate did not differ from in vivo fertilization, but the rate of live young from IVF was significantly lower than from in vivo. The results of this experiment showed that ejaculated rabbit sperm could be capacitated efficiently after treatment of mDM and mHIS, and rabbit IVF embryos achieved great development in mM199 in vitro.     

An efficient method for gene disruption in Neurospora crassa

The frequency with which transforming DNA undergoes homologous recombination at a chromosomal site can be quite low in some fungal systems. In such cases, strategies for gene disruption or gene replacement must either select against ectopic integration events or provide easy screening to identify homologous site, double-crossover insertion events. A protocol is presented for efficient isolation of Neurospora crassa strains carrying a definitive null allele in a target gene. The protocol relies on the presence of a selectable marker flanking a disrupted plasmid-borne copy of the gene, and in the case presented led to a seven-fold enrichment for putative homologous site replacement events. In addition, a polymerase chain reaction assay is utilized for rapid identification of homologous recombinants among the remaining candidates. This protocol was used to identify 3 isolates, out of 129 primary transformants, which have a disruption in the Neurospora ccg-1 gene. The method should be applicable to a variety of fungal systems in which two selectable markers can be expressed, including those in which homologous recombination rates are too low to allow easy identification of homologous site insertions by the more traditional molecular method of Southern analysis. In addition to disrupting target genes for the purpose of generating null mutations, this method is useful for the targeting of reporter gene fusions to a native chromosomal site for the purpose of studying gene regulation.     

Glycerol extracting dealcoholization for the biodiesel separation process

By means of utilizing sunflower oil and Jatropha oil as raw oil respectively, the biodiesel transesterification production and the multi-stage extracting separation were carried out experimentally. Results indicate that dealcoholized crude glycerol can be utilized as the extracting agent to achieve effective separation of methanol from the methyl ester phase, and the glycerol content in the dealcoholized methyl esters is as low as 0.02 wt.%. For the biodiesel separation process utilizing glycerol extracting dealcoholization, its technical and equipment information were acquired through the rigorous process simulation in contrast to the traditional biodiesel distillation separation process, and results show that its energy consumption decrease about 35% in contrast to that of the distillation separation process. The glycerol extracting dealcoholization has sufficient feasibility and superiority for the biodiesel separation process.     

An efficient and cost-effective isotope labeling protocol for proteins expressed in shape Escherichia coli

A cost-effective protocol for uniform ¹⁵N and/or¹³ C isotope labeling of bacterially expressed proteins is presented. Unlike most standard protocols, cells are initially grown in a medium containing nutrients at natural abundance and isotopically labeled nutrients are only supplied at the later stages of growth and during protein expression. This permits the accumulation of a large cell mass without the need to employ expensive isotopically labeled nutrients. The abrupt decrease in oxygen consumption that occurs upon complete exhaustion of essential nutrients is used to precisely time the switch between unlabeled and labeled nutrients. Application of the protocol is demonstrated for wild-type and a mutant of the N-terminal zinc-binding domain of HIV-1 integrase.     

An efficient molecular docking method for adsorbent screening

In this study, with flavonol glycosides (FG) and terpene lactones (TL) in ginkgo biloba extract (GBE) as the targets for separation, we investigated the effectiveness of molecular docking in adsorbent screening. Several polyamine-modified methyl acylate-co-divinylbenzene (MA-co-DVB) adsorbent models were built, and their affinity to rutin, quercetin and ginkgolide B (GB) was evaluated via molecular docking. The model of ethylenediamine-modified adsorbent showed the largest difference in affinity between to GB and to quercetin as well as rutin, and thus this adsorbent could have the best separation performance. The results of the subsequently conducted static adsorption and dynamic adsorption experiments correlated well with docking results. Finally, using ethylenediamine-modified MA-co-DVB adsorbent, nearly complete separation of the FG and TL in GBE was simply achieved by one step of adsorption-desorption. Thus, the reported molecular docking method is expected to be helpful for rapid adsorbent screening.     

An efficient method for explant sterilization for reduced contamination

An improved method for sterilization of explants was tested and found to be suitable for plants with elongated internodes, such as bamboos. Final cutting of the explants into single nodal segments for inoculation was done only after surface sterilization of multi-nodal explants in a stoppered glass measuring cylinder. This minimized penetration of the contaminants and the sterilizing agents into the exposed intercellular spaces and vascular cavities at the cut ends, thereby minimizing their harmful effects. The method was experimented upon three different plants, viz., bamboo, tea and rose. Through this method the number of cultures getting contaminated was substantially reduced as compared to the conventional means where single nodal explants were used, employing identical treatments. Moreover, in this method, the number of cultures showing bud-break also showed a marked increase thereby resulting in a tremendous increase in the percentage of successfully established proliferating cultures.     

An efficient method for the isolation and separation of basolateral-membrane and luminal-membrane vesicles from rabbit kidney cortex.

A procedure for isolation and separation of purified luminal-membrane and basolateral-membrane vesicles from adult and newborn rabbit renal cortex by using Ca2+/Mg2+ precipitation, differential centrifugation and a self-orienting Percoll-gradient centrifugation is described. The purity of the membrane-vesicle suspensions was examined by electron microscopy and by measuring the activity of several marker enzymes. The activity of Na+ + K+-stimulated ATPase in the fraction mainly containing adult rabbit basolateral-membrane vesicles was enriched 16-fold, and the activity of alkaline phosphatase in the fraction mainly containing luminal-membrane vesicles was increased 13-fold, compared with the homogenate. Similar results were obtained with kidneys from newborn rabbits. Uptake studies, with a rapid filtration technique and the spectrophotometric method described in an accompanying paper [Kragh-Hansen, Jørgensen & Sheikh (1982) Biochem. J. 208, 359-368], showed that both adult and newborn rabbit luminal-membrane vesicles, in contrast with the basolateral-membrane preparations, possess an Na+-dependent electrogenic transport system for L-proline. Adult rabbit luminal-membrane vesicles take up citrate and L-malate by Na+-dependent electrogenic processes, whereas adult rabbit basolateral membrane vesicles do not exhibit electrogenic uptake of citrate. By contrast, these vesicles show Na+-dependent electrogenic uptake of L-malate.     

An efficient staining method for ultrathin sections.

A staining method to handle simultaneously as many as 20 electron microscope grids is described. The devices used are easily constructed of readily obtained inexpensive materials. The volumes of stain and wash water required are very small and drying grids is simplified.     

Rapid and efficient method for the size separation of homogeneous fluorescein-encapsulating liposomes

Liposomes are colloidal structures formed by the self-assembly of lipid molecules in solution into spherical, self-closed structures through their amphiphilic properties. All liposome preparation protocols reported consist of several steps of preparation, homogenization, and purification, which are labor-intensive, arduous, and lengthy to execute. In this work, a new procedure has been developed to reduce the time of the postrehydration sizing of liposomes from multilamellar vesicles, while improving the uniformity of the resulting liposomes produced and achieving high encapsulation efficiencies. For the homogenization step, the typically used method of filter extrusion was substituted by centrifugation. Purification of liposomes to eliminate nonencapsulated molecules and lipids is routinely carried out via gel permeation chromatography, an extremely lengthy procedure, and in the method we report, this lengthy step was replaced by the use of molecular-weight cut-off filters. Using this novel method, large unilamellar vesicles were produced and the time required, postrehydration, was dramatically reduced from almost 48 to less than 2 hours, with a highly uniformly sized population of liposomes being produced—the homogeneity of the liposome population achieved using our method was 99%, as compared to 88% attained by using the traditional method of production. We have used this approach to encapsulate fluorescein isothiocyanate (FITC), and 160,000 FITC molecules were encapsulated and the liposomes were demonstrated to be stable for at least 10 weeks at 4°C.