V‐antigen homologs in pathogenic gram‐negative bacteria

Gram‐negative bacteria cause many types of infections in animals from fish and shrimps to humans. Bacteria use Type III secretion systems (TTSSs) to translocate their toxins directly into eukaryotic cells. The V‐antigen is a multifunctional protein required for the TTSS in Yersinia and Pseudomonas aeruginosa. V‐antigen vaccines and anti‐V‐antigen antisera confer protection against Yersinia or P. aeruginosa infections in animal models. The V‐antigen forms a pentameric cap structure at the tip of the Type III secretory needle; this structure, which has evolved from the bacterial flagellar cap structure, is indispensable for toxin translocation. Various pathogenic gram‐negative bacteria such as Photorhabdus luminescens, Vibrio spp., and Aeromonas spp. encode homologs of the V‐antigen. Because the V‐antigens of pathogenic gram‐negative bacteria play a key role in toxin translocation, they are potential therapeutic targets for combatting bacterial virulence. In the USA and Europe, these vaccines and specific antibodies against V‐antigens are in clinical trials investigating the treatment of Yersinia or P. aeruginosa infections. Pathogenic gram‐negative bacteria are of great interest because of their ability to infect fish and shrimp farms, their potential for exploitation in biological terrorism attacks, and their ability to cause opportunistic infections in humans. Thus, elucidation of the roles of the V‐antigen in the TTSS and mechanisms by which these functions can be blocked is critical to facilitating the development of improved anti‐V‐antigen strategies.     

Immunohistochemical evidence of serotoninergic regulation of vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus

By applying a double-immunolabeling technique to preembedded tissue preparations, we demonstrated the existence of serotoninergic innervation to neurons containing vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus (SCN). Immunoreactivity for serotonin and VIP was revealed by the presence of diaminobenzidine (DAB) reaction products and silver-intensified DAB reaction products, respectively; in a further stage, the silver grains were substituted with gold particles. DAB reaction products were precipitated on the surface of vesicular structures, while gold particles were scattered diffusely throughout the neuroplasma at various densities. Serotoninergic axons were numerous and closely packed together, occasionally forming synaptic junctions with gold-labeled VIP-containing neurons. At these synaptic junctions, small vesicular structures accumulated to form a coat under the presynaptic membrane, and the postsynaptic membrane was lined with a homogeneous accumulation of fine deposits. This postsynaptic apparatus varied in appearance; some parts were flat and thin, while others were of irregular thickness. Serotoninergic fibers also formed synaptic junctions with unidentified neurons, in which postsynaptic membrane specialization was also observable. As VIP-containing neurons are known to be synapsed by somatostatin (SRIH)-containing neurons, their regulation must involve both serotonin and SRIH at least.     

Involvement of the Binuclear Copper Site in the Proteolytic Activity of Polyphenol Oxidase

Plant polyphenol oxidase (PPO) is apt to degrade during andeven after purification. We developed a method to stabilizePPO by 0.3 M NaCl, 0.1% (w/v) Tween 20, and 50% (w/v) ethyleneglycol at pH 6.5. The protein slowly degraded by itself whenthe stabilizing reagents were removed. Ascorbate and/or H₂O₂accelerated the degradation. The ascorbate-induced degradationwas inhibited by catalase, suggesting that H₂O₂ is generatedthrough reduction of PPO by ascorbate. It is likely that dissolvedoxygen is converted to peroxide through two-electron reductionby the reaction center of PPO, binuclear Cu site, and a Fenton-typereaction occurred on it. This understanding was supported bythe finding that the H₂O₂-induced degradation was inhibitedby metal-chelators as well as by polyphenolic substrate of PPO.Considering the postulated mechanism of the self-degradationof PPO, we re-examined the degradation of the 23-kDa proteinof PSII by PPO [Kuwabara et al. (1997) Plant Cell Physiol. 38:179]. The obtained results suggested that the 23-kDa proteintriggers the active oxygen production by the binuclear Cu site,probably as reductant, and receives the radical species preferentiallyto the polypeptide moiety of PPO. (Received April 15, 1999; Accepted July 21, 1999)     

Subnuclear localization and antitransforming activity of N-myc:beta-galactosidase fusion proteins.

N-myc expression is under stage- and tissue-specific regulation in mammalian development, but its function is totally unknown. We sought agents to block N-myc activity in order to infer from the effect the possible function of N-myc in the apparently complex processes. As candidates for such agents, we tested fusion genes encoding N-myc:beta-galactosidase fusion proteins for their effects on the formation of transformed foci of rat embryo primary fibroblasts as the result of transfection with N-myc and activated H-ras. One of the gene constructs very efficiently antagonized N-myc activity, as assessed by its effect on focus formation, but did not appreciably affect cell viability. The product of this gene was not only targeted to the nucleus but also accumulated in subnuclear loci which may represent the sites where normal N-myc proteins reside. The occurrence of antagonistic effect at a low stoichiometric ratio suggested that the fusion protein gene competed with the N-myc gene in a fashion analogous to a dominant negative mutation.     

Chloramphenicol is an inhibitor of photosynthesis.

Chloramphenicol inhibited significantly but incompletely photosynthesis in leaf segments of rice. Fluorescence and polarographic experiments indicated that chloramphenicol competes with the CO2 reducing cycle for electrons from photosystem I because it serves as an electron acceptor of photosystem I and its reduction intermediate transfers its electron to molecular oxygen.     

Discovery of Molecular Markers to Discriminate Corneal Endothelial Cells in the Human Body

The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.