Effects of thermoradiation on bacteria.

A 60Co source was used to determine the effects of thermoradiation on Achromobacter aquamarinus, Staphylococcus aureus, and vegetative and spore cells of Bacillus subtilis var. globigii. The rate of inactivation of these cultures, except vegetative-cell populations of B. subtilis, was exponential and in direct proportion to temperature. The D10 (dose that inactivates 90% of the microbial population) value for A. aquamarinus was 8.0 Krad at 25 degrees C and 4.9 Krad at 35 degrees C. For S. aureus, D10 was 9.8 and 5.3 Krad at 35 and 45 degrees C, respectively. Vegetative cells of B. subtilis demonstrated a rapid initial inactivation followed by a steady but decreased exponential rate. The D10 at 25 degrees C was 10.3 Krad, but at 35 and 45 degrees C this value was 6.2 and 3.8 Krad, respectively. Between 0 and 95 Krad, survival curves for B. subtilis spores at 75 degrees C showed slight inactivation, increasing in rat at and above 85 degrees C. The D10 values for spores at 85 and 90 degrees C were 129 and 92 Krad, respectively. Significant synergism between heat and irradiation was noted at 35 degrees C for A. aquamarinus and 45 degrees C for S. aureus. The presence of 0.1 mM cysteine in suspending media afforded protection to both cultures at these critical temperatures. On the other hand, cysteine sensitized B. subtilis spores at radiation doses greater than 100 Krad. The combined effect of heat and irradiation was more destructive to bacteria than either method alone.     

Large-Scale Production of Nanographite by Tube-Shear Exfoliation in Water

The number of applications based on graphene, few-layer graphene, and nanographite is rapidly increasing. A large-scale process for production of these materials is critically needed to achieve cost-effective commercial products. Here, we present a novel process to mechanically exfoliate industrial quantities of nanographite from graphite in an aqueous environment with low energy consumption and at controlled shear conditions. This process, based on hydrodynamic tube shearing, produced nanometer-thick and micrometer-wide flakes of nanographite with a production rate exceeding 500 gh^-1 with an energy consumption about 10 Whg^-1. In addition, to facilitate large-area coating, we show that the nanographite can be mixed with nanofibrillated cellulose in the process to form highly conductive, robust and environmentally friendly composites. This composite has a sheet resistance below 1.75 Ω/sq and an electrical resistivity of 1.39×10^-4 Ωm and may find use in several applications, from supercapacitors and batteries to printed electronics and solar cells. A batch of 100 liter was processed in less than 4 hours. The design of the process allow scaling to even larger volumes and the low energy consumption indicates a low-cost process.     

Glyphosate complexation to aluminium(III). An equilibrium and structural study in solution using potentiometry, multinuclear NMR, ATR-FTIR, ESI-MS and DFT calculations

The stoichiometries and stability constants of a series of Al³⁺-N-phosponomethyl glycine (PMG/H₃L) complexes have been determined in acidic aqueous solution using a combination of precise potentiometric titration data, quantitative ²⁷Al and ³¹P NMR spectra, ATR-FTIR spectrum and ESI-MS measurements (0.6 M NaCl, 25 °C). Besides the mononuclear AlH₂L²⁺, Al(H₂L)(HL), and Al(HL)L²⁻, dimeric Al₂(HL)L⁺ and trinuclear complexes have been postulated.¹H and ³¹P NMR data show that different isomers co-exist in solution and the isomerization reactions are slow on the ³¹P NMR time scale. The geometries of monomeric and dimeric complexes likely double hydroxo bridged and double phosphonate bridged isomers have been optimized using DFT ab initio calculations starting from rational structural proposals. Energy calculations using the PCM solvation method also support the co-existence of isomers in solutions.     

Vectorially oriented membrane protein monolayers: profile structures via x-ray interferometry/holography.

X-ray interferometry/holography was applied to meridional x-ray diffraction data to determine uniquely the profile structures of a single monolayer of an integral membrane protein and a peripheral membrane protein, each tethered to the surface of a solid inorganic substrate. Bifunctional, organic self-assembled monolayers (SAMs) were utilized to tether the proteins to the surface of Ge/Si multilayer substrates, fabricated by molecular beam epitaxy, to facilitate the interferometric/holographic x-ray structure determination. The peripheral membrane protein yeast cytochrome c was covalently tethered to the surface of a sulfhydryl-terminated 11-siloxyundecanethiol SAM via a disulfide linkage with residue 102. The detergent-solubilized, photosynthetic reaction center integral membrane protein was electrostatically tethered to the surface of an analogous amine-terminated SAM. Optical absorption measurements performed on these two tethered protein monolayer systems were consistent with the x-ray diffraction results indicating the reversible formation of densely packed single monolayers of each fully functional membrane protein on the surface of the respective SAM. The importance of utilizing the organic self-assembled monolayers (as opposed to Langmuir-Blodgett) lies in their ability to tether specifically both soluble peripheral membrane proteins and detergent-solubilized integral membrane proteins. The vectorial orientations of the cytochrome c and the reaction center molecules were readily distinguishable in the profile structure of each monolayer at a spatial resolution of 7 A.     

Structure and Mode-of-Action of the Two-Peptide (Class-IIb) Bacteriocins

This review focuses on the structure and mode-of-action of the two-peptide (class-IIb) bacteriocins that consist of two different peptides whose genes are next to each other in the same operon. Optimal antibacterial activity requires the presence of both peptides in about equal amounts. The two peptides are synthesized as preforms that contain a 15–30 residue double-glycine-type N-terminal leader sequence that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter that concomitantly transfers the bacteriocin peptides across cell membranes. Two-peptide bacteriocins render the membrane of sensitive bacteria permeable to a selected group of ions, indicating that the bacteriocins form or induce the formation of pores that display specificity with respect to the transport of molecules. Based on structure–function studies, it has been proposed that the two peptides of two-peptide bacteriocins form a membrane-penetrating helix–helix structure involving helix–helix-interacting GxxxG-motifs that are present in all characterized two-peptide bacteriocins. It has also been suggested that the membrane-penetrating helix–helix structure interacts with an integrated membrane protein, thereby triggering a conformational alteration in the protein, which in turn causes membrane-leakage. This proposed mode-of-action is similar to the mode-of-action of the pediocin-like (class-IIa) bacteriocins and lactococcin A (a class-IId bacteriocin), which bind to a membrane-embedded part of the mannose phosphotransferase permease in a manner that causes membrane-leakage and cell death.     

Variation in quantity and extractability of the 148-kilodalton cartilage protein with age.

The occurrence of non-collagenous matrix proteins was studied in samples of tracheal cartilage from steers of different ages. The amounts of the 148 kDa cartilage protein in 4M-guanidinium chloride extracts and in subsequent trypsin digests of the extraction residues were determined by radioimmunoassay. Surprisingly, the 148 kDa-protein antigenicity was not changed by tryptic digestion, even though the protein was extensively degraded. The amount of the 148 kDa protein increased dramatically with age, both in the guanidinium chloride extract and in the subsequent tryptic digest, and reached maximal values at about 3 and 8 years respectively. The increase of the guanidinium chloride-soluble pool preceded that of the trypsin-digestible pool, possibly indicating a metabolic relationship. The ratio between the trypsin-digestible and guanidinium chloride-soluble pools increased continuously, and at 12 years of age close to 90% of the total 148 kDa protein detected was insoluble in guanidinium chloride. At all ages, the bulk of the cartilage collagen was insoluble both to extraction with guanidinium chloride and to tryptic digestion. The decreasing extractability of the 148 kDa protein was therefore not secondary to changes in the solubility of the collagen network. Other cartilage proteins, such as the link proteins and the 36 kDa protein, showed much smaller quantitative variations of a different character.     

Properties of spin and fluorescent labels at a receptor-ligand interface.

Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently attached to an engineered cysteine in position 140 in sTF, a position normally occupied by a Phe residue previously characterized as an important contributor to the sTF:FVIIa interaction. Two spin labels, IPSL [N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide] and MTSSL [(1-oxyl-2,2,5, 5-tetramethylpyrroline-3-methyl)methanethiosulfonate], and two fluorescent labels, IAEDANS [5-((((2-iodoacetyl)amino) ethyl)amino)naphthalene-1-sulfonic acid] and BADAN [6-bromoacetyl-2-dimethylaminonaphthalene], were used. Spectral data from electron paramagnetic resonance (EPR) and fluorescence spectroscopy showed a substantial change in the local environment of all labels when the sTF:FVIIa complex was formed. However, the interaction was probed differently by each label and these differences in spectral appearance could be attributed to differences in label properties such as size, polarity, and/or flexibility. Accordingly, molecular modeling data suggest that the most favorable orientations are unique for each label. Furthermore, line-shape simulations of EPR spectra and calculations based on fluorescence depolarization measurements provided additional details of the local environment of the labels, thereby confirming a tight protein-protein interaction between FVIIa and sTF when the complex is formed. The tightness of this local interaction is similar to that seen in the interior of globular proteins.     

Norepinephrine Metabolism in Man Using Deuterium Labeling: Turnover of 4-Hydroxy-3-Methoxymandelic Acid

Abstract: 4-Hydroxy-3-methoxymandelic acid (HMMA; VMA) labeled with three deuterium atoms was used to study the turnover and fate of HMMA following intravenous injection. Five healthy men were given a pulse dose of 5.0 μmol of labeled HMMA. Plasma and urinary levels of both endogenous and labeled HMMA were subsequently followed by gas chromatography-mass spectrometry using selected ion detection. The kinetic parameters were determined both with and without compensation for the pool expansion caused by the injection of labeled HMMA. The urinary recovery of labeled HMMA was 85 × 10% (mean ± SD). No conversion of HMMA t o 4-hydroxy-3-methoxyphenyl glycol (HMPG) occurred. The biological half-life of HMMA was 0.54 ± 0.22 h. The apparent volume of distribution was 0.36 ± 0.11 L/kg. The production rate or body turnover was 1.27 ± 0.51 μmol HMM/h and urinary excretion rate was 0.82 ± 0.22 μmol/h. These results show that HMMA is turning over rapidly in a relatively small volume of distribution and that, unlike HMPG, it is an end metabolite of norepinephrine in man.     

Specific interaction of one subunit of eukaryotic initiation factor eIF-3 with 18S ribosomal RNA within the binary complex, eIF-3 small ribosomal subunit, as shown by cross-linking experiments.

Initiation factor eIF-3 from rat liver forms a binary complex with the small ribosomal subunit. Within this complex, 18S ribosomal RNA can be cross-linked to the 66 000 dalton subunit of eIF-3 by treating the complex with a short bifunctional reagent, diepoxybutane, with a distance of 4A between the reactive groups. In binary complexes containing eIF-3 premodified with the heterobifunctional reagent, methyl-p-azido-benzoylaminoacetimidate (10A), the 66 000 dalton subunit of eIF-3 became covalently bound to 18S rRNA after irradiation of the complex with ultraviolet light. The involvement of only one of the eight eIF-3 subunits in the formation of the covalent RNA-protein complexes indicates a highly specific interaction between 18S rRNA and eIF-3 at the attachment site of the factor on the 40S subunit.     

The Tyrosine Sulfate-rich Domains of the LRR Proteins Fibromodulin and Osteoadherin Bind Motifs of Basic Clusters in a Variety of Heparin-binding Proteins, Including Bioactive Factors

The small leucine-rich repeat proteins, fibromodulin and osteoadherin, have N-terminal extensions with a variable number of O-sulfated tyrosine residues. This modification combined with a number of aspartic and glutamic acid residues results in a highly negatively charged domain of less than 30 amino acids. We hypothesized that this domain shares functional properties with heparin regarding binding to proteins and polypeptides containing clusters of basic amino acids. Two other family members, PRELP and chondroadherin, have distinctly different clusters of basic amino acids in their N and C termini, respectively, and PRELP is known to bind to heparin via this domain. Another heparin-binding protein is the cytokine Oncostatin M, with a different cluster of basic amino acids in its C terminus. We used polypeptides representing these basic domains in solid phase assays and demonstrate interactions with the negatively charged N-terminal domain of fibromodulin and full-length osteoadherin. The tyrosine sulfate domains also bound heparin-binding proteins such as basic fibroblast growth factor-2, thrombospondin I, MMP13, the NC4 domain of collagen IX, and interleukin-10. Fibronectin with large heparin-binding domains did not bind, neither did CILP containing a heparin-binding thrombospondin type I motif without clustered basic amino acids. Affinity depends on the number and position of the sulfated tyrosine residues shown by different binding properties of 10-kDa fragments subfractionated by ion-exchange chromatography. These interactions may sequester growth factors, cytokines, and matrix metalloproteinases in the extracellular matrix as well as contribute to its organization.The integrity of the extracellular matrix depends on a multitude of interactions between molecular constituents leading to the formation of major macromolecular assemblies important for tissue functions. A major component in most types of extracellular matrix is the network of fibrillar structures primarily composed of collagen I in fibrous tissues and bone, whereas cartilage contains the similar collagen II.These collagen fibrils contain a number of associated molecules, often bound to their surface. One such molecule is the distinct collagen IX, containing three triple helical domains each surrounded by non-triple helical domains. Another set of molecules binding to triple helical collagen is the members of the small leucine-rich repeat protein family, such as fibromodulin (1), lumican (2), decorin (3), biglycan (4), PRELP (5), chondroadherin (6), and possibly osteoadherin. The typical LRR³ protein contains 10–11 repeats of some 25 amino acids with leucine residues at conserved locations. This domain represents a common denominator for the family and contains structures providing for interaction with, e.g. triple helical collagen (7–9). The LRR proteins contain an extension at either the N- or C-terminal end or, in a few cases, at both ends. These extensions may contribute to a second function exemplified by PRELP, where the N-terminal with a stretch of clustered arginine residues provides binding to heparin/heparan sulfate containing optimally five or more disaccharides with three sulfate groups each (10). In decorin and biglycan, the N-terminal extension have substituents of glycosaminoglycan chains of dermatan/chondroitin sulfate that can contribute to collagen binding (11) as well as provide putative self interactions with a similar chain on another molecule. In particular, it has been shown that decorin and biglycan will bind via their protein core to the N-terminal globular domain of collagen VI (4) and direct the formation of the collagen VI-beaded filament network, provided that the glycosaminoglycan chains are intact.There are a number of proteins known to interact with heparin. Whereas heparin is not present in the extracellular matrix, these proteins may bind to stretches within the heparan sulfate chains enriched in disaccharides having high sulfate content. The heparan sulfate is found particularly as a component of cell surface proteoglycans such as glypicans (12) and syndecans (13) and of the extracellular matrix proteoglycans perlecan (14) and agrin (15). Important ligands for these chains are growth factors exemplified by members of the FGF family. Other molecules that bind to heparan sulfate include fibronectin, having two such domains with molecular weights of around 20,000 (16). Also several members of the metalloproteinase family contain heparin-binding motifs as do many cytokines.The most common heparin-binding sequence contains clusters of basic amino acids, often with additional proline residues. PRELP and chondroadherin as well as the other proteins mentioned represent examples having such sequences. A different type of motif, first found in thrombospondin I, contains consecutive repeats of a WXZ sequence, where the tryptophan may be mannosylated (17, 18). This is referred to as the thrombospondin type I motif heparin-binding structure. Thrombospondin I in addition contains a heparin-binding basic cluster of amino acids (19). CILP on the other hand only contains the thrombospondin type 1 motif. These domains can bind to heparin with high affinity, an interaction that can be disrupted by high salt.A very different type of extension is found in the N-terminal part of fibromodulin and osteoadherin. These proteins contain a number of tyrosine residues, which may and often do, carry a sulfate group. Thus, fibromodulin contains up to nine such residues and osteoadherin as many as eight, where six are located in the N-terminal and two in the C-terminal extension (20). Any given preparation contains molecules within the same species with a range of levels of O-sulfate-substituted tyrosine. The functional significances of these domains have been unknown. We now show that these domains can mimic heparin in several interactions.