Reproducibility and usability of chronic virus infection model using agent-based simulation; comparing with a mathematical model

We created agent-based models that visually simulate conditions of chronic viral infections using two software. The results from two models were consistent, when they have same parameters during the actual simulation. The simulation results comprise a transient phase and an equilibrium phase, and unlike the mathematical model, virus count transit smoothly to the equilibrium phase without overshooting which correlates with actual biology in vivo of certain viruses. We investigated the effects caused by varying all the parameters included in concept; increasing virus lifespan, uninfected cell lifespan, uninfected cell regeneration rate, virus production count from infected cells, and infection rate had positive effects to the virus count during the equilibrium period, whereas increasing the latent period, the lifespan-shortening ratio for infected cells, and the cell cycle speed had negative effects. Virus count at the start did not influence the equilibrium conditions, but it influenced the infection development rate. The space size had no intrinsic effect on the equilibrium period, but virus count maximized when the virus moving speed was twice the space size. These agent-based simulation models reproducibly provide a visual representation of the disease, and enable a simulation that encompasses parameters those are difficult to account for in a mathematical model.     

An interspecific linkage map of SSR and intronic polymorphism markers in tomato

Despite the collection and availability of abundant tomato genome sequences, PCR-based markers adapted to large scale analysis have not been developed in tomato species. Therefore, using public genome sequence data in tomato, we developed three types of DNA markers: expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers (TES markers), genome-derived SSR markers (TGS markers) and EST-derived intronic polymorphism markers (TEI markers). A total of 2,047 TES, 3,510 TGS and 674 TEI markers were established and used in the polymorphic analysis of a cultivated tomato (Solanum lycopersicum) ‘LA925’ and its wild relative Solanum pennellii ‘LA716’, parents of the Tomato-EXPEN 2000 mapping population. The polymorphic ratios between parents revealed by the TES, TGS and TEI markers were 37.3, 22.6 and 80.0%, respectively. Those showing polymorphisms were used to genotype the Tomato-EXPEN 2000 mapping population, and a high-density genetic linkage map composed of 1,433 new and 683 existing marker loci was constructed on 12 chromosomes, covering 1,503.1 cM. In the present map, 48% of the mapped TGS loci were located within heterochromatic regions, while 18 and 21% of TES and TEI loci, respectively, were located in heterochromatin. The large number of SSR and SNP markers developed in this study provide easily handling genomic tools for molecular breeding in tomato. Information on the DNA markers developed in this study is available at http://www.kazusa.or.jp/tomato/.     

Type 2 diabetes mellitus in a non-obese mouse model induced by Meg1/Grb10 overexpression

We assessed the possibility of C57BL/6-Tg (Meg1/Grb10)isn(Meg1 Tg) mice as a non-obese type 2 diabetes (2DM) animal model. Meg1 Tg mice were born normal, but their weight did not increase as much as normal after weaning and showed about 85% of normal size at 20 weeks of age. Body mass index of Meg1 Tg mice was also smaller than that of control mice. The glucose tolerance test and insulin tolerance test showed that Meg1 Tg mice had reduced ability to normalize the blood glucose level. Blood urea nitrogen (BUN) in Meg1 Tg mice (19.6 +/- 1.2 mg/dl) was significantly lower than in controls (22.0 +/- 0.8 mg/dl), while plasma triglyceride, insulin, adiponectin, and resistin levels were significantly higher (202.0 +/- 23.4 mg/dl vs 146.3 +/- 23.4 mg/dl, 152.4 +/- 16.3 pg/ml vs 88.1 +/- 16.9 pg/ml, 74.4 +/- 10.9 microg/ml vs 48.3 +/- 7.0 microg/ml, and 4.0 +/- 0.2 ng/ml vs 3.6 +/- 0.2 ng/ml, respectively). Body, visceral fat weight and liver weights were significantly lower (19.6 +/- 0.4 g vs 24.3 +/- 0.3 g, 376.7 +/- 29.6 mg to 507.5 +/- 23.0 mg, and 906.0 +/- 41.8 mg to 1,001.0 +/- 15.1 mg, respectively). Thus, hyperinsulinemia observed in Meg1 Tg mice indicates that their insulin signaling pathway is somehow inhibited. With high fat diet, the diabetes onset rate of Meg1 Tg mice increased up to 60%. These results suggest that Meg1 Tg mice resemble human 2DM.     

Effects of growth temperature and winter duration on leaf phenology of a spring ephemeral (Gagea lutea) and a summergreen forb (Maianthemum dilatatum)

Effects of growth temperature and winter duration on leaf longevity were compared between a spring ephemeral, Gagea lutea, and a forest summergreen forb, Maianthemum dilatatum. The plants were grown at day/night temperatures of 25/20°C and 15/10°C after a chilling treatment for variable periods at 2°C. The temperature regime of 25/20°C was much higher than the mean air temperatures for both species in their native habitats. Warm temperature of 25/20°C and/or long chilling treatment shortened leaf longevity in G. lutea, but not in M. dilatatum. The response of G. lutea was consistent with that reported for other spring ephemerals. Air temperature increases as the vegetative season progresses. The decrease in leaf longevity in G. lutea under warm temperature condition ensures leaf senescence in summer, an unfavorable season for its growth. This also implies that early leaf senescence could occur in years with early summers. Warm spring temperatures have been shown to accelerate the leafing-out of forest trees. The decrease in leaf longevity due to warm temperature helps synchronize the period of leaf senescence roughly with the time of the forest canopy leaf-out. Prolonged winter due to late snowmelt has been shown to shorten the vegetative period for spring ephemerals. The decrease in leaf longevity due to long chilling treatment would correspond with this shortened vegetative period.     

Subcellular localization of overexpressed maize AChE gene in rice plant

The ACh-mediated system consisting of acetylcholine (ACh), acetylcholine receptor (AChR) and acetylcholinesterase (AChE) is fundamental for nervous system function in animals and insects. Although plants lack a nervous system, both ACh and ACh-hydrolyzing activity have been widely recognized in the plant kingdom. The function of the plant ACh-mediated system is still unclear, despite more than 30 years of research. To understand ACh-mediated systems in plants, we previously purified maize AChE and cloned the corresponding gene from maize seedlings (Plant Physiology). In a recent paper in Planta, we also purified and cloned AChE from the legume plant siratro (Macroptilium atropurpureum). In comparison with electric eel AChE, both plant AChEs showed enzymatic properties of both animal AChE and animal butyrylcholinesterase. On the other hand, based on Pfam protein family analysis, both plant AChEs contain a consensus sequence of the lipase GDSL family, while the animal AChEs possess a distinct alpha/beta-hydrolase fold superfamily sequence, but no lipase GDSL sequence. Thus, neither plant AChE belongs to the well-known AChE family, which is distributed throughout the animal kingdom. To address the possible physiological roles of plant AChEs, we herein report our data from the immunological analysis of the overexpressed maize AChE gene in plants.Key words: acetylcholinesterase activity, maize AChE gene, overexpression, rice, subcellular localizationIn the animal ACh-mediated system, ACh serves to propagate an electrical stimulus across the synaptic junction. At the presynaptic neuron end, an electrical impulse triggers the release of ACh, which accumulates in vesicles into the synaptic cleft via exocytosis. ACh then binds to an ACh receptor (AChR) on the postsynaptic neuron surface, and the ACh-AChR binding induces subsequent impulses to the postsynaptic neuron. Finally, ACh, which is released again by the receptor into the synaptic cleft, is rapidly degraded by acetylcholinesterase (AChE; E.C.3.1.1.7).^1,2 ACh and AChE,^3,4,5 and choline acetyltransferase activity that takes part during synthesis of ACh^6,7 have been recognized in plants. AChR has not been identified in plant cells so far. However, so-called “ACh-binding sites” were detected in membrane fractions from some bean seedlings^8,9 and evidence was also detected in plant organelles, such as chloroplasts¹⁰ and tonoplasts¹¹ using pharmacological methods.Concerning the function of the ACh-mediated system in plants, Momonoki^12,13 has proposed that it results in an asymmetric distribution of hormones and substances due to gravity stimuli, as well as changes in ACh content, AChE activity and Ca²⁺ level in response to heat stress. However, all these phenomena have been investigated using indirect techniques. Thus, to understand the plant ACh-mediated system, we purified AChEs and cloned the AChE genes from maize¹⁴ and siratro¹⁵ seedlings. The maize AChE was found to exist as two types of 88-kDa homodimers, which in turn consisted of disulfide-linked and noncovalently-linked 42- to 44-kDa subunits.¹⁴ The siratro AChE might exist as a disulfide-linked 125-kDa homotrimer consisting of 41- or 42-kDa subunits.¹⁵ The plant AChEs apparently from various quaternary structures, depending on the plant species, similar to animal AChEs. Furthermore, maize and siratro AChEs showed enzymatic properties of both animal AChE and animal butyrylcholinesterase, compared with electric eel AChE.¹⁵In this addendum, we report our recent immunohistochemical study using an antibody against maize AChE. In order to overexpress the maize AChE gene in rice plants, we constructed a plasmid for the sense expression of the AChE gene by cloning it into the pT7 Blue vector. The maize AChE gene¹⁴ was introduced behind the maize ubiquitin 1 promoter (Ubi) in the p2K-1⁺ plant expression vector. The Ubi::maize AChE and control (p2K-1⁺ only) plasmid were introduced into Agrobacterium tumefaciens EHA 101, which was transformed into rice (Nihonbare) via Agrobacterium-mediated transformation methods.¹⁶ The maize AChE transgenic plants exhibited approximately 650-fold higher AChE activity than was observed in the control plants but no phenotypic changes between transgenic and control plants. The subcellular localization of AChE was observed by immunofluorescence in paraffin-embedded leaf and stem tissues of transgenic rice plants. The maize AChE protein was detected in extracellular spaces in the leaf and stem of the plants (Fig. 1). Therefore, plant AChEs may function in the extracellular space, similar to some isoforms of animal AChE.^2,17Open in a separate windowFigure 1Subcellular localization of maize AChE in leaf and stem of transgenic rice. (A) Leaf cross-section of transgenic rice; (B) leaf cross-section of control; (C) stem cross-section of transgenic rice; (D) stem cross-section of control. Each section was probed with maize AChE antibody and then visualized with Alexa Fluor 488-conjugated secondary antibody. Control indicates rice plants transfected with p2K-1⁺ vector only. Arrowheads indicate localization of maize AChE.Most of the AChE activity in the root was associated with cell wall materials.¹⁸ The computer-assisted cellular sorting prediction program TargetP presumed that our purified maize AChE¹⁴ is targeted to the secretory pathway via the endoplasmic reticulum. Furthermore, the SOSUI program (http://sosui.proteome.bio.tuat.ac.jp / sosuiframe0.html), which discriminates between membrane and soluble proteins, showed that the maize AChE does not contain any likely transmembrane helical regions, which are features of proteins that associate with the lipid bilayers of the cell membrane. These findings suggested that the maize AChE might be localized at the cell wall. However, in an earlier work,¹³ we speculated that AChE is localized at the plasmodesmatal cell-to-cell interface and that it plays a role in regulation of the plasmodesmatal channel as a constituent of the ACh-mediated system. We improved our hypothesis of the role of the ACh-mediated system in a paper in Plant Physiol.¹⁴The results based on fluorescence-immunohistochemistry in transgenic rice plants reported in this paper confirmed that the maize AChE is localized at the cell wall. Here we propose again our hypothesis of an ACh-mediated system including this new finding; the system might be localized to the extracellular region around the plasmodesmatal channel and might conduct cell-to-cell trafficking by channel gating regulation. Adjoining cells in plant tissues are interconnected via plasmodesmata, which allow the trafficking of low-molecular-mass materials across the cell wall between two cells. According to a recent model,¹⁹ transport of these substances could be regulated by the opening and/or closing of conductive channels to prevent infection by pathogens and to selectively control trafficking through the plasmodesmata. Furthermore, it has been speculated that morphoregulatory proteins around the plasmodesmata could be involved in channel regulation.²⁰ Therefore, the ACh-mediated system might regulate the opening and/or closing of channels by interactions with morphoregulatory proteins at the cell wall matrix surrounding the plasmodesmata. Further research will be required to clarify the precise physiological roles of plant AChEs.     

Taurine Alters Respiratory Gas Exchange and Nutrient Metabolism in Type 2 Diabetic Rats

Objective: To assess the effect of taurine supplementation on respiratory gas exchange, which might reflect the improved metabolism of glucose and/or lipid in the type 2 diabetic Otsuka Long‐Evans Tokushima Fatty (OLETF) rats. Research Methods and Procedures: Male OLETF rats (16 weeks of age) were randomly divided into two groups: unsupplemented group and taurine‐supplemented (3% in drinking water) group. After 9 weeks of treatment, indirect calorimetry and insulin tolerance tests were conducted. The amounts of visceral fat pads, tissue glycogen, the blood concentrations of glucose, triacylglycerol, taurine, and electrolytes, and the level of hematocrit were compared between groups. A nondiabetic rat strain (Long‐Evans Tokushima Otsuka) was used as the age‐matched normal control. Results: The indirect calorimetry showed that the treatment of OLETF rats with taurine could reduce a part of postprandial glucose oxidation possibly responsible for the increase of triacylglycerol synthesis in the body. Taurine supplementation also improved hyperglycemia and insulin resistance and increased muscle glycogen content in the OLETF rats. Supplementation with taurine increased the blood concentration of taurine and electrolyte and fluid volume, all of which were considered to be related to the improvement of metabolic disturbance in OLETF rats. Discussion: Taurine supplementation may be an effective treatment for glucose intolerance and fat/lipid accumulation observed in type 2 diabetes associated with obesity. These metabolic changes might be ascribed, in part, to the alteration of circulating blood profiles, where the improved hyperglycemia and/or the blood accumulation of taurine itself would play roles.     

Glucokinase is concentrated in insulin-secretory granules of pancreatic B-cells

We immunohistochemically examined the distribution of glucokinase (GK) in the B-cells of pancreatic islets of normal rats. GK was stained punctately in the cytoplasm of B-cells when examined under the light microscope. By use of a double-immunostaining technique, most of the GK immunoreactivity was observed to be colocalized with insulin immunoreactivity. Electron microscopic examination by the immunogold method revealed that GK immunoreactivity was predominantly located within insulin-secretory granules of pancreatic B-cells. Accepted: 20 April 1999     

Birth of offspring after transfer of Mongolian gerbil (Meriones unguiculatus) embryos cryopreserved by vitrification

The Mongolian gerbil (Meriones unguiculatus) has been used as a laboratory species in many fields of research, including neurology, oncology, and parasitology. Although the cryopreservation of embryos has become a useful means to protect valuable genetic resources, its application to the Mongolian gerbil has not yet been reported. In this study, we investigated the in vitro and in vivo developmental competence of Mongolian gerbil embryos cryopreserved by vitrification. In vivo-fertilized embryos were vitrified on the day of collection using the ethylene glycol (EG)-based solutions EFS20 and EFS40, which contained 20% and 40% EG, respectively, in PB1 containing 30% (w/v) Ficoll 70 and 0.5 M sucrose. First, we compared one-step and two-step vitrification protocols. In the one-step method, the embryos were directly transferred into the vitrification solution (EFS40), whereas in the two-step method, the embryos were exposed serially to EFS20 and EFS40 and then vitrified. After liquefying (thawing), late two-cell embryos (collected on day 3) vitrified by the two-step method showed significantly better rates of in vitro development to the morula stage compared to those vitrified by the one-step method (65% vs. 5%, P < 0.0001). We then examined whether the same two-step method could be applied to early two-cell embryos (collected on day 2), four-cell embryos (day 4), morulae (day 5), and blastocysts (day 6). After liquefying, 87%-100% of the embryos were morphologically normal in all groups, and 23% and 96% developed to the compacted morula stage from early two- and four-cell embryos, respectively. After transfer into recipient females, 3% (4/123), 1% (1/102), 5% (4/73), and 10% (15/155) developed to full-term offspring from vitrified and liquefied early two-cell embryos, late two-cell embryos, morulae, and blastocysts, respectively. This demonstrates that Mongolian gerbil embryos can be safely cryopreserved using EG-based vitrification solutions.     

Increase of total homocysteine concentration in cerebrospinal fluid in patients with Alzheimer's disease and Parkinson's disease

We determined the concentrations of free homocysteine (HC) and total HC in the cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD) or Parkinson's disease (PD) in order to elucidate whether HC is related to the pathogenesis of these neurodegenerative diseases. The concentration of free HC did not differ significantly from that of the normal controls, while the concentration of total HC was significantly higher in the AD and PD patients (+31% in AD,+31% in PD; p<0.05). These findings suggest that an increase of total HC concentration in the brain is commonly seen in patients with AD and PD, and this may be related to the pathogenesis of these two diseases.