Protein phosphatase 1 activity in Drosophila mutants with abnormalities in mitosis and chromosome condensation

A Drosophila gene encoding a protein phosphatase 1 (PP1) has been sequenced, and lethal mutations in this locus (87B) analysed. Two mutants (ck19e211 and ck19hs46), which disrupt mitosis, lack the 87B isoenzyme and express only approximately 20% of wild type PP1 activity. The promoter region of the gene is deleted in the ck19e211 mutant. A third mutant (ck19e078), which shows suppression of position effect variegation, but has little effect on mitosis, possesses approximately 35% of wild type PP1 activity. The results indicate that the PP1 87B isoenzyme is involved in regulation of chromosome condensation at interphase as well as mitosis.     

UVA-mediated down-regulation of MMP-2 and MT1-MMP coincides with impaired angiogenic phenotype of human dermal endothelial cells

UVA irradiation, dose-dependently (5-20 J/cm2), was shown to impair the morphogenic differentiation of human microvascular endothelial cells (HMECs) on Matrigel. Parallely, UVA down-regulated the expression of MMP-2 and MT1-MMP, both at the protein and the mRNA levels. On the contrary, the production of MMP-1 and TIMP-1 by HMECs increased following UVA treatment. The inhibitory effect of UVA on MMP expression and pseudotubes formation was mediated by UVA-generated singlet oxygen (1O2). The contribution of MT1-MMP, but not TIMP-1, to the regulation of HMECs' angiogenic phenotype following UVA irradiation was suggested using elastin-derived peptides and TIMP-1 blocking antibody, respectively.     

Characterization of acid phosphatase-1 null activity mutants of Drosophila melanogaster

A complementation analysis was performed on 15 Acph-1 ⁿ alleles. Only three of these alleles proved to be nonleaky and to exhibit no evidence of complementation. These were then tested immunologically to determine their level of antigenically cross-reacting material (CRM). Their CRM levels were virtually zero, as compared to low, but positive, levels for two other homozygous Acph-1 ⁿ mutants and considerably higher levels for heteroallelic combinations of some of the leaky alleles. Acid phosphatase-1 enzyme subunits, formed by dissociating native enzyme, have close to 100% CRM activity in tests with antibodies elicited by native enzyme.Submitted by John Bell in partial fulfillment of the requirements for a Doctor of Philosophy degree from Cornell University.     

Persistent locus-specific instability of yellow 2-717 and Notch Uc-1 in Drosophila melanogaster coincides with hobo multiplication

The presence of multiple copies of the hobo element in unstable yellow and Notch loci in y ^2-717 and Uc-1 Drosophila melanogaster stocks, respectively, was found according to FISH data. Locus-specific instability in these strains is caused by hobo multiplication in the respective loci and its subsequent recombination with neighboring hobo copies rather than its insertion (excision). Original Russian Text L.P. Zakharenko, L.V. Kovalenko, S.Mai, I.K. Zakharov, 2007, published in Tsitologiya, Vol. 49, No. 6, 2007.     

Mating activity of yellow and sepia Drosophila willistoni mutants

By analyzing the mating activity of newly isolated yellow and sepia mutants of Drosophila willistoni no difference in behavior between sepia and wild-type flies were observed, whereas yellow males were less successful than wild-type males when competing for females. These results are in agreement with those reported for other Drosophila species. D. willistoni was different in the 'females-competing' crosses because wild-type males mated more frequently with wild-type females whereas yellow males mated successfully with both phenotypes. These results indicate the complexity of the courtship behavior in D. willistoni and provide data for comparative and evolutionary research into the genus.     

The protein phosphatases of Drosophila melanogaster and their inhibitors

Protein phosphatases-1, 2A and 2B have been identified in membrane and soluble fractions of Drosophila melanogaster heads. Similarities between Drosophila and mammalian protein phosphatase-1 included specificity for the beta subunit of phosphorylase kinase, sensitivity to inhibitor-1 and inhibitor-2, inhibition by protamine, retention by heparin-Sepharose and selective interaction with membranes. In addition, an inactive form of protein phosphatase-1, termed protein phosphatase-1I, was detected in the soluble fraction that could be activated by preincubation with MgATP and mammalian glycogen synthase kinase-3. Inhibitor-2 partially purified from Drosophila had an identical molecular mass to its mammalian counterpart, and recombined with mammalian protein phosphatase-1 to form a hybrid protein phosphatase-1I. Similarities between Drosophila and mammalian protein phosphatase-2A included preferential dephosphorylation of the alpha subunit of phosphorylase kinase, insensitivity to inhibitors-1 and -2, activation by protamine, exclusion from heparin-Sepharose and apparent molecular mass. A Ca2+-dependent calmodulin-stimulated protein phosphatase (protein phosphatase-2B) that was inhibited by trifluoperazine was identified in the soluble fraction. The remarkable similarities between Drosophila protein phosphatases and their mammalian counterparts are indicative of strict phylogenetic conservation and demonstrate that the procedures used to classify mammalian protein phosphatases have a wider application. Characterisation of the Drosophila phosphatases will facilitate genetic analysis of dephosphorylation systems and their possible roles in neuronal and behavioural plasticity in Drosophila.     

A protein kinase gene complements the lytic phenotype of Saccharomyces cerevisiae lyt2 mutants

By genetic analysis of a thermosensitive autolytic mutant whose phenotype was complemented by osmotic stabilization with sorbitol, we identified gene LYT2 of Saccharomyces cerevisiae, which is probably involved in cell wall formation. A yeast gene complementing lyt2 strains was cloned and shown to carry an open reading frame coding for a 484-amino-acid protein exhibiting all the characteristic domains of serine/threonine protein kinases and highly homologous to other yeast protein kinases involved in control of the mitotic cycle. Mutants disrupted in the cloned gene also displayed an autolytic phenotype complemented by osmotic stabilization with sorbitol. However, genetic comparison of lyt2 mutants and disruptants of the protein kinase gene revealed that the cloned gene is not the structural gene LYT2 but a suppressor of the lytic phenotype, named gene SLT2, that was mapped to chromosome V. The product of gene SLT2 is the first protein kinase to be described in relation to the yeast cell-wall functions.     

The genetics of abnormal abdomen, incomplete abdomen, and bobbed in Drosophila buzzatii

Five stocks of Drosophila buzzatii with superficially similar abdominal disruptions including partial tergite and sternite loss were isolated by inbreeding. Three of the stocks have indistinguishable phenotypes, the inheritance of which is maternally influenced. This phenotype and its mode of inheritance bear similarities with those of Abnormal abdomen in D. melanogaster. The phenotype in the fourth stock is slightly different and is due to a single autosomal recessive gene, which we denote incomplete abdomen. In the fifth stock the trait is limited to females, and in appearance and mode of inheritance resembles bobbed in D. melanogaster. Furthermore, only in this stock are rDNA deletions evident. The combined frequencies of the three types of abdominal aberration were found to be around 1% in several samples from wild and laboratory populations of D. buzzatii.     

三叶因子Bm-TFF2突变体在原核体系中的表达及促细胞迁移活性

大蹼铃蟾三叶因子Bm-TFF2具有较人TFF2更强的促细胞迁移和抗凋亡活性。该研究利用RT-PCR方法扩增得到野生型Bm-TFF2的基因,然后分别构建N端、C端和分子中两个精氨酸突变的突变体,最后连接表达载体产生pET32a(+)/Bm-TFF2突变型重组质粒,转入大肠杆菌中,经37℃培养,IPTG诱导,其融合蛋白主要存在于包涵体中,用组氨酸标签的亲合柱纯化溶解后的包涵体上清,进一步用RP-HPLC纯化得到硫氧还蛋白(TRX)/Bm-TFF2突变型融合蛋白。通过SDS-PAGE和Westernblotting检测分析其纯度和特异性。最终,从1L培养基中得到20mg纯度为95%的三种重组突变型融合蛋白。三种突变型重组蛋白都具有剂量依赖性的促细胞迁移活性,并且其活性无显著差异。该研究为进一步研究Bm-TFF2结构和功能的关系以及揭示其作用的分子机制奠定了基础。     

Folding and activity of cAMP-dependent protein kinase mutants

The catalytic subunit of cAMP-dependent protein kinase (PKA) can easily be expressed in Escherichia coli and is catalytically active. Four phosphorylation sites are known in PKA (S10, S139, T197 and S338), and the isolated recombinant protein is a mixture of different phosphorylated forms. Obtaining uniformly phosphorylated protein requires separation of the protein preparation leading to significant loss in protein yield. It is found that the mutant S10A/S139D/S338D has similar properties as the wild-type protein, whereas additional replacement of T197 with either E or D reduces protein expression yield as well as folding propensity of the protein. Due to its high sequence homology to Akt/PKB, which cannot easily be expressed in E. coli, PKA has been used as a surrogate kinase for drug design. Several mutations within the ATP binding site have been described to make PKA even more similar to Akt/PKB. Two proteins with Akt/PKB-like mutations in the ATP binding site were made (PKAB6 and PKAB8), and in addition S10, S139 and S338 phosphorylation sites have been removed. These proteins can be expressed in high yields but have reduced activity compared to the wild-type. Proper folding of all proteins was analyzed by 2D 1H, 15N-TROSY NMR experiments.     

Induction of in vitro nuclear apoptosis activity coincides with the production of 50 kDa cytosolic protein

Human monocytic leukemia U937 cells undergo apoptosis when cells are treated with the anticancer drug etoposide. To study the mechanism of drug-induced apoptosis, we used an in vitro apoptosis system with cytosol from etoposide-treated U937 cells. The cytosol from apoptotic U937 cells showed activity to induce morphologic changes and oligonucleosomal DNA fragmentation in isolated nuclei in vitro; both are typical features of apoptosis. We generated monoclonal antibodies to the proteins in the etoposide-treated U937 cytosol. We found that a 50 kDa protein, recognized by SN-1 monoclonal antibody, appeared in the cytosol of U937 cells, in accordance with its cell-free apoptosis activity. Z-Asp, an inhibitor of interleukin-1beta converting enzyme (ICE) family proteases, inhibited the appearance of the 50 kDa protein and the emergence of the cell-free apoptosis activity in the etoposide-treated U937 cytosol. These results indicate that the 50 kDa protein is produced by the activation of ICE family protease during apoptosis and suggest some roles of the protein in the development of apoptosis.     

Alterations in both acetylcholinesterase activity and synaptic scaffolding protein localization in the nervous system of Drosophila presenilin mutants

Presenilins are one of two types of critical genetic factors in familial Alzheimer's disease, and they regulate various cellular functions such as intracellular Ca²⁺ homeostasis, the endoplasmic reticulum (ER) stress response, apoptosis, and synaptic transmission. We utilized Drosophila presenilin (psn) mutants as a model for studying the role of this gene in regulating acetylcholinesterase activity (AChE) and synaptic plasticity. Several lines of biochemical evidence indicated that AChE activity in a functionally null psn mutant (psn^B3) was significantly reduced. In addition, we also found that psn^B3 mutant neuromuscular junctions (NMJs) had smaller synaptic boutons and altered localization of Discs large, a synaptic scaffolding protein at the synaptic terminals compared to wild-type controls. These phenotypic defects were completely rescued in transgenic lines expressing the long form of wild-type Psn under an endogenous psn promoter cassette (PEPC-Psn^WT;psn^B3 lines). Taken together, these results indicate that Psn is important for regulating AChE activity, the size of synaptic boutons, and the localization of DLG at synaptic terminals.