Novel ligands for the affinity-chromatographic purification of antibodies.

Affinity chromatography represents one of the most powerful fractionation techniques for the large-scale purification of biotechnological products. Despite its potential, the use of this methodology is limited by the availability of specific ligands for each target. Combinatorial chemistry and molecular modeling, often combined, have become interesting and innovative methods for generating novel ligands, tailored to specific biotechnological needs. One of the greatest area of application has been the discovery of novel ligands for the purification of antibodies, which represent an emerging but very important class of innovative therapeutic agents for the treatment of a vast array of diseases. Naturally available affinity ligands, such as Protein A or G for IgG purification or lectins for IgA and IgM purification, which are obtained from microorganisms or genetically modified bacteria through complex and expensive procedures, are not well suited for large-scale purification and require moreover time-consuming analytical controls to check for the presence of contaminants which may affect the safety of the purified antibody for clinical purposes. Recent results suggest that the application of combinatorial technologies and molecular modeling for the discovery of synthetic ligands may open new avenues for the development of more efficient, less expensive and--more importantly--safer procedures for antibody purification at the industrial level.     

Physico-chemical characterization of proteins by capillary electrophoresis

The electrophoretic mobility of proteins was successfully determined by means of capillary electrophoresis (CE) with various background electrolytes (BGEs). The objective was focused on the variation in BGE physico-chemical composition and the consequential impact on the observed protein charge. Experimental and calculated mobilities, according to Henry's equation, versus ionic strength have been compared. For positively-charged lysozyme, a good agreement between observed and calculated mobilities was observed using triethanolamine chloride at pH 7.0 as the BGE. Mobility close to zero was shown using borate (pH 8.0) and phosphate (pH 7.0) at a low ionic strength of about 20 mmol l⁻¹, and as a consequence, specific adsorption of oxyanions was evidenced. Lysozyme retention in the case of reversed-phase high-performance liquid chromatography (RP-HPLC) was decreased by the presence of phosphate ions. CE and HPLC are complementary tools for characterizing the behaviour of lysozyme. On the other hand, the mobility of the negatively-charged α-lactalbumin remained constant as regards phosphate at pH 7.0 in the 20–200 mmol l⁻¹ range, contrary to the decrease that had been expected with the increasing ionic strength. β-Lactoglobulin exhibited increasingly lower mobilities than those expected of boric acid/borate at pH 7.0 and 8.0 (I=20 mmol l⁻¹).     

Systematics,reproductive isolation and species boundaries in monogonont rotifers

The typological concept of rotifer species and the morphological basis of rotifer systematics is reviewed and alternatives proposed. Occasional sexuality in the cyclical parthenogenetic life cycle of monogononts permits application of the biological species concept to this group. Data from cross-mating experiments with Asplanchna, Brachionus and Epiphanes illustrate the usefulness of reproductive isolation as a criterion for species boundaries. Populations from different geographic regions are often interfertile indicating that rotifer species are genetically integrated over wide areas. The main types of isolating mechanisms operating in monogononts are reviewed. The role of behavioral reproductive isolation in maintaining species boundaries is examined. The use of a mate recognition bioassay which estimates the probability of copulation and quantifies the degree of isolation is described. Recent work of the mechanism of mate recognition is reviewed. It is concluded that the biological species concept is applicable to rotifers and that a more experimental approach to determining species boundaries is both feasible and desirable.     

Reproductive isolation and the maintenance of species boundaries in two serpentine endemic Jewelflowers

Speciation occurs when reproductive barriers substantially reduce gene flow between lineages. Understanding how specific barriers contribute to reproductive isolation offers insight into the initial forces driving divergence and the evolutionary and ecological processes responsible for maintaining diversity. Here, we quantified multiple pre‐ and post‐pollination isolating barriers in a pair of closely related California Jewelflowers (Streptanthus, Brassicaceae) living in an area of sympatry. S. breweri and S. hesperidis are restricted to similar serpentine habitats; however, populations are spatially isolated at fine‐scales and rarely co‐occur in intermixed stands. Several intrinsic postzygotic barriers were among the strongest we quantified, yet, postzygotic barriers currently contribute little to overall reproductive isolation due to the cumulative strength of earlier‐acting extrinsic barriers, including spatial isolation, and flowering time and pollinator differences. Data from multiple years suggest that pre‐pollination barriers may have different strengths depending on annual environmental conditions. Similarly, crossing data suggest that the strength of intrinsic isolation may vary among different population pairs. Estimates of total reproductive isolation in S. breweri and S. hesperidis are robust to uncertainty and variability in individual barrier strength estimates, demonstrating how multiple barriers can act redundantly to prevent gene flow between close relatives living in sympatry.     

A one-step miniprep for the isolation of plasmid DNA and lambda phage particles

Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs) have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E. coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.     

Development and evaluation of a simple, one-step salmonella isolation test

R.H. DAVIES AND C. WRAY. 1996. A one-step test unit was developed which allowed migration of salmonellas to selective medium and indicated the presence of salmonella by motility inhibition using polyvalent flagella antiserum. Evaluation of the method using 1038 naturally contaminated samples led to identification of more contaminated samples than a standard method within 24 h as opposed to 96 h.     

Compartments and organising boundaries in the Drosophila eye: the role of the homeodomain Iroquois proteins.

The Drosophila eye is patterned by a dorsal-ventral organising centre mechanistically similar to those in the fly wing and the vertebrate limb bud. Here we show how this organising centre in the eye is initiated - the first event in retinal patterning. Early in development the eye primordium is divided into dorsal and ventral compartments. The dorsally expressed homeodomain Iroquois genes are true selector genes for the dorsal compartment; their expression is regulated by Hedgehog and Wingless. The organising centre is then induced at the interface between the Iroquois-expressing and non-expressing cells at the eye midline. It was previously thought that the eye develops by a mechanism distinct from that operating in other imaginal discs, but our work establishes the importance of lineage compartments in the eye and thus supports their global role as fundamental units of patterning.     

Detection and isolation of minisatellite Pc-1 binding proteins.

Minisatellites (MNs) are arrays of 5-100 nucleotide repeats that are dispersed throughout the genome of vertebrates. They demonstrate alteration in tumors and in cells exposed to various carcinogens, but the molecular mechanisms underlying the induction of mutations at MNs are largely unknown. Hypervariable MN Pc-1 isolated from the mouse genome consists of tandem repeats of d(GGCAG) flanked with locus-specific sequences at both ends. We have found that MN mutations are induced in NIH3T3 cells by treatment with okadaic acid using a Pc-1 MN fragment as a probe. In order to shed light on the molecular mechanisms, we isolated six MN Pc-1 binding proteins, pA, pB, pD, pE, pF and pG, from nuclear extracts of NIH3T3 cells treated with okadaic acid. While pA and pB bound to the G-rich strand of Pc-1, pD, pE, pF and pG bound to the complementary C-rich strand. Sequence specificities for DNA binding were revealed and one base substitution and insertion into the Pc-1 repeat unit dramatically changed the affinity of each protein, suggesting that they bind to Pc-1 and Pc-1-like MNs in vivo.     

Simultaneous isolation of major viral proteins in one step.

We have developed a rapid and simple technique for the simultaneous isolation of all the major viral proteins from RNA tumor viruses. The basis for this procedure is analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using dansylated virus as internal marker it is possible to follow the migration of unlabeled viral proteins since dansylation does not change the mobility of labeled proteins (8). The method results in approximately 80% recovery of starting protein and is very reproducible. Using radioimmunoassay no alteration of the purified proteins is detectable.     

Corticosteroidogenesis in the isolated Mongolian gerbil adrenal gland during continuous and discontinuous superfusion

Corticosteroid (C) release by adrenals of male Mongolian gerbils (Meriones unguiculatus) has been studied during continuous and discontinuous (flow stop) superfusion. Flow stops of superfusion for 1, 5, 10 or 20 min resulted in a significant accumulation of C within adrenal tissue and superfusion flask. Amounts of C in the first 2-min samples after re-start of superfusion were positively correlated with the amounts secreted during continuous superfusion (5 min: r = 0.97, 10 min: r = 0.97, 15 min: r = 0.74, 20 min: r = 0.84, all p less than 0.001) and with the length of flow stops (1-20 min: r = 0.92, p less than 0.001). However, C concentrations in superfusates were significantly lower than values calculated from secretion during continuous superfusion and the length of flow stops (0 min = 100%, 1 min: 92%, 5 min: 65%, 10 min: 49%, 15 min: 39%, 20 min: 35%). As is evident from the very similar C amounts secreted by adrenals incubated for 15 min without or with 95%O2/5%CO2 (234 vs 256% of basal secretion), flow stop-induced inhibition of corticosteroidogenesis was not due to a lack of oxygen during flow stops. The results demonstrate that superfusion of sliced adrenal tissue gives insights into regulatory mechanisms, including the rapid changes of corticosteroidogenesis during short-lasting flow stops, which cannot be studied in static incubation of either tissue slices or isolated cells. The possibility that the observed decline in steroidogenesis during flow stops may be due to a local feedback inhibition resulting from C accumulating in the microenvironment of adrenal cells is discussed.     

A one-step method for the isolation and determination of leaf ribulose-1,5-diphosphate carboxylase

A convenient method is described for the isolation in one step of Rumex leaf ribulose-1,5-diphosphate carboxylase (RuDPCase) by sucrose density gradient centrifugation. The amount of RuDPCase in a sample of leaf tissue is determined by quantification of the enzyme peak. RuDPCase prepared by this method is shown to be highly pure by criteria including light absorption at 260 and 280 nm, RuDPCase enzyme activity, and polyacrylamide gel electrophoresis at pH 9.2 and at pH 7.1 in the presence of sodium dodecylsulfate. The procedure has been successfully applied to various additional species including bean, maize, and spinach with little or no modification. This method should prove useful for determination of in vivo levels of RuDPCase protein in physiological studies, for measurements of synthesis and breakdown of RuDPCase using radioisotope labeling, and for the preparation of milligram amounts of RuDPCase for further purification and in vitro studies of enzyme structure and function.     

Efficient one-step production of astaxanthin by the microalga Haematococcus pluvialis in continuous culture

The performance of Haematococcus pluvialis in continuous photoautotrophic culture has been analyzed, especially from the viewpoint of astaxanthin production. To this end, chemostat cultures of Haematococcus pluvialis were carried out at constant light irradiance, 1,220 microE/m2.s, and dilution rate, 0.9/d, but varying the nitrate concentration in the feed medium reaching the reactor, from 1.7 to 20.7 mM. Both growth and biomass composition were affected by the nitrate supply. With saturating nitrate, the biomass productivity was high, 1.2 g/L.d, but astaxanthin accumulation did not take place, the C/N ratio of the biomass being 5.7. Under moderate nitrate limitation, biomass productivity was decreased, as also did biomass concentration at steady state, whereas accumulation of astaxanthin developed and the C/N ratio of the biomass increased markedly. Astaxanthin accumulation took place in cells growing and dividing actively, and its extent was enhanced in response to the limitation in nitrate availability, with a recorded maximum for astaxanthin cellular level of 0.8% of dry biomass and of 5.6 mg/L.d for astaxanthin productivity. The viability of a significant continued generation of astaxanthin-rich H. pluvialis cells becomes thus demonstrated, as also does the continuous culture option as an alternative to current procedures for the production of astaxanthin using this microalga. The intensive variable controlling the behavior of the system has been identified as the specific nitrate input, and a mathematical model developed that links growth rate with both irradiance and specific nitrate input. Moreover, a second model for astaxanthin accumulation, also as a function of irradiance and specific nitrate input, was derived. The latter model takes into account that accumulation of astaxanthin is only partially linked to growth, being besides inhibited by excess nitrate. Simulations performed fit experimental data and emphasize the contention that astaxanthin can be efficiently produced under continuous mode by adjustment of the specific nitrate input, predicting even higher values for astaxanthin productivity. The developed models represent a powerful tool for management of such an astaxanthin-generating continuous process, and could allow the development of improved systems for the production of astaxanthin-rich Haematococcus cells.     

Studies on groundnut proteins. V. Physico-chemical properties of arachin prepared by different methods.

ORD,¹ CD, and fluorescence spectra of arachin prepared by Tombs' (Biochem. J., 96, 119; 1965), Dawson's (Anal. Biochem., 41, 305; 1971) or Shetty and Rao's (Anal. Biochem., 62, 108; 1974) procedure were measured; the effect of denaturants such as SDS, GuHCl, and acid was also determined. ORD and CD spectra showed differences, whereas fluorescence spectra did not show any difference. The effect of the denaturants was the same on the three arachins. At low concentrations of GuHCl (<2 m), the denaturant was bound by the protein molecule without causing any conformational change. The binding affinity varied among the arachins.     

A rapid one-step extraction procedure for the isolation of ubiquitin from human erythrocytes for antibody production.

A procedure is described that employs 5% perchloric acid extraction to isolate ubiquitin from human erythrocytes. The procedure is rapid and economical as it requires no specialized equipment. The extracted protein appeared to be highly purified as judged by electrophoresis and was identified as ubiquitin by immunoblotting and total amino acid analysis. The extraction yields about 78% of the ubiquitin in the hemolysate, which is a higher yield than is obtained with other procedures. The purified ubiquitin was used to make a polyclonal antiserum. As ubiquitin is a small and highly conserved protein, it is necessary to couple it to a larger immunogen to elicit an immune response. This ubiquitin antiserum was produced using an immunogen system that produces an immune response to the ubiquitin, but not to the carrier protein.     

Phase separation in the isolation and purification of membrane proteins

Phase separation is a simple, efficient, and cheap method to purify and concentrate detergent-solubilized membrane proteins. In spite of this, phase separation is not widely used or even known among membrane protein scientists, and ready-to-use protocols are available for only relatively few detergent/membrane protein combinations. Here, we summarize the physical and chemical parameters that influence the phase separation behavior of detergents commonly used for membrane protein studies. Examples for the successful purification of membrane proteins using this method with different classes of detergents are provided. As the choice of the detergent is critical in many downstream applications (e.g., membrane protein crystallization or functional assays), we discuss how new phase separation protocols can be developed for a given detergent buffer system.     

Two-step affinity-chromatographic purification of cathepsin D from pig myometrium with high yield.

Cathepsin D was purified by two-step affinity chromatography on concanavalin A-- and pepstatin--Sepharose. The main purification was achieved by washing the enzyme bound to the pepstatin--Sepharose column with buffered 6 M-urea. This step separated cathepsin D from all low- and high-molecular-weight impurities. Although the 1700-fold purified acid proteinase was homogeneous on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it still showed microheterogeneity.     

Structured population dynamics: continuous size and discontinuous stage structures

A nonlinear stochastic model for the dynamics of a population with either a continuous size structure or a discontinuous stage structure is formulated in the Eulerian formalism. It takes into account dispersion effects due to stochastic variability of the development process of the individuals. The discrete equations of the numerical approximation are derived, and an analysis of the existence and stability of the equilibrium states is performed. An application to a copepod population is illustrated; numerical results of Eulerian and Lagrangian models are compared.      

Coupled,continuous and discontinuous fluorometric assays for trehalase activity

Continuous and discontinuous coupled fluorometric assays which couple trehalose hydrolysis to peroxidation of the fluorogenic compounds eugenol (4-allyl-2-methoxyphenol) or p-hydroxyphenylacetic acid using glucose oxidase (EC 1.1.3.4) and peroxidase (EC 1.11.1.7) as ancillary enzymes have been developed for the measurement of trehalase (α,α′-trehalose-1-d-glucohydrolase, EC 3.2.1.28) activity from the cellular slime mold, Dictyostelium discoideum. With these methods, product formation was linear with time and the coupled reaction rate was directly proportional to the level of enzyme assayed. The validity of both the discontinuous and continuous fluorometric assays was confirmed by comparative studies with discontinuous spectrophotometric assays for glucose. Levels of glucose as low as 0.02 nmol were measurable with the discontinuous fluorometric procedures, thereby making the latter about 500-fold more sensitive than routine spectrophotometric assays. With the continuous fluorometric trehalase assays, the lower limits of sensitivity correspond to enzyme levels of the order of 5 to 25 μunits. The high level of sensitivity achieved with these assays makes them ideally well suited for: (i) elucidation of the regulatory mechanisms underlying the dramatic changes in trehalase activity that occur during spore germination and cellular aggregation in Dictyostelium and (ii) characterization studies involving electrophoresis or isoelectric focusing of trehalase in solid matrices in which enzymatic activity is measured either quantitatively in gel eluates or qualitatively by the in situ localization of the enzyme histochemically.