Molecular cloning and characterization of CALP/KChIP4, a novel EF-hand protein interacting with presenilin 2 and voltage-gated potassium channel subunit Kv4

Presenilin (PS) genes linked to early-onset familial Alzheimer's disease encode polytopic membrane proteins that are presumed to constitute the catalytic subunit of gamma-secretase, forming a high molecular weight complex with other proteins. During our attempts to identify binding partners of PS2, we cloned CALP (calsenilin-like protein)/KChIP4, a novel member of calsenilin/KChIP protein family that interacts with the C-terminal region of PS. Upon co-expression in cultured cells, CALP was directly bound to and co-localized with PS2 in endoplasmic reticulum. Overexpression of CALP did not affect the metabolism or stability of PS complex, and gamma-cleavage of betaAPP or Notch site 3 cleavage was not altered. However, co-expression of CALP and a voltage-gated potassium channel subunit Kv4.2 reconstituted the features of A-type K(+) currents and CALP directly bound Kv4.2, indicating that CALP functions as KChIPs that are known as components of native Kv4 channel complex. Taken together, CALP/KChIP4 is a novel EF-hand protein interacting with PS as well as with Kv4 that may modulate functions of a subset of membrane proteins in brain.     

Evaluation of self-similar features in time series of serum growth hormone and prolactin levels by fractal analysis: Effect of delayed sleep and complexity of diurnal variation

We assayed the diurnal concentrations of growth hormone (GH) and prolactin (PRL) in 6 healthy male volunteers to evaluate the self-similar features in the time series of each hormone on the basis of fractal theory and to determine the fractal dimension as an index of the complexity of the diurnal variation. In addition, we assessed the effects of a 6-hour delay in the sleep period on the complexity of the diurnal variaton of these hormones. There was a statistically significant fractal feature in the serum levels of GH both under the nocturnal-sleep and delayed-sleep conditions in all subjects. The time series of the serum PRL concentrations also showed a statistically significant fractal feature under the nocturnal-sleep and delayed-sleep conditions in all subjects. The fractal dimensions of the patterns of the GH or PRL levels were 1.879 and 1.929 or 1.754 and 1.785 under the nocturnal-sleep and delayed-sleep conditions, respectively. Two-way ANOVA revealed no significant difference in the fractal dimension between the two sleep conditions but did reveal a significant difference between the fractal dimensions of the GH and PRL levels. These results showed (1) that delayed sleep had no significant effect on the complexity of the diurnal pattern of these hormones, and (2) that the diurnal pattern of the GH levels was more complex than that of the PRL levels.     

Concentrated Protein Body Product Derived from Rice Endosperm as an Oral Tolerogen for Allergen-Specific Immunotherapy—A New Mucosal Vaccine Formulation against Japanese Cedar Pollen Allergy

The endoplasmic reticulum-derived type-I protein body (PB-I) from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1) and Cry j 2 were concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The modified Cry j 1 and Cry j 2 antigens in this concentrated PB product were more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. The high resistance of these allergens could be attributed to changes in protein physicochemical properties induced by the high temperature concentration process, as suggested by the decreased solubility of the antigens and seed proteins in PBs in step-wise-extraction experiments. Confocal microscopy showed that the morphology of antigen-containing PB-Is was preserved in the concentrated PB product. The concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered, supporting its potential use as a novel oral tolerogen formulation.     

Crystallographic study of a site-specifically cross-linked protein complex with a genetically incorporated photoreactive amino acid

The benzophenone photophore is widely used to photo-cross-link macromolecules. Recent developments in genetic code expansion have allowed the biosynthesis of proteins with p-benzoyl-L-phenylalanine (pBpa) at defined sites, for covalent bonding with interacting proteins. However, the structure of a photo-cross-linked protein complex had not been revealed, and thus neither the actual structure of the "photobridge" in a complex nor the influence of this covalent bridge on the overall complex structure was known. In this study, we determine the crystal structure of the cross-linked complex of the liver oncoprotein gankyrin and the C-terminal domain of S6 proteasomal protein (S6C), at 2.05 ? resolution. First, the photoreactive amino acid was separately incorporated into gankyrin at 16 sites on the protein surface, and two variants that efficiently formed a covalent bond with S6C were found. The yield of one of the cross-linked products, with pBpa in place of Arg85 in gankyrin, was maximized for crystallization via optimization of the duration of complex exposure to 365 nm light. The structure revealed that the carbonyl group of the benzophenone of pBpa85 formed a covalent bond exclusively with the Cγ atom of Glu356 in S6C, showing the high selectivity of formation of cross-links by pBpa. In addition, the cross-linked structure exhibited little structural distortion from the native complex structure. Our results demonstrated that cross-linking with site-specifically incorporated pBpa preserves the native binding mode and is useful for probing protein-protein interactions.     

Nε-Methylated Lysines from Formaldehyde-treated Casein

Phospholipase A activity of Pseudomonas aeruginosa was found only in the membrane fraction under normal conditions, but a part of that activity was converted into soluble form after polymyxin B treatment. This soluble phospholipase A was further activated by addition of 80% saturated (NH₄)₂SO₄ or incubation at 70 ± 5°C for 5 minutes. The phenomenon was confirmed in terms of kinetic parameters. Phospholipase A activity in the sonicates of P. aeruginosa was also markedly enhanced by (NH₄)₂SO₂ at 80% saturated concentration. The mechanism of the activation is discussed.     

Cloning and functional analysis of the K+ transporter, PhaHAK2, from salt-sensitive and salt-tolerant reed plants

We isolated PhaHAK2 cDNAs from salt-tolerant and salt-sensitive reed plants. PhaHAK2 belongs to group II by phylogenetic analysis, and was predicted to be a high-affinity plasma membrane K⁺ transporter. Yeast transformed with the PhaHAK2-u from salt-sensitive reed plants (Phragmites australis) had a decreased ability to take up K⁺ in the presence of NaCl and showed a higher Na⁺ permeability than yeast transformed with PhaHAK2-n or PhaHAK2-e from two salt-tolerant reed plants. These results suggest a possibility that the continuous K⁺ uptake by PhaHAK2 and maintenance of high K⁺/Na⁺ ratio under salt stress condition is one of the causes of the salt-tolerance in reed plants.     

Cadmium,Lead, and Selenium in Cord Blood and Thyroid Hormone Status of Newborns

Cadmium (Cd), lead (Pb), and selenium (Se) concentrations in cord whole blood, sampled from 24 women at the time of delivery in a hospital in Tokyo in 2005, were determined by inductively coupled plasma mass spectrometry with a reaction cell. Signal enhancement caused by nonspectroscopic interference for Se was evident and the standard addition technique was essential for correcting the interference. Median concentration in cord bloods was 0.20 ng/g, 6.7 ng/g (0.67 μg/dL), and 191 ng/g for Cd, Pb and Se, respectively. Lead concentration was lower, whereas Se concentration was higher, than those reported in other countries. The trace element concentration was related to the levels of thyroid stimulating hormone (TSH) and free thyroxin (fT4) in the neonatal blood sampled at 4–6 days postpartum. A significantly negative correlation was observed between Cd concentrations in cord blood and TSH concentration in neonatal blood. The result indicated the possible effect of in utero Cd exposure on thyroid hormone status of newborns and that Cd exposure level should be assessed as a covariate in the survey on the relationship between in utero chemicals (e.g., PCBs) exposure and thyroid hormone status.     

High-affinity K<Superscript>+</Superscript> transporter <Emphasis Type="Italic">PhaHAK5</Emphasis> is expressed only in salt-sensitive reed plants and shows Na<Superscript>+</Superscript> permeability under NaCl stress

To understand the mechanism of ion homeostasis in salt tolerant and sensitive plants, we isolated cDNAs for K⁺ transporter PhaHAK1-u and PhaHAK5-u from reed plants. PhaHAK1-u belongs to group I and PhaHAK5-u belongs to group IV by phylogenetic analysis, respectively. PhaHAK5-u is predicted to be a plasma membrane transporter, and shows high-affinity K⁺ transporter. Expression of PhaHAK5 was found in salt-sensitive reed plants, but not in any parts of salt-tolerant reed plants maintained under both control and K⁺ starvation conditions. Under the NaCl stress, the K⁺ uptake ability of the yeast strain expressing PhaHAK5-u was remarkably lower than that of the yeast strain expressing PhaHAK1-u, and PhaHAK5-u showed Na⁺ permeability. These results suggest that PhaHAK5 is one of the routes by which Na⁺ enters cells.     

Chlorine Sensitivity of Feline Calicivirus, a Norovirus Surrogate

The sensitivity to free chlorine of feline calicivirus (FCV), a norovirus surrogate, was examined relative to chlorine demand. When a crude suspension of FCV was treated with a sodium hypochlorite solution containing 10 μg/ml free chlorine, the extent of the decrease of viral infectivity clearly depended on the volume of the reaction mixture. The apparent sensitivity of FCV to free chlorine increased with the reduction of host cell debris, indicating that chlorine demand must be minimized to know the true sensitivity of the virus. We therefore partially purified the viruses from the host cell components and found that the infectivity of FCV was reduced by more than log 4.6 by 5 min of treatment with 300 ng/ml free chlorine.