Acquisition of meiotic competence in growing pig oocytes correlates with their ability to activate Cdc2 kinase and MAP kinase

Meiotic maturation of mammalian oocytes is under the control of cell cycle molecules Cdc2 kinase and MAP kinase (mitogen-activated protein kinase). In the present study, we investigated the relationship between the ability to activate Cdc2 kinase and MAP kinase and the acquisition of meiotic competence during pig oocyte growth. Growing and fully grown pig oocytes were collected from four groups of antral follicles of various diameters (A, 0.5-0.7 mm; B, 1.0-1.5 mm; C, 2.0-2.5 mm; D, 4.0-6.0 mm) and cultured in vitro. Fully grown oocytes from class D follicles, which have full competence to mature to metaphase II, had the ability to activate both Cdc2 kinase and MAP kinase. In contrast, growing oocytes from class A follicles, which have limited competence to resume meiosis, had no such ability. Cyclin B1 molecules did accumulate, however, with phosphorylated 35 and 36 kDa bands of p34cdc2 appearing in the cultured oocytes. Of the growing oocytes from class B follicles, 60% resumed meiosis but arrested at metaphase I. Some of the oocytes in this class were capable of activating Cdc2 kinase, although they did not appear to have established a MAP kinase-activating pathway or the ability to activate MEK. These results suggest that limited meiotic competence in growing oocytes from class A follicles is due to their inability to activate Cdc2 kinase and their incomplete MEK-MAP-kinase pathway, although the oocytes are capable of accumulating cyclin B1 molecules. During the final growth phase, pig oocytes acquire the ability to activate Cdc2 kinase and then establish the MEK-MAP-kinase pathway for full meiotic competence.     

Stereochemical studies of chiral resolving agents, M9PP and H9PP acids

The stereoselective Grignard reaction of (1R,2S,5R)-(-)-2-isopropyl-5-methylcyclohexyl pyruvate (menthyl pyruvate) with 9-phenanthrylmagnesium bromide yielded diastereomeric hydroxy-esters, where intramolecular OH em leader O=C hydrogen bond was observed in IR and (1)H NMR spectra. The alkaline hydrolysis of the major product gave (+)-2-hydroxy-2-(9-phenanthryl)propionic acid (H9PP acid (3)), whose absolute configuration was assigned as S based on the chemical correlation with (1R,2S,5R)-2-isopropyl-5-methylcyclohexyl ester of (S)-2-methoxy-2-(9-phenanthryl)propionic acid (M9PP acid (2)); the absolute configuration of 2 had been previously established by X-ray crystallography. The enantioresolution of (+/-)-6-methyl-5-hepten-2-ol, sulcatol, an insect pheromone, was carried out using (S)-(+)-M9PP acid 2.     

Absolute configuration of the thyroid hormone analog KAT-2003 as determined by the 1H NMR anisotropy method with a novel chiral auxiliary, MalphaNP acid

The absolute configuration of the chiral thyroid hormone analog KAT-2003 (+)-2, showing hypocholesterolemic activities, decreases of hepatic triglyceride contents with lowering cardiac side effects, and significant inhibitory effect for the second primary hepatocellular carcinoma, was determined as S by the (1)H NMR anisotropy method using a novel chiral auxiliary, 2-methoxy-2-(1-naphtyl)propionic acid (MalphaNP acid).     

Gelling properties of soybean beta-conglycinin having different subunit compositions

The effects of protein concentration, and heating temperature and time on the gelling properties of soybean beta-conglycinin (7S globulins) lacking the alpha or alpha' subunit were compared with those of 7S containing all three subunits (alpha, alpha', and beta) to determine whether each subunit contributes equally. In most of the conditions, the relative order of gel hardness was alpha'-lacking > 7S > alpha-lacking. From Fourier transform infrared studies, the secondary structure change after heating was very similar among the three samples; thus, the secondary structural change is not the reason for the differences in gel hardness. By using scanning electron microscopy, we observed differences in strand thickness and the density of the gel network among the three samples. These differences correlated well with the differences in gel hardness.     

Possible relationship between Helicobacter pylori infection and cap polyposis of the colon

BACKGROUND: Cap polyposis is a rarely encountered disease characterized by multiple distinctive inflammatory colonic polyps located from the rectum to the distal colon. The etiology of this disease is still unknown, and no specific treatment has been established. AIM: We report three cases of cap polyposis that were cured following eradication therapy for Helicobacter pylori infection. METHODS AND RESULTS: Three women were referred to Shinshu University Hospital because of mucoid and/or bloody diarrhea. Laboratory data showed hypoproteinemia in all cases; markers of inflammation such as C-reactive protein were negative. Colonoscopy revealed multiple sessile polyps with mucus adherent on the apices of the mucosal folds in the rectum and/or the sigmoid colon. The intervening mucosa was normal. Microscopic examinations of biopsy specimens taken from sessile polyps revealed inflamed mucosa with elongated tortuous crypts attenuated towards the mucosal surface. A granulation tissue 'cap' was observed on the surface of the mucosa. Various treatments were unsuccessful, including administration of metronidazole or prednisolone, avoidance of straining at defecation, and surgical or endoscopic resection. All were diagnosed with H. pylori infection in the stomach. Helicobacter pylori was not detected in the biopsy specimens from the colonic inflammatory polyps by immunohistochemical study using polyclonal anti-H. pylori antibody. After successful eradication therapy the clinical symptoms improved. Disappearance of cap polyposis was confirmed by colonoscopy in all three cases. CONCLUSION: We speculate that H. pylori infection might play a role in the pathogenesis of cap polyposis.     

Expression of flap endonuclease-1 during meiosis in a basidiomycete, Coprinus cinereus

In the basidiomycete Coprinus cinereus (C. cinereus), which shows a highly synchronous meiotic cell cycle, the meiotic prophase I cells demonstrate flap endonuclease-1 activity. To investigate its role during meiosis, we isolated a C. cinereus cDNA homolog of flap endonuclease-1 (CcFEN-1), 1377bp in length with the open reading frame (ORF) encoding a predicted molecular mass of 51 kDa. At amino-acid residues Glu276-Pro345, a specific inserted sequence composed of 70 amino acids rich in polar forms was found to exist, without sequence identity to other eukaryotic FEN-1 or the polar amino acid rich sequences found in C. cinereus PCNA and C. cinereus DNA ligase IV, although the lengths and percentages of polar amino acids were similar. Northern hybridization analysis indicated CcFEN-1 to be expressed not only in the pre-meiotic S phase but also in meiotic prophase I. The roles of CcFEN-1 during meiosis are discussed.     

Eukaryotic proteasomes cannot digest polyglutamine sequences and release them during degradation of polyglutamine-containing proteins

Long glutamine sequences (polyQ) occur in many cell proteins, and several neurodegenerative diseases result from expansion of these sequences. PolyQ-containing proteins are degraded by proteasomes, whose three active sites prefer to cleave after hydrophobic, basic, or acidic residues. We tested whether these particles can digest a polyQ chain. Eukaryotic 26S and 20S proteasomes failed to cut within stretches of 9-29Q residues in peptides. While digesting a myoglobin Q(35) fusion protein, the proteasomes spared the polyQ sequence. In contrast, archaeal proteasomes, whose 14 active sites are less specific, rapidly digested such polyQ repeats. Therefore, when degrading polyQ proteins, eukaryotic proteasomes must release aggregation-prone polyQ-containing fragments for further hydrolysis by unidentified peptidases. In polyQ diseases, such polyQ sequences (38-300Qs) exceed the lengths of normal proteasome products (2-25 residues). Occasional failure of these long undegradable sequences to exit may interfere with proteasome function and help explain why longer polyQ expansions promote early disease onset.     

Evolutionary History and Mode of the <Emphasis Type="Italic">amylase</Emphasis> Multigene Family in <Emphasis Type="Italic">Drosophila</Emphasis>

Previous studies indicate that the tandemly repeated members of the amylase (Amy) gene family evolved in a concerted manner in the melanogaster subgroup and in some other species. In this paper, we analyzed all of the 49 active and complete Amy gene sequences in Drosophila, mostly from subgenus Sophophora. Phylogenetic analysis indicated that the two types of diverged Amy genes in the Drosophila montium subgroup and Drosophila ananassae, which are located in distant chromosomal regions from each other, originated independently in different evolutionary lineages of the melanogaster group after the split of the obscura and melanogaster groups. One of the two clusters was lost after duplication in the melanogaster subgroup. Given the time, 24.9 mya, of divergence between the obscura and the melanogaster groups (Russo et al. 1995), the two duplication events were estimated to occur at about 13.96 ± 1.93 and 12.38 ± 1.76 mya in the montium subgroup and D. ananassae, respectively. An accelerated rate of amino acid changes was not observed in either lineage after these gene duplications. However, the G+C contents at the third codon positions (GC3) decreased significantly along one of the two Amy clusters both in the montium subgroup and in D. ananassae right after gene duplication. Furthermore, one of the two types of the Amy genes with a lower GC3 content has lost a specific regulatory element within the montium subgroup species and D. ananassae. While the tandemly repeated members evolved in a concerted manner, the two types of diverged Amy genes in Drosophila experienced frequent gene duplication, gene loss, and divergent evolution following the model of a birth-and-death process.     

Addition of a peptide tag at the C terminus of AtHKT1 inhibits its Na+ transport

Eight transmembrane segments in the Arabidopsis Na+ transporter, AtHKT1, have been proposed to be arranged around the axis of the pore. We report here that the addition of a GFP or a FLAG tag to the C terminus of AtHKT1 impairs the Na+ transport in Xenopus ooytes as detected by electrophysiological measurements.     

Abnormal blood vessel development in mice lacking presenilin-1

Presenilin-1 (PS1) is a gene responsible for the development of early-onset familial Alzheimer's disease. To explore the potential roles of PS1 in vascular development, we examined the vascular system of mouse embryos lacking PS1. PS1-deficient embryos exhibited cerebral hemorrhages and subcutaneous edema by mid gestation. Immunohistochemical analysis revealed vascular remodeling failure in the stomach and trunk dorsal median region of the skin and insufficient formation of the perineural plexus around the spinal cord of the PS1 mutant embryos. The number of capillary sprouting sites reduced and the capillary diameter increased in the mutant brains, especially at the amygdaloid and striatal regions. Endothelial cells in the sprouting capillaries of the mutant mice showed abnormal morphologies such as multiplication, apoptotic and necrotic images, in contrast to pericytes showing a normal appearance. An in vitro assay using para-aortic splanchnopleural mesoderm (P-Sp) revealed aberrant angiogenesis in the explant culture from the mutant. These findings suggest the essential roles of PS1 in angiogenesis.