Amino acid sequence and carbohydrate-binding analysis of the N-acetyl-D-galactosamine-specific C-type lectin, CEL-I, from the Holothuroidea, Cucumaria echinata

CEL-I is one of the Ca2+-dependent lectins that has been isolated from the sea cucumber, Cucumaria echinata. This protein is composed of two identical subunits held by a single disulfide bond. The complete amino acid sequence of CEL-I was determined by sequencing the peptides produced by proteolytic fragmentation of S-pyridylethylated CEL-I. A subunit of CEL-I is composed of 140 amino acid residues. Two intrachain (Cys3-Cys14 and Cys31-Cys135) and one interchain (Cys36) disulfide bonds were also identified from an analysis of the cystine-containing peptides obtained from the intact protein. The similarity between the sequence of CEL-I and that of other C-type lectins was low, while the C-terminal region, including the putative Ca2+ and carbohydrate-binding sites, was relatively well conserved. When the carbohydrate-binding activity was examined by a solid-phase microplate assay, CEL-I showed much higher affinity for N-acetyl-D-galactosamine than for other galactose-related carbohydrates. The association constant of CEL-I for p-nitrophenyl N-acetyl-beta-D-galactosaminide (NP-GalNAc) was determined to be 2.3 x 10(4) M(-1), and the maximum number of bound NP-GalNAc was estimated to be 1.6 by an equilibrium dialysis experiment.     

Diethylstilbestrol attenuates antioxidant activities in testis from male mice

It has been reported that acute exposure to diethylstilbestrol (DES) induces apoptosis in the testis, and antioxidants play a role in preventing DES-induced tissue damage. In this study, the effect of chronic exposure to DES on the antioxidants was examined in the testis and liver. Eight-week old male ICR mice were treated subcutaneously with various doses of DES for 20 days. Morphologically apparent apoptotic changes, 4-hydroxy-2-nonenal-positive cells and TUNEL-positive DNA-fragmentation, were demonstrated in the testis, but were minimal in the liver. Activities of antioxidants such as glutathione (GSH) peroxidase and GSH S -transferase decreased in both the liver and testis. The activity of Mn-superoxide dismutase (SOD) decreased in the liver but increased in the testis. The activity of Cu, Zn-SOD decreased in the liver but was unchanged in the testis. On Western and Northern blots, gamma-glutamylcysteine synthetase ( γ-GCS), a rate limiting enzyme of GSH synthesis, was increased in the liver dependent on the dose of DES. However, the expression of γ-GCS was reduced in the testis. Since quinones, metabolites of DES, generate reactive oxygen species, which damage DNA, antioxidants are important to prevent the damage. The data suggest that antioxidant activities are impaired by DES, and the levels of GSH are related to DES-induced apoptosis in the testis.     

Possible involvement of Fas system in the induction of apoptosis in human astrocytic brain tumors

1. For a better understanding of the biological features of astrocytic tumors, we investigated apoptosis and its pathway, especially in the interaction between Fas and Fas ligand (FasL).2. We examined the presence of apoptosis in human astrocytic brain tumors by terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end labeling (TUNEL) and then apoptotic index (AI) was calculated. We also examined the distribution of Fas and FasL-positive tumor cells immunohistochemically. Labeling index (LI) for Fas and FasL was calculated as Fas-LI and FasL-LI, respectively, and compared to AI.3. Tumor cells expressing both Fas and FasL were TUNEL positive. Such cells were distributed sparsely in low-grade astrocytomas, but focally in glioblastomas. There was a close correlation among AI, Fas-LI, and FasL-LI, and astrocytic tumors with higher AI were associated with a longer survival time than that with lower AI.4. It was concluded that the Fas system may be involved in the apoptosis of astrocytic tumors, and AI can be a useful parameter for assessing prognosis of astrocytic tumors.     

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 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.     

Molecular analysis of X-linked ichthyosis in Japan

BACKGROUND: X-linked ichthyosis (XLI) is an inherited skin disorder caused by a deficiency of steroid sulfatase (STS). The gene and protein of STS were examined in 19 Japanese patients with XLI. RESULTS: In Western blotting analysis, no cross-reacting peptide was detected in the patients' placenta, although a single band (63 kD) corresponding to STS in a normal subject was observed. Southern blotting was performed using EcoRI digests of cellular DNA from 13 XLI patients and full-length human STS cDNA as a probe. Normal males had bands of 20, 15, 10, 9.0, 6.1, 4.2, 2.6, and 1.5 kb. Twelve of the 19 patients had only 20- and 1.5-kb bands. Only one patient had the same band pattern as that of normal males. The STS gene was analyzed by PCR in 6 of the 19 patients. PCR amplification products were sequenced to analyze the STS gene. Two cases with one-base change in the STS gene and variation in amino acids H444R and E560P were found. Mutant STS cDNA was transfected into COS-1 cells and the STS enzyme activity was assayed. The enzyme activities were less than the minimum detection value of the detection system. CONCLUSIONS: These results suggest that XLI is mainly caused by an extensive deletion of the STS gene and that the PCR method is useful for detection of STS point mutations.