βVI Turns in peptides and proteins: A model peptide mimicry

To investigate the role of proline in defining β turn conformations within cyclic hexa- and pentapeptides we synthesized and determined the conformations of a series of L - and D -proline-containing peptides by means of 2D NMR spectroscopy and restrained molecular dynamics simulations. Due to cis/trans isomerism the L -proline peptides adopt at least two different conformations that are analyzed and compared to the structures of the corresponding D -proline peptides. The cis conformations of the compounds cyclo(-Pro-Ala-Ala-Pro-Ala-Ala-), cyclo(-Arg-Gly-Asp-Phe-Pro-Gly-), cyclo(-Arg-Gly-Asp-Phe-Pro-Ala-), cyclo(-Pro-Ala-Ala-Ala-Ala--), and cyclo(-Pro-Ala-Pro-Ala-Ala-) form uncommon βVI turns that mimic the turn geometries found in crystallographically refined protein structures at such a detailed level that even preferred side chain orientations are reproduced. The ratios of the cis/trans isomers are analyzed in terms of the steric demand of the proline-following residue. The conformational details derived from this study illustrate the importance of the examination of small model compounds derived from protein loop regions, especially if bioactive recognition sequences, such as RGD (Arg-Gly-Asp), are incorporated. © 1993 Wiley-Liss, Inc.     

A comparison of transplantable bicoid activity and partial bicoid homeobox sequences in several Drosophila and blowfly species (Calliphoridae)

In order to test for bicoid-like activity in insects other than Drosophila melanogaster, anterior egg cytoplasm from the following species was injected into cleavage stage embryos from mutant D. melanogaster lacking a functional bicoid (bcd) product: six other Drosophila species, the housefly, three blowfly species, the primitive cyclorrhaphic dipteran Megaselia, and the honeybee Apis mellifera; preliminary tests were made with four lower dipterans (Nematocera). Rescue effects were only observed with the drosophilids, housefly, and two of the three blowfly species. Rescue was stronger with the drosophilids than with the other flies as donors. Where checked (D. pseudoobscura), a positive correlation was found between the amount of cytoplasm injected and the number of pattern elements formed, suggesting threshold effects upon target genes as with the endogenous bcd product. By polymerase chain reaction, fragments from a bcd-orthologous homeobox were cloned from the three blowfly species. The derived sequence of 43 amino acids was identical in all blowflies and the housefly but differed at 4 positions from the orthologous D. melanogaster sequence. Localization of the mRNA recognized by the respective fragments in the blowflies Lucilia and Phormia resembled that known from D. melanogaster, while Calliphora — the blowfly species lacking rescue activity —showed remarkable differences of localization in both ovarian follicles and the deposited egg cell. This surprising divergence within a morphologically rather uniform family of cyclorrhaphic dipterans should be of interest from both functional and evolutionary points of view.     

Using ecological networks to answer questions in global biogeography and ecology

Ecological networks have classically been studied at site and landscape scales, yet recent efforts have been made to collate these data into global repositories. This offers an opportunity to integrate and upscale knowledge about ecological interactions from local to global scales to gain enhanced insights from the mechanistic information provided by these data. By drawing on existing research investigating patterns in ecological interactions at continental to global scales, we show how data on ecological networks, collected at appropriate scales, can be used to generate an improved understanding of many aspects of ecology and biogeography—for example, species distribution modelling, restoration ecology and conservation. We argue that by understanding the patterns in the structure and function of ecological networks across scales, it is possible to enhance our understanding of the natural world.     

Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.     

Biotin carboxylases in mitochondria and the cytosol from skeletal and cardiac muscle as detected by avidin binding

Biotin carboxylases in mammalian cells are regulatory enzymes in lipogenesis and gluconeogenesis. In this study, endogenous biotin in skeletal and cardiac muscle was detected using avidin conjugated with alkaline phosphatase and applied in high concentrations to muscle sections. The avidin binding was subsequently visualized by histochemical demonstration of the alkaline phosphatase activity. All cardiac muscle cells showed high affinity for avidin with only the nuclei and the intercalated discs remaining unstained. In skeletal muscle a diffuse reaction could be detected in the sarcoplasm of the muscle fibres. A granular reaction was noted in the same fibres that showed activity for succinic dehydrogenase. The specificity of the coloured reaction product in the muscle sections was investigated and is suggested to be caused by avidin binding to biotin moieties in mitochondria and the cytosol. Mitochondrial and cytosolic preparations of skeletal muscle were electrophoresed in sodium dodecyl sulphate gels. After blotting and incubation with conjugated avidin, two bands with molecular weights of 75 kDa and 130 kDa respectively were evident in the mitochondrial preparation. It is suggested that the 75-kDa band represents comigration of the biotin-containing subunits of propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The 130-kDa band may represent the biotin-containing pyruvate carboxylase. In the cytosolic preparation a 270-kDa band was stained in blots that had been incubated with conjugated avidin; this band is suggested to represent acetyl-CoA carboxylase. A 190-kDa cytosolic band might be a cleavage product of acetyl-CoA carboxylase. We propose that using alkaline phosphatase-conjugated avidin it is possible to detect the mitochondrial and cytosolic biotin-dependent carboxylases in striated muscle.     

Inhibition of bacteriophage Mu transposition by Mu repressor and Fis

In this paper we show that the Escherichia coli protein Fis has a regulatory function in Mu transposition in the presence of Mu repressor. Fis can lower the transposition frequency of a mini-Mu 3–80-fold, but only if the Mu repressor is expressed simultaneously. In this novel type of regulation of transposition by the concerted action of Fis and repressor, the IAS, the internal activating sequence, is also involved as deletion of this site leads to the loss of the Fis effect. As the IAS contains strong repressor binding sites these are probably the target for the repressor in the observed negative regulation by Fis and repressor. However, the role of Fis and repressor is not only to inactivate the IAS, since a 4bp insertion in the IAS, which changes the spacing of the repressor-binding site, abolishes the enhancing function of the IAS but leaves the repressor-Fis effect intact. A likely target for Fis in this regulation is a strong Fis-binding site, which is located adjacent to the L2 transposase-binding site. However, when this Fis-binding sequence was substituted by a random sequence and Fis no longer showed specific binding to this site, the Fis effect was still observed. Although it is still possible that Fis can function by binding to this non-specific site in a particular complex, it seems more likely that Fis is directly or indirectly involved in determining the level of the repressor.     

A Mycobacterium leprae-specific gene encoding an immunologically recognized 45 kDa protein

By screening a Mycobacterium leprae lambda gt11 expression library with a serum from an Ethiopian lepromatous leprosy (LL) patient a clone was isolated (LL4) belonging to hybridization group III of a panel of previously isolated M. leprae clones. Members of this hybridization group encode a serologically recognized 45 kDa protein. The complete DNA sequences of the partially overlapping clones LL4 and L1 (hybridization group III) are presented and these revealed the presence of an open reading frame (ORF) predicting a protein with a molecular size of 42 448 Da. Southern hybridizations on total genomic DNA of M. Ieprae, Mycobacterium tuberculosis and eight atypical mycobacteria showed that the LL4 DNA fragment is specific for M. Ieprae DNA even under low-stringency conditions. The M. Ieprae specificity of LL4 DNA was further confirmed by the polymerase chain reaction using four different sets of primers. Western blotting analyses showed that the M. Ieprae 45 kDa protein is frequently recognized by antibodies from leprosy patients and that this recognition is specific since no antibodies could be detected in sera of tuberculosis patients. T-cell proliferation assays also demonstrated T-cell recognition by leprosy patients and healthy contacts of the M. Ieprae 45 kDa protein. The specificity of the LL4 DNA region and the 45 kDa antigen that is encoded by hybridization group III could provide unique tools for the development of M. Ieprae-specific immunological and DNA reagents.     

Sensitivity of a two-dimensional biomorphoelastic model for post-burn contraction

We consider a two-dimensional biomorphoelastic model describing post-burn scar contraction. This model describes skin displacement and the development of the effective Eulerian strain in the tissue. Besides these mechanical components, signaling molecules, fibroblasts, myofibroblasts, and collagen also play a significant role in the model. We perform a sensitivity analysis for the independent parameters of the model and focus on the effects on features of the relative surface area and the total strain energy density. We conclude that the most sensitive parameters are the Poisson’s ratio, the equilibrium collagen concentration, the contraction inhibitor constant, and the myofibroblast apoptosis rate. Next to these insights, we perform a sensitivity analysis where the proliferation rates of fibroblasts and myofibroblasts are not the same. The impact of this model adaptation is significant.

     

BLOEM: A spatially explicit model of bioenergy and carbon capture and storage,applied to Brazil

Bioenergy could play a major role in decarbonizing energy systems in the context of the Paris Agreement. Large-scale bioenergy deployment could be related to sustainability issues and requires major infrastructure investments. It, therefore, needs to be studied carefully. The Bioenergy and Land Optimization Spatially Explicit Model (BLOEM) presented here allows for assessing different bioenergy pathways while encompassing various dimensions that influence their optimal deployment. In this study, BLOEM was applied to the Brazilian context by coupling it with the Brazilian Land Use and Energy Systems (BLUES) model. This allowed investigating the most cost-effective ways of attending future bioenergy supply projections and studying the role of recovered degraded pasture lands in improving land availability in a sustainable and competitive manner. The results show optimizing for limiting deforestation and minimizing logistics costs results in different outcomes. It also indicates that recovering degraded pasture lands is attractive from both logistics and climate perspectives. The systemic approach of BLOEM provides spatial results, highlighting the trade-offs between crop allocation, land use and the logistics dynamics between production, conversion, and demand, providing valuable insights for regional and national climate policy design. This makes it a useful tool for mapping sustainable bioenergy value chain pathways.