Extracellular Zinc Ion Regulates Transient Receptor Potential Melastatin 5 (TRPM5) Channel Activation through Its Interaction with a Pore Loop Domain

The transient receptor potential melastatin 5 (TRPM5) channel is a monovalent cation channel activated by intracellular Ca²⁺. Expression of this channel is restricted to taste cells, the pancreas and brainstem, and is thought to be involved in controlling membrane potentials. Its endogenous ligands are not well characterized. Here, we show that extracellular application of Zn²⁺ inhibits TRPM5 activity. In whole-cell patch-clamp recordings, extracellular application of ZnCl₂ inhibited step-pulse-induced TRPM5 currents with 500 nm free intracellular Ca²⁺ in a dose-dependent manner (IC₅₀ = 4.3 μm at −80 mV). ZnSO₄ also inhibited TRPM5 activity. Extracellular application of ZnCl₂ inhibited TRPM5 activation at several temperatures. Furthermore, inhibition by 30 μm ZnCl₂ was impaired in TRPM5 mutants in which His at 896, and Glu at 926 and/or Glu at 939 in the outer pore loop were replaced with Gln. From these results, we conclude that extracellular Zn²⁺ inhibits TRPM5 channels, and the residues in the outer pore loop of TRPM5 are critically involved in the inhibition.     

Aberrant Assembly of RNA Recognition Motif 1 Links to Pathogenic Conversion of TAR DNA-binding Protein of 43 kDa (TDP-43)

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant β-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS.     

A Novel Bivalent Vaccine Based on a PB2-Knockout Influenza Virus Protects Mice from Pandemic H1N1 and Highly Pathogenic H5N1 Virus Challenges

Vaccination is an effective means to protect against influenza virus. Although inactivated and live-attenuated vaccines are currently available, each vaccine has disadvantages (e.g., immunogenicity and safety issues). To overcome these problems, we previously developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2 protein-expressing cells. Here, we generated two PB2-KO viruses whose PB2-coding regions were replaced with the HA genes of either A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1). The resultant viruses comparably, or in some cases more efficiently, induced virus-specific antibodies in the serum, nasal wash, and bronchoalveolar lavage fluid of mice relative to a conventional formalin-inactivated vaccine. Furthermore, mice immunized with these PB2-KO viruses were protected from lethal challenges with not only the backbone virus strain but also strains from which their foreign HAs originated, indicating that PB2-KO viruses with antigenically different HAs could serve as bivalent influenza vaccines.     

Activin A regulates growth of gastro‐intestinal epithelial cells by mediating epithelial‐mesenchymal interaction

The importance of epithelial–mesenchymal interaction on the development of gastro‐intestinal (GI) organs has been repeatedly reported, but its molecular mechanism has not been fully understood though several factors including hepatocyte growth factor and endothelin‐3 have been shown to mediate it. Activins have been demonstrated to play important roles in the regulation of organogenesis in vertebrates, but their roles in the regulation of growth and differentiation of GI organs remain to be solved. In the present study, we examined expression of activins in developing rat GI tract, and found that inhibin bA encoding activin A was specifically expressed by GI mesenchymes, while inhibin bB encoding activin B was expressed by both epithelial and mesenchymal components. We then examined the effect of activin A on the growth of fetal rat GI epithelial cells in primary culture. We found that activin A inhibited the growth of forestomach and glandular stomach epithelial cells while it stimulated the growth of colonic epithelial cells. These results suggest that activin A secreted from GI mesenchymes region‐specifically regulates the growth of attaching epithelial cells. We thus conclude that activin A mediates epithelial‐mesenchymal interaction in the developing GI tract.     

Synthesis and evaluation of 2,3-dinorprostaglandins: Dinor-PGD1 and 13-epi-dinor-PGD1 are peroxisome proliferator-activated receptor α/γ dual agonists

2,3-Dinorprostaglandins (dinor-PGs) have been regarded as β-oxidation products of arachidonic-acid-derived prostaglandins, but their biological activities in mammalian cells remain unclear. On the other hand, C18 polyunsaturated fatty acids (PUFAs), such as γ-linolenic acid (GLA), have various biological activities, and dinor-PGs are speculated to be biosynthesized from GLA. Here, we synthesized dinor-PGs that may possibly be derived from GLA and examined their activities towards peroxisome proliferator-activated receptors (PPARs). Dinor-PGD₁ (1) and its epimer 13-epi-dinor-PGD₁ (epi-1) were found to be dual agonists for PPARα/γ, whereas PGD₂ derived from arachidonic acid is selective for PPARγ. Thus, GLA-derived dinor-PGs may have unique biological roles.     

Effect of Gamma Irradiation on the Amino Acid of Potatoes

The free amino acids of potatoes irradiated with the doses of 7,000, 15,000 and 30,000 rad were determined by ion-exchange chromatography.

After 15 days storage following irradiation, it was shown that the concentration of asparatic acid, proline and aliphatic amino acids increased with increasing irradiation doses, while that of basic amino acids and glutamic acid especially decreased. However, after 105 days of storage, the similarity of the free amino acid content of irradiated potatoes to that of non-irradiated and non-stored potatoes was observed.

On the concentration of protein-bound amino acids, there were no significant differences between non-irradiated and 15,000 rad irradiated potatoes.     

α-Aminoisobutyric Acid as the Constituent Amino Acid of Protein

α-Aminoisobutyric acid is the only tertiary amino acid which is reported to occur in the proteins. Nevertheless, this amino acid has not been yet isolated from the proteins. Recently we succeeded in isolating this amino acid as white prismy crystalline substance from both acid and pepsin hydrolysate of horse hind leg muscle proteins, and this crystal was identified to be α-amino-isobutyric acid by elementary analysis, properties of this derivates, etc.     

Effect of Glucose on the Uricase Formation by Streptomyces sp

The effect of glucose on the formation of uricase by a strain of Streptomyces sp. incubated under conditions of nitrogen limitation was investigated. Glucose stimulated uricase formation in the presence of potassium ion and inhibited it in the absence of the ion. Glucose metabolism by the organism was altered in the absence of the ion, and this appeared to cause the inhibition of the enzyme formation. The stimulatory effect of glucose in the presence of potassium ion was to shorten the lag period. Comparisons of the enzyme formation with and without urate in the presence and absence of glucose revealed that glucose promoted the utilization of exogenous urate as the inducer. The effect of glucose appeared to require protein synthesis, since it was prevented by chloramphenicol. Cyclic adenosine 3′,5′-mono-phosphate showed apparently no effect on uricase formation of this organism.     

Effects of Lipids on the Rheological Properties of Aqueous Soybean Protein Dispersions

The flow properties of soybean protein–lipid–water suspension systems and coagulated gels were related to a protein–lipid interaction. For powdered soybean lecithin-added soybean protein suspension systems and their heat-induced gels, the yield stress (σ_y) and the consistency index (K) increased with increasing amounts of added lipid, but the flow behavior index (n) and the thixotropy index (TI) decreased. On the other hand, there were only small changes in the magnitudes of the thixotropic parameters and the viscometric parameters (σ_y, K and n) after adding soybean oil at various concentrations to soybean protein dispersions. These facts suggested that the formation of a protein–phospholipid complex increased the effective particle size, and that the intermolecular entanglements and linkages among the protein molecules or among the protein-phospholipid complexes were weakened by the addition of polar lipids. The thixotropy index defined in this study is available for characterizing the stress decay that occurs within soybean protein dispersions and heat-induced gels as they are sheared.