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At least 685 records · Page 38Linked to original sources
PubMed2003

Kep1 interacts genetically with dredd/caspase-8, and kep1 mutants alter the balance of dredd isoforms.

The Drosophila kep1 gene encodes an RNA binding protein related to the murine QUAKING apoptotic inducer. We have previously shown that kep1 can induce apoptosis when transfected into different cell lines. To better define the role of Kep1 in apoptosis, we generated kep1 null flies. These flies were viable, but females displayed reduced fertility, with approximately half of the eggs laid from kep1- homozygotes failing to hatch. In addition, loss of kep1 suppressed GMR-rpr-mediated apoptosis in the Drosophila eye, and kep1 mutant flies had increased susceptibility to Escherichia coli infection. We found that Kep1 bound dredd RNA in vitro, and that extracts prepared from kep1 mutant ovaries had markedly reduced proteolytic cleavage activity toward the caspase-8 target substrate IETD-7-amino-4-trifluoromethyl coumarin. We observed increased levels of the beta isoform of dredd mRNA in kep1 mutants as compared with wild-type. Taken together, our results suggest that Kep1 regulates apoptosis by influencing the processing of dredd RNA.

Animals↗
PubMed1975

Temperature-sensitive mutants of chemically transformed epithelial cells.

The first temperature-sensitive mutants of epithelial cells transformed with chemical carcinogens have been isolated. Like the wild-type transformed parental cells, the mutants readily grow in agar suspension at 36 degrees, but in contrast to the wild type, they do not do so at 40 degrees. Detailed studies of one of these mutants, TS-223, indicate that at high temperature it also has reduced cloning efficiency in monolayer culture and a lower saturation density. Scanning electron microscopy revealed that at 40 degrees confluent cultures of TS-223 consist of a monolayer of generally flat polygonal cells, whereas 36 degrees cultures contain many patches of piled-up cells that are spherical and have rougher surface membranes. All of these cellular changes are reversible with upward or downward temperature shifts. The temperature-sensitive lesion appears to reside in a host cell gene which modulates expression of the transformed cell phenotype. These mutants may provide a useful system for elucidating the minimal biochemical changes required for expression of the transformed phenotype in epithelial cells.

Animals↗
PubMed1977

Role of cell surface carbohydrates and proteins in cell behavior: studies on the biochemical reversion of an N-acetylglucosamine-deficient fibroblast mutant.

AD6, a mutant derived from 3T3 Balb/c cells, is characterized by low adhesion to substratum, round shape, increase in surface microvilli, increase in agglutinability by concanavalin A, and loss of directional motility. These properties are often observed in transformed cells. However, the mutant has normal growth properties and anchorage-dependence of growth, and it does not form tumors. In AD6, the biosynthesis of complex carbohydrates and glycoproteins is impaired because of a block in the acetylation of GlcN-6-P. This defect is responsible for all the surface alterations because feeding of GlcNAc to AD6 cells corrects the defects in the synthesis of complex carbohydrates and the exposure of glycoproteins at the outer surface of the plasma membrane. Parallel to this biochemical reversion, there is full restoration of the altered biological properties. In contrast, GlcNAc has no effect on the morphologic features of two lines of transformed cells. Our results suggest that the carbohydrate portion of cell surface proteins has an important role in adhesion and related aspects of cell behavior. The fact that a defined alteration of the cell surface induces many properties often encountered in transformed cells, without affecting control of cell division, strongly suggests that these alterations in properties are not sufficient to account for the loss of growth regulation.

Acetylglucosamine↗
PubMed1995

Requirement for a phospholipase C in odor response: overlap between olfaction and vision in Drosophila.

A central problem in sensory system biology is the identification of the signal transduction pathways used in different sensory modalities. Genetic analysis of transduction mutants provides a means of studying in vivo the contributions of different pathways. This report shows that odorant response in one olfactory organ of Drosophila melanogaster depends on the norpA phospholipase C (EC 3.1.4.3) gene, providing evidence for use of the inositol 1,4,5-trisphosphate (IP3) signal transduction pathway. Since the norpA gene is also essential to phototransduction, this work demonstrates overlap in the genetic and molecular underpinnings of vision and olfaction. Genetic and molecular data also indicate that some olfactory information flows through a pathway which does not depend on norpA.

Animals↗
PubMed1998

The involvement of the fibronectin type II-like modules of human gelatinase A in cell surface localization and activation.

Recombinant collagen-binding domain (rCBD) comprising the three fibronectin type II-like modules of human gelatinase A was found to compete the zymogen form of this matrix metalloproteinase from the cell surface of normal human fibroblasts in culture. Upon concanavalin A treatment of cells, the induced cellular activation of gelatinase A was markedly elevated in the presence of the rCBD. Therefore, the mechanistic aspects of gelatinase A binding to cells by this domain were further studied using cell attachment assays. Fibroblasts attached to rCBD-coated microplate wells in a manner that was inhibited by soluble rCBD, blocking antibodies to the beta1-integrin subunit but not the alpha2-integrin subunit, and bacterial collagenase treatment. Addition of soluble collagen rescued the attachment of collagenase-treated cells to the rCBD. As a probe on ligand blots of octyl-beta-D-thioglucopyranoside-solubilized cell membrane extracts, the rCBD bound 140- and 160-kDa protein bands. Their identities were likely procollagen chains being both bacterial collagenase-sensitive and also converted upon pepsin digestion to 112- and 126-kDa bands that co-migrated with collagen alpha1(I) and alpha2(I) chains. A rCBD mutant protein (Lys263 --> Ala) with reduced collagen affinity showed less cell attachment, whereas a heparin-binding deficient mutant (Lys357 --> Ala), heparinase treatment, or heparin addition did not alter attachment. Thus, a cell-binding mechanism for gelatinase A is revealed that does not involve the hemopexin COOH domain. Instead, an attachment complex comprising gelatinase A-native type I collagen-beta1-integrin forms as a result of interactions involving the collagen-binding domain of the enzyme. Moreover, this distinct pool of cell collagen-bound proenzyme appears recalcitrant to cellular activation.

Antibodies↗
PubMed2001

Binding of antigenic peptide to the endoplasmic reticulum-resident protein gp96/GRP94 heat shock chaperone occurs in higher order complexes. Essential role of some aromatic amino acid residues in the peptide-binding site.

Vaccination with heat shock protein gp96-antigenic peptide complexes produces a powerful specific immune response against cancers and infectious diseases in some experimental animal models, and gp96-peptide complexes are now being tested in human clinical trials. gp96 appears to serve as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. A fundamental issue that needs to be addressed is the mechanism of binding of antigenic peptide to gp96. Here, we show using scanning transmission electron microscopy that recombinant gp96 binds peptide in stable multimeric complexes, which may have biological significance. To open the possibility for genetically engineering gp96 for improved immunogenicity and to understand if molecular recognition plays a role in the binding of antigenic peptide, we mutagenized some specific aromatic amino acids in the presumed peptide-binding pocket. Replacement of Tyr-667 or Tyr-678 to Ala reduced affinity for peptide whereas conversion of Trp-654 to Tyr increased peptide binding. Similarly, changing Trp-621 to Phe or Leu or Ala or Ile negatively affected peptide binding whereas changing Trp-621 to Tyr or Val positively affected peptide binding. Probing the peptide microenvironment in gp96-peptide complexes, suggested that hydrophobic interactions (and perhaps hydrogen bonding/stacking interactions) may play a role in peptide loading by gp96.

Antigens, Neoplasm↗
PubMed2002

New insights into the tPA-annexin A2 interaction. Is annexin A2 CYS8 the sole requirement for this association?

Annexin A2 has been described as an important receptor for tissue-type plasminogen activator in endothelium and other cell types. Interaction between tissue-type plasminogen activator and its cellular receptor is critical for many of the functions of this protease. The annexin A2 motif that mediates tissue plasminogen activator interaction has been assigned to the hexapeptide LCKLSL in the amino-terminal domain of the protein, and it has been proposed that Cys(8) of this sequence is essential for tPA binding. In an attempt to identify other amino acids critical for tPA-annexin A2 interaction, we have analyzed a set of peptides containing several modifications of the original hexapeptide, including glycine scans, alanine scans, d-amino acid scans, conservative mutations, cysteine blocking, and enantiomer and retroenantiomer sequences. Using a non-radioactive competitive binding assay, we have found that all cysteine-containing peptides, independently of their sequence, compete the interaction between tPA and annexin A2. Cysteine-containing peptides also inhibit tPA binding to the surface of cultured human umbilical vein endothelial cells (HUVEC). Mass spectrometry demonstrates that the peptides bind through a disulfide bond to a cysteine residue of annexin A2, the same mechanism that has been suggested for the inhibition mediated by homocysteine. These data call for a revision of the role of the LCKLSL sequence as the sole annexin A2 structural region required to bind tPA and indicate that further studies are necessary to better define the annexin A2-tPA interaction.

Amino Acid Sequence↗
PubMed2004

Post-translational formylglycine modification of bacterial sulfatases by the radical S-adenosylmethionine protein AtsB.

C(alpha)-Formylglycine (FGly) is the catalytic residue of sulfatases. FGly is generated by post-translational modification of a cysteine (prokaryotes and eukaryotes) or serine (prokaryotes) located in a conserved (C/S)XPXR motif. AtsB of Klebsiella pneumoniae is directly involved in FGly generation from serine. AtsB is predicted to belong to the newly discovered radical S-adenosylmethionine (SAM) superfamily. By in vivo and in vitro studies we show that SAM is the critical co-factor for formation of a functional AtsB.SAM.sulfatase complex and for FGly formation by AtsB. The SAM-binding site of AtsB involves (83)GGE(85) and possibly also a juxtaposed FeS center coordinated by Cys(39) and Cys(42), as indicated by alanine scanning mutagenesis. Mutation of these and other conserved cysteines as well as treatment with metal chelators fully impaired FGly formation, indicating that all three predicted FeS centers are crucial for AtsB function. It is concluded that AtsB oxidizes serine to FGly by a radical mechanism that is initiated through reductive cleavage of SAM, thereby generating the highly oxidizing deoxyadenosyl radical, which abstracts a hydrogen from the serine-C(beta)H(2)-OH side chain.

Alanine↗
PubMed2002

Concentration of Ran on chromatin induces decondensation, nuclear envelope formation and nuclear pore complex assembly.

Nuclear envelope (NE) formation can be studied in a cell-free system made from Xenopus eggs. In this system, NE formation involves the small GTPase Ran. Ran associates with chromatin early in nuclear assembly and concentration of Ran on inert beads is sufficient to induce NE formation. Here, we show that Ran binds to chromatin prior to NE formation and recruits RCC1, the nucleotide exchange factor that generates Ran-GTP. In extracts prepared by high-speed centrifugation, increased concentrations of Ran are sufficient to induce chromatin decondensation and NE assembly. Using field emission in-lens scanning electron microscopy (FEISEM), we show that Ran promotes the formation of smoothed membranes and the assembly of nuclear pore complexes (NPCs). In contrast, RanT24N, a mutant that fails to bind GTP and inhibits RCC1, does not support efficient NE assembly, whereas RanQ69L, a mutant locked in a GTP-bound state, permits some membrane vesicle recruitment to chromatin, but inhibits vesicle fusion and NPC assembly. Thus, binding of Ran to chromatin, followed by local generation of Ran-GTP and GTP hydrolysis by Ran, induces chromatin decondensation, membrane vesicle recruitment, membrane formation and NPC assembly. We propose that the biological activity of Ran is determined by its targeting to structures such as chromatin as well as its guanine nucleotide bound state.

Animals↗
PubMed2002

Purine composition of the crystalline cytoplasmic inclusions of Paramecium tetraurelia.

Crystalline cytoplasmic inclusions were isolated by differential centrifugation from mass cultures of Paramecium tetraurelia feeding on Klebsiella pneumonia. Physical and chemical measurements of intact and solubilized crystals determined that they consist primarily of guanine and hypoxanthine with traces of xanthine. Crystals from the mutant sombre consist primarily of xanthine, suggesting there is a disorder of purine metabolism in this mutant.

Animals↗
PubMed2004

Genomic organisation of the chicken ghrelin gene and its single nucleotide polymorphisms detected by denaturing high-performance liquid chromatography.

(1) Ghrelin is a novel endogenous ligand for the growth hormone secretagogue receptor (GHS-R) and is expressed primarily in the stomach and hypothalamus with the probable function of stimulating GH secretion and food intake both in mammals and poultry. The complete sequences of ghrelin gene have been reported in humans and mice; however, that of chickens remains unclear. (2) Here, we report the complete sequence of chicken ghrelin gene (submitted to Genbank; accession number AY303688), which consists of 5 exons and 4 introns. As in mice, the first exon of chicken ghrelin gene does not encode any amino acid. (3) Scanning point mutations with denaturing high-performance liquid chromatography (DHPLC) using WAVE DNA Fragment Analysis Systems and confirmed with direct sequencing for polymerase chain reaction (PCR) products, we analysed the single nucleotide polymorphisms (SNPs) in the entire gene of chicken ghrelin. (4) Results showed that there were 19 SNPs in chicken ghrelin gene, and most of these SNPs were scattered in the 4 introns. In these SNPs, one mutation in exon 5 (A2355G) led to the change of amino acid from glutamine to arginine (Gln 113 Arg): as a result a different ghrelin precursor instead of a mature peptide was produced. In addition, one SNP in 5'UTR (C223G) determined the presence or absence of a potential binding site of transcription factor serum response factor (SRF), which might affect the expression of chicken ghrelin gene. Some of the SNPs detected in the present study could be used in quantitative trait loci (QTL) mapping for growth characters in chickens. (5) Because one SNP is located in a polymorphic site of restriction enzyme PagI of intron 4, it was possible to design a PCR-RFLP procedure and analyse the diversity of 10 chicken populations. Results showed the allelic frequencies of C2100T differ among these breeds, however, no significant difference was observed between imported breeds and Chinese native ones, nor between egg layers and meat type chickens. A chi-square test showed that 4 chicken populations of Beijing Fat, Xinghua, Recessive White and Silky followed the Hardy-Weinberg equilibrium.

Amino Acid Sequence↗
PubMed1990

Comparative morphological and biological studies on the itraconazole- and ketoconazole-resistant mutants of Cryptococcus neoformans.

Studies were carried out on the resistance in vitro of Cryptococcus neoformans to the oral antifungal drugs itraconazole and ketoconazole. None of the six sensitive strains tested developed resistance to itraconazole or ketoconazole by serial transfer on Sabouraud's glucose agar plates containing increasing concentrations of either drug. One mutant resistant to itraconazole, and one mutant resistant to ketoconazole, were isolated from the progenies of yeast cells surviving after treatment with a mutagenic substance, N-methyl-N'-nitro-N-nitroso-guanidine. These mutants were capable of growing in the presence of high concentrations of the drugs to which they were resistant. The itraconazole- and ketoconazole-resistant mutants obtained by mutagenesis were compared morphologically and biologically. The itraconazole-resistant mutant was characterized by the formation of very rough colonies which varied in size and shape, production of a large number of cell clusters, complete loss of capsule formation, and major degenerative changes in the cells, while in the ketoconazole-resistant mutant these changes were less pronounced and no cell clusters were formed. The acquisition of resistance was more stable in the itraconazole-resistant mutant than in the ketoconazole-resistant mutant. Both mutants showed partial cross-resistance and complete loss of virulence for mice.

Animals↗
PubMed1995

F actin bundles in Drosophila bristles. I. Two filament cross-links are involved in bundling.

Transverse sections though Drosophila bristles reveal 7-11 nearly round, plasma membrane-associated bundles of actin filaments. These filaments are hexagonally packed and in a longitudinal section they show a 12-nm periodicity in both the 1.1 and 1.0 views. From earlier studies this periodicity is attributable to cross-links and indicates that the filaments are maximally cross-linked, singed mutants also have 7-11 bundles, but the bundles are smaller, flattened, and the filaments within the bundles are randomly packed (not hexagonal); no periodicity can be detected in longitudinal sections. Another mutant, forked (f36a), also has 7-11 bundles but even though the bundles are very small, the filaments within them are hexagonally packed and display a 12-nm periodicity in longitudinal section. The singed-forked double mutant lacks filament bundles. Thus there are at least two species of cross-links between adjacent actin filaments. Hints of why two species of cross-links are necessary can be gleaned by studying bristle formation. Bristles sprout with only microtubules within them. A little later in development actin filaments appear. At early stages the filaments in the bundles are randomly packed. Later the filaments in the bundles become hexagonally packed and maximally cross-linked. We consider that the forked proteins may be necessary early in development to tie the filaments together in a bundle so that they can be subsequently zippered together by fascin (the singed gene product).

Actins↗
PubMed1998

Why are two different cross-linkers necessary for actin bundle formation in vivo and what does each cross-link contribute?

In developing Drosophila bristles two species of cross-linker, the forked proteins and fascin, connect adjacent actin filaments into bundles. Bundles form in three phases: (a) tiny bundles appear; (b) these bundles aggregate into larger bundles; and (c) the filaments become maximally cross-linked by fascin. In mutants that completely lack forked, aggregation of the bundles does not occur so that the mature bundles consist of <50 filaments versus approximately 700 for wild type. If the forked concentration is genetically reduced to half the wild type, aggregation of the tiny bundles occurs but the filaments are poorly ordered albeit with small patches of fascin cross-linked filaments. In mutants containing an excess of forked, all the bundles tend to aggregate and the filaments are maximally crossbridged by fascin. Alternatively, if fascin is absent, phases 1 and 2 occur normally but the resultant bundles are twisted and the filaments within them are poorly ordered. By extracting fully elongated bristles with potassium iodide which removes fascin but leaves forked, the bundles change from being straight to twisted and the filaments within them become poorly ordered. From these observations we conclude that (a) forked is used early in development to aggregate the tiny bundles into larger bundles; and (b) forked facilitates fascin entry into the bundles to maximally cross-link the actin filaments into straight, compact, rigid bundles. Thus, forked aligns the filaments and then directs fascin binding so that inappropriate cross-linking does not occur.

Actins↗
PubMed1996

The role of O-acetylation in the metabolism of peptidoglycan in Providencia stuartii.

The gentamicin 2'-N-acetyltransferase [EC 2.3.1.59; AAC(2')-Ia] of Providencia stuartii was shown to contribute to the O-acetylation of peptidoglycan and mutants that either under- or overexpress the aac(2')-Ia gene was characterized phenotypically to possess either lower or higher levels of peptidoglycan O-acetylation, respectively, compared to the wild-type. These mutants were subjected to scanning electron microscopy. P. stuartii PR100, with 42-44% peptidoglycan O-acetylation compared to 54% for the wild-type, appeared as irregular rods. In direct contrast, strains PR50.LM3 and PR51, with increased levels of peptidoglycan O-acetylation (63 and 65%, respectively), appeared as coccobacilli or chain formers, respectively. Zymogram analysis of the autolysins produced by another member of the closely related Proteeae group of bacteria, Proteus mirabilis, indicated the presence of three classes of enzymes: one that acts preferentially on native, O-acetylated peptidoglycan, a second that hydrolyses non-O-acetylated peptidoglycan, and a third that is not distinguished by the two forms of substrate. On the basis of the apparent morphological changes directly related to levels of O-acetylation combined with the presence of different classes of autolysins, a model is proposed that invokes the role of this modification in the control of autolysins for the maintenance of the structure of the peptidoglycan sacculus.

Acetylation↗
PubMed2003

Involvement of the AP-1 adaptor complex in early steps of phagocytosis and macropinocytosis.

The best described function of the adaptor complex-1 (AP-1) is to participate in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes. Here, we show that AP-1 is also localized to phagocytic cups in murine macrophages as well as in Dictyostelium amoebae. AP-1 is recruited to phagosomal membranes at this early stage of phagosome formation and rapidly dissociates from maturing phagosomes. To establish the role of AP-1 in phagocytosis, we made used of Dictyostelium mutant cells (apm1(-) cells) disrupted for AP-1 medium chain. In this mutant, phagocytosis drops by 60%, indicating that AP-1 is necessary for efficient phagocytosis. Furthermore, phagocytosis in apm1(-) cells is more affected for large rather than small particles, and cells exhibiting incomplete engulfment are then often observed. This suggests that AP-1 could participate in the extension of the phagocytic cup. Interestingly, macropinocytosis, a process dedicated to fluid-phase endocytosis and related to phagocytosis, is also impaired in apm1(-) cells. In summary, our data suggest a new role of AP-1 at an early stage of phagosome and macropinosome formation.

Animals↗
PubMed1999

The role of RBF in the introduction of G1 regulation during Drosophila embryogenesis.

The first appearance of G1 during Drosophila embryogenesis, at cell cycle 17, is accompanied by the down-regulation of E2F-dependent transcription. Mutant alleles of rbf were generated and analyzed to determine the role of RBF in this process. Embryos lacking both maternal and zygotic RBF products show constitutive expression of PCNA and RNR2, two E2F-regulated genes, indicating that RBF is required for their transcriptional repression. Despite the ubiquitous expression of E2F target genes, most epidermal cells enter G1 normally. Rather than pausing in G1 until the appropriate time for cell cycle progression, many of these cells enter an ectopic S-phase. These results indicate that the repression of E2F target genes by RBF is necessary for the maintenance but not the initiation of a G1 phase. The phenotype of RBF-deficient embryos suggests that rbf has a function that is complementary to the roles of dacapo and fizzy-related in the introduction of G1 during Drosophila embryogenesis.

Animals↗
PubMed2003

Critical role of active repression by E2F and Rb proteins in endoreplication during Drosophila development.

E2F transcription factors can activate or actively repress transcription of their target genes. The role of active repression during normal development has not been analyzed in detail. dE2F1(su89) is a novel allele of dE2F1 that disrupts dE2F1's association with RBF [the Drosophila retinoblastoma protein (Rb) homolog] but retains its transcription activation function. Interestingly, the dE2F1(su89) mutant, which has E2F activation by dE2F1(su89) and active repression by dE2F2, is viable and fertile with no gross developmental defects. In contrast, complete removal of active repression in de2f2;dE2F1(su89) mutants results in severe developmental defects in tissues with extensive endocycles but not in tissues derived from mitotic cycles. We show that the endoreplication defect resulted from a failure to downregulate the level of cyclin E during the gap phase of the endocycling cells. Importantly, reducing the gene dosage of cyclin E partially suppressed all the phenotypes associated with the endoreplication defect. These observations point to an important role for E2F-Rb complexes in the downregulation of cyclin E during the gap phase of endocycling cells in Drosophila development.

Animals↗