Two Types of Apoptotic Cell Death of Rat Central Nervous System-Derived Neuroblastoma B50 and B104 Cells: Apoptosis Induced During Proliferation and After Differentiation

Abstract: We describe here two types of apoptotic cell death observed in the rat CNS-derived neuroblastoma B50 and B104 cells. One type was induced by dibutyryl cyclic AMP (DBcAMP) after differentiation, and the other was induced by treatment of proliferating cells with cycloheximide. When B50 and B104 cells were treated with 1 m M DBcAMP in the presence of 0.5% fetal calf serum, they began to extend neurites within 12 h and differentiated into neurons at 24 h, as reported previously. However, further cultivation with DBcAMP for up to 72 h led to flotation and, finally, death. Death was by apoptosis as shown by chromatin condensation and DNA fragmentation. Addition of a protein kinase A inhibitor or removal of DBcAMP after differentiation suppressed apoptosis, indicating the involvement of cyclic AMP and protein kinase A in apoptotic cell death. Cell death was also induced in proliferating cells without neurite outgrowth by treatment with cycloheximide. The death was also judged to be by apoptosis based on chromatin condensation and apoptotic body formation, although DNA fragmentation into small sizes was not detected. Both types of cell death showed similar responses to inhibitors for protein kinases and protein phosphatases.     

Completion of first meiosis by sperm penetration in vitro of bovine oocytes inhibited at metaphase-I with dimethylsulphoxide

The effects of dimethylsulphoxide (DMSO) on immature oocytes during maturation in culture and following penetration by spermatozoa were examined. Germinal vesicle breakdown (GVBD) was observed in all oocytes cultured in the maturation medium supplemented with 2, 4 and 8% DMSO. When the oocytes were cultured in medium with 8% DMSO, 95% (57 60 ) of them were inhibited at prometaphase-I. Cumulus cells were significantly (P<0.05) beneficial for resumption of oocyte nuclear maturation during further culture in the maturation medium for 4, 8 and 24 h after DMSO treatment. When the oocytes were additionally cultured for 4 and 8 h in the maturation medium after DMSO treatment, the proportions of oocytes reaching metaphase-II were significantly (P<0.05) higher in those cultured with spermatozoa than without (68 vs 49% and 84 vs 56%, respectively). These results indicate that 8% DMSO does not affect GVBD of oocytes, but conversely it inhibits oocytes at prometaphase-I, and that cumulus cells are important for recovery from DMSO inhibition and for the resumption of nuclear maturation of oocytes. Sperm penetration was also found to stimulate the completion of meiotic maturation of oocytes inhibited at metaphase-I with 8% DMSO.     

The importance of NaCl concentration in a chemically defined medium for the development of bovine oocytes matured and fertilized in vitro

Bovine oocytes matured and fertilized in vitro were cultured in a chemically defined medium (modified Tyrode's solution) without glucose. When different concentrations of NaCl were added to the medium, the proportions of embryos developed to the >/=8-cell, morula and blastocyst stages 96, 144 and 192 h post insemination, respectively, were significantly higher at 89 to 114 mM than 64 to 76 and 126 to 139 mM NaCl. A high proportion (28%) of blastocyst-stage embryos 192 h post insemination was obtained at 89 mM NaCl. When calculated osmolarity in the medium with 64 mM NaCl was varied by adding D-sorbitol, significantly higher proportions of morula-stage embryos were obtained at 265 to 315 mOsm (27 to 38%) than 215 (9%) and 365 (2%) mOsm, but the development to the blastocyst stage was difficult at any osmolarities (215 to 365 mOsm) tested. In the medium with a fixed osmolarity (315 mOsm) but with different concentrations (64 to 114 mM) of NaCl, there were no differences in the proportions (29 to 33%) of morula-stage embryos among different NaCl concentrations. However, significantly higher proportions of embryos developed to the blastocyst stage at 89 to 101 mM (22 to 23%) than 64 to 76 (0 to 9%) and 114 (11%) mM NaCl. When Cl- concentration in the medium with 64 mM NaCl was adjusted by adding choline chloride, significantly higher proportions of embryos developed to the morula stage at 97 to 122 mM (32 to 40%) than 72 (6%) and 147 (2%) mM Cl-, but few embryos developed to the blastocyst stage at any Cl- concentrations (72 to 147 mM) tested. In the medium with 64 or 114 mM NaCl and each with 2 different Na (+)K (+) ratios, there were no differences in the proportions of morula- and blastocyst-stage embryos between different Na+ K+ ratios (31 and 39 at 64 mM NaCl, and 39 and 47 at 114 mM NaCl) at each NaCl concentration. When glucose was added to the medium with 89 mM NaCl 120 h postinsemination, there were no significant differences in the proportions (40 to 48%) of morula-stage embryos 144 h post insemination among different concentrations (0 to 6.95 mM) of glucose. The proportion (33%) of blastocysts 192 h post insemination at 2.78 mM glucose was significantly higher than the values at 0 (22%), 5.56 (19%) and 6.95 (15%) mM but not different compared with the values at 1.39 (23%) and 4.17 (28%) mM. In conclusion, NaCl concentration in a defined medium is one of the most important factors for the development of bovine embryo to the blastocyst stage, but the development of embryos up to the morula stage is also regulated by osmolarity and/or Cl-concentration.     

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.     

Transmembrane signal transduction by the Escherichia coli osmotic sensor, EnvZ: intermolecular complementation of transmembrane signalling

Summary
The Escherichia coli regulatory proteins, EnvZ and OmpR, are crucially involved in expression of the outer membrane proteins OmpF/OmpC in response to the medium osmolarity. The EnvZ protein is presumably a membrane-located osmotic sensor (or signal transducer), which exhibits both kinase and phosphatase activities specific for the OmpR protein. To examine the functional importance of the membrane-spanning segments (named TM1 and TM2) of EnvZ molecules in transmembrane signalling, a set of EnvZ mutants, each having amino acid substitutions within the membrane-spanning regions, was characterized in terms of both their in vivo phenotype and in vitro catalytic activities. One of them, characterized further, has an amino acid change (Pro-41 to Ser or Leu) In TM1, and appeared to be defective in its phosphatase activity but not in its kinase activity. This EnvZ mutant conferred a phenotype of OmpF⁻/OmpC-constitutive. For this EnvZ(P41S or P41L) mutant, a set of intragenic suppressors, each exhibiting a wild-type phenotype of OmpF⁺/OmpC⁺, was isolated. These suppresor mutants were revealed to have an additional amino acid change within either TM1 or TM2. Furthermore, they exhibited restored phosphatase activity (i.e., both kinase⁺ and phosphatase⁺ activities). It was further demonstrated that one of the suppressors, EnvZ(Arg-180 to Trp in TM2), was able to suppress the defects in both the in vivo phenotype and the in vitro catalytic activities caused by EnvZ(P41S), through intermolecular complementation. These results are best interpreted as meaning that an intimate intermolecular interaction between the membrane–spanning segments of EnvZ is crucial for transmembrane signalling per se in response to an external osmotic stimulus.     

Codons AGA and AGG are read as glycine in ascidian mitochondria

Summary A 1.2-kb DNA fragment of the cytochrome oxidase subunit I (CO I) gene of mitochondria isolated from an ascidian,Halocynthia roretzi, was amplified by polymerase chain reaction (PCR) and sequenced. Codons AGA and AGG appeared in its reading frame, indicating that these are sense codons in this organelle. Sequence comparisons with the corresponding regions of other animal mitochondrial CO I genes suggest that codons AGA and AGG correspond to glycine in the ascidian mitochondrial genome, but not to serine as in most invertebrate genomes, nor to stops as in vertebrate genomes. The other codons are identical to those of vertebrate mitochondria.     

LIFE HISTORY AND SYSTEMATIC POSITION OF PSEUDOCHORDA GRACILIS SP. NOV. (LAMINARIALES,PHAEOPHYCEAE)1

Pseudochorda gracilis sp. Nov. (Pseudochordacease, Laminariales) is described from the Japan Sea coast of Hokkaido/ the species is subtidal, epilithic and annual, appearing in spring and maturing in winter. Erect thalli grow solitary or in tufts on a small discoid holdfast. They are simple, cord-shaped and hollow, with inner hyphal filaments, cylindrical medullary cells and paraphyses consisting of 3–6 cells. Hair tufts are observed only in young thalli. Unilocular sporangia are sessile and narrowly ovate. In culture, P. gracilis shows a heteromorphic life history with oogamy, characteristic of the order Laminariales. Gametophytes are dioecious and dimorphic. Gametophytes mature under lower temperature conditions (usually below 10°C), and sporophytes mature under low temperature and short-day conditions (5°C, SD). The seasonal growth pattern of the species results from the photoperiod-temperature conditions controlling saprophyte maturation.     

Phosphorylation of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) by Proline-Directed Protein Kinases and Its Dephosphorylation

Abstract: Excitatory amino acid (EAA) neurotransmitters may play a role in the pathophysiology of traumatic injury to the CNS. Although NMDA receptor antagonists have been reported to have therapeutic efficacy in animal models of brain injury, these compounds may have unacceptable toxicity for clinical use. One alternative approach is to inhibit the release of EAAs following traumatic injury. The present study examined the effects of administration of a novel sodium channel blocker and EAA release inhibitor, BW1003C87, or the NMDA receptor-associated ion channel blocker magnesium chloride on cerebral edema formation following experimental brain injury in the rat. Animals (n = 33) were subjected to fluid percussion brain injury of moderate severity (2.3 atm) over the left parietal cortex. Fifteen minutes after injury, the animals received a constant infusion of BW1003C87 (10 mg/kg, i.v.), magnesium chloride (300 µmol/kg, i.v.), or saline over 15 min (2.75 ml/kg/15 min). In all animals, regional tissue water content in brain was assessed at 48 h after injury, using the wet weight/dry weight technique. In saline-treated control animals, fluid percussion brain injury produced significant regional brain edema in injured left parietal cortex ( p < 0.001), the cortical area adjacent to the site of maximal injury ( p < 0.001), left hippocampus ( p < 0.001), and left thalamus ( p = 0.02) at 48 h after brain injury. Administration of BW1003C87 15 min postinjury significantly reduced focal brain edema in the cortical area adjacent to the site of maximal injury ( p < 0.02) and left hippocampus ( p < 0.01), whereas magnesium chloride attenuated edema in left hippocampus ( p = 0.02). These results suggest that excitatory neurotransmission may play an important role in the pathogenesis of posttraumatic brain edema and that pre- or post-synaptic blockade of glutamate receptor systems may attenuate part of the deleterious sequelae of traumatic brain injury.