Role of protein synthesis and proteases in production and inactivation of maturation-promoting activity during meiotic maturation of starfish oocytes

In starfish oocytes, activity of the maturation-promoting factor (MPF) and that of a major cAMP-independent protein kinase dropped at the time of meiotic cleavage, and rose again after the first but not the second meiotic cleavage. Protein synthesis was required before the first meiotic cleavage for both MPF and protein kinase activity to rise again after the first meiotic cleavage. Microinjection of either leupeptin or soybean trypsin inhibitor early enough prior to first polar body emission suppressed both the meiotic cleavage and the associated drop of MPF activity. Microinjection of leupeptin or soybean trypsin inhibitor during the 10-min period before the first meiotic cleavage also suppressed cytokinesis but did not prevent a decrease in MPF activity at the normal time of cytokinesis. The lysosomotropic inhibitor ammonia neither suppressed cytokinesis nor the drop of MPF activity at the time of first meiotic cleavage. Activity of neutral proteases sensitive to leupeptin and soybean trypsin inhibitor was demonstrated in oocyte homogenates prepared at the time of first meiotic cleavage. It is proposed that such proteases might be involved in degradation of protein kinase(s) and in the drop of MPF activity at the time of first meiotic cleavage.     

Parthenogenetic activation decreases the polyphosphoinositide content of frog eggs

Polyphosphoinositides were quantified in metaphase II-arrested eggs of the amphibian Xenopus laevis and 8-10 min later in eggs activated by pricking. The content of phosphatidylinositol 4,5-biphosphate (PIP2) was remarkably high in metaphase II-arrested eggs with respect to that of phosphatidylinositol 4-phosphate (PIP). It was found to drop dramatically at activation. In contrast PIP content did not change significantly.     

Cell-type preference of immunoglobulin kappa and lambda gene promoters.

Immunoglobulin gene constant regions are known to be associated with strictly tissue-specific enhancer elements. Until recently the promoter of the variable region, which becomes linked to the constant region by somatic rearrangement, could have been viewed as a passive recipient of the enhancer stimulus. Here we show that the promoters of the immunoglobulin kappa and lambda light chain genes are approximately 20-30 times more active in lymphoid cells than in non-lymphoid cells. To avoid the problem of differential mRNA stability upon transfection of immunoglobulin genes into non-lymphoid cells we have constructed chimeric genes. All kappa mRNA sequences were progressively deleted to fuse the kappa gene promoter to a globin gene coding body. A similar chimeric gene was constructed with the promoter of the lambda gene. The cell-type preference of the promoter may be exploited during B-lymphocyte differentiation to regulate the immunoglobulin gene promoter independently from the enhancer.     

Occurrence of glutamate dehydrogenase isoenzymes during growth of Oocystis alga

Oocystis sp., a unicellular green alga, contained two glutamate dehydrogenase isoenzymes: one was specific for NADH and the other for NADPH. Activity staining after gel electrophoresis indicated that one component in NADH-GDH was not specific for the cofactor and three components in NADPH-GDH. The optimal concentration of substrate, purification procedure and kinetic properties of both glutamate dehydrogenase (GDH) enzymes in vitro are presented. The kinetics of growth, nutrient removal and enzyme activities for Oocystis growing in wastewater showed that ammonia was preferentially utilized over nitrate and the medium was depleted before the maximum population was obtained in indoor culture. There was a sharp increase in NADPH-GDH activity following the exhaustion of ammonia from the medium but NADH-GDH activity remained unchanged. The NADPH-GDH activity at the outset increased exponentially with time in greenhouse culture but then decreased sharply accompained by a rapid increase in biomass and nitrite concentration. The K(m) values for ammonia in this algal GDH was high, while glutamate synthase activity was not detected; this suggests that Oocystis may adapt to conditions of ammonia limitation by producing large quantities of NADPH-GDH instead of using glutamate synthase pathway.     

Changes in chromosomes and in development of cells of Sciara ocellaris induced by microsporidian infections

Two species of microsporidia distinguished by the shape of their spores were found to infect cells of various tissues of Sciara ocellaris. Although the infection affects profoundly the development of the infected cells, the interaction between the infective agents and the host cells are often well-balanced and the infected cells may survive longer than the uninfected of the same tissue. The infected cells, including their nuclei and chromosomes, increase greatly. The general reaction of the chromosomes of cells of S. ocellaris infected by microsporidia is the increase of their volume by an increase in the polyteny and the accumulation of chromosomal products between their chromonemata. The cells of the fat bodies, on the other hand, have peculiar types of reactions. Some show an increase in polyteny, frequently showing asynapsis of the entire or parts of the chromosome. Other cells show increased chromosome polyteny associated with different degrees of polyploidy. Still other infected cells develop a new type of chromosome morphology called brachy-polytene which may or may not be associated with polyploidy. Special emphasis must be given to the fact that in Diptera, which have polytene chromosomes, the relationships of the infection and the host cell may in many cases be studied thoroughly, starting with the reaction of the genes to the infective agent. It was shown that in the infected cells of the salivary gland of S. ocellaris the pattern of puffs is greatly changed; hence, this change may be the probable cause of their change in development. Infected cells of the salivary gland with enlarged and active polytene chromosomes were found in adult flies, a situation which never occurs in non-infected animals. Since the microsporidia when entering the cells of S. ocellaris do not cause degeneration of the infected cells but determine a new pattern of development, their association with host cells offers great possibilities in the study of basic problems in cell biology, mainly related to chromosomal morphology, physiology and differentiation.This paper is dedicated to our dear friend Professor Sally Hughes-Schrader for her outstanding contribution to Biology.Work supported by Grants of the Public Health Service (GM 15769), Oonselho Nacional de Pesquisas and Pundação de Amparo á Pesquisa do Estado de São Paulo Brazil.     

Characterization of the deamidated forms of recombinant hirudin.

Recombinant hirudin variant rHV2-Lys 47 (MW = 6906.5) was intentionally deamidated by incubation in pH 9 phosphate buffer at 37 degrees C. Anion-exchange HPLC analysis showed that 11 forms could be generated. These were isolated and purified by combined anion-exchange and reversed-phase HPLC. Acid-catalyzed carboxyl methylation was used to introduce a mass shift of +15 amu per deamidated residue present in the molecule before analysis by liquid secondary ion mass spectrometry (LSIMS). Methylation enhanced, in particular, the abundance of the sequence ions in the LSIMS spectra. This permitted the determination of both the number (three) and the localization of the deamidated residues: Asn 52, Asn 53, and a residue located in the N-terminal 1-39 domain. Complementary sequencing techniques proved that the latter residue was Asn 33. Altogether four mono-, three di-, and four tri-deamidated forms were identified. The heterogeneity of the forms having identical deamidation positions but being chromatographically separable is thought to arise from the generation of alpha- and beta-aspartyl iso forms during the nonenzymatic deamidation process.     

Functional regions of the mouse thrombopoietin receptor cytoplasmic domain: evidence for a critical region which is involved in differentiation and can be complemented by erythropoietin.

Thrombopoietin (TPO) is the major regulator of growth and differentiation of megakaryocytes. To identify functionally important regions in the cytoplasmic domain of the TPO receptor, mpl, we introduced wild-type mpl and deletion mutants of murine mpl into the granulocyte-macrophage colony-stimulating factor (GM-CSF)- or erythropoietin (EPO)-dependent human cell line UT7. TPO induced differentiation of UT7-Wtmpl cells, not parental UT7 cells, along the megakaryocytic lineage, as evidenced by decreased proliferation, changes in cell morphology, and increased surface expression and mRNA levels of megakaryocytic markers CD41, CD61, and CD42b. When UT7-mpl cells were cultured long-term in EPO instead of GM-CSF, the TPO effect was dominant over that of EPO. Moreover, the differentiation induced by TPO was more pronounced for cells shifted from EPO to TPO than for cells shifted from GM-CSF to TPO, as shown by the appearance of polyploid cells. Mutational analysis of the cytoplasmic domain of mpl showed that proliferation and maturation functions of mpl can be uncoupled. Two functional regions were identified: (i) the first 69 amino acids comprising the cytokine receptor motifs, box I and box 2, which are necessary for both TPO-induced mitogenesis and maturation; and (ii) amino acids 71 to 94, which are dispensable for proliferation but required for differentiation. Surprisingly, however, EPO could complement this latter domain for TPO-induced differentiation, suggesting a close relationship between EPO and TPO signaling.     

Cell cycle dependent distribution of a centrosomal antigen at the perinuclear MTOC or at the kinetochores of higher plant cells

Compelling evidence has been obtained in favour of the idea that the nuclear surface of higher plant cells is a microtubule-nucleating and/or organizing site (MTOC), in the absence of defined centrosomes. How these plant MTOC proteins are redistributed and function during the progression of the cell cycle remains entirely unknown. Using a monoclonal antibody (mAb 6C6) raised against isolated calf thymus centrosomes and showing apparent reaction with the plant nuclear surface, we followed the targeted antigen distribution during mitosis and meiosis of higher plants. Immunoblot analysis of protein fractions from Allium root meristematic cell extracts probed with mAb 6C6 reveals a polypeptide of an apparent Mr of 78000. In calf centrosome extracts, a polypeptide of comparable molecular mass is found in addition to a major antigen of Mr 180000 after mAb 6C6 immunoblotting. During mitotic initiation, the plant antigen is prominent on the periphery of the prophase nucleus. When the nuclear envelope breaks down, the antigen suddenly becomes associated with the centromere-kinetochores until late anaphase. In telophase, when the nuclear envelope is being reconstructed, it is no longer detected at the kinetochores but is solely associated again with the nuclear surface. This antigen displays a unique spatial and temporal distribution, which may reflect the pathway of plant protein(s) between the nuclear surface and the kinetochores under cell cycle control. So far, such processes have not been described in higher plant cells. These observations shed light on the putative activity of the plant kinetochore as a protein transporter. They also suggest that a plant centrosome-like antigen may have different cytoskeletal related functions depending on cell cycle regulated changes in its subcellular distribution.Abbreviations mAb monoclonal antibody - MSB microtubule stabilizing buffer - TBS Tris buffered saline - MTOC microtubule organizing centre