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C E Sunkel

Publications and source records attributed to C E Sunkel.

At least 37 records · Page 2Linked to original sources
PubMed1996

The recombinant alpha isoform of protein kinase CK1 from Xenopus laevis can phosphorylate tyrosine in synthetic substrates.

The cDNA coding for protein kinase CK1 alpha has been cloned from a Xenopus laevis cDNA library. The derived amino acid sequence of the protein contains 337 amino acids and has a calculated molecular mass of 38874 Da. The sequence is identical to that of the human CK1 alpha and to the bovine CK1 alpha, except that it is 12 amino acids longer than the latter protein. Southern blotting with a 264-bp probe demonstrates that four or more fragments are obtained upon digestion of genomic DNA with EcoR1 and Hind3, suggesting that X. laevis possesses a family of related CK1 genes. CK1 alpha was expressed in Escherichia coli as a glutathione transferase fusion protein (GT-CK1 alpha) and certain of its characteristics were determined. The recombinant GT-CK1 alpha fusion protein was found to have apparent Km values for ATP (12 microM), casein (1.5 mg/ml) and the specific peptide substrate RRKDLHDDEEDEAMSITA (180 microM) which are similar to those of the rat liver CK1 enzyme. The recombinant CK1 alpha activity is weakly inhibited by heparin, but strongly inhibited by poly(Glu80:Tyr20). This inhibition is competitive and shows an approximate K1 of 5 microM. CK1 alpha can phosphorylate the tyrosine residues of poly(Glu80:Tyr20) and the tyrosine residue in the synthetic peptide RRREEEYEEEE. This kinase preparation also autophosphorylates in serine, threonine and weakly in tyrosine.

Amino Acid Sequence↗
PubMed1996

The conserved mitotic kinase polo is regulated by phosphorylation and has preferred microtubule-associated substrates in Drosophila embryo extracts.

The Drosophila gene polo encodes a protein kinase required for progression through mitosis. Wild-type polo protein migrates as a tight doublet of 67 kDa which is converted to a single band by phosphatase treatment, which also inactivates the kinase. We have determined putative polo substrates in a cell-free system derived from mutant embryos. Exogenous polo protein kinase phosphorylates proteins of sizes 220 kDa, 85 kDa and 54 kDa, to a greater extent when added to extracts of polo(1)-derived embryos compared with extracts of wild-type embryos, which in both cases have been subject to mild heat treatment to inactivate endogenous kinases. Proteins of the same size are predominantly phosphorylated by the endogenous kinases present in wild-type extracts, and are either not phosphorylated or are poorly phosphorylated in extracts of polo(1)-derived embryos. We show that a specific monoclonal antibody to beta-tubulin precipitates the phosphorylated 54 kDa protein together with an associated 85 kDa protein also phosphorylated by polo protein kinase. Moreover polo binds to an 85 kDa protein which is enriched in microtubule preparations. We discuss the extent to which these in vitro phosphorylation results reflect the effects of mutations in polo on microtubule behaviour during the mitotic cycle.

Animals↗
PubMed1996

Abnormal development of the locomotor activity in yellow larvae of Drosophila: a cuticular defect?

The yellow (y) mutation of Drosophila melanogaster affects the development of behavior and morphology. We have analyzed some behavioral and morphological parameters during the development of y mutants. Wild-type third instar larvae move in straighter paths than larvae of the same age homozygous for the y mutation. At 96 h of age, the tracks of y larvae have 10 times as many loops as tracks of wild-type larvae, and at 120 h of age, y larvae show bending behavior about 2.5 times more frequently than do wild-type. Consequently, they do not disperse as much as wild-type larvae. Concomitant with the behavioral changes, the larvae present a defect in the morphology of large chaetae in the larval denticle belts, particularly of 2nd and 3rd instars, both with light and scanning electron microscopes. These results suggest that a cuticular defect is probably involved in the abnormal locomotor activity observed in y larvae of Drosophila melanogaster.

Animals↗
PubMed1996

The casein kinase 1 alpha gene of Drosophila melanogaster is developmentally regulated and the kinase activity of the protein induced by DNA damage.

We report the molecular cloning and characterisation of the first CK1(casein kinase) gene of Drosophila melanogaster (dmCK1). The protein sequence (DMCK1) shares significant homology with other mammalian CK1 protein kinases of the alpha sub-class. The dmCK1 gene is expressed only in adult females and during early embryonic development as a single transcript. Western blot analysis of total protein extracts of different stages of development show that the gene product is likewise present during early embryogenesis and in adult females. Kinase activity studies show that DMCK1 is active when in vitro translated but inactive when immunoprecipitated from total early embryo extracts. However, after dephosphorylation treatment the immunoprecipitates show high kinase activity. More significantly, DMCK1 kinase activity present in the immunoprecipitates can be specifically activated by gamma-irradiation of early embryos. Also, when DMCK1 is immunoprecipitated after irradiation it appears to undergo phosphorylation. Immunolocalization of DMCK1 in early embryos shows that the protein is predominantly cytoplasmic but after irradiation there is a significant relocalization to the interphase nucleus. The results suggest a possible requirement of the Drosophila CK1 alpha for mechanisms associated with DNA repair during early embryogenesis.

Amino Acid Sequence↗
PubMed1996

Identification of Porto-1, a new repeated sequence that localises close to the centromere of chromosome 2 of Drosophila melanogaster.

We have used the polymerase chain reaction (PCR) technique to search the Drosophila melanogaster genome for the presence of sequences with homology to mammalian and yeast centromeric DNA. Using primers based on the human CENP-B box present in alpha-satellite DNA and part of the Saccharomyces cerevisiae CDEIII centromeric sequence, a number of specific DNA fragments were amplified from total genomic DNA. In situ hybridization to polytene and mitotic chromosomes showed these fragments to localise to centromeric and pericentromeric regions. Direct cloning of the amplified fragments into conventional plasmids proved unsuccessful. However, a recombinant P1 clone containing D. melanogaster genomic DNA that supports PCR amplification by the primers was identified. Molecular characterisation of this clone revealed a DNA fragment that localises primarily to the centromere of chromosome 2. Sequence analysis indicated that this fragment contains at least four different repeats, including Rsp, transposable elements, Bari-1 and a new AT-rich repeated sequence that we have designated Porto-1. Detailed fluorescence in situ hybridization analysis shows that Porto-1 is localised very close to the primary constriction of chromosome 2. Sequence analysis suggests that this repeat was specifically amplified by our primers, although limited homology to the CENP-B box or CDEIII elements was found. In situ hybridization to a number of Drosophila species shows Porto-1 to be present only in D. melanogaster.

Amino Acid Sequence↗
PubMed1995

Gamma-tubulin is required for the structure and function of the microtubule organizing centre in Drosophila neuroblasts.

We report that in Drosophila, gamma-tubulin is required for the structure as well as the function of microtubule organizing centres (MTOCs). This conclusion is based on the identification and phenotypic characterization of a mutant allele of the gamma-tubulin gene located at region 23C of the polytene chromosome map. This mutation, which we have called gamma-tub23CPl, is caused by the insertion of a P-element within the 5' untranslated leader of the gamma-tubulin transcript. Northern and Western analysis show that gamma-tub23CPl is either a null or a very severe hypomorph as no gamma-tubulin mRNA or protein can be detected in mutant individuals. Visualization of DNA, MTOCs and microtubules by confocal laser scanning microscopy of cells from individuals homozygous for gamma-tub23CPl reveals a series of phenotypic abnormalities. Some of these are similar to those observed after disruption of gamma-tubulin function in other organisms, including mitotic arrest and a dramatic decrease in the number of microtubules, but, in addition, we have observed that mutation in this gene also results in highly abnormal MTOCs which show a variety of shapes and sizes which we never observed in wild type cells. These results show that gamma-tubulin is required for both structural and functional roles in the MTOCs.

Animals↗
PubMed1995

The elusive centromere: sequence divergence and functional conservation.

The centromere is an essential cis-acting structure present in the chromosomes of all eukaryotes, central to the mechanism that ensures proper segregation during meiosis and mitosis. Molecular characterization of centromeres in the budding and fission yeasts has advanced significantly over the last few years due to their relatively small size and the availability of functional assays. However, identification and characterization of centromeric sequences from multicellular organisms has proven to be slow and difficult in the absence of direct functional tests. Molecular data have recently become available on the centromere of Drosophila, making it possible to bridge a long-standing gap in our knowledge on the general structure of centromeres. An evaluation of the available data from yeast to man suggests that centromere sequence and centromere sequence organization have diverged significantly, even amongst different chromosomes of a single organism; however, overall centromere organization and kinetochore components might be significantly more conserved than thought previously.

Animals↗
PubMed1994

Isolation of chromosome-associated proteins from Drosophila melanogaster that bind a human centromeric DNA sequence.

The molecular mechanism involved in packaging centromeric heterochromatin is still poorly understood. CENP-B, a centromeric protein present in human cells, is though to be involved in this process. This is a DNA-binding protein that localizes to the central domain of the centromere of human and mouse chromosomes due to its association with the 17-bp CENP-B box sequence. We have designed a biochemical approach to search for functional homologues of CENP-B in Drosophila melanogaster. This strategy relies upon the use of DNA fragments containing the CENP-B box to identify proteins that specifically bind this sequence. Three polypeptides were isolated by nuclear protein extraction, followed by sequential ion exchange columns and DNA affinity chromatography. All three proteins are present in the complex formed after gel retardation with the human alphoid satellite DNA that contains the CENP-B box. Footprinting analysis reveals that the complex occupies both strands of the CENP-B box, although it is still unclear which of the polypeptides actually makes contact with the DNA. Localization of fluorescein-labeled proteins after microinjection into early Drosophila embryos shows that they associate with condensed chromosomes. Immunostaining of embryos with a polyclonal serum made against all three polypeptides also shows chromosomal localization throughout mitosis. During metaphase and anaphase the antigens appear to localize preferentially to centromeric heterochromatin. Immunostaining of neuroblasts chromosome spreads confirmed these results, though some staining of chromosomal arms is also observed. The data strongly suggests that the polypeptides we have identified are chromosomal binding proteins that accumulate mainly at the centromeric heterochromatin. Furthermore, DNA binding assays clearly indicate that they have a high specific affinity for the human CENP-B box. This would suggest that at least one of the three proteins isolated might be a functional homologue of the human CENP-B.

Animals↗
PubMed1993

Interference of the PAF receptor antagonist, PCA 4248, with the rat pleurisy evoked by inflammatory mediators or allergen.

This study investigated the effect of the platelet-activating factor (PAF) receptor antagonist, PCA 4248, on the rat pleurisy caused by PAF, serotonin, bradykinin, histamine or allergen. The pleurisy was assessed by measuring liquid extravasation and leucocyte infiltration. Oral pretreatment with PCA 4248 (2.5-20 mg/kg) completely inhibited the pleural exudation caused by intrathoracic (i.t.) injection of PAF (1 microgram/cavity) (ED50 = 6.1 mg/kg), partially (42% reduction) the one induced by serotonin (100 micrograms/cavity), but was inactive against histamine (200 micrograms/cavity) or bradykinin (50 micrograms/cavity). PCA 4248 blocked the increase in the number of neutrophils, eosinophils and mononuclear cells observed 6 h after the i.t. injection of PAF, as well as the selective eosinophil accumulation noted 24 h later. In actively sensitized rats, PCA 4248 (20 mg/kg) failed to modify the increase in the total leucocyte counts noted 4 h after ovalbumin (12 micrograms/cavity), but dose dependently inhibited the pleural exudation observed within 1 h and the late eosinophil infiltration noted 24 h post-antigen. These observations led us to suggest that PCA 4248 is a potent PAF antagonist with anti-serotoninergic properties. Its interference with exudation and eosinophil infiltration caused by allergen is consistent with the interpretation that PCA 4248 may be useful in the management of allergic dysfunctions.

Allergens↗
PubMed1993

The 55 kd regulatory subunit of Drosophila protein phosphatase 2A is required for anaphase.

The gene encoding the Drosophila protein phosphatase 2A 55 kd regulatory subunit (PR55) is located at 85F and directs the synthesis of differentially spliced transcripts. Maternal RNAs are present at very high levels in early embryos and decline around cellularization. Zygotic transcripts are present mainly in the developing embryonic nervous system and gonads. Transcripts are uniformly distributed in third instar larval discs and testes and at lower levels in the proliferative centers of the brain. Mutations in abnormal anaphase resolution (aar) are rescued by the wild-type gene for PR55. aar mutants display intact lagging chromatids that have undergone separation from their sisters, but that remain at the position formerly occupied by the metaphase plate, as well as anaphase figures that show bridging chromatin having two centromeric regions.

Alternative Splicing↗
PubMed1993

Abnormal anaphase resolution (aar): a locus required for progression through mitosis in Drosophila.

We describe a new mitotic locus of Drosophila melanogaster required for the progression through mitosis in the syncytial embryo and in late larval development. The locus aar (abnormal anaphase resolution) maps to the cytological interval 85E7-F16 and was identified by two alleles. The aar1 allele causes pupal lethality. Larval neuroblasts show an elevated mitotic index with high chromosome condensation and stretched and lagging chromatids during anaphase. aar2 produces fully viable but sterile females. aar1/aar2 females lay eggs that develop mitotic figures with similar abnormalities to those observed in neuroblasts. Indirect immunofluorescence of these embryos indicates that the centrosome cycle appears normal, although some abnormal spindle microtubules can be seen during mitosis.

Alleles↗
PubMed1992

Synthesis of 3-[(2,3-dihydro-1,1,3-trioxo-1,2-benzisothiazol-2-yl)alkyl] 1,4-dihydropyridine-3,5-dicarboxylate derivatives as calcium channel modulators.

1,4-Dihydropyridine (DHP) derivatives with a 1,2-benzisothiazol-3-one 1,1-dioxide group, linked through an alkylene bridge to the C-3 carboxylate of the DHP ring, with both vasoconstricting and vasorelaxant properties were obtained. In blocking Ca(2+)-evoked contractions of K(+)-depolarized rabbit aortic strips, compounds 12 and 41 were 10-fold more potent than nifedipine; 27 other compounds were 1-4-fold more potent. Their vascular versus cardiac selectivity was very pronounced; for instance, the selectivity index for compound 41 was 70-fold higher than that of nifedipine. This was also true for the vasoconstricting compound 22, which was as potent as Bay K 8644 in enhancing the Ca(2+)-evoked contractions of rabbit aorta strips, yet it had poor inotropic activity in rabbit left atria. Oral administration of compounds 38, 40, 43, and 53 (20 mg/kg) caused a 35-37% decrease in systolic blood pressure in spontaneously hypertensive rats (SHR); these effects were similar to those of nifedipine. However, iv administration of these compounds to anesthetized SHR caused a decrease in blood pressure which was more pronounced and long-lasting than that of nifedipine. When administered iv at 100 micrograms/kg, the vasoconstricting compound 22 caused a 40% increase in systolic and diastolic blood pressure. Compound 22 exhibited an unusually interesting feature over the other five Ca2+ DHP agonists: it had diester substitutions at the C-3 and C-5 positions of the DHP ring. Overall, compounds possessing these properties might be useful in treating clinical cardiovascular conditions in which DHP Ca2+ antagonists or agonists are indicated.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Systemic lupus erythematosus murine monoclonal DNA-binding antibodies recognize cytoplasmic and nuclear phosphorylated antigens that display cell cycle redistribution in HEp-2 cells.

The immunological basis for the production of autoantibodies characteristic of systemic lupus erythematosus (SLE) against a wide range of antigens remains obscure. The specificity of (NZB x NZW)F1 (BWF1) or MRL/Mp-lpr/lpr (MRL/lpr) mouse monoclonal antibodies (mAb) was examined by immunofluorescence, immunoblotting and immunoprecipitation techniques. Using non-synchronized HEp-2 cells as substrate, the murine mAb were classified by indirect immunofluorescence into five groups on the basis of their staining patterns of subcellular components in interphase and mitotic stages of the cell cycle. The nature of the antigens recognized by the murine lupus was assessed by immunoblotting experiments in total, cytoplasmic and nuclear cell extracts from HEp-2 cells. The six antibodies used recognized in total cell extracts a range of polypeptides with apparent molecular weights from 25,000 to 210,000. Three polypeptides of 130,000, 110,000 and 45,000 MW were recognized by all six antibodies in both nuclear and cytoplasmic extracts. Immunoprecipitation of total cellular extracts labelled with [35S]methionine showed almost the same pattern as obtained in the immunoblotting assay. The labelling in vivo of HEp-2 cells with [32P], followed by the immunoprecipitation of the [32P]cell lysate showed that these mAb recognized phosphorylated proteins. The progressive decrease in reactivity of these mAb following treatment with higher concentrations of alkaline phosphatase in both [32P]cell lysate or nitrocellulose membranes indicates that these mAb recognize phosphorylated epitopes.

Animals↗
PubMed1991

The spindle is required for the process of sister chromatid separation in Drosophila neuroblasts.

We have studied two aspects of the process of sister chromatid separation in the Drosophila melanogaster neuroblasts. First, we analyzed the requirement of a functional spindle for sister chromatid separation to take place using microtubule depolymerizing drugs such as colchicine or a reversible analogue (MTC). Incubation of this tissue in colchicine causes the cells to block irreversibly at metaphase and no significant levels of sister chromatid separation were observed even after long periods of incubation. Exposure of neuroblasts to MTC also causes cells to block at metaphase, but after reversion most of the cells enter anaphase and are thus able to complete sister chromatid separation. These results imply that a functional spindle is required for sister chromatid separation. Second, we studied the role of heterochromatin during chromatid pairing and subsequent separation in chromosomes which carry either one or two extra pieces of heterochromatin. The results indicate that sister chromatids establish strong pairing along the translocated heterochromatin. During the early stages of anaphase, these chromosomes separate first the centromeric region and later the regions bearing extra heterochromatin. These results indicate that constitutive heterochromatin plays an important role for sister chromatid pairing and might be involved in the process of separation.

Animals↗
PubMed1991

polo encodes a protein kinase homolog required for mitosis in Drosophila.

We show that mutation in polo leads to a variety of abnormal mitoses in Drosophila larval neuroblasts. These include otherwise normal looking mitotic spindles upon which chromosomes appear overcondensed; normal bipolar spindles with polyploid complements of chromosomes; bipolar spindles in which one pole can be unusually broad; and monopolar spindles. We have cloned the polo gene from a mutant allele carrying a P-element transposon and sequenced cDNAs corresponding to transcripts of the wild-type locus. The sequence shows that polo encodes a 577-amino-acid protein with an amino-terminal domain homologous to a serine-threonine protein kinase. polo transcripts are abundant in tissues and developmental stages in which there is extensive mitotic activity. The transcripts show no obvious spatial pattern of distribution in relation to the mitotic domains of cellularized embryos but are specifically concentrated in dividing cells in larval discs and brains. In the cell cycles of both syncytial and cellularized embryos, the polo kinase undergoes cell cycle-dependent changes in its distribution: It is predominantly cytoplasmic during interphase; it becomes associated with condensed chromosomes toward the end of prophase; and it remains associated with chromosomes until telophase, whereupon it becomes cytoplasmic.

Amino Acid Sequence↗
PubMed1990

Properties and partial characterization of the heat-shock factor from Tetrahymena pyriformis.

A heat-shock-factor-binding activity was identified in Tetrahymena pyriformis whole-cell extracts and was further purified by sequential heparin-agarose and sequence-specific oligonucleotide affinity chromatography. Tetrahymena heat-shock factor (HSF) was able to bind to the heat-shock elements (HSE) both before and after thermal stress, although heat shock altered both the HSE-binding affinity and the protein.DNA-complex mobility on polyacrylamide gels. The mobility difference was significantly reduced by treatment of the proteins with phosphatase. The HSE-binding proteins, isolated by oligonucleotide-affinity chromatography, migrated on SDS/polyacrylamide gels as a closely spaced doublet to about 70 kDa. Polypeptides with similar molecular mass were recovered from preparative band-shift gels indicating that both are components of the protein.DNA complex.

Animals↗
PubMed1990

4-Alkyl-1,4-dihydropyridines derivatives as specific PAF-acether antagonists.

A new series of 4-alkyl-1,4-dihydropyridines (1,4-DHP) were synthesized and evaluated for their ability to inhibit washed rabbit platelet aggregation induced by PAF-acether (1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glycero-3-phosphorylcholine) and to reverse PAF-induced hypotension in anesthetized rats. Additionally, compounds were evaluated for their ability to inhibit the binding of radiolabeled PAF to its receptor on rabbit platelets. Among these compounds, 6I and 6L were the most potent and specific antagonists. At concentrations up to 100 microM, neither compound 6I nor compound 6L caused platelet aggregation nor did they inhibit platelet aggregation induced by collagen or adenosine diphosphate. Compound 6L did not show in vitro calcium channel blocker activity measured on vascular smooth muscle preparations of rabbit aorta and on [3H]nitrendipine binding assays. The compound did not show any cardiovascular effects in anesthetized rat at iv doses up to 1000 micrograms/kg, and the Ki value was 568.62 nmol. These results indicate that compound 6L is a potent and specific PAF antagonist with 1,4-dihydropyridine structure but devoid of a significant cardiovascular activity related to calcium-antagonist properties.

Adenosine Triphosphate↗