Evidence supporting the hypothesis that one of the main functions of the aryl hydrocarbon receptor is mediation of cell stress responses

We have previously proposed that one of the major consequences of activation of the aryl hydrocarbon receptor (AhR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could be elicitation of 'cell stress response' reactions [Matsumura, Biochem. Pharmacol. 66 (2003), 527-540]. This hypothesis was based mainly on the similarity between the toxic symptoms, particularly those related to the wasting syndrome, and those induced by bacterial endotoxins, namely lipopolysaccharides (LPS) in vivo, as well as the biochemical and molecular consequences of their toxic actions in vitro. Since the basic action mechanism of LPS as an inducer of cell stress responses (CSR) is known to some extent, including knowledge of their specific receptors (i.e., toll-like receptors) and their signaling process through the inflammatory response messengers, the above comparison offered a good point of reference to this subject. Furthermore, the process of constructing this hypothesis itself has provided us with a good opportunity to give a fresh view on the toxic action patterns of TCDD.     

Defects in the cytochrome b6/f complex prevent light-induced expression of nuclear genes involved in chlorophyll biosynthesis

Mutants with defects in the cytochrome (cyt) b6/f complex were analyzed for their effect on the expression of a subgroup of nuclear genes encoding plastid-localized enzymes participating in chlorophyll biosynthesis. Their defects ranged from complete loss of the cytb6/f complex to point mutations affecting specifically the quinone-binding QO site. In these seven mutants, light induction of the tetrapyrrole biosynthetic genes was either abolished or strongly reduced. In contrast, a normal induction of chlorophyll biosynthesis genes was observed in mutants with defects in photosystem II, photosystem I, or plastocyanin, or in wild-type cells treated with 3-(3'4'-dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-methyl-6-isopropyl benzoquinone. We conclude that the redox state of the plastoquinone pool does not control light induction of these chlorophyll biosynthetic genes. The signal that affects expression of the nuclear genes appears to solely depend on the integrity of the cytb6/f complex QO site. Since light induction of these genes in Chlamydomonas has recently been shown to involve the blue light receptor phototropin, the results suggest that cytb6/f activity regulates a plastid-derived factor required for their expression. This signaling pathway differs from that which regulates state transitions, since mutant stt7, lacking a protein kinase involved in phosphorylation of the light-harvesting complex II, was not altered in the expression of the chlorophyll biosynthetic genes.     

Protection by sucrose against heat-induced lethal and sublethal injury of Lactococcus lactis: an FT-IR study

The heat inactivation of Lactococcus lactis was studied by determination of cell counts, and by FT-IR spectroscopy recording the average structure of cell proteins. Cell counts were measured after incubation milk buffer or milk buffer with 1. 5 M sucrose, and FT-IR spectra were recorded in (2)H(2)O or (2)H(2)O with 1. 5 M sucrose in the range of 6-75 degrees Celsius. Sucrose protected L. lactis against heat inactivation. The cell counts differed by up to 6-log cycles after treatment in milk buffer as compared to milk buffer with sucrose. The (1)H/(2)H exchange in proteins, and secondary structure elements were detected by the analysis of amide I', amide II and amide II' bands. A reduced (1)H/(2)H exchange as well as a lower content of disordered structural elements was observed when sucrose was present. Conformational fluctuations of native proteins as indicated by the (1)H/(2)H exchange were apparent already at sublethal temperatures. The loss of viability of L. lactis occurred in the same temperature range as the loss of the protein secondary structure. These results demonstrate that sucrose protects L. lactis against heat inactivation, and that the increased heat stability of proteins in the presence of sucrose contributed to this enhanced heat resistance.     

Comparative analysis of the human angiotensin II type 1a receptor heterologously produced in insect cells and mammalian cells

Angiotensin II type 1a receptor (AT1aR) is a member of GPCR superfamily and it plays crucial roles in the regulation of blood pressure, hormone secretion and renal functions. Here, we report functional overexpression and characterization of the human AT1aR in insect cells using the baculovirus system and in mammalian cells using the Semliki Forest virus system. The recombinant receptor was expressed at a level of 29-32 pmol/mg and it binds to angiontensin II with high affinity (Kd=0.98-1.1 nM). Angiotensin II stimulated accumulation of inositol phosphate and phosphorylation of MAP kinase was also observed, which indicated that the recombinant AT1aR could couple to endogenous Galphaq protein. Confocal laser scanning microscopy revealed that the recombinant receptor was predominantly localized in the plasma membrane and agonist induced internalization of the recombinant AT1aR was also observed. The recombinant AT1aR was expressed in glycosylated form and in vivo inhibition of glycosylation suppressed its surface expression.