Uptake of transmitter amino acids by glial plasmalemmal vesicles from different regions of rat central nervous system

From rat hippocampal homogenate, we recently isolated a novel subcellular fraction richly containing glial plasmalemmal vesicles (GPV), which takes up glutamate remarkably as a synaptosomal fraction [Y. Nakamura et al. (1993) Glia, 9, 48–56]. In the present study, we prepared GPV from different regions of rat CNS, namely olfactory bulb (Ob), cerebral cortex (Cx), caudatoputamen (Cp), hippocampus (Hp), cerebellum (Ce) and spinal cord (Sc), and analyzed their activities of Na⁺-dependent uptake of following neurotransmitters and a related compound; glutamate, -aminobutyrate (GABA), glycine, dopamine and choline. The uptake activities of these amino acids were not significantly different between GPV and synaptosomes in each region. Regionally, however, the activities were varied considerably. The activities of glutamate uptake revealed in the following rank order: Cx, Hp, Cp>Ce, Ob>Sc. GABA uptake activities were: Ce>Ob, Cx, Hp>Cp, while glycine uptake activities were: Sc, Ce>Ob, Cp, Cx, Hp. On the other hand, the uptake activities of dopamine and choline were quite different between GPV and synaptosomes. Synaptosomal fraction from Cp took up dopamine in a high activity; however, GPV from the same tissue hardly showed the uptake activity. Choline was taken up by synaptosomes prepared from Hp but not by GPV.     

Work at cold storage

1. 1. Work activities in cold storage rooms were assessed by a mailed questionnaire survey of cold storage facilities in Japan.

2. 2. There are nearly 4000 cold storage facilities and about 80% are being kept at temperatures below −20°C.

3. 3. The chief items of stock in storage were marine products, livestock products, frozen food and agricultural products.

4. 4. Methods used for loading and unloading in cold storage rooms are forklift, manual handling, and automatic machines.

5. 5. Use of forklifts appeared to be widespread.

6. 6. Working time differed according to the ambient temperature of the cold storage rooms.

7. 7. Common ailments of cold storage workers are lumbago, bronchitis, neuralgia etc.

A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.

The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection.     

Analysis of cell viability and differential activity of mouse hepatocytes under 3D and 2D culture in agarose gel

Three-dimensional (3D) and two-dimensional (2D) cultures of hepatocytes in various concentrations (0.3–0.7%) of agarose gel revealed that the hepatocytes under 3D cultures in 0.3% agarose gel possess long-term (>3 weeks) viability, significant self-assembly to form tissue like aggregates, low lactate dehydrogenase release and high albumin synthesis. These were in contrast to 2D culture of hepatocytes. Our results suggest that the 3D culture of hepatocytes in agarose gel favors aggregate formation of functionally active cells and would be useful for liver transplantation as well as to analyze hepatocytes biology.     

Defective maintenance of intracellular Ca^2＋ homeostasis is linked to increasedmuscle fatigability in the MG29 null mice

Mitsugumin 29 (MG29) is a transmembrane protein that is normally found in the triad junction of skeletal muscle. Our previous studies have shown that targeted deletion of rag29 from the skeletal muscle resulted in abnormality of the triad junction structure, and also increased susceptibility to muscle fatigue. To elucidate the basis of these effects, we investigated the properties of Ca^2 uptake and -release in toxin-skinned Extensor Digitorium Longus (EDL) muscle fibers from control and rag29 knockout mice. Compared with the control muscle, submaximal Ca^2 uptake into the sarcoplasmic reticulum (SR) was slower and the storage of Ca^2 inside the SR was less in the mutant muscle, due to increased leakage process of Ca^2 movement across the SR. The leakage pathway is associated with the increased sensitivity of Ca^2 /caffeine -induced Ca^2 release to myoplasmic Ca^2 . Therefore, the increased fatigability of mutant EDL muscles can result from a combination of a slowing of Ca^2 uptake, modification of Ca^2 -induced Ca^2 release (CICR), and a reduction in total SR Ca^2 content.     

Isolation and characterization of three novel serine protease genes from Xenopus laevis

Three novel cDNAs encoding serine proteases, that may play a role in early vertebrate development, have been identified from Xenopus laevis. These Xenopus cDNAs encode trypsin-like serine proteases and are designated Xenopus embryonic serine protease (Xesp)-1, Xesp-2, and XMT-SP1, a homolog of human MT-SP1. Xesp-1 is likely to be a secreted protein that functions in the extracellular space. Xesp-2 and XMP-SP1 are likely to be type II membrane proteases with multidomain structures. Xesp-2 has eight low density lipoprotein receptor (LDLR) domains and one scavenger receptor cysteine-rich (SRCR) domain, and XMT-SP1 has four LDLR domains and two CUB domains. The temporal expressions of these serine protease genes show distinct and characteristic patterns during embryogenesis, and they are differently distributed in adult tissues. Overexpression of Xesp-1 caused no significant defect in embryonic development, but overexpression of Xesp-2 or XMT-SP1 caused defective gastrulation or apoptosis, respectively. These results suggest that these proteases may play important roles during early Xenopus development, such as regulation of cell movement in gastrulae.     

Molecular cloning of cDNA encoding a drosophila ryanodine receptor and functional studies of the carboxyl-terminal calcium release channel

Ryanodine is a plant alkaloid that was originally used as an insecticide. To study the function and regulation of the ryanodine receptor (RyR) from insect cells, we have cloned the entire cDNA sequence of RyR from the fruit fly Drosophila melanogaster. The primary sequence of the Drosophila RyR contains 5134 amino acids, which shares approximately 45% identity with RyRs from mammalian cells, with a large cytoplasmic domain at the amino-terminal end and a small transmembrane domain at the carboxyl-terminal end. To characterize the Ca(2+) release channel activity of the cloned Drosophila RyR, we expressed both full-length and a deletion mutant of Drosophila RyR lacking amino acids 277-3650 (Drosophila RyR-C) in Chinese hamster ovary cells. For subcellular localization of the expressed Drosophila RyR and Drosophila RyR-C proteins, green fluorescent protein (GFP)-Drosophila RyR and GFP-Drosophila RyR-C fusion constructs were generated. Confocal microscopic imaging identified GFP-Drosophila RyR and GFP-Drosophila RyR-C on the endoplasmic reticulum membranes of transfected cells. Upon reconstitution into the lipid bilayer membrane, Drosophila RyR-C formed a large conductance cation-selective channel, which was sensitive to modulation by ryanodine. Opening of the Drosophila RyR-C channel required the presence of microM concentration of Ca(2+) in the cytosolic solution, but the channel was insensitive to inhibition by Ca(2+) at concentrations as high as 20 mM. Our data are consistent with our previous observation with the mammalian RyR that the conduction pore of the calcium release channel resides within the carboxyl-terminal end of the protein and further demonstrate that structural and functional features are essentially shared by mammalian and insect RyRs.     

Comparison of Ca(2+) sparks produced independently by two ryanodine receptor isoforms (type 1 or type 3)

The molecular determinants of a Ca(2+) spark, those events that determine the sudden opening and closing of a small number of ryanodine receptor (RyR) channels limiting Ca(2+) release to a few milliseconds, are unknown. As a first step we investigated which of two RyR isoforms present in mammalian embryonic skeletal muscle, RyR type 1(RyR-1) or RyR type 3 (RyR-3) has the ability to generate Ca(2+) sparks. Their separate contributions were investigated in intercostal muscle cells of RyR-1 null and RyR-3 null mouse embryos. A comparison of Ca(2+) spark parameters of RyR-1 null versus RyR-3 null cells measured at rest with fluo-3 showed that neither the peak fluorescence intensity (DeltaF/F(o) = 1.25 +/- 0.7 vs. 1.55 +/- 0.6), spatial width at half-max intensity (FWHM = 2.7 +/- 1.2 vs. 2.6 +/- 0.6 microm), nor the duration at half-max intensity (FTHM = 45 +/- 49 vs. 43 +/- 25 ms) was significantly different. Sensitivity to caffeine (0.1 mM) was remarkably different, with sparks in RyR-1 null myotubes becoming brighter and longer in duration, whereas those in RyR-3 null cells remained unchanged. Controls performed in double RyR-1/RyR-3 null cells obtained by mice breeding showed that sparks were not observed in the absence of both isoforms in >150 cells imaged. In conclusion, 1) RyR-1 and RyR-3 appear to be the only intracellular Ca(2+) channels that participate in Ca(2+) spark activity in embryonic skeletal muscle; 2) except in their responsiveness to caffeine, both isoforms have the ability to produce Ca(2+) sparks with nearly identical properties, so it is rather unlikely that a single RyR isoform, when others are also present, would be responsible for Ca(2+) sparks; and 3) because RyR-1 null cells are excitation-contraction (EC) uncoupled and RyR-3 null cells exhibit a normal phenotype, Ca(2+) sparks result from the inherent activity of small clusters of RyRs regardless of the participation of these RyRs in EC coupling.     

Distribution of dorsal-forming activity in precleavage embryos of the Japanese newt, Cynops pyrrhogaster: effects of deletion of vegetal cytoplasm, UV irradiation, and lithium treatment

Two types of axis-deficient embryos developed after deletion of the vegetal cytoplasm: wasp-shaped embryos and permanent-blastula-type embryos. In situ hybridization revealed that neither type of axis-deficient embryo expressed goosecoid or pax-6. brachyury was expressed in the constricted waist region of the wasp-shaped embryos but was not expressed in the permanent-blastula-type embryos. Further, we examined the effect of UV irradiation on Japanese newt embryos. Surprisingly, UV-irradiated Japanese newt eggs formed hyperdorsalized embryos. These embryos gastrulated in an irregular circular fashion with goosecoid expression in the circular equatorial region. At tailbud stage, these embryos formed a proboscis which is very reminiscent of that formed in hyperdorsalized Xenopus embryos. Transplantation of the marginal region of the UV-irradiated embryos revealed that the entire marginal zone had organizer activity. Thus we conclude that UV hyperdorsalizes Japanese newt embryos. Finally, lithium treatment of normal embryos at the 32-cell stage also resulted in hyperdorsalization. Lithium treatment of vegetally deleted embryos had two distinct results. Lithium treatment of permanent-blastula-type embryos did not result in the formation of dorsal axial structures, while the same treatment reinduced gastrulation and dorsal axis formation in the wasp-shaped embryos. Based on these results, we propose a model for early axis specification in Japanese newt embryos. The model presented here is fundamentally identical to the Xenopus model, with some important modifications. The vegetally located determinants required for dorsal development (dorsal determinants, DDs) are distributed over a wider region at fertilization in Japanese newt embryos than in Xenopus embryos. The marginal region of the Japanese newt embryo at the beginning of development overlaps with the field of the DDs. Gastrulation is very likely to be a dorsal marginal-specific property, while self-constriction is most probably a ventral marginal-specific property in Japanese newt embryos.