Single crystals of bovine heart cytochrome c oxidase at fully oxidized resting, fully reduced and CO-bound fully reduced states are isomorphous with each other.

Fully reduced and CO-bound fully reduced forms of cytochrome c oxidase from beef heart muscle were crystallized in the presence of sodium ascorbate under N2 or CO atmosphere. Hexagonal bipyramidal and tetragonal crystals were obtained for both forms depending on buffer species. The hexagonal bipyramidal crystals, as large as 0.6 mm in the largest dimension, diffracted X-rays at 7 A resolution, showing an identical space group and cell dimension, P6(2) or P6(4) and a = b = 209 A, c = 283 A, respectively. These parameters coincide with those for crystals of the fully oxidized resting enzyme. This result suggests that a large conformational change, like a subunit arrangement, is not induced by the redox change and/or binding of CO (and possibly O2) to heme a3.     

Transforming growth factor-beta3 promotes mesenchymal cell proliferation and angiogenesis mediated by the enhancement of cyclin D1, Flk-1, and CD31 gene expression during CL/Fr mouse lip fusion

BACKGROUND: Cleft lip with or without cleft palate is the most common congenital anomaly in the craniofacial region. Knowledge of the molecular mechanisms behind normal lip fusion can contribute to better intervention and improved functional clinical outcome. Transforming growth factor-beta3 (TGF-beta3) has been implicated in lip morphogenesis. Therefore, we hypothesized that TGF-beta3 functions during lip fusion through regulation of angiogenesis and mesenchymal cell cycle progression during early developmental stages. METHODS: To test this hypothesis we used the CL/Fraser mouse model, which has a high incidence of cleft lip. Lips isolated from embryonic day (ED) 11.5 mouse embryos were allowed to develop in serum-free organ cultures in the presence or absence of TGF-beta3. The lips that developed in these cultures fused in 2 days. RESULTS: During normal development, we detected positive immunoreactions for TGF-beta3 at the site of fusion. We also detected mesenchymal cells that were immunopositive for Flk-1 and CD31, which are markers for endothelial cell precursors. Exogenous TGF-beta3 accelerated lip fusion in culture. This enhancement was associated with an increase in the number of capillary blood vessels in the lips cultured in the presence of TGF-beta3, in comparison with controls. In tandem, TGF-beta3 increased the level of expression of both Flk-1 and CD31. Our data suggest that an elevated level of TGF-beta3 may promote angiogenesis in developing lips that is mediated by increased Flk-1 and CD31 expression. We also detected increased cyclin D1 expression (a marker for cell proliferation) in the presence of TGF-beta3, which suggests that TGF-beta3 promoted cell proliferation. CONCLUSIONS: TGF-beta3 promoted cell proliferation and angiogenesis in lip mesenchymal tissues. These events led to enhanced lip fusion in the presence of TGF-beta3.     

Piperine analogs in a hydrophobic fraction from Piper ribersoides (Piperaceae) and its insect antifeedant activity

Piper ribersoides Wall. (Piperaceae), which is called “Khua Sa khan” in the local language, is mainly grown in Laos. This plant is used as a food in Laos, but no report on its metabolites exists. Crushed stems were immersed in methanol. The ethyl acetate fraction showed potential insect antifeedant activity for Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), and some of the active compounds were piperine analogs. Piperine and its geometrical isomer independently showed potent antifeedant activities, and piperine was presumably the main active compound in the methanol extract. Interestingly, we discovered that the antifeedant activity was reduced when they were mixed (50:50).     

Sequence of an intestinal cDNA encoding human motilin precursor

A cDNA clone encoding the human motilin precursor was isolated from an intestinal library using synthetic oligonucleotide probes. The predicted amino acid sequence indicates that the motilin precursor consists of 115 amino acids and includes a 25-residue N-terminal signal peptide followed by the 22-amino-acid motilin sequence and a long, 68-residue C-terminal peptide. The amino acid sequence of human motilin predicted from the cDNA sequence is identical to its porcine counterpart, which has been determined by protein sequencing. Proteolytic processing of promotilin to motilin occurs at the sequence, Lys-Lys, this being the first reported instance of processing occurring at a pair of Lys residues. In other precursors it occurs at Lys-Arg, Arg-Arg, Arg, or very rarely Lys.     

The p250GAP gene is associated with risk for schizophrenia and schizotypal personality traits

Background

Hypofunction of the glutamate N-Methyl-d-aspartate (NMDA) receptor has been implicated in the pathophysiology of schizophrenia. p250GAP is a brain-enriched NMDA receptor-interacting RhoGAP. p250GAP is involved in spine morphology, and spine morphology has been shown to be altered in the post-mortem brains of patients with schizophrenia. Schizotypal personality disorder has a strong familial relationship with schizophrenia. Several susceptibility genes for schizophrenia have been related to schizotypal traits.

Methods

We first investigated the association of eight linkage disequilibrium-tagging single-nucleotide polymorphisms (SNPs) that cover the p250GAP gene with schizophrenia in a Japanese sample of 431 schizophrenia patients and 572 controls. We then investigated the impact of the risk genetic variant in the p250GAP gene on schizotypal personality traits in 180 healthy subjects using the Schizotypal Personality Questionnaire.

Results

We found a significant difference in genotype frequency between the patients and the controls in rs2298599 (χ² = 17.6, p = 0.00015). The minor A/A genotype frequency of rs2298599 was higher in the patients (18%) than in the controls (9%) (χ² = 15.5, p = 0.000083). Moreover, we found that subjects with the rs2298599 risk A/A genotype, compared with G allele carriers, had higher scores of schizotypal traits (F_1,178 = 4.08, p = 0.045), particularly the interpersonal factor (F_1,178 = 5.85, p = 0.017).

Discussion

These results suggest that a genetic variation in the p250GAP gene might increase susceptibility not only for schizophrenia but also for schizotypal personality traits. We concluded that the p250GAP gene might be a new candidate gene for susceptibility to schizophrenia.     

Deformation of lipid droplets in fixed samples

Nile red, Sudan III, and oil red O have been used to stain lipid droplets (LDs) for fluorescence microscopy. We noticed that LDs labeled by Nile red are different in appearance from those stained by the latter two dyes. To understand the cause of the difference, we used sequential labeling procedures (first LD stain-photography-quenching-second LD stain-photography), and examined the effect of several factors. Immunofluorescence labeling for adipose differentiation-related protein (ADRP), an LD marker, was also observed comparatively with the lipid stains. As a result, we found that ethanol and isopropanol used for Sudan III and oil red O staining, respectively, and glycerol used for mounting, cause fusion of adjacent LDs even in glutaraldehyde-fixed samples. By the same treatment, immunofluorescence labeling for ADRP was dislocated to the rim of large LDs that were formed as a result of the artifactual fusion. The result indicates that the LD structure can be better observed with Nile red than with Sudan III or oil red O.     

Molecular characterization of ENU mouse mutagenesis and archives

The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.     

Impaired hippocampal long-term potentiation and failure of learning in β1,4-N-acetylgalactosaminyltransferase gene transgenic mice

Gangliosides (sialic acid-containing glycosphingolipids) play important roles in many physiological functions, including synaptic plasticity in the hippocampus, which is considered as a cellular mechanism of learning and memory. In the present study, three types of synaptic plasticity, long-term potentiation (LTP), long-term depression (LTD) and reversal of LTP (depotentiation, DP), in the field excitatory post-synaptic potential in CA1 hippocampal neurons and learning behavior were examined in β1,4-N-acetylgalactosaminyltransferase (β1,4 GalNAc-T; GM2/GD2 synthase) gene transgenic (TG) mice, which showed a marked decrease in b-pathway gangliosides (GQ1b, GT1b and GD1b) in the brain and isolated hippocampus compared with wild-type (WT) mice. The magnitude of the LTP induced by tetanus (100 pulses at 100?Hz) in TG mice was significantly smaller than that in control WT mice, whereas there was no difference in the magnitude of the LTD induced by three short trains of low-frequency stimulation (LFS) (200 pulses at 1?Hz) at 20?min intervals between the two groups of mice. The reduction in the LTP produced by delivering three trains of LFS (200 pulses at 1?Hz, 20?min intervals) was significantly greater in the TG mice than in the WT mice. Learning was impaired in the four-pellet taking test (4PTT) in TG mice, with no significant difference in daily activity or activity during the 4PTT between TG and WT mice. These results suggest that the overexpression of β1,4 GalNAc-T resulted in altered synaptic plasticity of LTP and DP in hippocampal CA1 neurons and learning in the 4PTT, and this is attributable to the shift from b-pathway gangliosides to a-pathway gangliosides.     

Bisphenol A Does Not Affect Memory Performance in Adult Male Rats

Bisphenol A (BPA) is an estrogenic endocrine disruptor used for producing polycarbonate plastics and epoxy resins. This study investigated the effects of oral BPA administration on memory performance, general activity, and emotionality in adult male Sprague Dawley rats using a battery of behavioral tests, including an appetite-motivated maze test (MAZE test) used to assess spatial memory performance. In addition, in order to confirm the effects of BPA on spatial memory performance, we examined whether intrahippocampal injection of BPA affects spatial memory consolidation. In the MAZE test, although oral BPA administration at 10 mg/kg significantly altered the number of entries into the incorrect area compared to those of vehicle-treated rats, male rats given BPA through either oral administration or intrahippocampal injection failed to show significant differences in latencies to reach the reward. Also, oral BPA administration did not affect fear-motivated memory performance in the step-through passive avoidance test. Oral BPA administration at 0.05 mg/kg, the lowest dose used in this study, was correlated with a decrease in locomotor activity in the open-field test, whereas oral administration at 10 mg/kg, the highest dose used in this study, was correlated with a light anxiolytic effect in the elevated plus-maze test. The present study suggests that BPA in adulthood has little effect on spatial memory performance in male rats.