Chlorophyll-sensitized Photooxidation Products of Spinach Galactolipids

Spinach monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were oxidized with singlet molecular oxygen by the use of chlorophyll a as the photosensitizer. The oxidation products were separated from the unoxidized MGDG and DGDG by reverse-phase high performance liquid chromatography (HPLC). The products separated by HPLC were identified to be mono- and di-hydroperoxides formed by ¹O₂ oxidation of the 16:3 or 18:3 component of MGDG and DGDG. Each unsaturated fatty acid moiety in the MGDG and DGDG produced isomeric hydroperoxides in a manner similar to the corresponding fatty acid methyl ester.     

Development and evaluation of stability and ultrasound response of DSPC-DPSG-based freeze-dried microbubbles

Abstract

It is known that Phosphatidyl choline-Phosphatidyl glycerol mixtures can be used for liposome formulations, making them less leaky than liposomes with only one lipid. We hypothesized that this might also be the case for bubbles, which can be used as ultrasound (US) contrast agents. Therefore, we have compared a series of mixed distearoyl phosphatidylcholine-distearoyl phosphatidylglycerol (DSPC-DPSG) bubbles and with bubbles containing either DSPC or DSPG (and distearoyl ethanolamine-polyethyleneglycol 2000, DSPE-PEG2k). Here, we describe the development, examination of stability in vitro and attenuation of broad frequency US pulses. Novel lipid-stabilized freeze-dried formulations for US applications, using the phospholipids DSPC, DSPG, and PEGylated DSPE-PEG2k and perfluoropropane gas were developed. It was found that the bubbles could effectively be preserved by freeze-drying and then re-constituted by addition of water. Average bubble sizes were around 2?µm for all bubbles after re-constitution. Bubble stability was assessed by evaluating the decay of the US backscattering signal in vitro. Bubbles containing DSPG were more stable than bubbles with only DSPC. The composition DSPC:DSPG:DSPE-PEG2k 30:60:10 (molar ratio) was the most stable with an effective half-life of 9.12?min, compared to bubbles without DSPG, which had half-life of 2.05?min. Bubble attenuation of US depended highly on the compositions. Bubbles without DSPG had the highest attenuation indicating higher oscillation the most but were also destroyed by higher energy US. No bubbles with DSPG showed any indication of destruction but all had increased attenuations to varying degrees, DSPC:DSPG:DSPE-PEG2k 45:45:10 showed the least attenuation.     

Transferase Action of α-Galactosidase from Tubers of Stachys affinis

Endogenous auxins in the shoots and ears of rice were investigated. Indole-3-acetic acid (IAA) and indole-3-carboxylic acid (ICA) was identified by GC-MS, the endogenous level of IAA being much higher than that of ICA. To analyze the fluctuation of endogenous IAA level throughout the life cycle of rice, a rapid and effective procedure, using HPLC and a fluorescence detector, was developed. The level of IAA in shoots was 10 —26ng/g fr. wt., while that in ears was 10 to 100 times higher. The level of IAA conjugate in ears was also much higher than that in shoots. These results show that the biosynthesis of IAA occurs at an early stage of seed development, and it is also suggested that IAA may play a role in regulating the reproductive growth of rice.     

Catalysis-Enhancement via Rotary Fluctuation of F1-ATPase

Protein conformational fluctuations modulate the catalytic powers of enzymes. The frequency of conformational fluctuations may modulate the catalytic rate at individual reaction steps. In this study, we modulated the rotary fluctuation frequency of F₁-ATPase (F₁) by attaching probes with different viscous drag coefficients at the rotary shaft of F₁. Individual rotation pauses of F₁ between rotary steps correspond to the waiting state of a certain elementary reaction step of ATP hydrolysis. This allows us to investigate the impact of the frequency modulation of the rotary fluctuation on the rate of the individual reaction steps by measuring the duration of rotation pauses. Although phosphate release was significantly decelerated, the ATP-binding and hydrolysis steps were less sensitive or insensitive to the viscous drag coefficient of the probe. Brownian dynamics simulation based on a model similar to the Sumi-Marcus theory reproduced the experimental results, providing a theoretical framework for the role of rotational fluctuation in F₁ rate enhancement.     

Thermodynamic Analyses of Nucleotide Binding to an Isolated Monomeric β Subunit and the α3β3γ Subcomplex of F1-ATPase

Rotation of the γ subunit of the F₁-ATPase plays an essential role in energy transduction by F₁-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F₁-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α₃β₃γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K_d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH⁰ and ΔG⁰ values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α₃β(Y341W)₃γ subcomplex had similar K_d and ΔG⁰ values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.     

6-(4-Pyridyl)pyrimidin-4(3H)-ones as CNS penetrant glycogen synthase kinase-3β inhibitors

The discovery of a series of 6-(4-pyridyl)pyrimidin-4(3H)-ones derived from a hit compound with low molecular weight and sufficient chemical space is reported. Transformation of substituents led to subnanomolar potent inhibitors with in vivo tau phoshorylation lowering activity.     

Resampling Nucleotide Sequences with Closest-Neighbor Trimming and Its Comparison to Other Methods

A large number of nucleotide sequences of various pathogens are available in public databases. The growth of the datasets has resulted in an enormous increase in computational costs. Moreover, due to differences in surveillance activities, the number of sequences found in databases varies from one country to another and from year to year. Therefore, it is important to study resampling methods to reduce the sampling bias. A novel algorithm–called the closest-neighbor trimming method–that resamples a given number of sequences from a large nucleotide sequence dataset was proposed. The performance of the proposed algorithm was compared with other algorithms by using the nucleotide sequences of human H3N2 influenza viruses. We compared the closest-neighbor trimming method with the naive hierarchical clustering algorithm and -medoids clustering algorithm. Genetic information accumulated in public databases contains sampling bias. The closest-neighbor trimming method can thin out densely sampled sequences from a given dataset. Since nucleotide sequences are among the most widely used materials for life sciences, we anticipate that our algorithm to various datasets will result in reducing sampling bias.     

Tropisms of AAV for Subretinal Delivery to the Neonatal Mouse Retina and Its Application for In Vivo Rescue of Developmental Photoreceptor Disorders

Background

Adeno-associated virus (AAV) is well established as a vehicle for in vivo gene transfer into the mammalian retina. This virus is promising not only for gene therapy of retinal diseases, but also for in vivo functional analysis of retinal genes. Previous reports have shown that AAV can infect various cell types in the developing mouse retina. However, AAV tropism in the developing retina has not yet been examined in detail.

Methodology/Principal Findings

We subretinally delivered seven AAV serotypes (AAV2/1, 2/2, 2/5, 2/8, 2/9, 2/10, and 2/11) of AAV-CAG-mCherry into P0 mouse retinas, and quantitatively evaluated the tropisms of each serotype by its infecting degree in retinal cells. After subretinal injection of AAV into postnatal day 0 (P0) mouse retinas, various retinal cell types were efficiently transduced with different AAVs. Photoreceptor cells were efficiently transduced with AAV2/5. Retinal cells, except for bipolar and Müller glial cells, were efficiently transduced with AAV2/9. Horizontal and/or ganglion cells were efficiently transduced with AAV2/1, AAV2/2, AAV2/8, AAV2/9 and AAV2/10. To confirm the usefulness of AAV-mediated gene transfer into the P0 mouse retina, we performed AAV-mediated rescue of the Cone-rod homeobox gene knockout (Crx KO) mouse, which exhibits an outer segment formation defect, flat electroretinogram (ERG) responses, and photoreceptor degeneration. We injected an AAV expressing Crx under the control of the Crx 2kb promoter into the neonatal Crx KO retina. We showed that AAV mediated-Crx expression significantly decreased the abnormalities of the Crx KO retina.

Conclusion/Significance

In the current study, we report suitable AAV tropisms for delivery into the developing mouse retina. Using AAV2/5 in photoreceptor cells, we demonstrated the possibility of gene replacement for the developmental disorder and subsequent degeneration of retinal photoreceptors caused by the absence of Crx.     

A Lower Level of Forced Expiratory Volume in 1 Second Is a Risk Factor for All-Cause and Cardiovascular Mortality in a Japanese Population: The Takahata Study

Chronic obstructive pulmonary disease is a known risk factor for cardiovascular death in Western countries. Because Japan has a low cardiovascular death rate, the association between a lower level of forced expiratory volume in 1 s (FEV₁) and mortality in Japan’s general population is unknown. To clarify this, we conducted a community-based longitudinal study. This study included 3253 subjects, who received spirometry from 2004 to 2006 in Takahata, with a 7-year follow-up. The causes of death were assessed on the basis of the death certificate. In 338 subjects, airflow obstruction was observed by spirometry. A total of 127 subjects died. Cardiovascular death was the second highest cause of death in this population. The pulmonary functions of the deceased subjects were significantly lower than those of the subjects who were alive at the end of follow-up. The relative risk of death by all causes, respiratory failure, lung cancer, and cardiovascular disease was significantly increased with airflow obstruction. The Kaplan–Meier analysis showed that all-cause and cardiovascular mortality significantly increased with a worsening severity of airflow obstruction. After adjusting for possible factors that could influence prognosis, a Cox proportional hazard model analysis revealed that a lower level of FEV₁ was an independent risk factor for all-cause and cardiovascular mortality (per 10% increase; hazard ratio [HR], 0.89; 95% confidence interval [CI], 0.82–0.98; and HR, 0.72; 95% CI, 0.61–0.86, respectively). In conclusion, airflow obstruction is an independent risk factor for all-cause and cardiovascular death in the Japanese general population. Spirometry might be a useful test to evaluate the risk of cardiovascular death and detect the risk of respiratory death by lung cancer or respiratory failure in healthy Japanese individuals.