Mitochondrial membrane potential and intracellular ATP content after transient experimental ischemia in the cultured hippocampal neuron

Ischemia limits the delivery of oxygen and glucose to cells and disturbs the maintenance of mitochondrial membrane potential (MMP). MMP regulates the production of high-energy phosphate and apoptotic cascading. Thus, MMP is an important parameter determining the fate of neurons. Differences in the time course of MMP according to the grading of the ischemic impact have not been clarified. MMP and intracellular ATP contents were monitored before and after short-term oxygen-glucose deprivation. A primary hippocampal culture seeded in a 35 mm fenestrated dish for fluorescence microscopy was mounted in a sealed chamber for an anaerobic incubation. A continuous flow of 100% nitrogen into the chamber and a replacement of glucose-free medium allowed the condition of oxygen-glucose deprivation (OGD), thereby extrapolating ischemia. MMP was evaluated by the fluorescence of a voltage-dependent dye, JC-1, under fluorescence microscopy. The intracellular ATP content was evaluated in a hippocampal culture seeded in a 96-well plate by the luciferin-luciferase reaction after a designated period of OGD. During OGD, MMP decreased to 0.72+/-0.03 (normalized JC-1 fluorescence), then increased to the hyperpolarized level 1.99+/-0.12 during 60 min reoxygenation after 30 min OGD. MMP after 60 min OGD decreased and recovered occasionally during reoxygenation. After 90 min OGD and reoxygenation, MMP was reduced and never recovered. The intracellular ATP content was 8.1+/-6.6 and 3.2+/-1.9% after 30 min OGD and 30 min reoxygenation following 30 min OGD, respectively; 60 min OGD did not significantly change these levels (7.1+/-5.8, 2.6+/-0.5%). Hyperpolarization after OGD did not accompany ATP production. This observation suggests the inhibition of electron reentry into an inner membrane during reoxygenation and the disturbance of FoF1-ATP synthase. This pathological finding of an energy-producing system after OGD may provide a clue to explain post-ischemic energy failure.     

The PAR-1-activating peptide facilitates pepsinogen secretion in rats

Protease-activated receptor-2 (PAR-2) is abundantly expressed in gastric mucosal chief cells, facilitating pepsinogen secretion. In the present study, we investigated whether PAR-1, a thrombin receptor, could modulate pepsinogen secretion in rats. The PAR-1-activating peptide TFLLR-NH(2) as well as the PAR-2-activating peptide SLIGRL-NH(2), administered i.v. repeatedly at 1-h intervals, significantly increased gastric pepsinogen secretion over 2-4 h (after two to four doses). In contrast, the control peptide FTLLR-NH(2), given in the same manner, had no such effect. Thus, PAR-1, like PAR-2, might function to facilitate pepsinogen secretion, suggesting a novel role of the thrombin-PAR-1-pathway in the stomach.     

The replication fork barrier site forms a unique structure with Fob1p and inhibits the replication fork

The replication fork barrier site (RFB) is an approximately 100-bp DNA sequence located near the 3' end of the rRNA genes in the yeast Saccharomyces cerevisiae. The gene FOB1 is required for this RFB activity. FOB1 is also necessary for recombination in the ribosomal DNA (rDNA), including increase and decrease of rDNA repeat copy number, production of extrachromosomal rDNA circles, and possibly homogenization of the repeats. Despite the central role that Foblp plays in both replication fork blocking and rDNA recombination, the molecular mechanism by which Fob1p mediates these activities has not been determined. Here, I show by using chromatin immunoprecipitation, gel shift, footprinting, and atomic force microscopy assays that Fob1p directly binds to the RFB. Fob1p binds to two separated sequences in the RFB. A predicted zinc finger motif in Fob1p was shown to be essential for the RFB binding, replication fork blocking, and rDNA recombination activities. The RFB seems to wrap around Fob1p, and this wrapping structure may be important for function in the rDNA repeats.     

Carcinoembryonic antigen-related cell adhesion molecule 10 expressed specifically early in pregnancy in the decidua is dispensable for normal murine development

The carcinoembryonic antigen (CEA) family consists of a large group of evolutionarily and structurally divergent glycoproteins. The murine CEACAM9 and CEACAM11-related proteins as well as the pregnancy-specific glycoproteins (PSG) are secreted members of the CEA family which are differentially expressed in fetal trophoblast cell populations during placental development. PSG are essential for a successful pregnancy, possibly by protecting the semiallotypic fetus from the maternal immune system. In contrast, Ceacam10 mRNA, coding for a protein identical in structure with CEACAM11-related proteins, is expressed in the maternal decidua surrounding the implantation site of the conceptus only during early stages of gestation between day 6.5 and day 10.5 postcoitum. To determine its role during murine development, we inactivated Ceacam10. Ceacam10(-/-) mice developed, like the previously established Ceacam9(-/-) mice, indistinguishably from wild-type littermates with respect to sex ratio, weight gain, and fertility. However, a small but significant reduction of the litter size by 23% was observed in Ceacam10(-/-) matings. Furthermore, combining the Ceacam9 and Ceacam10 null alleles, both located on chromosome 7, by meiotic recombination and subsequent mating of heterozygotes carrying both knockout alleles on one chromosome yielded wild-type and double knockout offspring at the expected Mendelian ratio. Taken together, both Ceacam10 and Ceacam9, alone or in combination, are not essential for either murine placental and embryonic development or for adult life.     

Presence of free D-amino acids in microalgae

Several species of microalgae (phytoplankton), 4 species of freshwater algae and 4 species of marine diatoms, were cultured germ-free in the laboratory. The presence of free D-amino acids was verified in these species by a reversed-phase HPLC analysis. D-Aspartate was detected in all the microalgae examined, but D-alanine was only present in the marine diatoms. The D-amino acid content in Asterionella sp. of the marine diatoms increased from the exponential phase to the stationary phase and then decreased to the phase of decline.     

Overexpression, purification, and characterization of Bacillus subtilis N-acetylmuramoyl-L-alanine amidase CwlC

N-acetylmuramoyl-L-alanine amidase CwlC of Bacillus subtilis was overproduced in Escherichia coli and purified 21-fold. The amidase hydrolyzed type A cell walls such as B. subtilis. The amidase bound slightly to the Microbacterium lacticum cell wall (type B), but did not entirely hydrolyze it. The presence of calcium or magnesium ion increased the resistance of the amidase to heat denaturation.     

Inhibition of N-linked glycosylation causes apoptosis in hamster BHK21 cells

The tsBN7 cell line is one of the temperature-sensitive mutants for cell proliferation derived from hamster BHK21 cell line. It has a mutation in the DAD1 gene and enters apoptosis at the restrictive temperature of 39 degrees C. The defect of Dad1p causes a loss of N-linked glycosylation; therefore, it was thought that an inhibition of N-linked glycosylation induced apoptosis.However, tunicamycin, a potent inhibitor of N-linked glycosylation, had not caused apoptosis in wild-type BHK21 cells. In order to clarify this discrepancy, wild-type BHK21 cells treated with tunicamycin and tsBN7 cells incubated at 39.5 degrees C were examined by the annexin V staining and TUNEL methods. Both methods showed that tunicamycin induces apoptosis in wild-type BHK21 cells, similar to the defect of Dad1p. Thus, we concluded that loss of N-linked glycosylation causes apoptosis.     

Analysis of soluble interleukin 6 receptor in cerebrospinal fluid in inflammatory and non-inflammatory conditions

The objective of this study was to investigate the pathophysiological roles of soluble interleukin 6 receptor (sIL-6R) in cerebrospinal fluid (CSF). CSF was obtained from patients suspected with meningitis. Eight patients without any meningeal signs or symptoms were enrolled as controls. An additional 34 CSF samples were collected to measure both biologically active and immunoreactive sIL-6R. All CSF samples were proven to be aseptic. IL-6 and sIL-6R were measured using specific ELISAs. Patients were divided into three groups on the basis of cell number in CSF; inflammatory group (cell number >5 microl, mean 241+/-363.1, n=61); non-inflammatory group (cell number < or =5 microl, mean=2.1+/-1.7, n=12) and controls (cell number < or =5 microl, mean=0.3+1.7, n=8). Among these three groups, the differences in protein (F (2,78)=8.274, P<0.0001) and IL-6 concentration (F (2,78)=6.475, P<0.001) were statistically significant but those of sIL-6R concentration were not. There were only weak correlations between log (sIL-6R) versus log (cell number) (r=0.23, P=0.0375), log (protein) (r=0.239, P=0.0358) and log (IL-6) (r=0.27, P=0.0167). Amounts of immunoreactive and biologically active sIL-6R were closely correlated (r=0.62, n=34, P<0.005). It was concluded that sIL-6R is present constitutively in CSF and its level may not increase significantly in inflammatory conditions; infiltrating cells in CSF are not the main source of sIL-6R; and sIL-6R in CSF can bind IL-6.     

Cloning and sequencing of the triacylglycerol lipase gene of Aspergillus oryzae and its expression in Escherichia coli

Aspergillus oryzae produces at least three extracellular lipolytic enzymes, L1, L2 and L3 (cutinase, mono- and diacylglycerol lipase, and triacylglycerol lipase, respectively). We cloned the triacylglycerol lipase gene (provisionally designated tglA) by screening a genomic library using a PCR product obtained with two degenerate oligonucleotide primers corresponding to amino acid sequences of L3 as probes. Nucleotide sequencing of the genomic DNA and cDNA revealed that the L3 gene (tglA) has an open reading frame comprising 954 nucleotides, which contains three introns of 47, 83 and 62 bp. The deduced amino acid sequence of the tglA gene corresponds to 254 amino acid residues including a signal sequence of 30 amino acids and, in spite of the difference in substrate specificity, it is homologous to those of cutinases from fungi. Three residues presumed to form the catalytic triad, Ser, Asp and His, are conserved. The cloned cDNA of the tglA gene was expressed in Escherichia coli, and enzyme assaying and zymography revealed that the cloned cDNA encodes a functional triacylglycerol lipase.