Immunological characterisation of the acrosomal filament in the marine shrimp Sicyonia ingentis

Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolase that cleaves the terminal alpha-galactosyl moieties of various glycoconjugates. Overexpression of the enzyme in Chinese hamster ovary (CHO) cells results in high intracellular enzyme accumulation and the selective secretion of active enzyme. Structural analysis of the N -linked oligosaccharides of the intracellular and secreted glycoforms revealed that the secreted enzyme's oligosaccharides were remarkably heterogeneous, having high mannose (63%), complex (30%), and hybrid (5%) structures. The major high mannose oligosaccharides were Man5-7GlcNAc2 species. Approximately 40% of the high mannose and 30% of the hybrid oligosaccharides had phosphate monoester groups. The complex oligosaccharides were mono-, bi-, 2,4-tri-, 2,6-tri- and tetraantennary with or without core-region fucose, many of which had incomplete outer chains. Approximately 30% of the complex oligosaccharides were mono- or disialylated. Sialic acids were mostly N -acetylneuraminic acid and occurred exclusively in alpha2, 3-linkage. In contrast, the intracellular enzyme had only small amounts of complex chains (7.7%) and had predominantly high mannose oligosaccharides (92%), mostly Man5GlcNAc2 and smaller species, of which only 3% were phosphorylated. The complex oligosaccharides were fucosylated and had the same antennary structures as the secreted enzyme. Although most had mature outer chains, none were sialylated. Thus, the overexpression of human alpha-Gal A in CHO cells resulted in different oligosaccharide structures on the secreted and intracellular glycoforms, the highly heterogeneous secreted forms presumably due to the high level expression and impaired glycosylation in the trans- Golgi network, and the predominately Man5-7GlcNAc2 cellular glycoforms resulting from carbohydrate trimming in the lysosome.     

Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption

The spindle assembly checkpoint keeps cells with defective spindles from initiating chromosome segregation. The protein kinase Mps1 phosphorylates the yeast protein Mad1p when this checkpoint is activated, and the overexpression of Mps1p induces modification of Mad1p and arrests wild-type yeast cells in mitosis with morphologically normal spindles. Spindle assembly checkpoint mutants overexpressing Mps1p pass through mitosis without delay and can produce viable progeny, which demonstrates that the arrest of wild-type cells results from inappropriate activation of the checkpoint in cells whose spindle is fully functional. Ectopic activation of cell-cycle checkpoints might be used to exploit the differences in checkpoint status between normal and tumor cells and thus improve the selectivity of chemotherapy.     

Two major autoantigen-antibody systems of the mitotic spindle apparatus

OBJECTIVE: To characterize human autoantigen-antibody systems related to the mitotic poles and spindles. METHODS: Thirty-seven human sera with autoantibodies staining mitotic poles and spindles in indirect immunofluorescence (IIF) studies were further characterized by immunofluorescence on mitotic cells and by immunoblotting and immunoprecipitation. Clinical diagnoses meeting the American College of Rheumatology criteria were based on chart review and interview with the corresponding physicians. RESULTS: Two autoantibody systems reactive with mitotic poles and spindles were defined. Type 1 nuclear mitotic apparatus (NuMA-1) antibodies were identified in the serum of 30 patients. Interphase cells showed a fine, speckled, nuclear staining, while mitotic cells had bright staining of the rim of the centrosomes and light staining of the spindles proximal to the centrosomes. In telophase, the staining shifted from the centrosomes to the reforming nuclei. On immunoblotting, anti-NuMA-1 sera reacted with a 210-kd protein. The reactivity of these sera was identified (with the aid of reference antibodies) as the previously described NuMA antigen-antibody system. Clinical information was available for only 17 of the 30 patients with anti-NuMA-1; of these, 17 (53%) had clinical and lip biopsy findings that met the criteria for Sj?gren's syndrome. NuMA-2 antibodies were found in the sera of 7 patients. Interphase cells showed no nuclear or cytoplasmic staining, but mitotic cells had brightly stained poles and spindles. At anaphase/telophase, staining shifted to the midbody and the intercellular bridge. Anti-NuMA-2 sera immunoprecipitated a protein of 116 kd. This group of patients was more heterogeneous and had both systemic and organ-specific autoimmune diseases. CONCLUSIONS: NuMA protein (here called NuMA-1) and a 116-kd protein (here called NuMA-2) are the major targets of the autoimmune response in the mitotic apparatus, since most of the selected sera (based on IIF staining of the mitotic spindles and poles) recognized 1 of these 2 antigens.     

Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle

The three dimensional organization of microtubules in mitotic spindles of the yeast Saccharomyces cerevisiae has been determined by computer-aided reconstruction from electron micrographs of serially cross-sectioned spindles. Fifteen spindles ranging in length from 0.6-9.4 microns have been analyzed. Ordered microtubule packing is absent in spindles up to 0.8 micron, but the total number of microtubules is sufficient to allow one microtubule per kinetochore with a few additional microtubules that may form an interpolar spindle. An obvious bundle of about eight interpolar microtubules was found in spindles 1.3-1.6 microns long, and we suggest that the approximately 32 remaining microtubules act as kinetochore fibers. The relative lengths of the microtubules in these spindles suggest that they may be in an early stage of anaphase, even though these spindles are all situated in the mother cell, not in the isthmus between mother and bud. None of the reconstructed spindles exhibited the uniform populations of kinetochore microtubules characteristic of metaphase. Long spindles (2.7-9.4 microns), presumably in anaphase B, contained short remnants of a few presumed kinetochore microtubules clustered near the poles and a few long microtubules extending from each pole toward the spindle midplane, where they interdigitated with their counterparts from the other pole. Interpretation of these reconstructed spindles offers some insights into the mechanisms of mitosis in this yeast.     

Cell-cell interactions that modulate neuronal development in the leech

Mitotic lineage has been found to determine the cellular identity of leech neurons (reviewed in Stent et al., 1992), Int. Rev. Neurobiol. 33:109-133. However, the details of the adult phenotype of many neurons in the central nervous system of the leech have been shown to be shaped by interactions either with other neurons or with non-neuronal tissues in the environment. Four effects of cell-cell interactions will be considered in this article: stimulation of mitosis that generates new neurons, modulation of cell death or axonal retraction, modification of neurotransmitter metabolism, and modification of other physiological properties. In all cases, the interactions that modify development are thought to occur at a location distant from the soma, requiring that signals be transmitted a significant distance from the site of interaction to the metabolic machinery in the soma.     

APC-mediated proteolysis of Ase1 and the morphogenesis of the mitotic spindle

The molecular mechanisms that link cell-cycle controls to the mitotic apparatus are poorly understood. A component of the Saccharomyces cerevisiae spindle, Ase1, was observed to undergo cell cycle-specific degradation mediated by the cyclosome, or anaphase promoting complex (APC). Ase1 was degraded when cells exited from mitosis and entered G1. Inappropriate expression of stable Ase1 during G1 produced a spindle defect that is sensed by the spindle assembly checkpoint. In addition, loss of ASE1 function destabilized telophase spindles, and expression of a nondegradable Ase1 mutant delayed spindle disassembly. APC-mediated proteolysis therefore appears to regulate both spindle assembly and disassembly.     

Activation of the protein kinase p38 in the spindle assembly checkpoint and mitotic arrest

The mitogen-activated protein kinase (MAPK) superfamily comprises classical MAPK (also called ERK), c-Jun amino-terminal or stress-activated protein kinase (JNK or SAPK), and p38. Although MAPK is essential for meiotic processes in Xenopus oocytes and the spindle assembly checkpoint in Xenopus egg extracts, the role of members of the MAPK superfamily in M phase or the spindle assembly checkpoint during somatic cell cycles has not been elucidated. The kinase p38, but not MAPK or JNK, was activated in mammalian cultured cells when the cells were arrested in M phase by disruption of the spindle with nocodazole. Addition of activated recombinant p38 to Xenopus cell-free extracts caused arrest of the extracts in M phase, and injection of activated p38 into cleaving embryos induced mitotic arrest. Treatment of NIH 3T3 cells with a specific inhibitor of p38 suppressed activation of the checkpoint by nocodazole. Thus, p38 functions as a component of the spindle assembly checkpoint in somatic cell cycles.     

Control of spindle orientation in Drosophila by the Par-3-related PDZ-domain protein Bazooka

BACKGROUND: The orientation of the mitotic spindle influences the asymmetric distribution of cytoplasmic determinants and the positioning of the sibling cell, and therefore has important influences on cell-fate determination and patterning of the embryo. Both the establishment of an axis of polarity and the adjustment of this axis with respect to the coordinates of the embryo have to be controlled. None of the genes identified so far that are involved in these processes seems to have been conserved between flies and nematodes. RESULTS: Here, we show that the bazooka gene encodes a protein with three putative protein-interaction motifs known as PDZ domains and is the first Drosophila representative of the par gene family of Caenorhabditis elegans, members of which are required for establishment of anterior-posterior polarity of the nematode embryo. The bazooka RNA and protein were found to be restricted to the apical cortical cytoplasm of epithelial cells and neuroblasts. Embryos that were mutant for bazooka frequently failed to coordinate the axis of cell polarity with that of the embryo. This was manifested as defective spindle orientation and mispositioning of the daughter cell after division. CONCLUSIONS: The Drosophila gene bazooka is likely to be part of a regulatory mechanism required to coordinate the axis of polarity of a cell with that of the embryo. The PDZ domains of Bazooka provide several protein-protein interfaces, which possibly participate in the assembly of a multiprotein complex at the apical pole.     

Prenatal estrogens and the development of homosexual orientation.

In psychobiological research on sexual orientation, the prenatal hormone theory has a central position. This article examines the hypothesis that prenatal estrogens contribute to the development of human sexual orientation. Several groups of women with a history of prenatal exposure to diethylstilbestrol (DES), a nonsteroidal synthetic estrogen, were compared with several samples of control women in the context of a comprehensive study of the psychiatric and psychologic effects of prenatal DES. Various aspects of sexual orientation were assessed by systematic interview. Consistently across samples, more DES-exposed women than controls were rated as bisexual or homosexual (scores 2–6 on Kinsey-format scales ranging from 0 to 6). The data are compatible with the hypothesis that prenatal estrogens may play a role in the development of human sexual orientation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Hyperglycaemic hormones inhibit protein and mRNA synthesis in in vitro-incubated ovarian fragments of the marine shrimp Penaeus semisulcatus

The specificity of many signal transduction pathways relies on the temporal coordination of different second messenger signals. Here we found a molecular mechanism which guarantees that conventional protein kinase C (PKC) isoforms are sequentially activated by calcium and diacylglycerol signals. Receptor stimuli that triggered repetitive calcium spikes induced a parallel repetitive translocation of GFP-tagged PKCgamma to the plasma membrane. While calcium acted rapidly, diacylglycerol binding to PKCgamma was initially prevented by a pseudosubstrate clamp, which kept the diacylglycerol-binding site inaccessible and delayed calcium- and diacylglycerol-mediated kinase activation. After termination of calcium signals, bound diacylglycerol prolonged kinase activity. The properties of this molecular decoding machine make PKCgamma responsive to persistent diacylglycerol increases combined with high- but not low-frequency calcium spikes.     

The let-99 gene is required for proper spindle orientation during cleavage of the C. elegans embryo

The orientation of cell division is a critical aspect of development. In 2-cell C. elegans embryos, the spindle in the posterior cell is aligned along the long axis of the embryo and contributes to the unequal partitioning of cytoplasm, while the spindle in the anterior cell is oriented transverse to the long axis. Differing spindle alignments arise from blastomere-specific rotations of the nuclear-centrosome complex at prophase. We have found that mutations in the maternally expressed gene let-99 affect spindle orientation in all cells during the first three cleavages. During these divisions, the nuclear-centrosome complex appears unstable in position. In addition, in almost half of the mutant embryos, there are reversals of the normal pattern of spindle orientations at second cleavage: the spindle of the anterior cell is aligned with the long axis of the embryo and nuclear rotation fails in the posterior cell causing the spindle to form transverse to the long axis. In most of the remaining embryos, spindles in both cells are transverse at second cleavage. The distributions of several asymmetrically localized proteins, including P granules and PAR-3, are normal in early let-99 embryos, but are perturbed by the abnormal cell division orientations at second cleavage. The accumulation of actin and actin capping protein, which marks the site involved in nuclear rotation in 2-cell wild-type embryos, is abnormal but is not reversed in let-99 mutant embryos. Based on these data, we conclude that let-99(+) is required for the proper orientation of spindles after the establishment of polarity, and we postulate that let-99(+) plays a role in interactions between the astral microtubules and the cortical cytoskeleton.     

Career and marriage orientation in the vocational development of college women.

The problem dealt with the construction of an attitude inventory and the comparison of selected factors in the vocational development of 3 groups of college girls grouped on the basis of their attitudes as measured by the inventory. 180 college girls were administered the Career and Marriage Attitude Inventory; 30 were selected for study on the basis of scores earned. A structured interview-case study was used to prepare summaries which were ranked by 3 impartial judges. Significance at the 1% level was determined by Kendall's Coefficient of Concordance and the application of the F test. Comparison among groups revealed that girls differ in respect to career-marriage attitudes and occupational interests. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Sex role orientation and division of labor early in marriage.

Examined the division of labor by newlyweds, including the extent to which spouses participated in the labor force and involved themselves in household work. 120 husbands (mean age 24.3 yrs) and wives (mean age 21.9 yrs) completed the Attitudes Toward Women Scale, the Personal Attributes Questionnaire, and a measure assessing their perceived skill in carrying out 26 household tasks. Data on household task participation were gathered via 9 separate phone interviews. Tasks were classified into 2 groups based on whether they were performed more often by the men or by the women. Husbands' and wives' sex-role attitudes were related to the extent to which wives, but not husbands, participated in the labor force. The more traditional the couple was in terms of their relative employment hours, the less the husband, relative to his wife, was involved in female sex-typed household tasks. No such connection was found for male sex-typed tasks. Spouses' perceived skill at performing tasks traditionally assigned to the spouse of the opposite sex was significantly related to how traditional they were in housework participation. Findings suggest that gender-related attitudes and skills of newlyweds influence the way their relationships come to be structured with regard to economic and instrumental activities. (23 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

PD 98059 prevents establishment of the spindle assembly checkpoint and inhibits the G2-M transition in meiotic but not mitotic cell cycles in Xenopus

Human mast cells and basophils play a key role in the pathogenesis of several immunological and inflammatory disorders, not only by producing inflammatory and fibrogenic mediators, but also by directly (CD40 ligand) and indirectly secreting various cytokines and chemokines. Studies carried out to evaluate the effects of drugs that modulate the release of mediators and cytokines from these cells have contributed to clarifying the biochemical mechanism by which immunological and non-immunological stimuli activate these cells. Significant differences have been documented between human mast cells and basophils as regard the pharmacological agents that modulate the release of mediators, between mast cells isolated from different anatomical sites, and between compounds of the same class of drugs. Efforts to gain insight into the biochemical events occurring during immunological activation of mast cells and basophils could lead to the identification of new biochemical targets for therapeutic interventions in several immunological disorders.     

Fission yeast bub1 is a mitotic centromere protein essential for the spindle checkpoint and the preservation of correct ploidy through mitosis

The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. We investigated the role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses. We find that bub1(+) is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis. We show that Bub1 also has a crucial role in normal, unperturbed mitoses. Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. Such genomic instability is even more dramatic in Deltabub1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that bub1(+ )function is essential to maintain correct ploidy through mitosis. As in larger eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis. However, unlike its vertebrate counterpart, a pool of Bub1 remains centromere-associated at metaphase and even until telophase. We discuss the possibility of a role for the Bub1 kinase after the metaphase-anaphase transition.     

论我国目前黄金矿业开采现状及发展方向

通过对我国目前黄金矿山开采的技术分析、资源的合理开发利用及设备工艺的应用，探讨了采矿技术的发展方向。     

Comparative pharmacology of site directed antithrombin agents. Implication in drug development

Beside the direct inhibition of thrombin and its regulatory functions, many of the newer antithrombin agents produce several additional effects, unrelated to their anticoagulant actions. Synthetic peptide inhibitors are capable of producing fibrinolytic compromise by virtue of their actions on fibrinolytic enzymes such as t-PA, plasmin, urokinase and protein Ca. In addition, the low molecular weight arginine-containing peptides are also known to produce hemodynamic and hemostatic deficits. The designs of the ongoing clinical trials are largely empirical because of the non-availability of valid pharmacologic and toxicologic data on thrombin inhibitors. In contrast to heparin, none of the thrombin inhibitors produce endogenous release of tissue factor pathway inhibitor (TFPI) in the experimental and clinical settings. These observations suggest that beside the direct inhibition of thrombin, these agents also produce multiple additional effects that can significantly contribute to their pharmacologic and toxicologic profile.