Structural analysis of the phototactic transducer protein HtrII linker region from Natronomonas pharaonis

Halobacterial pharaonis phoborhodopsin [ppR, also called Natronomonas pharaonis sensory rhodopsin II (NpSRII)] is a phototaxis protein which transmits a light signal to the cytoplasm through its transducer protein (pHtrII). pHtrII, a two-transmembrane protein that interacts with ppR, belongs to the group of methyl-accepting chemotaxis proteins (MCPs). Several mutation studies have indicated that the linker region connecting the transmembrane and methylation regions is necessary for signal transduction. However, the three-dimensional (3D) structure of an MCP linker region has yet to be reported, and hence, details concerning the signal transduction mechanism remain unknown. Here the structure of the pHtrII linker region was investigated biochemically and biophysically. Following limited proteolysis, only one trypsin resistant fragment in the pHtrII linker region was identified. This fragment forms a homodimer with a Kd value of 115 microM. The 3D structure of this fragment was determined by solution NMR, and only one alpha-helix was found between two HAMP domains of the linker region. This alpha-helix was significantly stabilized within transmembrane protein pHtrII as revealed by CW-EPR. The presence of Af1503 HAMP domain-like structures in the linker region was supported by CD, NMR, and ELDOR data. The alpha-helix determined here presumably works as a mechanical joint between two HAMP domains in the linker region to transfer the photoactivated conformational change downstream.     

Association of Lps gene with natural resistance of mouse macrophages against Legionella pneumophila

We have cloned a 1.6-kb region of chromosomal DNA from Thermoplasma acidophilum into Escherichia coli using as a probe part of the Methanococcus vannielii fus-gene. The sequence of the clone was highly homologous to part of the corresponding Methanococcus vannielii gene. By chromosome walking, a 4.7-kb EcoRI fragment containing the complete gene was isolated. Nucleotide sequencing revealed an open reading frame of 2196 nucleotides. The deduced amino acid sequence contains the known peptide sequence around the ADP-ribosylation site of T. acidophilum elongation factor 2, which unequivocally confirms that the fus-gene has been cloned. The amino acid sequence was compared to that of hamster and E. coli, as well as to known archaebacterial EF-2 sequences.     

Lack of interaction in recombinant human FSH receptor and both TSAb and TSBAb

Since cross-reactivity of TSH with the human FSH receptor has been reported, in this study we tested the effect of thyroid-stimulating antibody (TSAb) and thyroid stimulation-blocking antibody (TSBAb) on Chinese hamster ovary cells expressing human FSH receptor (CHO-hFSH-R cells). We examined the TSBAb activity of sera from hypothyroid patients who had a positive TBII to determine whether these sera also block the effect of FSH on CHO-hFSH-R cells. Although human FSH I-3 (0.25-16 ng/ml) stimulated the production of intracellular cAMP in CHO-hFSH-R cells with dose-responsive manner, neither TSAb nor TSBAb had such an effect on the cells.     

Enhanced survival of shrimp, Penaeus (Marsupenaeus) japonicus from white spot syndrome disease after oral administration of recombinant VP28 expressed in Brevibacillus brevis

White spot syndrome virus (WSSV) disease is a major threat to shrimp culture worldwide. Here, we assessed the efficacy of the oral administration of purified recombinant VP28, an envelope protein of WSSV, expressed in a Gram-positive bacterium, Brevibacillus brevis, in providing protection in shrimp, Penaeus japonicus, upon challenge with WSSV. Juvenile shrimp (2-3g in body weight) fed with pellets containing purified recombinant VP28 (50mug/shrimp) for 2weeks showed significantly higher survival rates than control groups when challenged with the virus at 3days after the last day of feeding. However, when shrimp were challenged 2weeks after the last day of feeding, survival rates decreased (33.4% and 24.93%, respectively). Survival rate was dose-dependent, increasing from 60.7 to 80.3% as the dose increased from 1 to 50mug/shrimp. At a dose of 50mug/shrimp, the recombinant protein provided protection as soon as 1day after feeding (72.5% survival). Similar results were obtained with larger-sized shrimp. These results show that recombinant VP28 expressed in a Gram-positive bacterium is a potential oral vaccine against WSSV.     

The beta-1,3-exoglucanase gene exgA (exg1) of Aspergillus oryzae is required to catabolize extracellular glucan, and is induced in growth on a solid surface

The biological role of ExgA (Exg1), a secretory beta-1,3-exoglucanase of Aspergillus oryzae, and the expression pattern of the exgA (exg1) gene were analyzed. The exgA disruptant and the exgA-overexpressing mutant were constructed, and phenotypes of both mutants were compared. Higher mycelial growth rate and conidiation efficiency were observed for the exgA-overexpressing mutant than for the exgA disruptant when beta-1,3-glucan was supplied as sole carbon source. On the other hand, no difference in phenotype was observed between them in the presence or absence of the inhibitors of cell wall beta-glucan remodeling when grown with glucose. exgA Expression was induced in growth on solid surfaces such as filter membrane and onion inner skin. A combination of poor nutrition and mycelial attachment to a hydrophobic solid surface appears to be an inducing factor for exgA expression. These data suggest that ExgA plays a role in beta-glucan utilization, but is not much involved in cell wall beta-glucan remodeling.     

Soybean resistant proteins interrupt an enterohepatic circulation of bile acids and suppress liver tumorigenesis induced by azoxymethane and dietary deoxycholate in rats

We found that azoxymethane and dietary deoxycholate induced liver tumors in rats. The incidence and the development of the tumor were closely related to the enterohepatic circulation of bile acids. The feeding of a high-molecular-weight fraction of soy protein digest (HMF) suppressed the tumorigenesis, probably due to the inhibitory effect of soybean resistant protein on reabsorption of bile acids in the intestine.     

Inulavosin and its benzo‐derivatives,melanogenesis inhibitors,target the copper loading mechanism to the active site of tyrosinase

Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure–activity relationship of inulavosin and its benzo‐derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper‐binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzo‐derivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo‐tyrosinase that has a conformational defect.     

Sulfuric acid and leaching requirements for reclaiming sodium-affected calcareous soils

Summary The changes in exchangeable sodium and total dissolved salts due to the application of sulfuric acid and water to sodium-affected calcareous soils were studied for the purpose of establishing a convenient way of determining acid and leaching water requirements. Conventional methods which ignore the effect of sodium removed from soil exchange sites are inadequate for estimating amounts of acid needed to obtain a desired level of exchangeable sodium, especially at high acid application rates. Modified equations are presented which predict the changes in exchangeable sodium and total dissolved salts using essentially the same input data needed for conventional methods, and appear advantageous for estimating acid and leaching water needs. Contribution from the Department of Soils, Water and Engineering, The University of Arizona, Tucson 82751, Arizona Agricultural Experiment Station Journal No. 2340. Supported in part by a grant from the Arizona Mining Association.