Hydrophobic cluster analysis (HCA) of the hormone-binding domain of receptor proteins

A new technique of protein sequence analysis, namely, Hydrophobic Cluster Analysis (HCA), has been used to align and compare the sequences of proteins belonging to the receptor superfamily (steroid, thyroid hormone and retinoic acid receptors) and serpin superfamily (corticosteroid binding globulin (CBG) and alpha 1-antitrypsin (alpha 1-AT]. By matching up clusters of hydrophobic amino-acids that oftenmost correspond to identifiable secondary structures (alpha-helices, beta-strands etc.), it has been possible to deduce the following information on the secondary structures of these proteins: CBG is structurally related to alpha 1-AT (HCA score greater than 80%), the structures of the hormone-binding domains of the steroid receptors that bind 3-keto-delta 4-steroids are closely interrelated (greater than 80%) but less closely related to that of the estrogen receptor (ER) (approximately 75%), vitamin D, retinoic acid and thyroid hormone receptors are structurally closely related (greater than or equal to 80%). Their secondary structures are, however, also related to that of the steroid receptors (approximately 70%), and a high degree of analogy exists between the structures of serpins and of the hormone-binding domains of members of the steroid superfamily (60-70%). HCA has clearly shown that a previous local sequence alignment of the estrogen receptor with other steroid receptors and cytochromes P450 has to be reconsidered. The published consensus steroid binding sequence previously identified in cytochromes is in fact 80 amino-acids upstream from its previously defined position. Other regions of contiguous sequence identity have also been identified which may be involved in the hydrophobic core of the protein or in steroid binding. Their positions have been indicated using the crystal structure of alpha 1-AT as a model.     

A new training method for hybrid models of bioprocesses

Modeling of bioprocesses for engineering applications is a very difficult and time consuming task, due to their complex nonlinear dynamic behavior. In the last years several propositions for hybrid models, and especially serial approaches, were published and discussed, in order to combine analytical prior knowledge with the learning capabilities of Artificial Neural Networks (ANN). These approaches often require synchronous and equidistant sampled training data. However, in practice concentrations are mostly off-line measured, rare, and asynchronous. In this paper a new training method especially suited for very few asynchronously sampled data is presented and applied for modeling animal cell cultures. The achieved model is able to predict the concentrations of the reaction components inside a stirred tank bioreactor.     

A simple method for the measurement of bacterial particle conductivities

A method was developed for the measurement of the bacterial particle conductivity, based on the measurement of the conductivity of a bacterial cell suspension sigma(s) and the suspending medium sigma(m). A line plotted through sigma(s) - sigma(m) versus sigma(m) crosses the x-axis at sigma(m) = sigma(p), independent of the bacterial cell concentration. The method does not require anything more complex than a centrifuge and a conductivity meter. Knowledge of the bacterial particle conductivity is of importance in, for example, the dielectrophoretic separation, manipulation and trapping of bacterial cells, as well as the study of their physiological state.     

A freeze-capture method for the study of platelet-sized particle distributions

A rapid freezing method was developed to study the distributions of fluorescent platelet-sized particles in flows of blood suspensions through thin-walled capillary tubes. Segments of frozen tubes were mounted in a refrigerated microtome on the stage of an epifluorescence microscope. Sections of tube were cut away, images of newly exposed cross-sections were recorded on video tape, and distances of the particles from the wall were measured from recorded images. The distance data were used to construct histograms that were proportional to the local concentration. Results indicated that this method is suitable for the study of the distribution of platelet-sized particles over a wide range of hematocrit, that the basic profile is reproducible to within 15%, and that the non-uniform profile is not a result of events at the tube entrance.     

A decomposition method for the stability analysis of large microbial systems

The primary objective of this paper is to present a method for the stability analysis of microbial systems consisting of a large number of different populations of microorganisms. The overall system is decomposed into easily analyzable subsystems and its stability characteristics are deduced from those of a properly constructed linear system of lower dimension. Several examples are provided which demonstrate the use of the method in studying the dynamics of some interacting microbial populations which grow under continuous flow conditions and, after a parametric analysis, the regions of the parameters which guarantee coexistence are found.     

Poly(A) nuclease interacts with the C-terminal domain of polyadenylate-binding protein domain from poly(A)-binding protein

The poly(A)-binding protein (PABP) is an essential protein found in all eukaryotes and is involved in an extensive range of cellular functions, including translation, mRNA metabolism, and mRNA export. Its C-terminal region contains a peptide-interacting PABC domain that recruits proteins containing a highly specific PAM-2 sequence motif to the messenger ribonucleoprotein complex. In humans, these proteins, including Paip1, Paip2, eRF3 (eukaryotic release factor 3), Ataxin-2, and Tob2, are all found to regulate translation through varying mechanisms. The following reports poly(A) nuclease (PAN) as a PABC-interacting partner in both yeast and humans. Their interaction is mediated by a PAM-2 motif identified within the PAN3 subunit. This site was identified in various fungal and animal species suggesting that the interaction is conserved throughout evolution. Our results indicate that PABP is directly involved in recruiting a deadenylase to the messenger ribonucleoprotein complex. This demonstrates a novel role for the PABC domain in mRNA metabolic processes and gives further insight into the function of PABP in mRNA maturation, export, and turnover.     

Structure of the chloroplast signal recognition particle (SRP) receptor: domain arrangement modulates SRP-receptor interaction

The signal recognition particle (SRP) pathway mediates co-translational targeting of nascent proteins to membranes. Chloroplast SRP is unique in that it does not contain the otherwise universally conserved SRP RNA, which accelerates the association between the SRP guanosine-5′-triphosphate (GTP) binding protein and its receptor FtsY in classical SRP pathways. Recently, we showed that the SRP and SRP receptor (SR) GTPases from chloroplast (cpSRP54 and cpFtsY, respectively) can interact with one another 400-fold more efficiently than their bacterial homologues, thus providing an explanation as to why this novel chloroplast SRP pathway bypasses the requirement for the SRP RNA. Here we report the crystal structure of cpFtsY from Arabidopsis thaliana at 2.0 Å resolution. In this chloroplast SR, the N-terminal “N” domain is more tightly packed, and a more extensive interaction surface is formed between the GTPase “G” domain and the N domain than was previously observed in many of its bacterial homologues. As a result, the overall conformation of apo-cpFtsY is closer to that found in the bacterial SRP•FtsY complex than in free bacterial FtsY, especially with regard to the relative orientation of the N and G domains. In contrast, active-site residues in the G domain are mispositioned, explaining the low basal GTP binding and hydrolysis activity of free cpFtsY. This structure emphasizes proper N-G domain arrangement as a key factor in modulating the efficiency of SRP-receptor interaction and helps account, in part, for the faster kinetics at which the chloroplast SR interacts with its binding partner in the absence of an SRP RNA.     

A simple method for midkine purification by affinity chromatography with a heavy chain variable domain (VH) fragment of antibody

A DNA fragment for a heavy chain variable domain (VH) was prepared from a hybridoma that produces a monoclonal antibody against human midkine (MK). The antibody fragment was produced in Escherichia coli and its affinity for chemically synthesized full length MK or recombinant midkine c-terminus (MKc-half) protein was confirmed by ELISA. An Escherichia coli cell lysate expressing MKc-half was applied to a VH fragment-coupled Sepharose 4B column and eluted with a buffer containing 0.5 M NaCl. SDS-PAGE analysis revealed a high degree of purity of the MKc-half protein in the eluent, showing the utility of a recombinant VH fragment in purification of proteins by affinity chromatography.     

A simplified strategy for the identification of Gram negative fermenting rods using a desk-top computer

A total of 845 strains of Gram negative, fermenting rods isolated from humans were routinely identified using a desk-top computer. A reduced matrix of 54 taxa x 29 tests was used for a multistep procedure. By an average number of 24 tests, 97–5% of strains reached an identification score of P < 0.9.     

A high-resolution 15,000(rad) radiation hybrid panel for the domestic cat

The current genetic and recombination maps of the cat have fewer than 3,000 markers and a resolution limit greater than 1 Mb. To complement the first-generation domestic cat maps, support higher resolution mapping studies, and aid genome assembly in specific areas as well as in the whole genome, a 15,000(Rad) radiation hybrid (RH) panel for the domestic cat was generated. Fibroblasts from the female Abyssinian cat that was used to generate the cat genomic sequence were fused to a Chinese hamster cell line (A23), producing 150 hybrid lines. The clones were initially characterized using 39 short tandem repeats (STRs) and 1,536 SNP markers. The utility of whole-genome amplification in preserving and extending RH panel DNA was also tested using 10 STR markers; no significant difference in retention was observed. The resolution of the 15,000(Rad) RH panel was established by constructing framework maps across 10 different 1-Mb regions on different feline chromosomes. In these regions, 2-point analysis was used to estimate RH distances, which compared favorably with the estimation of physical distances. The study demonstrates that the 15,000(Rad) RH panel constitutes a powerful tool for constructing high-resolution maps, having an average resolution of 40.1 kb per marker across the ten 1-Mb regions. In addition, the RH panel will complement existing genomic resources for the domestic cat, aid in the accurate re-assemblies of the forthcoming cat genomic sequence, and support cross-species genomic comparisons.     

A novel design of simulated moving bed (SMB) chromatography for separation of ketoprofen enantiomer

A simulated moving bed (SMB) chromatography system is a powerful tool for preparative scale separation, which can be applied to the separation of chiral compound. We have designed our own lab-scale SMB chromatography using 5 HPLC pumps, 6 stainless steel columns and 4 multi-position valves, to separate a racemic mixture of ketoprofen in to its enantiomers. Our design has the characteristics of the low cost for assembly for the SMB chromatography and easy repair of the unit, which differs from the designs suggested by other investigators. It is possible for the flow path through each column to be independently changed by computer control, using 4 multi-position rotary valves and 5 HPLC solvent delivery pumps. In order to prove the operability of our SMB system, attempts were made to separate the (S)-ketoprofen enantiomer from a ketoprofen racemic mixture. The operating parameters of the SMB chromatography were calculated for ketoprofen separation from a batch chromatography experiment as well as by the triangle theory. With a feed concentration of 1 mg/mL, (S)-ketoprofen was obtained with a purity of 96% under the calculated operating conditions.     

A new enzymatic method for the detachment of particle associated soil bacteria

A new enzymatic technique for the detachment of bacteria from soil particles was developed and applied to different soil samples taken at various sampling sites and depths. Many soil microorganisms are closely associated with the organic matrix of soil particles. They produce extracellular polymeric substances (EPS), which promote the irreversible adhesion of cells to soil particulates. To characterize the EPS, a prestaining of the soil samples with different lectins was performed. Samples from a sewage field, an urban park, a farmland, a mixed forest and garden mold were stained with a set of FITC-labelled lectins from Triticum vulgaris, Ulex europaeus, Concanavalin A and Pseudomonas aeruginosa. Based on the results, a combination of alpha-glucosidase, beta-galactosidase and a lipase was chosen for degradation of the EPS structures, followed by gentle mechanical and chemical dispersion in a modified sodium pyrophosphate buffer. The samples were fixed with formaldehyde and total cell counts were determined by DAPI staining. With the exception of the wheat field sample, this technique revealed up to 22-fold higher total cell counts for all investigated soil samples compared to the conventional detachment method, a simple dispersion with sodium pyrophosphate buffer. Efficiency of the technique was assessed by scanning electron microscopy. These images showed convincingly that the enzymatic treatment followed by sonication efficiently detached the bacteria and left the soil particles almost blank.     

A deformation gradient decomposition method for the analysis of the mechanics of morphogenesis

A new finite element model is proposed for the analysis of the mechanical aspects of morphogenesis and tested on the biologically well studied gastrulation phenomenon, in particular ventral furrow invagination of the Drosophila melanogaster embryo. A set of mechanisms are introduced in the numerical model, which lead to the observed deformed shapes. We split the total deformation into two parts: an imposed active deformation, and an elastic deformation superimposed onto the latter. The active deformation simulates the effects of apical constriction and apico-basal elongation. These mechanisms are associated with known gene expressions and so in this way we attempt to bridge the well explored signalling pathways, and their associated phenotypes in a mechanical model. While the former have been studied in depth, much less can be said about the forces they produce and the mechanisms involved. From the numerical results, we are able to test different plausible mechanical hypotheses that generate the necessary folding observed in the invagination process. In particular, we conclude that only certain ratios between both modes (apical constriction and apico-basal elongation) can successfully reproduce the invagination process. The model also supports the idea that this invagination requires the contribution of several mechanisms, and that their redundancy provides the necessary robustness.     

A public domain image-analysis program for the single-cell gel-electrophoresis (comet) assay

The single-cell gel electrophoresis (or comet) assay has gained widespread acceptance as a cheap and simple genotoxicity test, but it requires a computer-assisted image-analysis system. As commercial programs are expensive and inflexible, we decided to develop an image-analysis system based on public domain programs and make it publicly available for the scientific community. Our system is based on the scientific image-processing program NIH Image, and was written in its Pascal-like macro language. User interaction was kept as simple as possible, to enable the measurement of a large number of cells with a few keystrokes. Therefore, the time for image analysis is very low, even on slow computers. The comet macro can be obtained from http://mailbox.univie.ac.at/christoph.helma++ +/comet/, NIH Image is available at http://rsb.info.nih.gov/nih-image/. Both programs are free of charge.