Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.     

Morphological Parameters Associated with Ruptured Posterior Communicating Aneurysms

The rupture risk of unruptured intracranial aneurysms is known to be dependent on the size of the aneurysm. However, the association of morphological characteristics with ruptured aneurysms has not been established in a systematic and location specific manner for the most common aneurysm locations. We evaluated posterior communicating artery (PCoA) aneurysms for morphological parameters associated with aneurysm rupture in that location. CT angiograms were evaluated to generate 3-D models of the aneurysms and surrounding vasculature. Univariate and multivariate analyses were performed to evaluate morphological parameters including aneurysm volume, aspect ratio, size ratio, distance to ICA bifurcation, aneurysm angle, vessel angles, flow angles, and vessel-to-vessel angles. From 2005–2012, 148 PCoA aneurysms were treated in a single institution. Preoperative CTAs from 63 patients (40 ruptured, 23 unruptured) were available and analyzed. Multivariate logistic regression revealed that smaller volume (p = 0.011), larger aneurysm neck diameter (0.048), and shorter ICA bifurcation to aneurysm distance (p = 0.005) were the most strongly associated with aneurysm rupture after adjusting for all other clinical and morphological variables. Multivariate subgroup analysis for patients with visualized PCoA demonstrated that larger neck diameter (p = 0.018) and shorter ICA bifurcation to aneurysm distance (p = 0.011) were significantly associated with rupture. Intracerebral hemorrhage was associated with smaller volume, larger maximum height, and smaller aneurysm angle, in addition to lateral projection, male sex, and lack of hypertension. We found that shorter ICA bifurcation to aneurysm distance is significantly associated with PCoA aneurysm rupture. This is a new physically intuitive parameter that can be measured easily and therefore be readily applied in clinical practice to aid in the evaluation of patients with PCoA aneurysms.     

Mutational analysis of slyD, an Escherichia coli gene encoding a protein of the FKBP immunophilin family

slyD encodes a 196 amino acid polypeptide that is a member of the FKBP family of cis–trans peptidyl–prolyl isomerases (PPIases). slyD mutations affect plaque formation by the phage φX174 by blocking the action of the phage lysis protein E. Here we describe the selection of a set of spontaneous slyD mutations conferring resistance to the expression of gene E from a plasmid. These mutations occur disproportionately in residues of SlyD that, based on the structure of the prototype mammalian FKBP12, make ligand contacts with immunosuppressing drug molecules or are conserved in other FKBP proteins. A wide variation in the plating efficiency of φX174 on these E  ^R strains is observed, relative to the parental, indicating that these alleles differ widely in residual SlyD activity. Moreover, it is found that slyD mutations cause significant growth rate defects in Escherichia coli B and C backgrounds. Finally, overexpression of slyD causes filamentation of the host. Thus, among the FKBP genes found in organisms across the evolutionary spectrum, slyD is unique in having three distinct drug-independent phenotypes.     

Influences of temporal independence of data on modelling species distributions

Modelling species distributions has been widely used to understand present and future potential distributions of species, and can provide adaptation and mitigation information as references for conservation and management under climate change. However, various methods of data splitting to develop and validate functions of the models do not get enough attention, which may mislead the interpretation of predicted results. We used the Taiwanese endemic birds to test the influences of temporal independence of datasets on model performance and prediction. Training and testing data were considered to be independent if they were collected during different survey periods (1993–2004 and 2009–2010). The results indicated no significant differences of six model performance measures (AUC, kappa, TSS, accuracy, sensitivity, and specificity) among the combinations of training and testing datasets. Both species- and grid cell-based assessments differed significantly between predictions by the annual and pooled training data. We also found an average of 85.8% similarity for species presences and absences in different survey periods. The remaining dissimilarity was mostly caused by species observed in the late survey period but not in the early one. The method of data splitting, yielding training and testing data, is critical for resulting model species distributions. Even if similar model performance exists, different methods can lead to different species distributional maps. More attention needs to be given to this issue, especially when amplifying these models to project species distributions in a changing world.     

Association between misalignment of circadian rhythm and obesity in Korean men: Sixth Korea National Health and Nutrition Examination Survey

ABSTRACT

Background: The disruption of circadian rhythm has been found to associate with obesity in vivo and in vitro. Sleep duration, eating habits, total feeding time, and nightshift work can also affect circadian rhythms. This study investigated the association between misalignment of circadian rhythm and obesity in Korean men, using a cross-sectional database.

Methods: This study used data from the Korean National Health and Nutrition Examination Survey (KNHANES), whose study population was 3,658 men aged 18 to 60 years. General and abdominal obesity was defined as a body mass index (BMI) ≥ 25 kg/m² and waist circumference ≥ 90 cm, respectively. Circadian rhythm factors were determined with a self-report questionnaire and included breakfast frequency, sleep duration, and work time. Frequency of breakfast was divided into regular breakfast (five to seven times a week) and irregular breakfast (less than five times a week). Sleep duration was divided into less than 7 hours, 7–9 hours, and over 9 hours. Working time was defined as day/evening, night shift, and other type. The adjusted odds ratios (aORs) and 95% confidence intervals (CIs) for general and abdominal obesity were calculated using multivariable logistic regression according to the number of factors that disturb the circadian rhythm.

Results: Participants with 1 (aOR 1.34, 95% Cl 1.10–1.61) and ≥2 (aOR 1.62, 95% Cl 1.29–2.05) factors disturbing circadian rhythms were associated with elevated risk for general obesity. Similarly, those with 1 (aOR 1.33, 95% Cl 1.09–1.63) and ≥2 (aOR 1.70, 95% Cl 1.32–2.20) factors had elevated risk for abdominal obesity.

Conclusions: Factors disturbing the circadian rhythm were associated with general and abdominal obesity. Additional studies are needed, and associations with metabolic diseases should be investigated.     

Mannitol induces selective astroglial death in the CA1 region of the rat hippocampus following status epilepticus

In the present study, we addressed the question of whether treatment with mannitol, an osmotic diuretic, affects astrogliovascular responses to status epilepticus (SE). In saline-treated animals, astrocytes exhibited reactive astrogliosis in the CA1-3 regions 2-4 days after SE. In the mannitol-treated animals, a large astroglial empty zone was observed in the CA1 region 2 days after SE. This astroglial loss was unrelated to vasogenic edema formation. There was no difference in SE-induced neuronal loss between saline- and mannitol-treated animals. Furthermore, mannitol treatment did not affect astroglial loss and vasogenic edema formation in the dentate gyrus and the piriform cortex. These findings suggest that mannitol treatment induces selective astroglial loss in the CA1 region independent of vasogenic edema formation following SE. These findings support the hypothesis that the susceptibility of astrocytes to SE is most likely due to the distinctive heterogeneity of astrocytes independent of hemodynamics. [BMB Reports 2015; 48(9): 507-512]     

A Candidate Subspecies Discrimination System Involving a Vomeronasal Receptor Gene with Different Alleles Fixed in M. m. domesticus and M. m. musculus

Assortative mating, a potentially efficient prezygotic reproductive barrier, may prevent loss of genetic potential by avoiding the production of unfit hybrids (i.e., because of hybrid infertility or hybrid breakdown) that occur at regions of secondary contact between incipient species. In the case of the mouse hybrid zone, where two subspecies of Mus musculus (M. m. domesticus and M. m. musculus) meet and exchange genes to a limited extent, assortative mating requires a means of subspecies recognition. We based the work reported here on the hypothesis that, if there is a pheromone sufficiently diverged between M. m. domesticus and M. m. musculus to mediate subspecies recognition, then that process must also require a specific receptor(s), also sufficiently diverged between the subspecies, to receive the signal and elicit an assortative mating response. We studied the mouse V1R genes, which encode a large family of receptors in the vomeronasal organ (VNO), by screening Perlegen SNP data and identified one, Vmn1r67, with 24 fixed SNP differences most of which (15/24) are nonsynonymous nucleotide substitutions between M. m. domesticus and M. m. musculus. We observed substantial linkage disequilibrium (LD) between Vmn1r67 and Abpa27, a mouse salivary androgen-binding protein gene that encodes a proteinaceous pheromone (ABP) capable of mediating assortative mating, perhaps in conjunction with its bound small lipophilic ligand. The LD we observed is likely a case of association rather than residual physical linkage from a very recent selective sweep, because an intervening gene, Vmn1r71, shows significant intra(sub)specific polymorphism but no inter(sub)specific divergence in its nucleotide sequence. We discuss alternative explanations of these observations, for example that Abpa27 and Vmn1r67 are coevolving as signal and receptor to reinforce subspecies hybridization barriers or that the unusually divergent Vmn1r67 allele was not a product of fast positive selection, but was derived from an introgressed allele, possibly from Mus spretus.     

Determination of rat brain buffering in vivo by 31P-NMR

Buffering capacity of most tissues is composed of both rapid and slow phases, the latter presumably due to active acid extrusion. To examine the time course of brain buffering the brain pH of Sprague-Dawley rats was measured using 31P-nuclear magnetic resonance. The effect on brain pH of 30- or 58-min exposures to 20% CO2 followed by 30- or 38-min recovery periods, respectively, was studied. Brain pH reached its lowest value after a 15-min exposure to elevated CO2, thereafter slowly and steadily increasing. During recovery brain pH rose rapidly in the first 5 min exceeding control brain pH by 0.08 pH units. Brain pH fell during the next 30 min despite increases in blood pH and decreases in blood CO2 tension. Calculated intrinsic brain buffering rose steadily threefold during the last 40 min of CO2 exposure and during the final 30 min of recovery. These data show that in rat brain there is a temporally late buffering process, most likely active acid extrusion, requiring greater than 30 min for full activation and at least 30 min for discontinuation.