Inherent limitations to the problem of reducing the lysine microbiological assay time

A kinetic approach is proposed to shorten the microbiological assay time for the determination of unboundl-lysine. The present lysine bacterial assay takes from 16 to 24 h usingPediococcus cerevisiae P-60 ATCC 8042 (formerlyLeuconostoc mesenteroides P-60 ATCC 8042) and uses a medium in which lysine is the limiting substrate. Measurements of the final cell concentration are linearly correlated with the initial concentration of lysine,S*, to provide an indirect estimate ofS*. We propose to understand the limitations inherent to the reduction of the assay time to 4 h by focusing in our analysis on the bacterial late lag or early growth transient phases, rather than the stationary phase of growth. Generally, the Monod equation is expected to describe a hyperbolically increasing correlation between the bacterial specific growth rate at about 2–4 h and the initial lysine concentration. A hyperbolic correlation is obtained by 3 h, but the lysine region of interest falls in the saturated portion of the curve. Discriminations between different initial lysine levels are therefore difficult with this nearly flat curve. On the other hand, when the initial inoculum level is lowered, so that substrate inhibition becomes effective, a correlation with a large negative slope is obtained by 4 h. Limitations to using absorbance measurements for the rapid assay turn up in a lack of reproducibility and, hence, a large variance associated with the measurements. Alternative microbial measuring techniques, such as impedance methods, need to be examined in order to reduce that large variance.     

Biflavones from Ginkgo biloba as inhibitors of human thrombin

Ginkgo Biloba leaf extract has been widely used for the prevention and treatment of thrombosis and cardiovascular disease in both eastern and western countries, but the bioactive constituents and the underlying mechanism of anti-thrombosis have not been fully characterized. The purpose of this study was to investigate the inhibitory effects of major constituents in Ginkgo biloba on human thrombin, a key serine protease regulating the blood coagulation cascade and the processes of thrombosis. To this end, a fluorescence-based biochemical assay was used to assay the inhibitory effects of sixteen major constituents from Ginkgo biloba on human thrombin. Among all tested natural compounds, four biflavones (ginkgetin, isoginkgetin, bilobetin and amentoflavone), and five flavonoids (luteolin, apigenin, quercetin, kaempferol and isorhamnetin) were found with thrombin inhibition activity, with the IC₅₀ values ranging from 8.05 μM to 82.08 μM. Inhibition kinetic analyses demonstrated that four biflavones were mixed inhibitors against thrombin-mediated Z-GGRAMC acetate hydrolysis, with the K_i values ranging from 4.12 μM to 11.01 μM. Molecular docking method showed that the four biflavones could occupy the active cavity with strong interactions of salt bridges and hydrogen bonds. In addition, mass spectrometry-based lysine labeling reactivity assay suggested that the biflavones could bind on human thrombin at exosite I rather than exosite II. All these findings suggested that the biflavones in Ginkgo biloba were naturally occurring inhibitors of human thrombin, and these compounds could be used as lead compounds for the development of novel thrombin inhibitors with improved efficacy and high safety profiles.     

A comparison of three methods for the determination of available lysine in barley

The protein quality of barley was determined by the measurement of the protein efficiency ratio (PER). Available lysine in the same samples was determined chemically and by microbiological assay, and relationships were obtained between PER and either total or available lysine content.In the PER test, groundnut meal was found to be a suitable protein supplement to barley for the purpose of detecting differences in the protein quality of barley. The method was sufficiently sensitive to detect a reduction in protein quality of one variety as a result of micronization. Available lysine values obtained by the modified Tetrahymena method were significantly correlated (r = 0.99) with PER values; there was no significant correlation between total lysine content and the PER value.The available lysine values measured by the two chemical methods were higher than the Tetrahymena values. The Silcock method gave results better correlated (r = 0.93) with the Tetrahymena values than did the Carpenter method (r = 0.82). The results of chemical methods were not significantly related with PER values, but the methods ranked the barleys in an order similar to that obtained with the PER and Tetrahymena tests.     

A Comparison of Seven Strains of Lactobacillus casei var. rhamnosus in Relation to their Use in the Microbiological Assay of Serum Folate

S ummary . A comparison was made of 7 strains of Lactobacillus casei var. rhamnosus in relation to their use in the microbiological assay of serum folate. The problem of clumping by some strains could be overcome by the addition of extra salt, or chloramphenicol, to the medium. The strains showed differences in their tolerance to added salts which was measured as the Mean Inhibitory Salt Concentration. The highest salt concentration which could be used in the assay medium without affecting the serum folate results was about one-third of the Mean Inhibitory Salt Concentration and this could be less than the optimum concentration for growth. These differences in salt tolerance may provide a partial explanation of the variety of normal values reported from different laboratories.     

The determination of available lysine in barley using Tetrahymena pyriformis W.

Attempts to use the microbiological procedure based on growth of Tetrahymena for the assay of available lysine in barley led to several technical problems. These were mainly due to interference by carbohydrate with the measurement of the growth response of the organism, either by cell counting or by optical density.The assay was modified to suit the measurement of available lysine in barley grains. Fine grinding of samples, predigestion with papain and selection of appropriate N-concentrations in the medium were found to be key factors in adapting the technique for barley.The availability of lysine in barley, as assessed by the modified technique was found to range from 62 to 73%. Heating and micronization of barley grains reduced availability to 54 and 51%, respectively.     

Aspartokinase of Lemna paucicostata Hegelm. 6746

A sensitive and specific method was developed for assay of aspartokinase (EC 2.7.2.4) in crude extracts of Lemna paucicostata. Lysine inhibited approximately 93%, and threonine approximately 6%; together, these amino acids inhibited 99%. Inhibition by lysine was synergistically increased by S-adenosylmethionine, which by itself had no effect on activity. Essentially complete inhibition of threonine-resistant activity was obtained with lysine, and of lysine-resistant activity with threonine. Inhibition by lysine and threonine was additive, with no indication of concerted inhibition. Aspartate concentration had no effect on the relative proportions of lysine- and threonine-sensitive activities. Aspartokinase activity was in large excess of that reported by other workers, the maximum capacity (V_max) far exceeding the in vivo requirements. Estimations of rates of aspartokinase in vivo suggest that the step catalyzed by this enzyme may not be the overall `rate-limiting' one for entry of 4-carbon units into the aspartate family of amino acids, and that feedback inhibition of this enzyme by lysine and threonine may not be a major factor in regulating flux through this step.     

Mono-sulfonated tetrazolium salt based NAD(P)H detection reagents suitable for dehydrogenase and real-time cell viability assays

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and is important for several biological processes. For GDH inhibitor screening, we developed a novel mono-sulfonated tetrazolium salt (EZMTT), which can be synthesized using H₂O₂ oxidation and purified easily on silica gel in large quantities. The EZMTT detection method showed linear dose responses to NAD(P)H, dehydrogenase concentration and cell numbers. In E. coli GDH assay, the EZMTT method showed excellent assay reproducibility with a Z factor of 0.9 and caused no false positives in the presence of antioxidants (such as BME). Using the EZMTT-formazan-NAD(P)H system, we showed that EGCG is a potent E. coli GDH inhibitor (IC₅₀ 45 nM) and identified that Ebselen, a multifunctional thioredoxin reductase inhibitor, inactivated E. coli GDH (IC₅₀ 213 nM). In cell-based assays at 0.5 mM tetrazolium concentration, EZMTT showed essentially no toxicity after a 3-day incubation, whereas 40% of inhibition was observed for WST-8. In conclusion, EZMTT is a novel tetrazolium salt which provides improved features that are suitable for dehydrogenases and real-time cell-based high-throughput screening (HTS).     

Catalysis by the Tumor-Suppressor Enzymes PTEN and PTEN-L

Phosphatase and tensin homologue deleted from chromosome ten (PTEN) is a lipid phosphatase tumor suppressor that is lost or inactivated in most human tumors. The enzyme catalyzes the hydrolysis of phosphatidylinositol-(3,4,5)-trisphosphate (PIP₃) to form phosphatidylinositol-(4,5)-bisphosphate (PIP₂) and inorganic phosphate. Here, we report on the first continuous assay for the catalytic activity of PTEN. Using this assay, we demonstrate that human PTEN is activated by the reaction product PIP₂, as well as in solutions of low salt concentration. This activation is abrogated in the K13A variant, which has a disruption in a putative binding site for PIP₂. We also demonstrate that PTEN-L, which derives from alternative translation of the PTEN mRNA, is activated constitutively. These findings have implications for catalysis by PTEN in physiological environments and could expedite the development of PTEN-based chemotherapeutic agents.     

Methylated Polyl(L-lysine): Conformational Effects and Interactions with Polynucleotides

Abstract

Methylated lysine, arginine and histidine residues are found in a number of proteins (for example, histones, non-histone chromosomal proteins, ribosomal proteins, calmodulin, cytochrome C, etc.). We are studying the effects of methylation on the conformations of poly(lysine) and of the effects of methylation of poly(lysine) and poly(arginine) on interactions with polynucleotides. The conformational properties of e-amino-methylated poly(lysine) differ from those of unmodified poly(lysine). Methylation increases resistance to thermally- induced and NaCl-induced changes in the CD spectrum. Guanidinium chloride increases (proportional to the degree of methylation) the extent of approach to the conformation in dispute as to its being a random coil or an extended helix. Methylation enhances aggregation in the helix-inducing solvent 0.5 M Ca(ClO₄)₂. With increasing methylation of poly(lysine), the conformation in dodecyl sulfate changes from β, to 50% α, to random coil at the maximum methylation.

Increasing methylation of poly(lysine) weakens the interaction with polynucleotides in respect to dissociation by salt, linearly with methyl content. Complexes of (dAdT)_n·(dAdT)_n with the polypeptides are increasingly stabilized to heat denaturation by progressive methylation. However, with a series of synthetic double-stranded RNA's and DNA's a more complex situation exists, Tm increasing or decreasing, depending on the base composition, sequence and type of sugar. Methylation of poly(lysine) and poly(arginine) can have opposite effects on Tm based on results with complexes with (dI)_n·(dC)_n. Methylated poly(lysine) affects the CD spectrum of polynucleotides, in a manner dependent on base composition and sequence. In some cases large positive or negative ψ-spectra are induced, which, in the case of (dGdC)_n·(dGdC)_n, can be positive or negative depending on the degree of methylation of the polypeptide and the salt concentration.

It is suggested that the biological effects of methylated proteins may be evoked by salt changes in the cell cycle, and that methylation can affect local interactions with nucleic acids and larger scale structure, and interactions with lipids.     

Degradation of phenol by a halotolerant strain of Penicillium chrysogenum

The lowest 50% lethal (effective) concentration, L(E)C₅₀, of phenol in a battery of seven microbiotests with species representing different trophic levels was 1–10 mg l⁻¹, classifying it as “toxic”. A phenol-degrading microorganism was isolated from soil samples of the salt mine of Clona in Portugal, after enrichment in the presence of phenol and high salt concentration. Based on cultural and morphological characteristics, the strain CLONA2 was identified as belonging to Penicillium chrysogenum. It was found to be a halotolerant fungus able to grow in a nutrient-rich medium with 5.8% NaCl. It degraded at least 300 mg l⁻¹ phenol as sole source of carbon and energy, without accumulation of intermediates. The samples were also tested for toxicity using the Microtox^® assay. Data showed that P. chrysogenum CLONA2 could be effectively utilized to reduce phenol toxicity. The results suggest also that phenol under saline conditions can be successfully mineralized by P. chrysogenum CLONA2.     

Development of a radiometric metabolic viability testing method for human and porcine skin

A radiometric viability assay based upon the conversion of [¹⁴C]glucose into ¹⁴CO₂ by the viable cells on the dermal side of whole skin has been developed. The assay proved to be sensitive, reproducible, and practical, and was based upon the use of a microbiological growth detection system commonly used in many hospitals and laboratories. Relatively small samples of skin (0.25–1.00 g) were used in the test, and it was found that microbiological contamination did not interfere with the assay under normal conditions. The linear proportionality of the assay with both time and amount of skin assayed precluded the difficulties of nonlinear proportionality in other systems, allowing direct comparisons to be made between skin samples of different sizes and different incubation times. The assay could also detect ¹⁴CO₂ released from many radiolabeled substrates, including glucose, aspartate, glutamate, ornithine, orotic acid, and glycerol. Thus, the method could be used to test a number of cellular functions necessary for viability, including glycolysis, the functioning of the citric acid cycle and the pentose phosphate pathway, sugar and amino acid metabolism, pyrimidine biosynthesis, and cryopreservative agent metabolism. Since any of these tests could be performed in 4 hr, a viability assay based upon glycolysis alone, or in combination with any of the other tested substrates, could be carried out after allograft skin procurement before a decision needed to be made on skin cryopreservation.     

The β Subunit of Eukaryotic Translation Initiation Factor 2 Binds mRNA through the Lysine Repeats and a Region Comprising the C2-C2 Motif

Eukaryotic translation initiation factor 2 (eIF2) has been implicated in the selection of the AUG codon as the start site for eukaryotic translation initiation, since mutations in its three subunits in yeast that allow the recognition of a UUG codon by the anticodon of the initiator Met-tRNA^Met have been identified. All such mutations in the beta subunit of eIF2 (eIF2β) mapped to a region containing a putative zinc finger structure of the C₂-C₂ type, indicating that these sequences could be involved in RNA recognition. Another feature of eIF2β that could mediate an interaction with RNA is located in the amino-terminal sequences and is composed of three repeats of seven lysine residues which are highly conserved in other species. We show here the ability of eIF2β, purified from Escherichia coli as a fusion to glutathione S-transferase, to bind mRNA in vitro. Through a deletion analysis, mRNA binding was found to be dependent on the lysine repeats and a region encompassing the C₂-C₂ motif. Strong mRNA binding in vitro could be maintained by the presence of only one lysine or one arginine run but not one alanine run. We further show that only one run of lysine residues is sufficient for the in vivo function of eIF2β, probably through charge interaction, since its replacement by arginines did not impair cell viability, whereas substitution for alanines resulted in inviable cells. mRNA binding, but not GTP-dependent initiator Met-tRNA^Met binding, by the eIF2 complex was determined to be dependent on the presence of the lysine runs of the beta subunit.     

Crystal structure of the sodium–proton antiporter NhaA dimer and new mechanistic insights

Sodium–proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium–proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium–proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pK_a calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium–proton antiport in NhaA.     

A molecular approach to fertilization. II. Viability and artificial fertilization of Xenopus laevis gemetes

Conditions necessary for procuring, handling, and storing Xenopus laevis gametes have been investigated in order to develop a simple and reliable method for artificial fertilization. Temperature, sperm concentration, and ionic strength influence sperm and/or egg viability. Extension of the fertilizing capacity of sperm suspensions could be correlated with reversible repression of sperm motility. In the case of various salt solutions used for testes maceration, inhibition of sperm motility was primarily a function of the ionic strength. However, there was some specificity in the salt employed as KCl and CaCl₂ solutions gave anomalous results in comparison with those obtained with other salt solutions. The fertilizability of Xenopus laevis eggs could be extended by shedding the eggs in DeBoers solution. Using this information a simple reliable method for artificial fertilization of Xenopus laevis gametes has been described.     

A simple and continuous assay for decarboxylase activity using a carbon dioxide-selective electrode

A carbon dioxide electrode has been used for kinetic studies of decarboxylase activity. The methodology used requires only a sensitive pH meter operating in the millivolt mode with the electrode. The CO₂ release may be followed continuously. Compared to other assay procedures the method outlined offers the advantages of rapidity, continuity, specificity, and sufficient precision. Techniques for the calibration of the electrode response are discussed. The applicability of this assay was appraised by determining the K_m of lysine decarboxylase. The value obtained showed good agreement with that reported in literature. The procedure might be useful for the study of enzyme-inhibitor interactions.     

Lysine acetylation stabilizes SP2 protein in the silkworm Bombyx mori

Lysine acetylation (Kac) is a vital post-translational modification that plays an important role in many cellular processes in organisms. In the present study, the nutrient storage proteins in hemolymph were first found to be highly acetylated—particularly SP2 protein, which contains 20 potential Kac sites. Further results confirmed that lysine acetylation could stabilize and up-regulate the protein level of anti-apoptosis protein SP2, thereby improving the survival of H₂O₂-treated BmN cells and suppressing the apoptosis induced by H₂O₂. The potential mechanism involved in the inhibition of ubiquitin-mediated proteasomal degradation by crosstalk between lysine acetylation and ubiquitination. Our results showed that the increase in the acetylation level by TSA could decrease the ubiquitination and improve the protein level of SP2, indicating that lysine acetylation could influence the SP2 protein level through competition between ubiquitination and the suppression of ubiquitin-mediated proteasomal degradation, thereby stabilizing the protein. SP2 is a major nutrient storage protein from hemolymph for amino acid storage and utilization. The crosstalk between lysine acetylation and ubiquitination of SP2 might imply an important role of lysine acetylation for nutrient storage and utilization in silkworm.     

Ectopic over-expression of peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt stress tolerance in transgenic peanut (Arachis hypogaea)

Peroxisomal ascorbate peroxidase gene (SbpAPX) of an extreme halophyte Salicornia brachiata imparts abiotic stress endurance and plays a key role in the protection against oxidative stress. The cloned SbpAPX gene was transformed to local variety of peanut and about 100 transgenic plants were developed using optimized in vitro regeneration and Agrobacterium mediated genetic transformation method. The T₀ transgenic plants were confirmed for the gene integration; grown under controlled condition in containment green house facility; seeds were harvested and T₁ plants were raised. Transgenic plants (T₁) were further confirmed by PCR using gene specific primers and histochemical GUS assay. About 40 transgenic plants (T₁) were selected randomly and subjected for salt stress tolerance study. Transgenic plants remained green however non-transgenic plants showed bleaching and yellowish leaves under salt stress conditions. Under stress condition, transgenic plants continued normal growth and completed their life cycle. Transgenic peanut plants exhibited adequate tolerance under salt stress condition and thus could be explored for the cultivation in salt affected areas for the sustainable agriculture.     

Effects of NaCl on Metabolic Heat Evolution Rates by Barley Roots

The effect of salinity stress on metabolic heat output of barley (Hordeum vulgare L.) root tips was measured by isothermal microcalorimetry. Several varieties differing in tolerance to salinity were compared and differences quantified. Two levels of inhibition by increasing salt were found. Following the transition from the initial rate to the first level, inhibition remained at about 50% with further increases in salt concentration up to 150 millimolar. The concentration of salt required to inhibit to this level was cultivar dependent. At higher concentrations (>150 millimolar) of salt, metabolism was further decreased. This decrease was not cultivar dependent. The decreased rate of metabolic heat output at the first transition could be correlated with decreases in uptake of NO₃⁻, NH₄⁺, and Pi that occurred as the salt concentration was increased. The high degree of dependence of the inhibition of metabolic heat output on NaCl concentration points to a highly cooperative reaction responsible for the general inhibition of metabolism and nutrient uptake. The time required to attain the first level of salt inhibition is less than 20 minutes. Inhibition of root tips was not reversible by washing with salt free solutions. In addition to revealing these features of salt inhibition, isothermal microcalorimetry is a promising method for convenient and rapid determination of varietal differences in response to increasing salinity.     

Determination of urinary oxalic acid and of oxalate pool size by stable isotope dilution.

An isotope dilution procedure for oxalate based upon [1,2-¹³C₂]oxalic acid is described. For routine determinations of urinary concentration, a known quantity of sodium [1,2-¹³C]oxalate is admixed with the sample, total oxalate precipitated as the calcium salt, and converted by BF₃ catalysis to di-n-propyl esters for mass-spectrometric analysis. Selective ion monitoring provides ¹²C:¹³C ratios directly, thus precluding the necessity for quantitative recovery at any step of the rapid, single-tube assay. Following a bolus injection of sodium [1,2-¹³C]oxalate, whole body oxalate pools and their turnover rates can be determined by sequential sampling of urine. Biosynthetic rates calculated from the product of pool size and turnover are in excellent agreement with urinary excretion rates, confirming directly that urinary oxalate is a quantitative index of biosynthesis.