Static Stability and Swim Bladder Volume in the Bluegill Sunfish (Lepomis macrochirus).

Static stability is a property inherent to every organism. More stable bodies benefit from a lower energy cost associated with maintaining a desired orientation, while less stable bodies can be more maneuverable. The static stability of a fish is determined by the relative locations of its center of mass (COM) and center of buoyancy (COB), which may change with changes in swim bladder volume. We hypothesized, however, that fish would benefit from consistent static stability, and predicted that changes in swim bladder volume would not alter the overall pattern of COM and COB locations. We used micro-computed tomography to estimate the locations of the COM and COB in bluegill sunfish (Lepomis macrochirus). Using this technique, we were able to find a small but significant difference between the location of the COM and COB for a given orientation. We found that the swim bladder can change shape within the body cavity, changing relative locations of the COM and COB. At one extreme, the COB is located 0.441 ± 0.007 BL from the snout and 0.190 ± 0.010 BL from the ventral surface of the pelvic girdle, and that the COM is 0.0030 ± 0.0020 BL posterior and 0.0006 ± 0.0005 BL ventral to the COB, a pattern that causes a nose-up pitching torque. At the other extreme, the COM is anterior and dorsal to the COB, a pattern that causes the opposite torque. These changes in location seems to be caused by changes in the shape and centroid location of the swim bladder within the body: The centroid of the swim bladder is located significantly more posteriorly in fish oriented head-down. The air in the bladder "rises" while heavier tissues "sink," driving a change in tissue distribution and changing the location of the COM relative to the COB. Supporting our hypothesis, we found no correlation between swim bladder volume and the distance between the COM and COB. We conclude that bluegill are statically unstable, requiring them to expend energy constantly to maintain their normal orientation, but that the pitch angle of the body could alter the relative locations of COM and COB, changing their static stability.

Heparin-binding growth-associated molecule contains two heparin-binding beta -sheet domains that are homologous to the thrombospondin type I repeat.

Heparin-binding growth-associated molecule (HB-GAM) is an extracellular matrix-associated protein implicated in the development and plasticity of neuronal connections of brain. Binding to cell surface heparan sulfate is indispensable for the biological activity of HB-GAM. In the present paper we have studied the structure of recombinant HB-GAM using heteronuclear NMR. These studies show that HB-GAM contains two beta-sheet domains connected by a flexible linker. Both of these domains contain three antiparallel beta-strands. In addition to this domain structure, HB-GAM contains the N- and C-terminal lysine-rich sequences that lack a detectable structure and appear to form random coils. Studies using CD and NMR spectroscopy suggest that HB-GAM undergoes a conformational change upon binding to heparin, and that the binding occurs primarily to the beta-sheet domains of the protein. Search of sequence data bases shows that the beta-sheet domains of HB-GAM are homologous to the thrombospondin type I repeat (TSR). Sequence comparisions show that the beta-sheet structures found previously in midkine, a protein homologous with HB-GAM, also correspond to the TSR motif. We suggest that the TSR sequence motif found in various extracellular proteins defines a beta-sheet structure similar to that found in HB-GAM and midkine. In addition to the apparent structural similarity, a similarity in biological functions is suggested by the occurrence of the TSR sequence motif in a wide variety of proteins that mediate cell-to-extracellular matrix and cell-to-cell interactions, in which the TSR domain mediates specific cell surface binding.

Spatially Inhomogeneous Metal-Insulator Transition in Doped Manganites.

Scanning tunneling spectroscopy was used to investigate single crystals and thin films of La(1-x)Ca(x)MnO(3) (with x of about 0.3), which exhibit colossal magnetoresistance. The different spectroscopic signatures of the insulating (paramagnetic) and metallic (ferromagnetic) phases enable their spatial extent to be imaged down to a lateral scale of the order of 10 nanometers. Above the bulk transition temperature T(c), the images show mostly insulating behavior. Below T(c), a phase separation is observed where inhomogeneous structures of metallic and more insulating areas coexist and are strongly field dependent in their size and structure. Insulating areas are found to persist far below T(c). These results suggest that the transition and the associated magnetoresistance behavior should be viewed as a percolation of metallic ferromagnetic domains.

Interaction of soluble and surface-bound heparin binding growth-associated molecule with heparin.

The interaction of heparin with heparin binding growth-associated molecule (HB-GAM) was studied using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). ITC studies showed that, in solution, heparin bound HB-GAM with a deltaH of -30 kcal/mole corresponding to a dissociation constant (Kd) of 460 nM. The stoichiometry of interaction was 3 moles of HB-GAM per mole of heparin, corresponding to a minimum heparin binding site for HB-GAM of 12-16 saccharide residues. Kinetic measurements of heparin interaction with HB-GAM made by SPR afforded a Kd of 4 nM, suggesting considerably tighter binding when HB-GAM was immobilized on a surface. Affinity chromatography of a sized mixture of heparin oligosaccharides, having a degree of polymerization (dp) of > 14 saccharide units, on HB-GAM-Sepharose demonstrated that oligosaccharides having more than 18 saccharide residues showed the tightest interaction.

Interaction of secretory leukocyte protease inhibitor with heparin inhibits proteases involved in asthma.

Protease inhibition by secretory leukocyte protease inhibitor (SLPI) is accelerated by the sulfated polysaccharides. The nature of the SLPI-polysaccharide interaction, explored with affinity chromatography, indicated that this interaction was sensitive to the charge and type of polysaccharide. Dextran and chondroitin had the lowest affinity for SLPI, followed by dermatan, heparan, and dextran sulfates. While heparin bound SLPI tightly, the highest affinity heparin chains unexpectedly contained a lower level of sulfation than more weakly interacting chains. Heparin oligosaccharides, prepared using heparin lyase I were SLPI-affinity fractionated. Surprisingly, undersulfated heparin oligosaccharides bound SLPI with the highest affinity, suggesting the importance of free hydroxyl groups for high affinity interaction. Isothermal titration calorimetry was used to determine the thermodynamics of SLPI interaction with a low molecular weight heparin, an undersulfated decasaccharide and a tetrasaccharide. The studies showed 12-14 saccharide units, corresponding to molecular weight of approximately 4,800, were required for a 1:1 (SLPI:heparin) binding stoichiometry. Furthermore, an undersulfated decasaccharide was able to bind SLPI tightly (Kd approximately 13 nM), resulting in its activation and the inhibition of neutrophil elastase and pancreatic chymotrypsin. The in vitro assessment of heparin and the decasaccharide and tetrasaccharide using stopped-flow kinetics suggested that heparin was the optimal choice to study SLPI-based in vivo protease inhibition. SLPI and heparin were co-administered by inhalation in therapy against antigen-induced airway hyperresponsiveness in a sheep bronchoprovocation model. Heparin, in combination with SLPI demonstrated in vivo efficacy reducing early and late phase bronchoconstriction. Heparin also increased the therapeutic activity of SLPI against antigen-induced airway hyperresponsiveness.

Genetic analysis of the colicin V secretion pathway.

Colicin V (ColV) is peptide antibiotic secreted by Escherichia coli through a dedicated exporter composed of three proteins, CvaA, CvaB, and TolC. ColV secretion is independent of the E. coli general secretory pathway (Sec) but requires an N-terminal export signal specific for the CvaAB/TolC exporter. ColV secretion was characterized using genetic and biochemical methods. When the ColV N-terminal extension is replaced with the OmpA signal sequence, the Sec system can localize ColV to the periplasm. Periplasmic ColV is lethal to cells lacking the ColV immunity protein, Cvi. Based on this result, a genetic assay was designed to monitor for the presence of periplasmic ColV during normal CvaAB/TolC mediated secretion. Results indicate that low levels of ColV may be present in the periplasm during secretion. Precursor and mature ColV were also characterized from the wild-type system and in various exporter mutant backgrounds using immunoprecipitation. ColV processing is rapid in wild-type cells, and CvaA and CvaB are critical for processing to occur. In contrast, processing occurs normally, albeit more slowly, in a TolC mutant.

Purification and characterization of colicin V from Escherichia coli culture supernatants.

The peptide antibiotic, colicin V (ColV), has been purified and characterized from Escherichia coli culture supernatants by precipitation with trichloroacetic acid (TCA) and high-performance liquid chromatography (HPLC). Polyacrylamide gel electrophoresis (PAGE) and Western analysis identifies ColV as a polypeptide with an apparent molecular mass of 5.8 kDa. The protein identified remains biologically active after purification and SDS-PAGE. A mutant form of ColV, ColV-1, removes the carboxy-terminal 21 amino acids and replaces them with eight heterologous residues. The ColV-1 mutant is also secreted into the extracellular medium, demonstrating that the carboxy-terminal 21 amino acids are not required for secretion by the dedicated ColV export system, CvaAB/TolC. N-Terminal amino acid sequencing shows that the primary translation product of cvaC, the ColV structural gene, is processed to remove the N-terminal 15 amino acids. The cleavage site is preceded by the sequence Ser-Gly-Gly, making it a potential substrate for leader peptidase. The ColV leader sequence has many characteristics in common with the amino-terminal leader sequences of the lactococcins, lactacins, and pediocins from Gram-positive bacteria. Mass spectroscopy of purified ColV shows that it has a mass of 8741.0 amu, consistent with the mass of the unmodified 88 amino acid polypeptide. The purification scheme provides a rapid and simple way to obtain ColV for further biochemical analysis.

ABC transporters: bacterial exporters.

The ABC transporters (also called traffic ATPases) make up a large superfamily of proteins which share a common function and a common ATP-binding domain. ABC transporters are classified into three major groups: bacterial importers (the periplasmic permeases), eukaryotic transporters, and bacterial exporters. We present a comprehensive review of the bacterial ABC exporter group, which currently includes over 40 systems. The bacterial ABC exporter systems are functionally subdivided on the basis of the type of substrate that each translocates. We describe three main groups: protein exporters, peptide exporters, and systems that transport nonprotein substrates. Prototype exporters from each group are described in detail to illustrate our current understanding of this protein family. The prototype systems include the alpha-hemolysin, colicin V, and capsular polysaccharide exporters from Escherichia coli, the protease exporter from Erwinia chrysanthemi, and the glucan exporters from Agrobacterium tumefaciens and Rhizobium meliloti. Phylogenetic analysis of the ATP-binding domains from 29 bacterial ABC exporters indicates that the bacterial ABC exporters can be divided into two primary branches. One branch contains the transport systems where the ATP-binding domain and the membrane-spanning domain are present on the same polypeptide, and the other branch contains the systems where these domains are found on separate polypeptides. Differences in substrate specificity do not correlate with evolutionary relatedness. A complete survey of the known and putative bacterial ABC exporters is included at the end of the review.

Functional complementation between bacterial MDR-like export systems: colicin V, alpha-hemolysin, and Erwinia protease.

The antibacterial protein Colicin V (ColV) is secreted from gram-negative bacteria by a signal sequence-independent pathway. The proteins that mediate the export of ColV share sequence similarities with components from other signal sequence-independent export systems such as those for alpha-hemolysin (Hly) and Erwinia protease (Prt). We report here that the intact HlyBD export system can export active ColV from Escherichia coli strains lacking the ColV export proteins CvaA and CvaB. The individual Hly export genes complement mutations in their respective ColV homologs, but do so at a lower efficiency. When CvaA or CvaB is expressed along with the intact HlyBD exporter, the Cva export protein interferes with export of ColV through the HlyBD system. Gene fusions and point mutations in the ColV structural gene were used to define signals in ColV recognized by the Hly exporter. An export signal in ColV recognized by HlyBD is localized to the amino-terminal 57 amino acids of the protein. In addition, mutations in the ColV export signal differentially affect export through CvaAB and HlyBD, suggesting differences in signal specificity between the Cva and Hly systems. The three Erwinia protease export proteins can also export active ColV, and interference is seen when CvaA or CvaB is expressed along with the intact Prt exporter. Functional complementation is not reciprocal; alpha-hemolysin is not exported through either the ColV system or the Prt system.

The secE gene encodes an integral membrane protein required for protein export in Escherichia coli.

Genetic screening and selection procedures employing a secA-lacZ fusion strain repeatedly have yielded mutations in four genes affecting the protein export pathway of Escherichia coli. These genes are secA, secD, prlA/secY, and secE. We discuss the significance of the failure to find new sec genes after extensive use of this approach. One of the genes, secE, has been characterized in some detail. From the DNA sequence of the gene and analysis of alkaline phosphatase fusions to the SecE protein, we propose that it is a 13,600-dalton integral cytoplasmic membrane protein. The data presented here and in the accompanying paper strongly suggest that secE has an important role in E. coli protein export.

Characterization of a purF operon mutation which affects colicin V production.

A mini-Tn10-kan insertion mutation identified a gene in the chromosome of Escherichia coli required for colicin V production from plasmid pColV-K30. With the complete restriction map of E. coli, the mutation was rapidly mapped to 50.0 min, within the purF operon. Sequence analysis showed that the insertion occurred in a gene with no previously known function which is located directly upstream of purF. We designated this gene cvpA for colicin V production. The mutant requires adenine for growth, probably because of a polar effect on purF expression. However, an adenine auxotroph showed no defect in colicin V production, suggesting that the cvpA mutation is responsible for the effect on colicin V production. Two possible models of cvpA1 allele function are discussed.