Genome sequencing of a single cell of the widely distributed marine subsurface Dehalococcoidia,phylum Chloroflexi

Bacteria of the class Dehalococcoidia (DEH), phylum Chloroflexi, are widely distributed in the marine subsurface, yet metabolic properties of the many uncultivated lineages are completely unknown. This study therefore analysed genomic content from a single DEH cell designated ‘DEH-J10'' obtained from the sediments of Aarhus Bay, Denmark. Real-time PCR showed the DEH-J10 phylotype was abundant in upper sediments but was absent below 160 cm below sea floor. A 1.44 Mbp assembly was obtained and was estimated to represent up to 60.8% of the full genome. The predicted genome is much larger than genomes of cultivated DEH and appears to confer metabolic versatility. Numerous genes encoding enzymes of core and auxiliary beta-oxidation pathways were identified, suggesting that this organism is capable of oxidising various fatty acids and/or structurally related substrates. Additional substrate versatility was indicated by genes, which may enable the bacterium to oxidise aromatic compounds. Genes encoding enzymes of the reductive acetyl-CoA pathway were identified, which may also enable the fixation of CO₂ or oxidation of organics completely to CO₂. Genes encoding a putative dimethylsulphoxide reductase were the only evidence for a respiratory terminal reductase. No evidence for reductive dehalogenase genes was found. Genetic evidence also suggests that the organism could synthesise ATP by converting acetyl-CoA to acetate by substrate-level phosphorylation. Other encoded enzymes putatively conferring marine adaptations such as salt tolerance and organo-sulphate sulfohydrolysis were identified. Together, these analyses provide the first insights into the potential metabolic traits that may enable members of the DEH to occupy an ecological niche in marine sediments.     

QRNAstruct: a method for extracting secondary structural features of RNA via regression with biological activity

Recent technological advances have enabled the generation of large amounts of data consisting of RNA sequences and their functional activity. Here, we propose a method for extracting secondary structure features that affect the functional activity of RNA from sequence–activity data. Given pairs of RNA sequences and their corresponding bioactivity values, our method calculates position-specific structural features of the input RNA sequences, considering every possible secondary structure of each RNA. A Ridge regression model is trained using the structural features as feature vectors and the bioactivity values as response variables. Optimized model parameters indicate how secondary structure features affect bioactivity. We used our method to extract intramolecular structural features of bacterial translation initiation sites and self-cleaving ribozymes, and the intermolecular features between rRNAs and Shine–Dalgarno sequences and between U1 RNAs and splicing sites. We not only identified known structural features but also revealed more detailed insights into structure–activity relationships than previously reported. Importantly, the datasets we analyzed here were obtained from different experimental systems and differed in size, sequence length and similarity, and number of RNA molecules involved, demonstrating that our method is applicable to various types of data consisting of RNA sequences and bioactivity values.     

Internal water molecules and H-bonding in biological macromolecules: a review of structural features with functional implications.

Conserved structural patterns of internal water molecules and/or H-bond chains were observed and are here correlated in this review, which then describes two functional properties: equilibration of hydrostatic pressure and proton transport. Available evidence in support of these hypotheses is presented, together with suggested experiments to test them. High-resolution crystal structures of a variety of proteins were studied with interactive computer graphics. Conserved H-bonding linkages may be used as a paradigm for a rationalization of proton transport in membranes. The concept of the "proton wire," which links buried active-site amino acids with the surface of the protein raises the more general question of the functional role of the various molecular components.     

Linking structural features of protein complexes and biological function

Protein–protein interaction (PPI) establishes the central basis for complex cellular networks in a biological cell. Association of proteins with other proteins occurs at varying affinities, yet with a high degree of specificity. PPIs lead to diverse functionality such as catalysis, regulation, signaling, immunity, and inhibition, playing a crucial role in functional genomics. The molecular principle of such interactions is often elusive in nature. Therefore, a comprehensive analysis of known protein complexes from the Protein Data Bank (PDB) is essential for the characterization of structural interface features to determine structure–function relationship. Thus, we analyzed a nonredundant dataset of 278 heterodimer protein complexes, categorized into major functional classes, for distinguishing features. Interestingly, our analysis has identified five key features (interface area, interface polar residue abundance, hydrogen bonds, solvation free energy gain from interface formation, and binding energy) that are discriminatory among the functional classes using Kruskal-Wallis rank sum test. Significant correlations between these PPI interface features amongst functional categories are also documented. Salt bridges correlate with interface area in regulator-inhibitors (r = 0.75). These representative features have implications for the prediction of potential function of novel protein complexes. The results provide molecular insights for better understanding of PPIs and their relation to biological functions.     

Problems of structural and functional identification of biological tissues

Theoretical methods for identification of topological and functional parameters of biological tissues (e.g., myocardium) are considered. Unknown topological parameters include the network dimension and connectivity, while unknown functional parameters include the resistivity of the cellular membrane and cytoplasm. The cell size (length and diameter), the input resistance of the tissue, and the fundamental electric potential field formed by a “point” current source (intracellular microelectrode) are treated as experimentally known parameters.     

Problems of structural and functional identification of biological tissues

The theoretical methods of identification of topological and functional parameters for biological tissues, such as myocardium, are considered. Among the unknown topological parameters are the dimension and connectivity of a network, and the unknown functional parameters include the electrical resistance of the cellular membrane and cytoplasm. Experimentally known data are the sizes of cells (length and diameter), the input resistance of tissue, and also the field of electrical potential formed by a "dot" current source (by an intracellular microelectrode).     

抗菌肽的结构特征、生物活性及应用

抗菌肽分子呈双亲性的仅一螺旋结构可使细菌、真菌、寄生虫的质膜形成离子通道,从而导致这些病原体死亡。抗菌肽分子也可通过影响它们的能量转运和代谢,损害它们的呼吸链的功能,抑制它们的蛋白质和DNA的合成,以及干扰病毒的侵染过程和抑制病毒的繁殖等机制,对病原体产生抑制或杀灭作用。此外,抗菌肽还可杀伤肿瘤细胞并提高机体的免疫功能。因此,抗菌肽有望开发为防治人和动物疾病的药物,应用前景广阔。     

A quantitative and qualitative RNA expression profiling assay for cell culture with single cell resolution

Cells are often characterized by their gene expression profile. However, commonly used methods to detect mRNA require cell pooling and could therefore mask differences in gene expression within heterogeneous cell populations. q²PISH allows for the analysis of both qualitative and quantitative (q²) gene expression on cultured cells for quality control measures with single cell resolution. q²PISH was optimized for the subsequent use of two alkaline phosphatase substrates in combination with a cell nucleus count to allow for accurate quantification of gene expression per cell and simultaneously qualitative assessment of potential culture population drift or heterogeneity. As proof of principle the assay was applied to cell lines derived from different areas of the bovine intervertebral disc, showing significant difference in the expression of Col1a1, Col2a1, Acan and Sox9. Furthermore, the assay served to explore a potential impact on cultured cells when substituting a critical media component, fetal bovine serum (FBS), suggesting no significant difference in gene expression for the biomarkers analyzed. As a tool, q²PISH serves as an accurate quality control with single cell resolution for cultured cells.     

Analysis of heat transfer in the biological tissue layer with distributed system of blood vessels

Processes of heat transfer in the skin layer with blood vessels were investigated using mathematical modeling. Analysis of influence of a pathological state of blood vessels on heterogeneity of thermal field of the skin surface was carried out. For each site of body surface, there is a certain difference of temperature between normal and pathological sites, being specific for differential diagnosis of diseases of dermal and hypodermic vessels.     

Geographical mapping of metabolites in biological tissue with quantitative bioluminescence and single photon imaging

Summary This article features a novel technique for measuring the spatial distribution of metabolites, such as ATP, glucose, and lactate, in rapidly frozen tissue. Concentration values are obtained in absolute terms and with a spatial resolution of single-cell dimension. The method is based on enzymatic reactions that link the metabolite of interest to luciferase with subsequent light emission. Using a specific array, cryosections are brought into contact with the enzymes in a well-defined, reproducible way inducing a distribution of light across the section with an intensity that is proportional to the metabolite concentration. The emitted light can be visualized through a microscope and an imaging photon counting system, and the respective image can be transferred to a computer for image analysis. Measurements in spherical cell aggregates with central necrosis demonstrate a close correlation between the distribution of ATP and of cellular viability at a microregional level. Similarly, ATP and glucose are correlated with the geometrical arrangement of more viable and more necrotic tissue regions in human melanomas xenografted in nude mice. Lactate did not show such a structure-related distribution in these tumours. Structure-related distributions of ATP, glucose, and lactate are found in cervix tumours of patients. In contrast to the heterogeneous distributions in tumours, the distribution patterns were much more homogeneous in normal tissues. Regional differences were present, but were much more gradual than in malignancies. This was illustrated for heart muscle where ATP concentrations were found that agreed with data in the literature, and that showed a decrease in periventricular areas.Presented as Histochemical Journal Lecture by W. Mueller-Klieser at the Annual Meeting of the Histochemistry Section of the Royal Microscopical Society in London on 6 January 1992.     

Tumor cell targeting of transferrin-PEG-TNF-alpha conjugate via a receptor-mediated delivery system: design, synthesis, and biological evaluation

PEGylation is a procedure of growing interest for enhancing the therapeutic and biotechnological potential of peptides and proteins. Transferrin (Tf) has been proposed to be useful for targeting cancer cells. The aim of this study was to modify PEGylated recombinant human tumor necrosis factor alpha (PEG-TNF-alpha) with Tf to form Tf-PEG-TNF-alpha conjugates, which would maintain the advantages of PEGylation and also achieve the function of active targeting to tumor cells. In PEGylation reactions with 5-, 20-, 40-, and 60-fold molar excess of 3.4 kDa N-hydroxysuccinimide-PEG-maleimide (PT1, PT2, PT3, and PT4, respectively), PEG-TNF-alpha conjugates with different PEG chains were synthesized. A perfusion chromatography technique using a cation-exchange column was introduced to purify PEG-TNF-alpha conjugates. PT4 with about five PEG chains was selected as a lead candidate due to highest extent of PEGylation and maximum reaction yield. Thiolated Tf was conjugated to the maleimide group at the distal end of the PEG chains on the PEG-TNF-alpha conjugates, with the resulting Tf-PEG-TNF-alpha conjugates after purification containing approximately one Tf ligand on one TNF-alpha molecule. The conjugate of Tf and PT4 (TPT4) was selected to assess the specificity and affinity to transferrin receptor (TfR) on two kinds of tumor cells, K562 and KB. Both the receptor binding assays and the competition experiments were performed using radioligand binding analysis. The results demonstrated that TPT4 as well as Tf bound specifically to the TfR on the tumor cell surface and the affinity of the conjugate to TfR was similar to that of native Tf. In contrast, PEG-TNF-alpha demonstrated no specificity. The biodistribution and antitumor effects were investigated in S-180 tumor-bearing mice. It was found that TPT4 could markedly alter in vivo behavioral characteristics of TNF-alpha. Compared with TNF-alpha and PT4, extravasated TPT4 in tumor tissues exhibited a significantly delayed blood clearance and the highest intratumoral TNF-alpha levels. Furthermore, the inhibitory rate of tumor of TPT4 enhanced 5.3- and 1.8-fold over that of TNF-alpha and PT4, indicating that TPT4 exhibited the highest antitumor activity. These results suggested that Tf-PEG-TNF-alpha was a useful long circulating conjugate with the capabilities of specific receptor binding resulting in enhanced antitumor activity of TNF-alpha.     

Kissper, a kiwi fruit peptide with channel-like activity: structural and functional features.

Kissper is a 39-residue peptide isolated from kiwi fruit (Actinidia deliciosa). Its primary structure, elucidated by direct protein sequencing, is identical to the N-terminal region of kiwellin, a recently reported kiwi fruit allergenic protein, suggesting that kissper derives from the in vivo processing of kiwellin. The peptide does not show high sequence identity with any other polypeptide of known function. However, it displays a pattern of cysteines similar, but not identical, to those observed in some plant and animal proteins, including toxins involved in defence mechanisms. A number of these proteins are also active on mammalian cells. Functional characterization of kissper showed pH-dependent and voltage-gated pore-forming activity, together with anion selectivity and channeling in model synthetic PLMs, made up of POPC and of DOPS:DOPE:POPC. A 2DNMR analysis indicates that in aqueous solution kissper has only short regions of regular secondary structure, without any evident similarity with other bioactive peptides. Comparative analysis of the structural and functional features suggests that kissper is a member of a new class of pore-forming peptides with potential effects on human health.     

Revealing the structural and functional diversity of plant cell walls

The extensive knowledge of the chemistry of isolated cell wall polymers, and that relating to the identification and partial annotation of gene families involved in their synthesis and modification, is not yet matched by a sophisticated understanding of the occurrence of the polymers within cell walls of the diverse cell types within a growing organ. Currently, the main sets of tools that are used to determine cell-type-specific configurations of cell wall polymers and aspects of cell wall microstructures are antibodies, carbohydrate-binding modules (CBMs) and microspectroscopies. As these tools are applied we see that cell wall polymers are extensively developmentally regulated and that there is a range of structurally distinct primary and secondary cell walls within organs and across species. The challenge now is to document cell wall structures in relation to diverse cell biological events and to integrate this knowledge with the emerging understanding of polymer functions.     

Temperature-dependent structural and functional features of a hyperthermostable enzyme using elastic neutron scattering

The dynamic behavior of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus was investigated using elastic neutron scattering. The temperature dependence of the atomic motions was correlated with conformational and functional characteristics of the enzyme. The onset of biological function at temperatures higher than approximately 25 degrees C (the hyperthermostable enzyme is essentially inactive at room temperature) was associated with a dynamical transition in the anharmonic motions domain. This transition from the nonactive to the enzymatically active conformation involved structurally similar conformational substates in the energy landscape. From the mean-square displacement of the protein atoms, the molecular flexibility and the effective force constants were calculated at different temperature zones. The results showed that the activity increases at higher temperatures where the intramolecular bonds are weakened and the overall rigidity of the protein is decreased. Further temperature increase resulted in significantly increased atomic fluctuations featuring heat denaturation of the protein.     

Gene disruption and gene replacement in Streptomyces via single stranded DNA transformation of integration vectors.

For the isolation of single stranded plasmid DNA, various E. coli and E. coli-Streptomyces shuttle plasmids were equipped with the phage f1 replication origin. The transformation of some representative Streptomyces species with plasmid vectors occurred irrespective of whether single or double stranded DNA was used. In contrast, the transformation of Streptomyces was 10 to 100 times more efficient when an integration vector was in the single stranded form as opposed to the double stranded form. Streptomyces viridochromogenes was transformed by single stranded DNA integration vectors in order to replace the pat by the tsr gene and generate mutants unable to synthesize phosphinothricin-tripeptide (PTT).     

The human THE-LTR(O) and MstII interspersed repeats are subfamilies of a single widely distributed highly variable repeat family.

Fifteen examples of the transposon-like human element (THE) LTR and thirteen examples of the MstII interspersed repeat are aligned to generate new consensus sequences for these human repetitive elements. The consensus sequences of these elements are very similar, indicating that they compose subfamilies of a single human interspersed repetitive sequence family. Members of this highly polymorphic repeat family have been mapped to at least 11 chromosomes. Seven examples of the THE internal sequence are also aligned to generate a new consensus sequence for this element. Estimates of the abundance of this repetitive sequence family, derived from both hybridization analysis and frequency of occurrence in GenBank, indicate that THE-LTR/MstII sequences are present every 100-3000 kb in human DNA. The widespread occurrence of members of this family makes them useful landmarks, like Alu, L1, and (GT)n repeats, for physical and genetic mapping of human DNA.     

Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen

We have optimized the expression level of 20 mammalian G protein-coupled receptors (GPCRs) in the methylotrophic yeast Pichia pastoris. We found that altering expression parameters, including growth temperature, and supplementation of the culture medium with specific GPCR ligands, histidine, and DMSO increased the amount of functional receptor, as assessed by ligand binding, by more than eightfold over standard expression conditions. Unexpectedly, we found that the overall amount of GPCR proteins expressed, in most cases, varied only marginally between standard and optimized expression conditions. Accordingly, the optimized expression conditions resulted in a marked fractional increase in the ratio of ligand binding-competent receptor to total expressed receptor. The results of this study suggest a general approach for increasing yields of functional mammalian GPCRs severalfold over standard expression conditions by using a set of optimized expression condition parameters that we have characterized for the Pichia expression system. Overall, we have more than doubled the number of GPCR targets that can be produced in our laboratories in sufficient amounts for structural studies.