A short consensus repeat-containing complement regulatory protein of lamprey that participates in cleavage of lamprey complement 3

The prototype of the short consensus repeat (SCR)-containing C regulatory protein is of interest in view of its evolutionary significance with regard to the origin of the C regulatory system. Lamprey is an agnathan fish that belongs to the lowest class of vertebrates. Because it does not possess lymphocytes, it lacks Ig and consequently the classical C pathway. We identified an SCR-containing C regulatory protein from the lamprey. The primary structure predicted from the cDNA sequence showed that this is a secretary protein consisting of eight SCRs. This framework is similar to the alpha-chain of C4b-binding protein (C4bp). SCR2 and -3 of human C4bp are essential for C4b inactivation, and this region is fairly well conserved in the lamprey protein. However, the other SCRs of this protein are similar to those of other human C regulatory proteins. The lamprey protein binds to the previously reported lamprey C3b/C3bi deposited on yeast and cleaves lamprey C3b-like C3 together with a putative serum protease. The scheme resembles the C regulatory system of mammals, where factor I and its cofactor inactivate C3b. Unlike human cofactors, the lamprey protein requires divalent cations for C3b-like C3 cleavage. Its artificial membrane-anchored form protects host cells from lamprey C attack via the lectin pathway. Thus, the target of this protein appears to be C3b and/or its family. We named this protein Lacrep, the lamprey C regulatory protein. Lacrep is a member of SCR-containing C regulators, the first of its kind identified in the lowest vertebrates.     

Importance of Trp59 and Trp60 in chitin-binding, hydrolytic, and antifungal activities of Streptomyces griseus chitinase C

The chitin-binding domain of Streptomyces griseus chitinase C (ChBD_ChiC) belongs to CBM family 5. Only two exposed aromatic residues, W59 and W60, were observed in ChBD_ChiC, in contrast to three such residues on CBD_Cel5 in the same CBM family. To study importance of these residues in binding activity and other functions of ChBD_ChiC, site-directed mutagenesis was carried out. Single (W59A and W60A) and double (W59A/W60A) mutations abolished the binding activity of ChiC to colloidal chitin and decreased the hydrolytic activity toward not only colloidal chitin but also a soluble high Mr substrate, glycol chitin. Interaction of ChBD_ChiC with oligosaccharide was eliminated by these mutations. The hydrolytic activity toward oligosaccharide was increased by deletion of ChBD but not affected by these mutations, indicating that ChBD interferes with oligosaccharide hydrolysis but not through its binding activity. The antifungal activity was drastically decreased by all mutations and significant difference was observed between single and double mutants. Taken together with the structural information, these results suggest that ChBD_ChiC binds to chitin via a mechanism significantly different from CBD_Cel5, where two aromatic residues play major role, and contributes to various functions of ChiC. Sequence comparison indicated that ChBD_ChiC-type CBMs are dominant in CBM family 5.     

Degradation of Tributyltin in Microcosm Using Mekong River Sediment

The degradation of tributyltin (TBT) and changes of bacterial number and community structures were investigated in microcosms using the sediment collected from the Mekong River, Vietnam. Concentrations of TBT in sediments were less than 0.62 ng/g (dry wt), lower than those reported from other areas. TBT-resistant bacteria were found in the three sampling sites, and the occurrence rates were 11–16% out of the total viable count. In this microcosm experiment, initial concentration of TBT [1.0–1.4 μg/g (dry wt)] decreased to 0.6 μg/g (dry wt) during 150 days, whereas that in the control microcosm with autoclaved sediment did not change, indicating that Mekong River sediment contains high TBT-degrading activity by microorganisms. The occurrence of TBT-resistant bacteria and the bacterial community structures monitored by denaturing gradient gel electrophoresis were almost the same between test and control groups, indicating that the addition of TBT had little influence on microbial community structure. Mekong River sediment seems to have a stable microbial community against TBT pollution.     

Assembly of the type III secretion apparatus of enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) secretes many Esps (E. coli-secreted proteins) and effectors via the type III secretion (TTS) system. We previously identified a novel needle complex (NC) composed of a basal body and a needle structure containing an expandable EspA sheath-like structure as a central part of the EPEC TTS apparatus. To further investigate the structure and protein components of the EPEC NC, we purified it in successive centrifugal steps. Finally, NCs with long EspA sheath-like structures could be separated from those with short needle structures on the basis of their densities. Although the highly purified NC appeared to lack an inner ring in the basal body, its core structure, composed of an outer ring and a central rod, was observed by transmission electron microscopy. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, Western blot, and immunoelectron microscopic analyses revealed that EscC was a major protein component of the outer ring in the core basal body. To investigate the mechanisms of assembly of the basal body, interactions between the presumed components of the EPEC TTS apparatus were analyzed by a glutathione S-transferase pulldown assay. The EscC outer ring protein was associated with both the EscF needle protein and EscD, a presumed inner membrane protein. EscF was also associated with EscJ, a presumed inner ring protein. Furthermore, escC, escD, and escJ mutant strains were unable to produce the TTS apparatus, and thereby the secretion of the Esp proteins and Tir effector was abolished. These results indicate that EscC, EscD, and EscJ are required for the formation of the TTS apparatus.     

Crosslinking of low-affinity glycoprotein ligands to galectin LEC-1 using a photoactivatable sulfhydryl reagent

The N-terminal lectin domain (Nh) of the tandem repeat-type nematode galectin LEC-1 has a lower affinity for sugars than the C-terminal lectin domain. To confirm that LEC-1 forms a complex with N-acetyllactosamine-containing glycoproteins, we used several mutants of LEC-1 in which a unique cysteine residue was introduced into the Nh domain and examined their binding to bovine asialofetuin with a photoactivatable sulfhydryl crosslinking reagent. A crosslinked product was formed with the Q38C mutant, strongly suggesting the low-affinity interaction of Nh with the glycoprotein could be detected with this system.     

Small molecule inhibitors of alpha-synuclein filament assembly

Alpha-synuclein is the major component of the filamentous inclusions that constitute defining characteristics of Parkinson's disease and other alpha-synucleinopathies. Here we have tested 79 compounds belonging to 12 different chemical classes for their ability to inhibit the assembly of alpha-synuclein into filaments in vitro. Several polyphenols, phenothiazines, porphyrins, polyene macrolides, and Congo red and its derivatives, BSB and FSB, inhibited alpha-synuclein filament assembly with IC(50) values in the low micromolar range. Many compounds that inhibited alpha-synuclein assembly were also found to inhibit the formation of Abeta and tau filaments. Biochemical analysis revealed the formation of soluble oligomeric alpha-synuclein in the presence of inhibitory compounds, suggesting that this may be the mechanism by which filament formation is inhibited. Unlike alpha-synuclein filaments and protofibrils, these soluble oligomeric species did not reduce the viability of SH-SY5Y cells. These findings suggest that the soluble oligomers formed in the presence of inhibitory compounds may not be toxic to nerve cells and that these compounds may therefore have therapeutic potential for alpha-synucleinopathies and other brain amyloidoses.     

Identification and characterization of ectosymbionts of distinct lineages in Bacteroidales attached to flagellated protists in the gut of termites and a wood-feeding cockroach

Bacterial attachments to nearly the entire surface of flagellated protists in the guts of termites and the wood-feeding cockroach Cryptocercus are often observed. Based on the polymerase chain reaction-amplified 16S rRNA gene sequences, we investigated the phylogenetic relationships of the rod-shaped, attached bacteria (ectosymbionts) of several protist species from five host taxa and confirmed their identity by fluorescence in situ hybridizations. These ectosymbionts are affiliated with the order Bacteroidales but formed three distinct lineages, each of which may represent novel bacterial genera. One lineage consisted of the closely related ectosymbionts of two species of the protist genus Devescovina (Cristamonadida). The second lineage comprised three phylotypes identified from the protist Streblomastix sp. (Oxymonadida). The third lineage included ectosymbionts of the three protist genera Hoplonympha, Barbulanympha and Urinympha in the family Hoplonymphidae (Trichonymphida). The ultrastructural observations indicated that these rod-shaped ectosymbionts share morphological similarities of their cell walls and their point of attachment with the protist but differ in shape. Elongated forms of the ectosymbionts appeared in all the three lineages. The protist cells Streblomastix sp. and Hoplonympha sp. display deep furrows and vane-like structures, but these impressive structures are probably evolutionarily convergent because both the host protists and their ectosymbionts are distantly related.     

Shigella flexneri YSH6000 induces two types of cell death, apoptosis and oncosis, in the differentiated human monoblastic cell line U937

Shigella flexneri, but not a non-invasive mutant derivative rapidly induced cell death in human monoblastic U937 cells as well as in differentiated cells pretreated with interferon-gamma (IFN gamma) or retinoic acid (RA). We investigated the morphological and biochemical characteristics of bacterial invasion-induced cell death in these differentiated U937 cells. IFN gamma-differentiated cells showed morphological changes typical of apoptosis and their DNA was cleaved giving a ladder-like electrophoretic pattern after infection by Shigellae. In contrast, swelling of the cytoplasm and blebbing of the plasma membrane were observed in RA-differentiated and undifferentiated cells invaded by the bacteria. No condensation of nuclei was observed in these cells by light microscopy, and no internucleosomal fragmentation of DNA was detected on agarose gels, which resembled the features of oncosis. Furthermore, cleavage of poly(ADP-ribose) polymerase, a substrate for apoptotic caspases, was seen only in IFN gamma-pretreated cells but not in RA-pretreated or undifferentiated cells. These findings suggested that virulent Shigella flexneri induces distinct types of cell death in U937 cells depending on their differentiation state.