Conformational stability of amyloid fibrils of beta2-microglobulin probed by guanidine-hydrochloride-induced unfolding

Although the stability of globular proteins has been studied extensively, that of amyloid fibrils is scarcely characterized. Beta2-microglobulin (beta2-m) is a major component of the amyloid fibrils observed in patients with dialysis-related amyloidosis. We studied the effects of guanidine hydrochloride on the amyloid fibrils of beta2-m, revealing a cooperative unfolding transition similar to that of the native state. The stability of amyloid fibrils increased on the addition of ammonium sulfate, consistent with a role of hydrophobic interactions. The results indicate that the analysis of unfolding transition is useful to obtain insight into the structural stability of amyloid fibrils.     

Identification of crucial histidines involved in carbon-nitrogen triple bond synthesis by aldoxime dehydratase

Aldoxime dehydratase (OxdA), which is a novel heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. The combination of site-directed mutagenesis of OxdA (mutation of all conserved histidines in the aldoxime dehydratase superfamily), estimation of the heme contents and specific activities of the mutants, and CD and resonance Raman spectroscopic analyses led to the identification of the proximal and distal histidines in this unique enzyme. The heme contents and CD spectra in the far-UV region of all mutants except for the H299A one were almost identical to those of the wild-type OxdA, whereas the H299A mutant lost the ability of binding heme, demonstrating that His(299) is the proximal histidine. On the other hand, substitution of alanine for His(320) did not affect the overall structure of OxdA but caused loss of its ability of carbon-nitrogen triple bond synthesis and a lower shift of the Fe-C stretching band in the resonance Raman spectrum for the CO-bound form. Furthermore, the pH dependence of the wild-type OxdA closely followed the His protonation curves observed for other proteins. These findings suggest that His(320) is located in the distal heme pocket of OxdA and would donate a proton to the substrate in the aldoxime dehydration mechanism.     

Contribution of comparative fish studies to general endocrinology: structure and function of some osmoregulatory hormones

Fish endocrinologists are commonly motivated to pursue their research driven by their own interests in these aquatic animals. However, the data obtained in fish studies not only satisfy their own interests but often contribute more generally to the studies of other vertebrates, including mammals. The life of fishes is characterized by the aquatic habitat, which demands many physiological adjustments distinct from the terrestrial life. Among them, body fluid regulation is of particular importance as the body fluids are exposed to media of varying salinities only across the thin respiratory epithelia of the gills. Endocrine systems play pivotal roles in the homeostatic control of body fluid balance. Judging from the habitat-dependent control mechanisms, some osmoregulatory hormones of fish should have undergone functional and molecular evolution during the ecological transition to the terrestrial life. In fact, water-regulating hormones such as vasopressin are essential for survival on the land, whereas ion-regulating hormones such as natriuretic peptides, guanylins and adrenomedullins are diversified and exhibit more critical functions in aquatic species. In this short review, we introduce some examples illustrating how comparative fish studies contribute to general endocrinology by taking advantage of such differences between fishes and tetrapods. In a functional context, fish studies often afford a deeper understanding of the essential actions of a hormone across vertebrate taxa. Using the natriuretic peptide family as an example, we suggest that more functional studies on fishes will bring similar rewards of understanding. At the molecular level, recent establishment of genome databases in fishes and mammals brings clues to the evolutionary history of hormone molecules via a comparative genomic approach. Because of the functional and molecular diversification of ion-regulating hormones in fishes, this approach sometimes leads to the discovery of new hormones in tetrapods as exemplified by adrenomedullin 2.     

Production of human growth hormone in transgenic rice seeds: co-introduction of RNA interference cassette for suppressing the gene expression of endogenous storage proteins

Rice seeds are potentially useful hosts for the production of pharmaceutical proteins. However, low yields of recombinant proteins have been observed in many cases because recombinant proteins compete with endogenous storage proteins. Therefore, we attempt to suppress endogenous seed storage proteins by RNA interference (RNAi) to develop rice seeds as a more efficient protein expression system. In this study, human growth hormone (hGH) was expressed in transgenic rice seeds using an endosperm-specific promoter from a 10 kDa rice prolamin gene. In addition, an RNAi cassette for reduction of endogenous storage protein expressions was inserted into the hGH expression construct. Using this system, the expression levels of 13 kDa prolamin and glutelin were effectively suppressed and hGH polypeptides accumulated to 470 μg/g dry weight at the maximum level in transgenic rice seeds. These results suggest that the suppression of endogenous protein gene expression by RNAi could be of great utility for increasing transgene products.     

Coexisting <Emphasis Type="Italic">Curtobacterium</Emphasis> Bacterium Promotes Growth of White-Rot Fungus <Emphasis Type="Italic">Stereum</Emphasis> sp.

White-rot basidiomycetes are the main decomposers of woody biomass in forest ecosystems. Little is known, however, about the interactions between white-rot fungi and other microorganisms in decayed wood. A wood-rotting fungus, Stereum sp. strain TN4F, was isolated from a fruit body, and its coexisting cultivable bacteria were isolated from its substrate; natural white-rot decayed wood. The effects of bacteria on fungal growth were examined by confrontational assay in vitro. A growth-promoting bacterium for this Stereum strain was identified as Curtobacterium sp. TN4W-19, using 16SrRNA sequencing. A confrontational assay revealed that Curtobacterium sp. TN4W-19 significantly promoted the mycelial growth of Stereum sp. TN4F in the direction of the bacterial colony, without direct contact between the mycelium and bacterial cells. This is the first report of a positive interaction between a white-rot fungus and a coexisting bacterial strain in vitro.     

Imiquimod Suppresses Propagation of Herpes Simplex Virus 1 by Upregulation of Cystatin A via the Adenosine Receptor A1 Pathway

Imiquimod is recognized as an agonist for Toll-like receptor 7 (TLR7) in immunocompetent cells. TLR7, as well as TLR3 and TLR8, triggers the immune responses, such as the production of type I interferons (IFNs) and proinflammatory cytokines via recognition of viral nucleic acids in the infected cells. In this study, we proposed that imiquimod has an IFN-independent antiviral effect in nonimmune cells. Imiquimod, but not resiquimod, suppressed replication of human herpes simplex virus 1 (HSV-1) in FL cells. We analyzed alternation of gene expression by treatment with imiquimod using microarray analysis. Neither type I IFNs, nor TLRs, nor IFN-inducible antiviral genes were induced in imiquimod-treated FL cells. Cystatin A, a host cysteine protease inhibitor, was strongly upregulated by imiquimod and took a major part in the anti-HSV-1 activity deduced by the suppression experiment using its small interfering RNA. Upregulation of cystatin A was suggested to be mediated by antagonizing adenosine receptor A(1) and activating the protein kinase A pathway. Imiquimod, but not resiquimod, was shown to interact with adenosine receptor A(1). Imiquimod-induced anti-HSV-1 activity was observed in other cells, such as HeLa, SiHa, and CaSki cells, in a manner consistent with the cystatin A induction by imiquimod. These results indicated that imiquimod acted as an antagonist for adenosine receptor A(1) and induced a host antiviral protein, cystatin A. The process occurred independently of TLR7 and type I IFNs.     

The advancing timberline on Mt. Fuji: natural recovery or climate change?

The alpine timberline on Mt. Fuji (central Japan) is at 2,400-2,500 m above sea level. Over a 21-year period (1978-1999), we tracked changes in this vegetation boundary on a transect at a site impacted by the 1707 volcanic eruption. The timberline advanced rapidly upwards during this time period. Dominant tree species at the timberline (Alnus maximowiczii, Salix reinii, and Larix kaempferi) colonized sites that were initially largely free of vegetation at higher altitudes. Seedlings of L. kaempferi were particularly abundant at the border of advancing vegetation. According to tree age, we found that this was the first canopy species in the colonized areas. L. kaempferi is drought resistant, and this probably contributes to its establishment capability in the high-altitude climate. Most seedlings of Abies veitchii invaded patches of herbs and shrubs. These vegetation patches in the upper kampfzone provide important shelter for seedlings of invading tree species. We predict that the upward advance of the alpine timberline is a recovery process following the volcanic eruption, and that climate change may accelerate this advance.