Biomolecular Characterization of Putative Antidiabetic Herbal Extracts

Induction of GLUT4 translocation in the absence of insulin is considered a key concept to decrease elevated blood glucose levels in diabetics. Due to the lack of pharmaceuticals that specifically increase the uptake of glucose from the blood circuit, application of natural compounds might be an alternative strategy. However, the effects and mechanisms of action remain unknown for many of those substances. For this study we investigated extracts prepared from seven different plants, which have been reported to exhibit anti-diabetic effects, for their GLUT4 translocation inducing properties. Quantitation of GLUT4 translocation was determined by total internal reflection fluorescence (TIRF) microscopy in insulin sensitive CHO-K1 cells and adipocytes. Two extracts prepared from purslane (Portulaca oleracea) and tindora (Coccinia grandis) were found to induce GLUT4 translocation, accompanied by an increase of intracellular glucose concentrations. Our results indicate that the PI3K pathway is mainly responsible for the respective translocation process. Atomic force microscopy was used to prove complete plasma membrane insertion. Furthermore, this approach suggested a compound mediated distribution of GLUT4 molecules in the plasma membrane similar to insulin stimulated conditions. Utilizing a fluorescent actin marker, TIRF measurements indicated an impact of purslane and tindora on actin remodeling as observed in insulin treated cells. Finally, in-ovo experiments suggested a significant reduction of blood glucose levels under tindora and purslane treated conditions in a living organism. In conclusion, this study confirms the anti-diabetic properties of tindora and purslane, which stimulate GLUT4 translocation in an insulin-like manner.     

Multiscale Characterization of Land-Use Patterns in China

This article explores the pattern of land use in China to understand the relationships between land use and factors that can be used to predict it. Such understanding is important for development of comprehensive models of land-use dynamics. Correlation and regression analyses are used to identify the most important explanatory variables from a large set of factors generally considered important in predicting the distribution of land use. We found that the spatial distribution of all land-use types in China is best described by an integrated set of biophysical and socioeconomic factors. Specific attention is given to the influence of the scale of analysis on study results. Both data resolution and the extent of the study area influence the discovered relationships. Relationships obtained at a certain scale of analysis therefore may not be directly applied at other scales or in other areas. The relevance of the systematic and quantitative characterization of the land-use patterns in China for the parameterization of spatially explicit land-use models is discussed. Received 20 July 1999; accepted 10 March 2000.     

A Multiscale Vibrational Spectroscopic Approach for Identification and Biochemical Characterization of Pollen

Background

Analysis of pollen grains reveals valuable information on biology, ecology, forensics, climate change, insect migration, food sources and aeroallergens. Vibrational (infrared and Raman) spectroscopies offer chemical characterization of pollen via identifiable spectral features without any sample pretreatment. We have compared the level of chemical information that can be obtained by different multiscale vibrational spectroscopic techniques.

Methodology

Pollen from 15 different species of Pinales (conifers) were measured by seven infrared and Raman methodologies. In order to obtain infrared spectra, both reflectance and transmission measurements were performed on ground and intact pollen grains (bulk measurements), in addition, infrared spectra were obtained by microspectroscopy of multigrain and single pollen grain measurements. For Raman microspectroscopy measurements, spectra were obtained from the same pollen grains by focusing two different substructures of pollen grain. The spectral data from the seven methodologies were integrated into one data model by the Consensus Principal Component Analysis, in order to obtain the relations between the molecular signatures traced by different techniques.

Results

The vibrational spectroscopy enabled biochemical characterization of pollen and detection of phylogenetic variation. The spectral differences were clearly connected to specific chemical constituents, such as lipids, carbohydrates, carotenoids and sporopollenins. The extensive differences between pollen of Cedrus and the rest of Pinaceae family were unambiguously connected with molecular composition of sporopollenins in pollen grain wall, while pollen of Picea has apparently higher concentration of carotenoids than the rest of the family. It is shown that vibrational methodologies have great potential for systematic collection of data on ecosystems and that the obtained phylogenetic variation can be well explained by the biochemical composition of pollen. Out of the seven tested methodologies, the best taxonomical differentiation of pollen was obtained by infrared measurements on bulk samples, as well as by Raman microspectroscopy measurements of the corpus region of the pollen grain. Raman microspectroscopy measurements indicate that measurement area, as well as the depth of focus, can have crucial influence on the obtained data.     

SWI/SNF Chromatin-remodeling Factors: Multiscale Analyses and Diverse Functions

Chromatin-remodeling enzymes play essential roles in many biological processes, including gene expression, DNA replication and repair, and cell division. Although one such complex, SWI/SNF, has been extensively studied, new discoveries are still being made. Here, we review SWI/SNF biochemistry; highlight recent genomic and proteomic advances; and address the role of SWI/SNF in human diseases, including cancer and viral infections. These studies have greatly increased our understanding of complex nuclear processes.     

New Methodology for Computer-Aided Modelling of Biomolecular Structure and Dynamics 1. Non-Cyclic Structures

Abstract

A general methodology is proposed for the conformational modelling of biomolecular systems. The approach allows one: (i) to describe the system under investigation by an arbitrary set of internal variables, i.e., torsion angles, bond angles, and bond lengths; it offers a possibility to pass from the free structure to a completely fixed one with the number of variables from 3N to zero, respectively, where N is the number of atoms; (ii) to consider both, a single molecule and a complex of many molecules, (e.g., proteins, water, ligands, etc.) in terms of one universal model; (iii) to study the dynamics of the system using explicit analytical Lagrangian equations of motion, thus opening up possibilities for investigations of slow concerted motions such as domain oscillations in proteins etc.; (iv) to calculate the partial derivatives of various functions of conformation, e.g., the conformatinal energy or external constraints imposed, using a standard efficient procedure regardless of the variables and the structure of the system. The approach is meant to be used in various investigations concerning the conformations and dynamics of biomacromolecules.     

Symbolic Extensions Applied to Multiscale Structure of Genomes

A genome of a living organism consists of a long string of symbols over a finite alphabet carrying critical information for the organism. This includes its ability to control post natal growth, homeostasis, adaptation to changes in the surrounding environment, or to biochemically respond at the cellular level to various specific regulatory signals. In this sense, a genome represents a symbolic encoding of a highly organized system of information whose functioning may be revealed as a natural multilayer structure in terms of complexity and prominence. In this paper we use the mathematical theory of symbolic extensions as a framework to shed light onto how this multilayer organization is reflected in the symbolic coding of the genome. The distribution of data in an element of a standard symbolic extension of a dynamical system has a specific form: the symbolic sequence is divided into several subsequences (which we call layers) encoding the dynamics on various “scales”. We propose that a similar structure resides within the genomes, building our analogy on some of the most recent findings in the field of regulation of genomic DNA functioning.     

Multiscale Characterization of Casein Micelles Under NaCl Range Conditions

Micellar casein (MC) dispersions were studied at a constant protein concentration of 5 wt % in high NaCl environment. The micellar edifices were characterized as to their morphology, size, and content of proteins in the supernatant after ultracentrifugation. Additionally, changes in secondary structures of the protein upon salt increase were followed by Fourier Transform Infrared Spectroscopy (FTIR). For the first time, the estimations of secondary structural elements (irregular, ß-sheet, ??-helix and turn) from Amide III assignments were correlated with results from Amide I. Casein micelles dispersions in water were characterized by Transmission Electron Microscopy (TEM) by a spherical shape and a size between 100 and 200 nm. A salt increase resulted to a destabilization of the micelle and the formation of mini-micelles more or less aggregated. The size of the new edifice was almost similar to the native micelle. These TEM observations were confirmed by a constant casein micelle hydrodynamic diameter determined by Dynamic Light Scattering (DLS) and ranging between 150 and 180 nm. Upon salt increase, FTIR revealed an increase in irregular structures and a concurrent decrease in ß-sheet structures. Secondary structural elements percentages were almost similar from Amide I and Amide III. The use of these multiscale techniques led to a better understanding of the micellar edifice under high salt environment. Around 3% NaCl addition, a good correlation was observed between destabilization of the micellar edifice, modifications of the caseins secondary structure and repartition of caseins between supernatant and pellet after ultracentrifugation.     

New Methodology for Computer-Aided Modelling of Biomolecular Structure and Dynamics 2. Local Deformations and Cycles

Abstract

A new methodology for the conformational modelling of biomolecular systems (1) is extended to local deformations of chain molecules and to flexible molecular rings. It is shown that these two cases may be reduced to considering an equivalent molecular model with a regular tree-like topology. A simple procedure is developed to analyze any flexible rings (the five- and six-membered suguar rings of carbohydrates and nucleic acids, in particular) and local deformation regions by energy minimization. Dynamic equations are also derived for such molecular systems. As a result, a unified approach is proposed for the efficient energy minimization and simulation of dynamic behavior of multimolecular systems having any set of variable internal coordinates, local deformation regions and cycles. Advantages and domains of applicability of the approach are discussed.     

Characterization of the Structure and Biological Functions of a Capsular Polysaccharide Produced by Staphylococcus saprophyticus

Staphylococcus saprophyticus is a common cause of uncomplicated urinary tract infections in women. S. saprophyticus strain ATCC 15305 carries two staphylococcal cassette chromosome genetic elements, SCC_15305RM and SCC_15305cap. The SCC_15305cap element carries 13 open reading frames (ORFs) involved in capsular polysaccharide (CP) biosynthesis, and its G+C content (26.7%) is lower than the average G+C content (33.2%) for the whole genome. S. saprophyticus strain ATCC 15305 capD, capL, and capK (capD_Ssp, capL_Ssp, and capK_Ssp) are homologous to genes encoding UDP-FucNAc biosynthesis, and gtaB and capI_Ssp show homology to genes involved in UDP-glucuronic acid synthesis. S. saprophyticus ATCC 15305 CP, visualized by immunoelectron microscopy, was extracted and purified using anionic-exchange and size exclusion chromatography. Analysis of the purified CP by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy and gas-liquid chromatography revealed two types of branched tetrasaccharide repeating units composed of the following: Sug represents two stereoisomers of 2-acetamido-2,6-dideoxy-hexos-4-ulose residues, one of which has an arabino configuration. The encapsulated ATCC 15305 strain was resistant to complement-mediated opsonophagocytic killing by human neutrophils, whereas the acapsular mutant C1 was susceptible. None of 14 clinical isolates reacted with antibodies to the ATCC 15305 CP. However, 11 of the 14 S. saprophyticus isolates were phenotypically encapsulated based on their resistance to complement-mediated opsonophagocytic killing and their failure to hemagglutinate when cultivated aerobically. Ten of the 14 clinical strains carried homologues of the conserved staphylococcal capD gene or the S. saprophyticus gtaB gene, or both. Our results suggest that some strains of S. saprophyticus are encapsulated and that more than one capsular serotype exists.Approximately 13 million women develop urinary tract infections (UTIs) annually in the United States, with a recurrence rate between 25% and 44% (45). Staphylococcus saprophyticus is second only to Escherichia coli as a cause of uncomplicated UTI in young women (45, 46). A novobiocin-resistant member of the coagulase-negative staphylococci (60), S. saprophyticus has rarely exhibited resistance to other antibiotics (25). However, a recent report (19) indicated that methicillin-resistant S. saprophyticus isolates have emerged in Japan. The gastrointestinal tract and the vagina are the major reservoirs of S. saprophyticus (18, 30) and the likely sources of recurrent infection (20, 37, 49). Approximately 40% of patients with S. saprophyticus UTI present with acute pyelonephritis (22, 30). These patients experience symptoms more severe than those of patients infected by E. coli (24), and they are more likely to develop recurrent infections (21).A number of potential virulence factors have been identified in S. saprophyticus. Gatermann et al. showed that in a rodent model of ascending UTI, the production of urease contributes to S. saprophyticus growth and pathogenicity in the bladder (10, 12). Other putative virulence factors of S. saprophyticus include a surface-associated lipase (11, 51, 53), the collagen binding protein SdrI (52), and a cell wall-anchored hemagglutinin protein that mediates the binding of S. saprophyticus to sheep erythrocytes, fibronectin, and human uroepithelial cells (14, 29, 34, 35). The hemagglutinin was dubbed UafA in the sequenced ATCC 15305 strain, and deletion of the uafA gene resulted in reduced S. saprophyticus hemagglutination (HA) and adherence to human bladder carcinoma cells (29). Kuroda et al. noted that UafA-mediated adherence of S. saprophyticus to the T24 cell line was inhibited by the presence of the ATCC 15305 polysaccharide capsule (29).Staphylococcal species produce a variety of extracellular glycopolymers that contribute to the surface properties and virulence of the bacterium, such as capsular polysaccharides (CP), teichoic acids, and poly-N-acetylglucosamine (PNAG). CP production renders Staphylococcus aureus resistant to opsonophagocytic killing; alanine modifications of teichoic acids promote bacterial resistance to antimicrobial peptides (40); and PNAG is involved in biofilm formation (4). Recently, the secretion of another anionic polymer (poly-γ-dl-glutamic acid) by certain other coagulase-negative staphylococci was reported (28). Polyglutamic acid production is enhanced under high-salt conditions and may contribute to the survival of Staphylococcus epidermidis on human skin.S. saprophyticus strain 15305 does not produce PNAG or polyglutamic acid (28, 29), but this uropathogenic species is encapsulated. CP are lacking in isolates of S. epidermidis, the most common of the coagulase-negative species, but genomic evidence indicates that Staphylococcus haemolyticus (7, 57), S. saprophyticus (29), and Staphylococcus carnosus (47) carry capsule loci with genetic similarity to the Staphylococcus aureus cap5 (cap8) gene locus. In this study, we purified and characterized the CP produced by S. saprophyticus ATCC 15305 and investigated the CP phenotype of S. saprophyticus clinical isolates.