Localization of Various Forms of the γ Subunit of G Protein in Neural and Nonneural Tissues

Abstract: For a study of the localization of various forms of the γ subunit of G proteins, antibodies were raised in rabbits against peptides that corresponded to partial amino acid sequences of bovine γ₂, γ₃, γ₅, and γ₇. Affinity-purified antibodies against γ₂, γ₃, and γ₅ reacted specifically with γ₂, γ₃, and γ₅, respectively, but the antibody against γ₇ reacted with γ₂, γ₃, and a novel γ subunit, designated γ_s1, as well as with γ₇. Because these antibodies reacted with the respective forms of the γ subunit from rat brain, we investigated the localization of γ subunits in the rat. γ₂ and γ₃ were abundant in all regions in the brain, whereas the concentration of γ₅ and γ₇ was relatively low with the single exception being a high concentration of γ₇ in the striatum. The concentration of γ₂ was consistently high during ontogenic development in the rat brain, whereas γ₃ appeared about a week after birth and their concentrations then increased until a month after birth. In tissues other than the brain, γ₃ was observed only in the pituitary gland, whereas γ₂, γ₅, and γ₇ were found in a variety of tissues. In addition, most tissues contained relatively high concentrations of some other γ subunit, which was detected with an antibody against a γ₇-derived peptide and appeared to be γ_s1. Among cloned cells tested, γ₃ was detected only in PC12 pheochromocytoma cells. Taken together, the results indicated that γ₃ was expressed specifically in neuronal cells, and γ_s1 was the major γ subunit in most nonneural cells. γ₂, γ₅, and γ₇ were distributed in a variety of tissues, but γ₂ was dominant in the brain.     

Development of nitrogen-removal bioreactor using poly(lactic acid) as an energy source.

To control nitrogen such as ammonia in a rearing water of aquatic animals, we developed new bioreactor capable of both nitrification and denitrification. It was consisted of gel-plate immobilized microorganisms and a biodegradable plastic plate composed of three kind of poly(lactic acid) as an energy source of denitrification. When batch treatment experiment by the bioreactor was continuously carried out with an artificial rearing water containing ammonia, nitrogen-removal rate of the bioreactor was approximately 3 g-N/d/m2-gel surface and the activity was maintained for over 3 month without additional energy source. Therefore, the bioreactor would be effective to control nitrogen concentration in rearing water of a closed water circulating system for aquatic animals.     

Artemisinin biosynthesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> and metabolic engineering: questions,challenges, and perspectives

Artemisinin-based combination therapy (ACT) forms the frontline treatment of malaria. Artemisinin, an endoperoxide sesquiterpenoid lactone biosynthesized by Artemisia annua, is the effective medicine that kills malarial parasites. Due to insufficient production of artemisinin for ACT, millions of people lost their lives in past years worldwide. To solve this severe problem, numerous studies have been undertaken to understand artemisinin biosynthesis and to innovate metabolic engineering technology to increase artemisinin yield. Here, we focus on reviewing progresses achieved in understanding biosynthetic pathway, genetic breeding, metabolic engineering, and synthetic biology. Furthermore, based on current knowledge, we discuss multiple fundamental questions and challenges.     

Developmental Changes in Distribution of Acidic Fibroblast Growth Factor in Rat Brain Evaluated by a Sensitive Two-Site Enzyme Immunoassay

We developed a sensitive two-site enzyme immunoassay (EIA) system for acidic fibroblast growth factor (aFGF), using a polyclonal antibody raised in rats. This assay is based on the sandwiching of the antigen between anti-aFGF antibody immunoglobulin G (IgG) coated on plates and biotinylated anti-aFGF antibody IgG; the detection of biotinylated IgG was performed by enzyme reaction of streptavidin-conjugated beta-D-galactosidase (beta-D-galactoside hydrolase; EC 3.2.1.23). Our system was specific for aFGF, because basic fibroblast growth factor, which shares a 55% homology of amino acid sequence with aFGF, hardly cross-reacted at all. The sensitivity of this system (0.2 ng/ml) enabled us to quantify endogenous immunoreactive aFGF in the CNS. Using this two-site EIA system, we examined the levels of aFGF in various regions of rat brain and their developmental changes. At the early stage of neonatal development, i.e., 2 days after birth, all brain regions registered low aFGF levels (less than 10 ng/g tissue). However, at the young adult stage (21- to 49-day-old animals), an extremely high level of aFGF (75-90 ng/g tissue) was found in the ponsmedulla; relatively high levels (30-40 ng/g tissue) were found in the diencephalon and mesencephalon; and comparatively low aFGF levels (5-15 ng/g tissue) were found in various other brain regions such as the frontal cortex, piriform cortex, hippocampus, olfactory bulb, cerebellum, and striatum. This marked change in the regional distribution of aFGF in the rat brain during postnatal development from 2 to 21 days after birth suggests that this factor plays a significant role in the brain during this period.     

Recovery from diabetes in neonatal mice after a low-dose streptozotocin treatment

Administration of streptozotocin (STZ) induces destruction of β-cells and is widely used as an experimental animal model of type I diabetes. In neonatal rat, after low-doses of STZ-mediated destruction of β-cells, β-cells regeneration occurs and reversal of hyperglycemia was observed. However, in neonatal mice, β-cell regeneration seems to occur much slowly compared to that observed in the rat. Here, we described the time dependent quantitative changes in β-cell mass during a spontaneous slow recovery of diabetes induced in a low-dose STZ mice model. We then investigated the underlying mechanisms and analyzed the cell source for the recovery of β-cells. We showed here that postnatal day 7 (P7) female mice treated with 50 mg/kg STZ underwent the destruction of a large proportion of β-cells and developed hyperglycemia. The blood glucose increased gradually and reached a peak level at 500 mg/dl on day 35–50. This was followed by a spontaneous regeneration of β-cells. A reversal of non-fasting blood glucose to the control value was observed within 150 days. However, the mice still showed impaired glucose tolerance on day 150 and day 220, although a significant improvement was observed on day 150. Quantification of the β-cell mass revealed that the β-cell mass increased significantly between day 100 and day 150. On day 150 and day 220, the β-cell mass was approximately 23% and 48.5% of the control, respectively. Of the insulin-positive cells, 10% turned out to be PCNA-positive proliferating cells. Our results demonstrated that, β-cell duplication is one of the cell sources for β-cell regeneration.     

Synchronous culture of Magnetospirillum sp. AMB-1 by repeated cold treatment

Abstract We established a synchronous culture of Magnetospirillum sp. AMB-1 by repeated cold treatment at 5 °C. This is the first reported synchronous culture of a magnetic bacterium. Cold treatment did not affect magnetic particle synthesis or cell morphology. Iron uptake was observed both before and during cell division. The amount of iron uptake was almost equivalent to that of magnetite formation. The proportion of magnetosensitive cells did not change during cell division.     

Fate maps of ventral and dorsal pancreatic progenitor cells in early somite stage mouse embryos

The origins of liver progenitor cells have been extensively studied, but evidence on the origin of pancreatic precursor cells is currently limited. Pancreatic and duodenal homeobox gene 1 (Pdx1) is one of the earliest known markers for the pancreas. A transgenic mouse line expressing green fluorescent protein (GFP) under the control of the Pdx1 promoter showed that Pdx1/GFP expression was first observed in the mid-region of the anterior intestinal portal (AIP) lip at embryonic day (E) 8.5 at the 5-6 somite stage (ss). The liver progenitors were confirmed to originate from separate domains at the lateral endoderm and the inner part of the medial AIP as previously reported (Tremblay and Zaret, 2005), which turned out to lie caudally to the Pdx1/GFP-expressing domain. To confirm if the early Pdx1/GFP-positive cells give rise to the pancreatic bud, we labeled the cells on the lip of the AIP using the carbocyanine dye CM-DiI and traced their fates in 1-4 ss, 5-6 ss and 7-9 ss E8.5 embryos using an ex utero whole embryo culture method. At 1 ss, the ventral pancreas progenitors were observed in the lateral endoderm, not yet being segregated from the liver or gut progenitors. Cells that contributed solely to the ventral pancreas first appeared at the AIP lip from 5 ss. At 5-6 ss, cells from the medial of the AIP lip contributed to the ventral pancreas. The pancreas fate region become narrower as development progresses. At 7-9 ss, the cells contributing to the ventral pancreas resided in a narrow region of the AIP lip. From 5 ss, the right flanking region contributes to the posterior gut, and the left flanking region contributes to the anterior gut. Dorsal pancreatic progenitors originate from the dorsal endoderm at the 3-6 somite level at 7-9 ss, though they have not yet diverged from the dorsal gut progenitors at this stage.