Metabolic engineering of coenzyme Q by modification of isoprenoid side chain in plant

Coenzyme Q (CoQ), an electron transfer molecule in the respiratory chain and a lipid-soluble antioxidant, is present in almost all organisms. Most cereal crops produce CoQ9, which has nine isoprene units. CoQ10, with 10 isoprene units, is a very popular food supplement. Here, we report the genetic engineering of rice to produce CoQ10 using the gene for decaprenyl diphosphate synthase (DdsA). The production of CoQ9 was almost completely replaced with that of CoQ10, despite the presence of endogenous CoQ9 synthesis. DdsA designed to express at the mitochondria increased accumulation of total CoQ amount in seeds.     

Mechanically induced intracellular calcium waves in osteoblasts demonstrate calcium fingerprints in bone cell mechanotransduction

An early response to mechanical stimulation of bone cells in vitro is an increase in intracellular calcium concentration ([Ca ²⁺]_i). This study analyzed the [Ca ²⁺]_i wave area, magnitude, duration, rise time, fall time, and time to onset in individual osteoblasts for two identical bouts of mechanical stimulation separated by a 30-min rest period. The area under the [Ca ²⁺]_i wave increased in the second loading bout compared to the first. This suggests that rest periods may potentiate mechanically induced intracellular calcium signals. Furthermore, many of the [Ca ²⁺]_i wave parameters were strongly, positively correlated between the two bouts of mechanical stimulation. For example, in individual primary osteoblasts, if a cell had a large [Ca ²⁺]_i wave area in the first bout it was likely to have a large [Ca ²⁺]_i wave area in the second bout (r ² = 0.933). These findings support the idea that individual bone cells have “calcium fingerprints” (i.e., a unique [Ca ²⁺]_i wave profile that is reproducible for repeated exposure to a given stimulus).     

Family Preference for Place of Death Mediates the Relationship between Patient Preference and Actual Place of Death: A Nationwide Retrospective Cross-Sectional Study

Background

Discrepancy between preferred and actual place of death is common in cancer patients. While previous research has elucidated the factors associated with congruence between patients'' preferred and actual place of death, it is not known how the perspective of the family influences the place of death. This study examined whether family preference for place of death mediates the relationship between patient preference and actual place of death.

Methods

A total of 258 cancer patients (home death, n = 142; hospital death, n = 116) who had received terminal care in Japan were analyzed. Measures included patients'' demographic variables, patient and family preferences for place for death, actual place of death, patients'' functional status, use and intensity of home care, availability of inpatient bed, living arrangement, and amount of extended family support.

Results

Patient-family congruence on preferred place of death was 66% in patients who died at home and 47% in patients who died at other places (kappa coefficient: 0.20 and 0.25, respectively). In a multiple logistic regression model, patients were more likely to die at home when patients were male (odds ratio [OR], 95% confidence interval [CI]: 2.53, 1.06–6.05) and when their family preferred death at home (OR, 95% CI: 37.37, 13.82–101.03). A Sobel test revealed that family preference mediated the relationship between patient preference and place of death (p<0.05).

Conclusions

This study is, to our knowledge, the first to unveil the role of the family in the relationship between patient preference and place of death in Japan. In order to honor patients'' wishes to die at home, supporting caregivers in the family may be an essential component of terminal care.     

Alterations of glucose metabolism in Escherichia coli mutants defective in respiratory-chain enzymes

The effects of reduced efficiency of proton-motive force (pmf) generation on glucose metabolism were investigated in Escherichia coli respiratory-chain mutants. The respiratory chain of E. coli consists of two NADH dehydrogenases and three terminal oxidases, all with different abilities to generate a pmf. The genes for isozymes with the highest pmf-generating capacity (NADH dehydrogenase-1 and cytochrome bo? oxidase) were knocked out singly or in combination, using a wild-type strain as the parent. Analyses of glucose metabolism by jar-fermentation revealed that the glucose consumption rate per cell increased with decreasing efficiency of pmf generation, as determined from the growth parameters of the mutants. The highest rate of glucose metabolism was observed in the double mutant, and the lowest was observed in the wild-type strain. The respiration rates of the single-knockout mutants were comparable to that of the wild-type strain, and that of the double mutant was higher, apparently as a result of the upregulation of the remaining respiratory chain enzymes. All of the strains excreted 2-oxoglutaric acid as a product of glucose metabolism. Additionally, all of the mutants excreted pyruvic acid and/or acetic acid. Interestingly, the double mutant excreted L-glutamic acid. Alterations of the fermentation profiles provide clues regarding the metabolic regulation in each mutant.     

Continuous stress-induced dopamine dysregulation augments PAP-I and PAP-II expression in melanotrophs of the pituitary gland

Under continuous stress (CS) in rats, melanotrophs, the predominant cell-type in the intermediate lobe (IL) of the pituitary, are hyperactivated to secrete α-melanocyte-stimulating hormone and thereafter degenerate. Although these phenomena are drastic, the molecular mechanisms underlying the cellular changes are mostly unknown. In this study, we focused on the pancreatitis-associated protein (PAP) family members of the secretory lectins and characterized their expression in the IL of CS model rats because we had identified two members of this family as up-regulated genes in our previous microarray analysis. RT-PCR and histological studies demonstrated that prominent PAP-I and PAP-II expression was induced in melanotrophs in the early stages of CS, while another family member, PAP-III, was not expressed. We further examined the regulatory mechanisms of PAP-I and PAP-II expression and revealed that both were induced by the decreased dopamine levels in the IL under CS. Because the PAP family members are implicated in cell survival and proliferation, PAP-I and PAP-II secreted from melanotrophs may function to sustain homeostasis of the IL under CS conditions in an autocrine or a paracrine manner.     

Efficient plant regeneration and Agrobacterium-mediated transformation of Begonia semperflorens-cultorum

Plant Cell, Tissue and Organ Culture (PCTOC) - Begonia semperflorens-cultorum, known as wax begonia, is one of the most popular Begonia species in which variable commercial cultivars have been...     

MutMap+: Genetic Mapping and Mutant Identification without Crossing in Rice

Advances in genome sequencing technologies have enabled researchers and breeders to rapidly associate phenotypic variation to genome sequence differences. We recently took advantage of next-generation sequencing technology to develop MutMap, a method that allows rapid identification of causal nucleotide changes of rice mutants by whole genome resequencing of pooled DNA of mutant F2 progeny derived from crosses made between candidate mutants and the parental line. Here we describe MutMap+, a versatile extension of MutMap, that identifies causal mutations by comparing SNP frequencies of bulked DNA of mutant and wild-type progeny of M3 generation derived from selfing of an M2 heterozygous individual. Notably, MutMap+ does not necessitate artificial crossing between mutants and the wild-type parental line. This method is therefore suitable for identifying mutations that cause early development lethality, sterility, or generally hamper crossing. Furthermore, MutMap+ is potentially useful for gene isolation in crops that are recalcitrant to artificial crosses.     

A Survey of Companion-Animal Owners Affected by the East Japan Great Earthquake in Iwate and Fukushima Prefectures,Japan

ABSTRACT

The unprecedented East Japan Great Earthquake in March 2011 impacted many humans as well as animals. To date, only national surveys that do not necessarily focus on the heavily impacted areas have been administered, and there is a lack of data on the situation for companion animals and their owners in these areas. This survey was administered between June and November 2012 to pet owners in Iwate (n = 140) and Fukushima (n = 149) Prefectures in north-eastern Japan, areas heavily affected by the disaster. It explored the types of disaster preparations for pets engaged in by owners; the situation on evacuation with pets; the use of, and need for, pet-related support after the disaster; and the associations between pet attachment and disaster-related behaviors of pet owners. In total, 41.2% (n = 119) of all respondents were able to evacuate with their pets, and evacuation rates were especially low in Fukushima. With the exception of preparation of pet food and other supplies, less than 50% of respondents engaged in different types of pet-related disaster preparations. Especially in Fukushima, those who evacuated with their pets were better prepared compared with those who could not. The rate of utilization of support was also low, with less than 50% of respondents utilizing each type of support, regardless of pet-evacuation status and area. The need for support was generally higher during the initial phase (immediately after the disaster; 30–40%) compared with the current phase (20–30%). However, in Fukushima the difference between the initial and current phases was not significant for both those who evacuated with their pets and those who could not. Bivariate analyses indicated mixed results for the association between disaster-related behaviors and pet attachment. Implications for future disaster-prevention measures are discussed.     

Effects of CFTR gene silencing by siRNA or the luminal application of a CFTR activator on fluid secretion from guinea-pig pancreatic duct cells

Aims

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP regulated chloride channel expressed in the apical plasma membrane of pancreatic duct cells where it plays an important role in fluid secretion. The purpose of this study was to elucidate the role of the CFTR chloride channel on ion and fluid secretion from the guinea-pig pancreas by manipulating the expression of CFTR by RNA interference or by luminal application of a CFTR selective activator, MPB91, in isolated cultured pancreatic ducts.

Materials and methods

Using cDNA isolated from the guinea-pig small intestine, fragments of the CFTR gene were generated by polymerase chain reaction and directly sequenced. Two different RNA duplexes for small interference RNA (siRNA) were designed from the sequence obtained. Fluid secretion from the isolated guinea-pig pancreatic ducts was measured using video-microscopy. The amount of CFTR chloride channel or AQP1 water channel expressed in pancreatic ducts was examined by immunoblotting with antibodies against CFTR or AQP1, respectively.

Results

Guinea-pig CFTR consists of 1481 amino acid residues. An additional glutamine residue was found to be inserted between amino acid residues 403 and 404 of human CFTR. Forskolin-stimulated fluid secretion from intact pancreatic ducts was significantly higher in the presence of MPB91 compared to fluid secretion in the absence of MPB91. Both basal and forskolin-stimulated fluid secretion in pancreatic ducts transfected with CFTR specific siRNAs were reduced by ∼50% compared to fluid secretion from ducts transfected with scrambled negative control dsRNAs. The amount of CFTR and AQP1 proteins was reduced to 34% and 45% of control, respectively.

Conclusions

The activity of the CFTR chloride channel or the amount of CFTR protein expressed determines the rate of fluid secretion from the isolated guinea-pig pancreatic ducts.     

Identification of 20-hydroxyecdysone-inducible genes from larval brain of the silkworm, Bombyx mori, and their expression analysis

The insect brain secretes prothoracicotropic hormone (PTTH), which stimulates the prothoracic gland to synthesize ecdysone. The active metabolite of ecdysone, 20-hydroxyecdysone (20E), works through ecdysone receptor (EcR) and ultraspiracle (USP) to initiate molting and metamorphosis by regulating downstream genes. Previously, we found that EcR was expressed in the PTTH-producing neurosecretory cells (PTPCs) in larval brain of the silkworm Bombyx mori, suggesting that PTPCs function as the master cells of development under the regulation of 20E. To gain a better understanding of the molecular mechanism of the 20E control of PTPCs, we performed a comprehensive screening of genes induced by 20E using DNA microarray with brains of day-2 fifth instar silkworm larvae. Forty-one genes showed greater than twofold changes caused by artificial application of 20E. A subsequent semiquantitative screening identified ten genes upregulated by 20E, four of which were novel or not previously identified as 20E-response genes. Developmental profiling determined that two genes, UP4 and UP5, were correlated with the endogenous ecdysteroid titer. Whole-mount in situ hybridization showed exclusive expression of these two genes in two pairs of cells in the larval brain in response to 20E-induction, suggesting that the cells are PTPCs. BLAST searches revealed that UP4 and UP5 are Bombyx homologs of vrille and tarsal-less, respectively. The present study identifies 20E-induced genes that may be involved in the ecdysone signal hierarchies underlying pupal-adult development and/or the 20E regulation of PTPCs.