Outer Membrane Permeabilization Is an Essential Step in the Killing of Gram-Negative Bacteria by the Lectin RegIIIβ

The C-type lectin RegIIIβ can kill certain Gram-positive and Gram-negative bacteria. The susceptibility of S. Typhimurium depends on the bacterial growth phase, i.e., bacteria from the logarithmic growth phase do bind RegIIIβ and are subsequently killed. Lipid A is one of the bacterial targets for RegIIIβ. However, at the molecular level, it is not understood how RegIIIβ interacts with and kills Gram-negative bacteria. Here, we show that RegIIIβ interacts with Gram-negative bacteria in two distinct steps. Initially, it binds to surface-exposed lipid A. The lipid A can be shielded by the O-antigen of lipopolysaccharide (LPS), as indicated by the exquisite susceptibility of wbaP mutants to RegIIIβ-mediated killing. Increased cell viability after incubation with an anti-lipid A antibody also supports this conclusion. This RegIIIβ-binding permeabilizes the outer membrane to hydrophobic dyes like Ethidium bromide or to bulky bacteriolytic enzymes like lysozyme. Conversely, compromising the outer membrane integrity by the mild detergent Triton X-100 enhances the antibacterial effect of RegIIIβ. Based on our observations, we conclude that RegIIIβ interacts with Gram-negative bacteria in two subsequent steps. Initially, it binds to the outer membrane thus leading to outer membrane permeabilization. This initial step is necessary for RegIIIβ to reach a second, still not well understood target site (presumably localized in the periplasm or the cytoplasmic membrane), thereby triggering bacterial death. This provides novel insights into the outer membrane-step of the bactericidal mechanism of RegIIIβ.     

Differential effects of various growth factors and cytokines on the syntheses of DNA,type I collagen,laminin, fibronectin,osteonectin/secreted protein,acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture

The purpose of this study is to differentiate roles of several growth factors and cytokines in proliferation and differentiation of pulp cells during development and repair. In human pulp cell cultures, laminin and type I collagen levels per cell remained almost constant during the whole culture period (22 days). On the other hand, secreted protein, acidic and rich in cysteine (SPARC/osteonectin) and alkaline phosphatase (ALPase) levels markedly increased after the cultures reached confluence. Laminin and type I collagen, as well as fibronectin, stimulated the spreading of pulp cells within 1 h. Adding transforming growth factor-β (TGF-β) decreased laminin and ALPase levels, whereas it increased SPARC and fibronectin levels 3- to 10-fold. Western and Northern blots showed that TGF-β enhanced SPARC synthesis at the protein and mRNA levels. Basic fibroblast growth factor (bFGF) decreased type I collagen, laminin, SPARC, and ALPase levels without changing the fibronectin level. Platelet-derived growth factor (PDGF) selectively decreased laminin, SPARC, and ALPase levels. Epidermal growth factor (EGF) also decreased SPARC and ALPase levels. Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) decreased type I collagen and laminin levels, and abolished SPARC and ALPase syntheses. Of these peptides, bFGF and PDGF showed the greatest stimulation of [³H]thymidine incorporation into DNA. TGF-β, EGF, and TNF-α had less effect on DNA synthesis, whereas IL-1β inhibited DNA synthesis. These findings demonstrated that TGF-β, bFGF, EGF, PDGF, TNF-α, and IL-1β have characteristically different patterns of actions on DNA, laminin, type I collagen, fibronectin, ALPase, and SPARC syntheses by pulp cells. J. Cell. Physiol. 174:194–205, 1998. © 1998 Wiley-Liss, Inc.     

Selection and structural analysis of de novo proteins from an α3β3 genetic library

The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding α3β3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP‐based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8‐anilino‐1‐naphthalenesulfonate (ANS) binding assay, and analytical size‐exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both α‐helix and β‐sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten‐globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three α‐helices and three β‐strands as intended by its design. Thus, it would appear that artificially generated α3β3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.     

Do Termites Avoid Carcasses? Behavioral Responses Depend on the Nature of the Carcasses

Background

Undertaking behavior is a significant adaptation to social life in enclosed nests. Workers are known to remove dead colony members from the nest. Such behavior prevents the spread of pathogens that may be detrimental to a colony. To date, little is known about the ethological aspects of how termites deal with carcasses.

Methodology and Principal Findings

In this study, we tested the responses to carcasses of four species from different subterranean termite taxa: Coptotermes formosanus Shiraki and Reticulitermes speratus (Kolbe) (lower termites) and Microcerotermes crassus Snyder and Globitermes sulphureus Haviland (higher termites). We also used different types of carcasses (freshly killed, 1-, 3-, and 7-day-old, and oven-killed carcasses) and mutilated nestmates to investigate whether the termites exhibited any behavioral responses that were specific to carcasses in certain conditions. Some behavioral responses were performed specifically on certain types of carcasses or mutilated termites. C. formosanus and R. speratus exhibited the following behaviors: (1) the frequency and time spent in antennating, grooming, and carcass removal of freshly killed, 1-day-old, and oven-killed carcasses were high, but these behaviors decreased as the carcasses aged; (2) the termites repeatedly crawled under the aging carcass piles; and (3) only newly dead termites were consumed as a food source. In contrast, M. crassus and G. sulphureus workers performed relatively few behavioral acts. Our results cast a new light on the previous notion that termites are necrophobic in nature.

Conclusion

We conclude that the behavioral response towards carcasses depends largely on the nature of the carcasses and termite species, and the response is more complex than was previously thought. Such behavioral responses likely are associated with the threat posed to the colony by the carcasses and the feeding habits and nesting ecology of a given species.     

Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4alpha-induced epithelial polarization

We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4alpha [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4alpha triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4alpha-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells. Induced expression of HNF-4alpha led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4alpha provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization.     

Classification of heterogeneous microarray data by maximum entropy kernel

Background  

There is a large amount of microarray data accumulating in public databases, providing various data waiting to be analyzed jointly. Powerful kernel-based methods are commonly used in microarray analyses with support vector machines (SVMs) to approach a wide range of classification problems. However, the standard vectorial data kernel family (linear, RBF, etc.) that takes vectorial data as input, often fails in prediction if the data come from different platforms or laboratories, due to the low gene overlaps or consistencies between the different datasets.     

Intracellular hydroxyproline-rich glycoprotein of suspension-cultured tobacco cells

Two soluble glycoproteins containing hydroxyproline were extractedfrom cultured tobacco cells (cell line XD-6S) and purified byion-exchange and gel-filtration chromatography. On DEAR-cellulosecolumn chromatography in the final step of the purification,one was eluted at 90 mM NaCl and the other at 120 mM as singlepeak. Both purified glycoproteins were also sedimented as singlepeak with an ultracentrifugation. The S_20,w values were 6.1for the former and 7.0 for the latter. These glycoproteins were composed of 94% polysaccharide and6% protein in the former, and 87% polysaccharide and 13% proteinin the latter. The sugar moiety consisted of galactose, arabinose,rhamnose, and uronic acid in both. Hydroxyproline accountedfor 12% in the former and 20% in the latter amino acid composition.A high content of alanine in both (14 and 15%) was one of thedistinctive characteristics of these soluble glycoproteins. These intracellular soluble hydroxyproline-containing glycoproteinswere not labelled within 30 min of incubation with ₃H-proline,although the radioactivity was rapidly incorporated (within15 min) into the intracellular macromolecules. (Received February 21, 1978; )     

Differential sensitivity between Fks1p and Fks2p against a novel beta -1,3-glucan synthase inhibitor,aerothricin3 [corrected

Fks1p and Fks2p are catalytic subunits of beta-1,3-glucan synthase, which synthesize beta-1,3-glucan, a main component of the cell wall in Saccharomyces cerevisiae. Although Fks1p and Fks2p are highly homologous, sharing 88.1% identity, it has been shown that Fks2p is more sensitive than Fks1p to one of echinocandin derivatives, which inhibits beta-1,3-glucan synthase activity. Here we show a similar differential sensitivity between Fks1p and Fks2p to a novel beta-1,3-glucan synthase inhibitor, aerothricin3 [corrected]. To investigate the molecular mechanism of this differential sensitivity, we constructed a series of chimeric genes of FKSs and examined their sensitivity to aerothricin3 [corrected]. As a result, it was shown that a region around the fourth extracellular domain of Fks2p, containing 10 different amino acid residues from those of Fks1p, provided Fks1p aerothricin3 [corrected] sensitivity when the region was replaced with a corresponding region of Fks1p. In order to identify essential amino acid residues responsible for the sensitivity, each of the 10 non-conserved amino acids of Fks1p was substituted into the corresponding amino acid of Fks2p by site-directed mutagenesis. Surprisingly, only one amino acid substitution of Fks1p (K1336I) conferred Fks1p hypersensitivity to aerothricin3 [corrected]. On the other hand, reverse substitution of the corresponding amino acid of Fks2p (I1355K) resulted in loss of hypersensitivity to aerothricin3 [corrected]. These results suggest that the 1355th isoleucine of Fks2p plays a key role in aerothricin3 [corrected] sensitivity.