Separation of biological variant insulin molecules from different species by reversed-phase high-performance liquid chromatography (HPLC)

Three different isocratic systems for the separation by reversed-phase high-performance liquid chromatography (HPLC) of different species of insulin have been investigated. The effect of different solvent compositions and temperatures on elution time and resolution have been studied. These studies have been used to devise a method for reversed-phase liquid chromatographic separation of bovine, porcine, and human insulin, as well as the A and B chains of bovine insulin. The method can also be used for the separation of the various products of the iodination of porcine insulin. 125I-A14 tyrosine-labeled porcine insulin can be readily separated from nonlabeled porcine insulin and from other iodinated constituents of the mixture. A flow-though gamma-counting system that was designed for this work is described.     

A strategy to investigate the plant meiotic proteome

The analysis of meiosis in higher plants has benefited considerably in recent years from the completion of the genome sequence of the model plant Arabidopsis thaliana and the development of cytological techniques for this species. A combination of forward and reverse genetics has provided important routes toward the identification of meiotic genes in Arabidopsis. Nevertheless identification of certain meiotic genes remains a challenge due to problems such as limited sequence conservation between species, existence of closely related gene families and in some cases functional redundancy between gene family members. Hence there is a requirement to develop new experimental approaches that can be used in conjunction with existing methods to enable a greater range of plant meiotic genes to be identified. As one potential route towards this goal we have initiated a proteomics-based approach. Unfortunately, the small size of Arabidopsis anthers makes an analysis in this species technically very difficult. Therefore we have initially focussed on Brassica oleracea which is closely related to Arabidopsis, but has the advantage of possessing significantly larger anthers. The basic strategy has been to use peptide mass-finger printing and matrix-assisted laser desorption ionization time of flight mass spectrometry to analyse proteins expressed in meiocytes during prophase I of meiosis. Initial experiments based on the analysis of proteins from staged anther tissue proved disappointing due to the low level of detection of proteins associated with meiosis. However, by extruding meiocytes in early prophase I from individual anthers prior to analysis a significant enrichment of meiotic proteins has been achieved. Analysis suggests that at least 18% of the proteins identified by this route have a putative meiotic function and that this figure could be as high as one-third of the total. Approaches to increase the enrichment of proteins involved in meiotic recombination and chromosome synapsis are also described.     

A robust purification strategy to accelerate membrane proteomics

The preparation of large quantities of purified membrane proteins for structural studies presents significant difficulties. Central among these are the frequent toxicity associated with over-expressing membrane targets and the difficulty associated with identifying the appropriate detergents for their solubilization and purification. To begin addressing these challenges, and lay the groundwork for membrane structural genomics efforts, we have developed a robust strategy for the expression and purification of large numbers of prokaryotic membrane proteins. Our approach rapidly identifies highly expressed targets and greatly simplifies their solubilization and purification. In this review, specific, hands-on protocols are provided for the expression and purification of CorA magnesium transporters. These methods form the basis for the expression and purification of many other membrane proteins, as discussed.     

Normal mode analysis with simplified models to investigate the global dynamics of biological systems

Dynamical properties of macromolecules are increasingly being recognized as significantly contributing to biological functions, including catalysis, regulation of activity, etc. In this review, theoretical approaches to the study of dynamics of biological systems and their application are discussed. In particular, simplified models for the normal mode analysis are described.     

Using HPLC pigment analysis to investigate phytoplankton taxonomy: the importance of knowing your species

Phytoplankton microscopic enumerations and HPLC analyses of their pigments were performed weekly for a complete year at a coastal station in the English Channel. The taxonomic composition of the phytoplankton community was assessed using the HPLC results combined with the mathematical tool CHEMTAX in two different ways. Firstly, without using the species level taxonomic information obtained at the microscopic level (blind analyses), and secondly by including the information from the microscopic taxonomic analysis (directed analyses). The results indicate that, due to the particular pigment composition of some species (for example, the dinoflagellate, Karenia mikimotoi and the haptophyte, Phaeocystis pouchetii), a blind analysis would result in very significant errors in the taxonomic determination of the bloom events at this station. Major blooms of Karenia mikimotoi and P. pouchetii were mistaken for blooms of diatoms on the basis of a blind HPLC-CHEMTAX analysis. Only with the information from the microscopic observations was it possible to obtain an accurate representation of the phytoplankton community.Communicated by H.-D. Franke     

Association of polyamines to different parts of various plant species

The variation in polyamine content in different plant species and in different parts within a plant can be considerable. To get general information about levels of polyamines in plants and about the association of polyamines to different types of tissue, 30 plants from 13 plant families were examined for their polyamine content before and after germination using high pressure liquid chromatography (HPLC) analysis. A marked increase in polyamine content occurs in the cotyledons or endosperms in the seeds on germination, i.e. in the nutrient storing and exporting part of the plant. In the radicle, hypocotyl or coleoptile, i.e. growing parts of the plant, an increase in polyamine content is rarely observed. Additionally, polyamine levels can be very low [below 1 nmol (g fresh weight)^-1] in different parts of various species. Obviously, levels in the pmol (g fresh weight)^-1 range satisfy the needs of many growing plant parts. The high levels of polyamines found especially in cotyledons cannot be explained by their postulated association with increased cell division rates.     

A method for determination of phosphatidylethanol from high density lipoproteins by reversed-phase HPLC with TOF-MS detection

Phosphatidylethanol (PEth) is a unique phospholipid that is formed in the body only in the presence of ethanol. According to a new hypothesis, blood high-density lipoprotein (HDL) particles may act as carriers of PEth and mediate part of the antiatherogenic effects of moderate alcohol drinking. Liquid chromatographic method using reversed-phase C8 column and negative ion mode electrospray ionization-mass spectrometry detection with time-of-flight (TOF) instrument was developed for the determination of very small amounts of PEth that might be present on blood HDL particles. The samples used in the current study were human HDL spiked with PEth and internal standard phosphatidylpropanol (PProp). The use of reversed-phase column enabled a short analysis time of 19 min/injection, which is only one-third of the earlier normal-phase methods reported. Because of the narrow bore column (2.1 mm i.d.) and short analysis time, the solvent consumption was decreased. The sensitivity of detection obtained with TOF-MS was better than that of previous methods, with the detection limit being as low as 1 ng/ml in injected sample (20 pg on-column approximately 28 fmol PEth), corresponding to approximately 6.7 ng of PEth in milliliter of unprepared HDL. Good linearity of detection was obtained for a range of 1-100 ng/ml of PEth, whereas all of the deviations in precision and accuracy were less than 15%.     

Sampling strategy matters to accurately estimate response curves' parameters in species distribution models

Aim

Assessing how different sampling strategies affect the accuracy and precision of species response curves estimated by parametric species distribution models.

Major Taxa Studied

Virtual plant species.

Location

Abruzzo (Italy).

Time Period

Timeless (simulated data).

Methods

We simulated the occurrence of two virtual species with different ecology (generalist vs specialist) and distribution extent. We sampled their occurrence following different sampling strategies: random, stratified, systematic, topographic, uniform within the environmental space (hereafter, uniform) and close to roads. For each sampling design and species, we ran 500 simulations at increasing sampling efforts (total: 42,000 replicates). For each replicate, we fitted a binomial generalised linear model, extracted model coefficients for precipitation and temperature, and compared them with true coefficients from the known species' equation. We evaluated the quality of the estimated response curves by computing bias, variance and root mean squared error (RMSE). Additionally, we (i) assessed the impact of missing covariates on the performance of the sampling approaches and (ii) evaluated the effect of incompletely sampling the environmental space on the uniform approach.

Results

For the generalist species, we found the lowest RMSE when uniformly sampling the environmental space, while sampling occurrence data close to roads provided the worst performance. For the specialist species, all sampling designs showed comparable outcomes. Excluding important predictors similarly affected all sampling strategies. Sampling limited portions of the environmental space reduced the performance of the uniform approach, regardless of the portion surveyed.

Main Conclusions

Our results suggest that a proper estimate of the species response curve can be obtained when the choice of the sampling strategy is guided by the species' ecology. Overall, uniformly sampling the environmental space seems more efficient for species with wide environmental tolerances. The advantage of seeking the most appropriate sampling strategy vanishes when modelling species with narrow realised niches.     

A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species

Advances in next generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS) is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs). This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM) and barley (Oregon Wolfe Barley) recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tagged sites becomes the reference. Alternatively, for kinship analyses in the absence of a reference genome, the sequence tags can simply be treated as dominant markers. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.     

A machine learning approach to investigate the reasons behind species extinction

Species extinction is one of the most important phenomena in conservation biology. Many factors are involved in the disappearance of species, including stochastic population fluctuations, habitat change, resource depletion, and inbreeding. Due to the complexity of the interactions between these various factors and the lengthy time period required to make empirical observations, studying the phenomenon of species extinction can prove to be very difficult in nature. On the other hand, an investigation of the various features involved in species extinction using individual-based simulation modeling and machine learning techniques can be accomplished in a reasonably short period of time. Thus, the aim of this paper is to investigate multiple factors involved in species extinction using computer simulation modeling. We apply several machine learning techniques to the data generated by EcoSim, a predator–prey ecosystem simulation, in order to select the most prominent features involved in species extinction, along with extracting rules that outline conditions that have the potential to be used for predicting extinction. In particular, we used five feature selection methods resulting in the selection of 25 features followed by a reduction of these to 14 features using correlation analysis. Each of the remaining features was placed in one of three broad categories, viz., genetic, environmental, or demographic. The experimental results suggest that factors such as population fluctuation, reproductive age, and genetic distance are important in the occurrence of species extinction in EcoSim, similar to what is observed in nature. We argue that the study of the behavior of species through Individual-Based Modeling has the potential to give rise to new insights into the central factors involved in extinction for real ecosystems. This approach has the potential to help with the detection of early signals of species extinction that could in turn lead to conservation policies to help prevent extinction.     

Comparative study of seed albumins in the Old-WorldLupinus species (Fabaceae) by reversed-phase HPLC

Seed albumins and 2S proteins isolated from the albumin fraction of 36 accessions representing 10 Old-WorldLupinus species (5 smooth- and 5 rough-seeded) were studied using reversed-phase high-performance liquid chromatography. In addition, the globulin fraction was analyzed to determine its 2S protein content. The performed separations showed the suitability of RP-HPLC technique in the analysis of variation of the seed albumin composition in lupins. In the group of rough-seeded lupins, 3 types of RP-HPLC elution profiles of albumins were distinguished: (1)L. atlanticus, (2)L. cosentinii andL. digitatus, (3)L. palaestinus andL. pilosus. All the species of this group were found to have proteins not observed in smooth-seeded species. Smooth-seeded species exhibited more abundant protein spectra, each species distinguishing by its specific RP-HPLC elution profile. It was found that 2S proteins classified as 2S albumins were responsible for the observed variation. Depending onLupinus species, the 2S albumin class consists of two to six proteins.     

Towards high resolution analysis of metabolic flux in cells and tissues

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Novel strategy to evaluate infectious salmon anemia virus variants by high resolution melting

Genetic variability is a key problem in the prevention and therapy of RNA-based virus infections. Infectious Salmon Anemia virus (ISAv) is an RNA virus which aggressively attacks salmon producing farms worldwide and in particular in Chile. Just as with most of the Orthomyxovirus, ISAv displays high variability in its genome which is reflected by a wider infection potential, thus hampering management and prevention of the disease. Although a number of widely validated detection procedures exist, in this case there is a need of a more complex approach to the characterization of virus variability. We have adapted a procedure of High Resolution Melting (HRM) as a fine-tuning technique to fully differentiate viral variants detected in Chile and projected to other infective variants reported elsewhere. Out of the eight viral coding segments, the technique was adapted using natural Chilean variants for two of them, namely segments 5 and 6, recognized as virulence-associated factors. Our work demonstrates the versatility of the technique as well as its superior resolution capacity compared with standard techniques currently in use as key diagnostic tools.     

Cellular models to investigate biochemical pathways in Parkinson's disease

Cellular models are instrumental in dissecting a complex pathological process into simpler molecular events. Parkinson's disease is multifactorial and clinically heterogeneous; the aetiology of the sporadic (and most common) form is still unclear and only a few molecular mechanisms have been clarified so far in the neurodegenerative cascade. In such a multifaceted picture, it is particularly important to identify experimental models that simplify the study of the different networks of proteins/genes involved. Cellular models that reproduce some of the features of the neurons that degenerate in Parkinson's disease have contributed to many advances in our comprehension of the pathogenic flow of the disease. In particular, the pivotal biochemical pathways (i.e. apoptosis and oxidative stress, mitochondrial impairment and dysfunctional mitophagy, unfolded protein stress and improper removal of misfolded proteins) have been widely explored in cell lines, challenged with toxic insults or genetically modified. The central role of α-synuclein has generated many models aiming to elucidate its contribution to the dysregulation of various cellular processes. In conclusion, classical cellular models appear to be the correct choice for preliminary studies on the molecular action of new drugs or potential toxins and for understanding the role of single genetic factors. Moreover, the availability of novel cellular systems, such as cybrids or induced pluripotent stem cells, offers the chance to exploit the advantages of an in vitro investigation, although mirroring more closely the cell population being affected.     

A human exposure study to investigate biological monitoring methods for 2-butoxyethanol

The enzymatic activity of acetylcholinesterase (AChE) has been shown to be altered by environmental contaminants such as metals. However, the available literature illustrates a background of contradictory results regarding these effects. Therefore, the main purpose of this study was to investigate the potential of five metal ions (nickel, copper, zinc, cadmium and mercury) to inhibit AChE activity in vitro. First, to accomplish this objective, the possible interference of metals as test toxicants in the Ellman's assay, which is widely used to assess AChE activity, was studied. The potential influence of two different reaction buffers (phosphate and Tris) was also determined. The results suggest that the selected metals react with the products of this photometric technique. It is impossible to ascertain the artefactual contribution of the interaction of the metals with the technique when measuring AChE inhibition. This constitutes a major obstacle in obtaining accurate data. The presence of phosphate ions also makes enzymatic inhibition difficult to analyse. Attending to this evidence, an assay using the substrate o-nitrophenyl acetate and Tris buffer was used to investigate the effects of metals on AChE activity. O-nitrophenyl acetate is also a substrate for esterases other than cholinesterases. It is therefore only possible to use it for the measurement of cholinesterase activity with purified enzymes or after a previous verification of the absence of other esterases in the sample tissue. Under these conditions, the results indicate that with the exception of nickel, all tested metals significantly inhibit AChE activity.     

A human exposure study to investigate biological monitoring methods for 2-butoxyethanol

2-Butoxyethanol is a glycol ether widely used in printing inks, varnishes and cleaning fluids. As skin absorption can be significant, biological monitoring is useful in monitoring worker exposure. A number of analytes and matrices have been used previously, including 2-butoxyethanol in blood and free and total 2-butoxyacetic acid in urine. Using a combination of a volunteer study and samples from exposed workers, we compared the applicability of some of the biological monitoring markers available. We conclude that 2-butoxyethanol in blood is not a suitable marker for biological monitoring due to sampling problems. In view of the low-level exposures reported in occupational surveys, 2-butoxyethanol in breath is also unsuitable because of a lack of sensitivity. Measuring 2-butoxyacetic acid in blood is possible, although non-invasive urine samples are preferred. Free 2-butoxyacetic acid in urine has previously been widely used; however, we found that the extent of conjugation of 2-butoxyacetic acid in urine varied from 0 to 100% both within and between individuals and is not related to time, concentration or urine pH. Data from 48 exposed workers suggested that an estimated 57% (95% confidence interval 44-70%) of the total 2-butoxyacetic acid is excreted in the conjugated form, and that conjugation may be activated above a certain exposure level. Using total 2-butoxyacetic acid significantly reduced inter-individual variation. Elimination half-lives for free and total 2-butoxyacetic acid were similar (∼6 h) and there was no delay in excretion of the conjugated metabolite (peak excretion for both free and total was between 6 and 12 h after the end of exposure). In conclusion, we propose that total butoxyacetic acid (after acid hydrolysis) in urine is the biomarker of choice for monitoring exposure to 2-butoxyethanol. Urine samples should be collected post-shift towards the end of the working week.     

Glucose 1,6-bisphosphate-overloaded erythrocytes: a strategy to investigate the metabolic role of the bisphosphate in red blood cells.

Human erythrocytes overloaded with glucose 1,6-bisphosphate were prepared in order to establish the metabolic significance of this phosphorylated sugar in the intact red cell. The intracellular glucose 1,6-bisphosphate concentration was increased six- and twofold over the normal level by encapsulating (i) the commercially available compound and (ii) the glucose 1,6-bisphosphate synthase obtained from rabbit skeletal muscle, respectively. In both experimental conditions, a reduction of glucose utilization by the loaded cells was observed after reequilibration to the steady state. At the steady state, the concentrations of the glycolytic intermediates and of the adenine nucleotides appeared substantially unmodified when compared with those of controls, with the exception of a 50% reduction of glucose and fructose 6-phosphate measured in erythrocytes encapsulated with exogenous glucose 1,6-bisphosphate. Under the considered experimental conditions, the elevated intracellular glucose 1,6-bisphosphate appears to display an inhibitory effect on hexokinase that overcomes the possible activation of phosphofructokinase or pyruvate kinase.     

Pathogenic properties of Yersinia pseudotuberculosis in different biological models

The study of the action of Y. pseudotuberculosis introduced into the cavity of the ligated intestinal loop of a rabbit, into the stomach of 2- to 4-day-old suckling mice or injected intradermally has made it possible to establish the importance of the invasive and toxic activity of this microbe in the pathogenesis of pseudotuberculosis infection. The lesion of the small intestine develops due to the penetration of the bacteria into the mucous membrane and the combined action of the microbial cells inhabiting the stroma, and secreting into the environment of cytotoxins, toxic substances with an enterotropic action or enterotoxins and factors increasing the permeability of the vessels. Y. pseudotuberculosis strains with high and low invasiveness have been isolated.