Light- and electron-microscopic study of the endolymphatic sac of the tree frog,Hyla arborea japonica

Summary The endolymphatic sac of the tree frog and its crystals were observed by light- and electron microscopy. Scanning electron microscopy revealed that the crystals have a faceted body and two pointed ends. Light- and transmission electron microscopy revealed that the endolymphatic sac is composed of many small chambers. In their lumina, numerous ghosts of crystals that resulted from decalcification were observed. The ghosts were demarcated by a linear dense material or embedded in a flocculent substance. The epithelium of the endolymphatic sac is simple squamous or cuboidal and peculiar cytoplasmic granules are found in most cells. The granules are surrounded by a limiting membrane and have varying electron density. Some granules contain a core and/or tubular structures. Vacuoles containing large ghosts are also found in the epithelial cells. These ghosts were quite similar to those in the lumen and sometimes coexist with cell debris. The fine structure of the endolymphatic sac and its crystals is discussed.     

Energy dispersive X-ray microanalysis of neurosecretory granules of mouse pituitary on fresh air-dried tissue spreads

Electron microscopy of fresh air dried spreads of unstained posterior lobe tissue from mouse pituitary disclosed neurosecretory granules. Each granule showed a seemingly homogeneous dense core surrounded by a halo and a bounding membrane. The area between granules in the cytoplasm was relatively well preserved. The energy dispersive X-ray microanalysis revealed peaks for sulfur, chlorine and potassium in two granules. The third granule displayed peaks for phosphorus and chlorine. These elements probably contribute to the high electron density of the granules. There was no peak for calcium, in contrast to the dense bodies of human blood platelets.     

Localization of angiotensin-converting enzyme,prolyl endopeptidase and other peptidases in cultured neuronal or glial cells

To determine the cellular localization of nervous tissue peptidases, 7 peptidases and 2 lysosomal marker enzyme activities were measured in cultured mouse and rat cells. Neuronal cells of both species exhibited higher activities of angiotensin-converting enzyme (ACE) and prolyl endopeptidase (Pro-EP) than glial cells did. In contrast, arginyl endopeptidase and lysosomal enzymes (acid phosphatase, β-glucuronidase) in the neuronal cell lines were lower than those in the glial cell lines. Other peptidases (alanyl aminopeptidase, arginyl aminopeptidase, leucyl aminopeptidase, dipeptidyl aminopeptidase) activities were not specifically localized in either cell lines. The effects of cellular differentiation on these peptidase activities in the PC 12h cell line and rat glioblasts were also examined using nerve growth factor (NGF) and glia maturation factor (GMF), respectively. Neuron specific peptidase (ACE and Pro-EP) activities were decreased in PC12h cells cultured with NGF, and Pro-EP activity was increased in the glioblast cells cultured with GMF. These results support the idea that some of the peptidases are differentially localized in neuronal or glial cells, and play physiological roles in central or peripheral neural tissues.     

Redox regulation of ascorbate and glutathione by a chloroplastic dehydroascorbate reductase is required for high-light stress tolerance in Arabidopsis

Chloroplasts are a significant site for reactive oxygen species production under illumination and, thus, possess a well-organized antioxidant system involving ascorbate. Ascorbate recycling occurs in different manners in this system, including a dehydroascorbate reductase (DHAR) reaction. We herein investigated the physiological significance of DHAR3 in photo-oxidative stress tolerance in Arabidopsis. GFP-fused DHAR3 protein was targeted to chloroplasts in Arabidopsis leaves. A DHAR3 knockout mutant exhibited sensitivity to high light (HL). Under HL, the ascorbate redox states were similar in mutant and wild-type plants, while total ascorbate content was significantly lower in the mutant, suggesting that DHAR3 contributes, at least to some extent, to ascorbate recycling. Activation of monodehydroascorbate reductase occurred in dhar3 mutant, which might compensate for the lack of DHAR3. Interestingly, glutathione oxidation was consistently inhibited in dhar3 mutant. These findings indicate that DHAR3 regulates both ascorbate and glutathione redox states to acclimate to HL.     

PACAP and NGF cooperatively enhance choline acetyltransferase activity in postnatal basal forebrain neurons by complementary induction of its different mRNA species

Both nerve growth factor (NGF) and pituitary adenylate cyclase activating polypeptide (PACAP) have neurotrophic effects on basal forebrain cholinergic neurons. They promote differentiation, maturation, and survival of these cholinergic neurons in vivo and in vitro. Here we report on the cooperative effects of NGF and PACAP on postnatal, but not embryonic, cholinergic neurons cultured from rat basal forebrain. Combined treatment with NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and PACAP induced an additive increase in choline acetyltransferase (ChAT) activity. There were no cooperative effects on the number of cholinergic neurons, suggesting that ChAT mRNA expression had been induced in each cholinergic neuron. Further analysis revealed that NGF and PACAP led to complementary induction of different ChAT mRNA species, thus enhancing total ChAT mRNA expression. These results explain the cooperative neurotrophic action of NGF and PACAP on postnatal cholinergic neurons.     

Conditional expression of MCM7 increases tumor growth without altering DNA replication activity

The minichromosome maintenance (MCM) 2-7 complex is a putative DNA helicase complex that facilitates the initiation of DNA replication. Here, we generated a cell line MCM7(+/-)/MCM7-FLAG, in which one allele of MCM7 is mutated whereas a tetracycline-repressible promoter could manipulate the expression of exogenous MCM7 protein. Overexpressed MCM7 protein supports efficient DNA replication of Epstein-Barr virus oriP and rapid formation of tumors in nude mice without altering the activity of cellular DNA replication. This system provides a unique setting for studying the function of MCM7 and for screening for potential therapeutics for malignant tumors.     

Comparison of the usability of an automatic sleep staging program via portable 1-channel electroencephalograph and manual sleep staging with traditional polysomnography

Sleep and Biological Rhythms - Automatic algorithms are a proposed alternative to manual assessment of polysomnography data for analyzing sleep structure; however, none are acceptably accurate for...     

Mutant firefly luciferase enzymes resistant to the inhibition by sodium chloride

Objectives

Firefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study was aimed at obtaining mutant luciferase enzymes resistant to the sodium chloride inhibition.

Results

We first obtained two mutant luciferase enzymes whose inhibition were alleviated and determined the mutations to be Val288Ile and Glu488Val. Under medical dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride.

Conclusions

The mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The luciferase CR may be extensively useful in any bioassay which includes firefly luciferase and is employed in the presence of sodium chloride.