Near-infrared observations of active asteroid (3200) Phaethon reveal no evidence for hydration

Asteroid (3200) Phaethon is an active near-Earth asteroid and the parent body of the Geminid Meteor Shower. Because of its small perihelion distance, Phaethon's surface reaches temperatures sufficient to destabilize hydrated materials. We conducted rotationally resolved spectroscopic observations of this asteroid, mostly covering the northern hemisphere and the equatorial region, beyond 2.5-micron to search for evidence of hydration on its surface. Here we show that the observed part of Phaethon does not exhibit the 3-micron hydrated mineral absorption (within 2-sigma). These observations suggest that Phaethon's modern activity is not due to volatile sublimation or devolatilization of phyllosilicates on its surface. It is possible that the observed part of Phaethon was originally hydrated and has since lost volatiles from its surface via dehydration, supporting its connection to the Pallas family, or it was formed from anhydrous material

Ki-Energy (Life-Energy) Protects Isolated Rat Liver Mitochondria from Oxidative Injury

We investigated whether ‘Ki-energy’ (life-energy) has beneficial effects on mitochondria. The paradigm we developed was to keep isolated rat liver mitochondria in conditions in which they undergo heat deterioration (39°C for 10 min). After the heat treatment, the respiration of the mitochondria was measured using a Clarke-type oxygen electrode. Then, the respiratory control ratio (RC ratio; the ratio between State-3 and State-4 respiration, which is known to represent the integrity and intactness of isolated mitochondria) was calculated. Without the heat treatment, the RC ratio was >5 for NADH-linked respiration (with glutamate plus malate as substrates). The RC ratio decreased to 1.86–4.36 by the incubation at 39°C for 10 min. However, when Ki-energy was applied by a Japanese Ki-expert during the heat treatment, the ratio was improved to 2.24–5.23. We used five preparations from five different rats, and the significance of the differences of each experiment was either P < 0.05 or P < 0.01 (n = 3–5). We analyzed the degree of lipid peroxidation in the mitochondria by measuring the amount of TBARS (thiobarbituric acid reactive substances). The amount of TBARS in heat-treated, no Ki-exposed mitochondria was greater than that of the control (no heat-treated, no Ki-exposed). However, the amount was reduced in the heat-treated, Ki-exposed mitochondria (two experiments; both P < 0.05) suggesting that Ki-energy protected mitochondria from oxidative stress. Calcium ions may play an important role in the protection by Ki-energy. Data also suggest that the observed Ki-effect involves, at least, near-infrared radiation (0.8–2.7 μm) from the human body

Five Decades of Research on Mitochondrial NADH-quinone Oxidoreductase (complex I)

NADH-quinone oxidoreductase (complex I) is the largest and most complicated enzyme complex of the mitochondrial respiratory chain. It is the entry site into the respiratory chain for most of the reducing equivalents generated during metabolism, coupling electron transfer from NADH to quinone to proton translocation, which in turn drives ATP synthesis. Dysfunction of complex I is associated with neurodegenerative diseases such as Parkinson’s and Alzheimer’s, and it is proposed to be involved in aging. Complex I has one non-covalently bound FMN, eight to 10 iron-sulfur clusters, and protein-associated quinone molecules as electron transport components. Electron paramagnetic resonance (EPR) has previously been the most informative technique, especially in membrane in situ analysis. The structure of complex 1 has now been resolved from a number of species, but the mechanisms by which electron transfer is coupled to transmembrane proton pumping remains unresolved. Ubiquinone-10, the terminal electron acceptor of complex I, is detectable by EPR in its one electron reduced, semiquinone (SQ) state. In the aerobic steady state of respiration the semi-ubiquinone anion has been observed and studied in detail. Two distinct protein-associated fast and slow relaxing, SQ signals have been resolved which were designated SQNf and SQNs. This review covers a five decade personal journey through the field leading to a focus on the unresolved questions of the role of the SQ radicals and their possible part in proton pumping

Characterization of recombinant rat cathepsin B and nonglycosylated mutants expressed in yeast. New insights into the pH dependence of cathepsin B-catalyzed hydrolyses.

The cysteine proteinase rat cathepsin B was expressed in yeast in an active form and was found to be heterogeneously glycosylated at the consensus sequence for N-linked oligosaccharide substitution. Purified enzyme fractions containing the highest levels of glycosylation were shown to have reduced activity. A glycosylation minus mutant constructed by site-directed mutagenesis (by changing the Ser to Ala in the consensus sequence) was still secreted by the yeast and was shown to be functionally identical with purified rat liver cathepsin B. Recombinant cathepsin B was used to further characterize the pH dependence of cathepsin B-catalyzed hydrolyses using 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA) substrates with arginine as the P1, and either arginine or phenylalanine as the P2 residue. The AMC and pNA groups give insights into the leaving group binding site (P') of cathepsin B. These studies show for the first time that at least seven dissociable groups are involved in substrate binding and hydrolysis in cathepsin B activity. Two of these groups, with pKa values of 6.9 and 7.7 in the recombinant enzyme, are in the leaving group binding site and are most likely His110 and His111. The same groups in rat liver cathepsin B have higher pKa values than in recombinant cathepsin B, but have identical function in the two enzymes. Two other groups are probably the active site Cys29 and His199 with pKa values of 3.6 and 8.6, respectively. A group with a pKa of 5.1 interacts with substrates containing Arg at P2, and the group is most likely Glu245. The remaining two groups, one with a pKa of about 4.9 and the other about 5.3, are most likely carboxyl residues possibly interacting with Arg at P1 in the substrate. The possible candidates on the basis of the x-ray structure are Asp22, Asp69, Glu171, and Glu122, all found within a 13 A radius from the active site thiol of Cys29

Serotonin Transporter Gene Polymorphism Modulates Activity and Connectivity within an Emotional Arousal Network of Healthy Men during an Aversive Visceral Stimulus.

Background and aimsThe 5-hydroxytryptamine transporter gene-linked polymorphic region (5-HTTLPR) has been linked to increased stress responsiveness and negative emotional states. During fearful face recognition individuals with the s allele of 5-HTTLPR show greater amygdala activation. We aimed to test the hypothesis that the 5-HTTLPR polymorphism differentially affects connectivity within brain networks during an aversive visceral stimulus.MethodsTwenty-three healthy male subjects were enrolled. DNA was extracted from the peripheral blood. The genotype of 5-HTTLPR was determined using polymerase chain reaction. Subjects with the s/s genotype (n = 13) were compared to those with the l allele (genotypes l/s, l/l, n = 10). Controlled rectal distension from 0 to 40 mmHg was delivered in random order using a barostat. Radioactive H2[15-O] saline was injected at time of distension followed by positron emission tomography (PET). Changes in regional cerebral blood flow (rCBF) were analyzed using partial least squares (PLS) and structural equation modeling (SEM).ResultsDuring baseline, subjects with s/s genotype demonstrated a significantly increased negative influence of pregenual ACC (pACC) on amygdala activity compared to l-carriers. During inflation, subjects with s/s genotype demonstrated a significantly greater positive influence of hippocampus on amygdala activity compared to l-carriers.ConclusionIn male Japanese subjects, individuals with s/s genotype show alterations in the connectivity of brain regions involved in stress responsiveness and emotion regulation during aversive visceral stimuli compared to those with l carriers

Growth Inhibition of Cultured Human Liver Carcinoma Cells by Ki-energy (Life-energy): Scientific Evidence for Ki-effects on Cancer Cells

‘Ki-energy’ (life-energy) is believed to increase the immune activity of its practitioners. It has also been shown to cause neuropsychological effects. We undertook this study to obtain objective and scientific evidence as to whether or not a ‘Ki-effect’ could inhibit the growth of cultured cancer cells. Cultured human liver carcinoma cells, HepG2, were used. A Japanese Ki-expert held his fingers toward the cells in culture dishes for 5 or 10 min. After culturing for 24 h, we measured cell numbers, protein concentration per cell, certain mRNA expressions and the synthesis of regucalcin. The results were compared with those for control cells (non-treated cells). We found that the number of cells in the Ki-exposed groups were less than those in the controls by 30.3 and 40.6% with 5 and 10 min Ki-exposure, respectively. The protein content per cell in the Ki-exposed groups (5 and 10 min) was higher than that in the control groups by 38.8 and 62.9%, respectively. These results were statistically significant. Using RT–PCR, we found that the mRNA expression for c-myc, a tumor stimulator gene, was decreased, while that for regucalcin, which suppresses DNA synthesis, was increased. Our molecular biological studies and mathematical model analysis demonstrated that Ki-energy inhibited cancer cell division. The data also indicate that the Ki-effects involve some form of infrared radiation from the human body. This study suggests the possibility that Ki-energy may be beneficial for cancer patients because it suppresses cancer cell growth, and at the same time, it stimulates immune functions of the patients

Light-stable rhodopsin. II. An opsin mutant (TRP-265→PHE) and a retinal analog with a nonisomerizable 11-cis configuration form a photostable chromophore

In order to prepare a completely light-stable rhodopsin, we have synthesized an analog, II, of 11-cis retinal in which isomerization at the C11-C12 cis-double bond is blocked by formation of a cyclohexene ring from the C10 to C13-methyl. We used this analog to generate a rhodopsin-like pigment from opsin expressed in COS-1 cells and opsin from rod outer segments (Bhattacharya, S., Ridge, K.D., Knox, B.E., and Khorana, H. G. (1992) J. Biol. Chem. 267, 6763-6769). The pigment (lambda max, 512 nm) formed from opsin and analog II (rhodospin-II) showed ground state properties very similar to those of rhodopsin, but was not entirely stable to light. In the present work, 12 opsin mutants (Ala-117&#8594;Phe, Glu-122&#8594;Gln(Ala, Asp), Trp-126&#8594;Phe(Leu, Ala), Trp-265&#8594;Ala(Tyr, Phe), Tyr-268&#8594;Phe, and Ala-292&#8594;Asp), where the mutations were presumed to be in the retinal binding pocket, were reconstituted with analog II. While all mutants formed rhodopsin-like pigments with II, blue-shifted (12-30 nm) chromophores were obtained with Ala-117&#8594;Phe, Glu-122&#8594;Gln(Ala), Trp-126&#8594;Leu(Ala), and Trp-265&#8594;Ala(Tyr, Phe) opsins. The extent of chromophore formation was markedly reduced in the mutants Ala-117&#8594;Phe and Trp-126&#8594;Ala. Upon illumination, the reconstituted pigments showed varying degrees of light sensitivity; the mutants Trp-126&#8594;Phe(Leu) showed light sensitivity similar to wild-type. Continuous illumination of the mutants Glu-122&#8594;Asp, Trp-265&#8594;Ala, Tyr-268&#8594;Phe, and Ala-292&#8594;Asp resulted in hydrolysis of the retinyl Schiff base. Markedly reduced light sensitivity was observed with the mutant Trp-265&#8594;Tyr, while the mutant Trp-265&#8594;Phe was light-insensitive. Consistent with this result, the mutant Trp-265&#8594;Phe showed no detectable light-dependent activation of transducin or phosphorylation by rhodopsin kinase

Ki-energy (Life-energy) Stimulates Osteoblastic Cells and Inhibits the Formation of Osteoclast-like Cells in Bone Cell Culture Models

Some practitioners of the Nishino Breathing Method (NBM) were found to have a higher bone density than the average values of age- and gender-matched non-practitioners. Using bone cell culture models, we investigated a possible mechanism behind this observation. For the study of bone mineralization, we performed the following two experiments using cultured osteoblastic MC3T3-E1 cells: (i) Kozo Nishino, a Japanese Ki expert, sent Ki-energy to the cells once for 5 or 10 min after they were seeded in culture dishes in the presence of 10% fetal bovine serum (FBS). They were incubated for 72 h and the cells were counted. The number in the dish with 10-min Ki-exposure was significantly greater than that in the control (P < 0.01 with n = 8). We performed a reverse transcription-polymerase chain reaction (RT–PCR) study using these cells, but the mRNA expressions did not change significantly. (ii) After cells were incubated for 72 h without Ki-exposure (in the presence of FBS), they were further cultured for 48 h (in the absence of FBS) to promote differentiation. At the beginning of the second culture stage, Ki was applied once for 10 min. After 48 h, RT–PCR was performed. The mRNA expressions which are related to bone mineralization, such as Runx2, α1(I) collagen, alkaline phosphatase and osteocalcin, increased significantly (P < 0.05 and n = 4 for all). For the bone resorption study, we used mouse marrow cultures, which can form osteoclast-like cells in the presence of (1–34) parathyroid hormone (PTH), and stimulate resorption. We exposed these cells to Ki-energy twice for the duration of 5 or 10 min on day 0 and day 4. On day 7, the cells were counted. The number of osteoclast-like cells in dishes with Ki exposure was significantly smaller than those in control dishes (P < 0.05 with n = 5). The difference between 5-min exposure and 10-min exposure was not statistically significant. All of our data suggest that the Ki-effect on osteoporosis should be further explored