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Biomedical subjects

A Ullmann

Publications and source records attributed to A Ullmann.

At least 19 recordsLinked to original sources
PubMed2003

Response to imatinib in patients who relapse after allogeneic stem cell transplantation for chronic myeloid leukemia.

We studied 128 patients with chronic myeloid leukemia (CML) relapsing after allogeneic stem cell transplantation (SCT). Disease at the time of treatment with Imatinib was in chronic phase (CP) in 51 patients, accelerated phase (AP) in 31 and blastic crisis (BC) in 46. Of the 51 patients in CP, 14 were in cytogenetic and two in molecular relapses. The median interval between relapse and Imatinib therapy was 5 months (0-65). A total of 50 patients had failed treatment with donor lymphocyte infusions prior to Imatinib. The overall hemato-logical response rate was 84% (98% for patients relapsing in CP). The complete cytogenetic response (CCR) was 58% for patients in CP, 48% for AP and 22% for patients in BC. Complete molecular responses were obtained in 25 patients (26%), of whom 21 were in CP or AP. With a median follow-up of 9 months, the estimated 2-year survival for CP, AP and BC patients was 100, 86 and 12%, respectively. Out of 79 evaluable patients, 45 (57%) achieved full donor and 11 (14%) mixed chimerism after Imatinib. We conclude that Imatinib has significant activity against CML in relapse after allogeneic SCT. Durable cytogenetic and molecular remissions are obtainable in patients in CP.

Adult↗
PubMed2001

Peptide aptamers targeting the hepatitis B virus core protein: a new class of molecules with antiviral activity.

A substantial proportion of the worldwide liver cancer incidence is associated with chronic hepatitis B virus (HBV) infection. The therapeutic management of HBV infections is still problematic and novel antiviral strategies are urgently required. Using the peptide aptamer screening system, we aimed to isolate new molecules, which can block viral replication by interfering with capsid formation. Eight peptide aptamers were isolated from a randomized expression library, which specifically bound to the HBV core protein under intracellular conditions. One of them, named C1-1, efficiently inhibited viral capsid formation and, consequently, HBV replication and virion production. Hence, C1-1 is a novel model compound for inhibiting HBV replication by blocking capsid formation and provides a new basis for the development of therapeutic molecules with specific antiviral potential against HBV infections.

Amino Acid Sequence↗
PubMed2001

Protein-protein interaction between Bacillus stearothermophilus tyrosyl-tRNA synthetase subdomains revealed by a bacterial two-hybrid system.

We have recently developed a bacterial two-hybrid system (BACTH), based on functional complementation between two fragments of the catalytic domain of Bordetella pertussis adenylate cyclase (AC), that allows an easy in vivo screening and selection of functional interactions between two proteins in Escherichia coli. In this work, we have further explored the potentialities of the BACTH system to study protein-protein interactions, using as a model, the interactions between various subdomains of the dimeric tyrosyl-tRNA synthetase (TyrRS) of Bacillus stearothermophilus. Using the BACTH system we confirmed the known interactions of the alpha/beta domains and those between the alpha/beta domain and the alpha domain that could be anticipated from the three-dimensional structure of TyrRS. Interestingly, the BACTH system revealed the unexpected interaction between the TyrRS alpha domains which is presumably mediated by a pseudo-leucine zipper motif. This study illustrates the interest of the bacterial two-hybrid system to delineate interacting domains of proteins and shows that it can reveal interactions that occur in vivo and that were not anticipated from the three-dimensional structure of the protein of interest.

Adenylyl Cyclases↗
PubMed2000

Induction of apoptosis in human papillomaviruspositive cancer cells by peptide aptamers targeting the viral E6 oncoprotein.

Certain types of human papillomaviruses (HPVs) are closely linked to the development of human cancers. Herein, it is shown that intracellular targeting of the HPV16 E6 oncoprotein by E6-binding peptide aptamers resulted in the apoptotic elimination of HPV16-positive cancer cells, whereas HPV-negative cells were not affected. These results provide direct experimental evidence that the HPV E6 oncoprotein has antiapoptotic activity in HPV-positive tumor cells that is required for their survival. The E6-targeting molecules identified herein have implications for the development of therapeutic strategies for the treatment of HPV-associated dysplasias and cancers.

Amino Acid Sequence↗
PubMed2000

Differential cell cycle response of nontumorigenic and tumorigenic human papillomavirus-positive keratinocytes towards transforming growth factor-beta1.

Human papillomaviruses (HPVs) are causative agents of a number of malignancies in humans, including cervical cancer. Their tumorigenic potential is linked to expression of the viral E6/E7 genes which can interfere with normal cell cycle control by targeting p53, p21WAF1, p27KIP1, and pRb. We show here that nontumorigenic and tumorigenic HPV-positive keratinocytes (HPK) exhibit striking differences in the response of cell cycle regulatory genes towards transforming growth factor beta-beta1. Treatment with this agent led to an efficient induction of p53 and the growth-inhibitory p15INK4 and p21WAF1 genes only in nontumorigenic HPKs and was linked to an efficient reduction in viral E6/E7 oncogene expression. This was associated with increased pRb levels, exhibiting sustained hypophosphorylation, and a permanent growth arrest in the G1 phase of the cell cycle. In contrast, tumorigenic HPKs exhibited only a modest rise in p53 protein levels and a substantially reduced induction of the p15INK4 and p21WAF1 genes, which was linked to a lesser degree of viral oncogene repression. In addition, tumorigenic HPKs rapidly resumed cell growth after a transient G1 arrest, concomitantly with the reappearance of hyperphosphorylated pRb. These results support the notion that the progression of HPV-positive cells to a malignant phenotype is associated with increased resistance to growth inhibition by transforming growth factor-beta1. This is linked in the tumorigenic cells to a lack of persistent G1 arrest, inefficient induction of several cell cycle control genes involved in growth inhibition, and inefficient repression of the growth-promoting viral E6/E7 oncogenes.

Blotting, Northern↗
PubMed2000

Bordetella pertussis adenylate cyclase toxin as a tool to analyze molecular interactions in a bacterial two-hybrid system.

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin (CyaA) that is able to enter into eukaryotic cells. We took advantage of the modular structure of the catalytic domain of CyaA to design a genetic system that can detect protein-protein interactions in Escherichia coli. This bacterial two-hybrid system is based on the functional complementation between two complementary fragments, T25 and T18, of the catalytic domain of CyaA, in an E. coli cya strain. This bacterial two-hybrid system could find applications in the studies of structure/function relationships of proteins, in functional analysis of genomes, in high-throughput screening of interacting ligands and in design of new therapeutic agents.

Adenylate Cyclase Toxin↗
PubMed2000

Sensitive genetic screen for protease activity based on a cyclic AMP signaling cascade in Escherichia coli.

We describe a genetic system that allows in vivo screening or selection of site-specific proteases and of their cognate-specific inhibitors in Escherichia coli. This genetic test is based on the specific proteolysis of a signaling enzyme, the adenylate cyclase (AC) of Bordetella pertussis. As a model system we used the human immunodeficiency virus (HIV) protease. When an HIV protease processing site, p5, was inserted in frame into the AC polypeptide, the resulting ACp5 protein retained enzymatic activity and, when expressed in an E. coli cya strain, restored the Cya(+) phenotype. The HIV protease coexpressed in the same cells resulted in cleavage and inactivation of ACp5; the cells became Cya(-). When the entire HIV protease, including its adjacent processing sites, was inserted into the AC polypeptide, the resulting AC-HIV-Pr fusion protein, expressed in E. coli cya, was autoproteolysed and inactivated: the cells displayed Cya(-) phenotype. In the presence of the protease inhibitor indinavir or saquinavir, AC-HIV-Pr autoproteolysis was inhibited and the AC activity of the fusion protein was preserved; the cells were Cya(+). Protease variants resistant to particular inhibitors could be easily distinguished from the wild type, as the cells displayed a Cya(-) phenotype in the presence of these inhibitors. This genetic test could represent a powerful approach to screen for new proteolytic activities and for novel protease inhibitors. It could also be used to detect in patients undergoing highly active antiretroviral therapy the emergence of HIV variants harboring antiprotease-resistant proteases.

Adenylyl Cyclases↗
PubMed1999

Induction of the p53-target gene GADD45 in HPV-positive cancer cells.

The E6 oncoprotein of human papillomaviruses (HPVs) has the potential to functionally antagonize p53. In several experimental model systems, ectopic expression of E6 can block the genotoxic induction of the growth inhibitory p53 target gene gadd45, suggesting that the inactivation of this pathway may play a major role for HPV-associated cell transformation. Here, we investigated whether this reflects the regulation of gadd45 expression in carcinoma-derived HPV-positive cells. We found that the gadd45 gene is efficiently induced by mitomycin C, cisplatin, and UV irradiation in a series of HPV-positive cervical cancer cell lines. Moreover, clear induction of gadd45 gene expression was also observed following treatment with gamma-irradiation, a pathway that is strictly dependent on functional p53. This contrasted with findings in human foreskin keratinocytes experimentally immortalized by expressing the HPV16 E6, E7, or E6/E7 oncogenes from the heterologous CMV promoter, where expression of the E6 gene was linked to a lack of gadd45 induction following gamma-irradiation. These results indicate (1) that the tumorigenic phenotype of HPV-positive cancer cells is not linked to an inability to induce the gadd45 gene following DNA damage, (2) that experimental model systems in which the E6 gene is expressed ectopically and/or in a different cellular context do not necessarily reflect the regulation of p53-associated pathways in HPV-positive cancer cells and (3) that a pathway strictly depending on functional p53 is inducible in HPV-positive cancer cells, providing direct evidence that the endogenous p53 protein in these cells is competent to activate a cellular target gene, despite coexpression of the viral E6 oncogene.

Carcinoma↗
PubMed1999

Therapy of murine tumors with recombinant Bordetella pertussis adenylate cyclase carrying a cytotoxic T cell epitope.

Bordetella pertussis secretes an invasive adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain into the cytosol of eukaryotic target cells directly through the cytoplasmic membrane. We have shown previously that recombinant CyaA can be used to deliver viral CD8+ T cell epitopes to the MHC-class I presentation pathway to trigger specific CTL responses in vivo. In the present study, we show that mice immunized with a detoxified but still invasive CyaA carrying a CD8+ T cell epitope of OVA developed strong epitope-specific CTL responses, which kill tumor cells expressing this Ag. Treating mice with this recombinant molecule after the graft of melanoma cells expressing OVA induced a strong survival advantage compared with control animals. To our knowledge, this study represents the first demonstration that a nonreplicative and nontoxic vector carrying a single CTL epitope can stimulate efficient protective and therapeutic antitumor immunity.

Adenylate Cyclase Toxin↗
PubMed1999

Direct delivery of the Bordetella pertussis adenylate cyclase toxin to the MHC class I antigen presentation pathway.

Among bacterial toxins, the adenylate cyclase toxin of Bordetella pertussis (CyaA) has a unique mechanism of entry that consists in the direct translocation of its catalytic domain across the plasma membrane of target cell, a mechanism supposed to be independent of any endocytic pathway. Here, we report that the CyaA toxin is delivered to the cytosolic pathway for MHC class I-restricted Ag presentation. Using peritoneal macrophages as APC, we show that the OVA 257-264 CD8+ epitope genetically inserted into a detoxified CyaA (CyaA-OVA E5) is presented to CD8+ T cells by a mechanism requiring 1) proteasome processing, 2) TAP, and 3) neosynthesis of MHC class I. We demonstrate that the presentation of CyaA-OVA E5, like the translocation of CyaA into eukaryotic cells, is dependent on extracellular Ca2+ and independent of vacuolar acidification. Moreover, inhibitors of the phagocytic and macropinocytic endocytic pathways do not affect the CyaA-OVA E5 presentation. The absence of specific cellular receptors for CyaA correlates with the ability of various APC to present the recombinant CyaA toxin, including dendritic cells, macrophages, splenocytes, and lymphoid tumoral lines. Taken together, our results show that the CyaA presentation pathway is not cell type specific and is unrelated to a defined type of endocytic mechanism. Thus, it represents a new and unconventional delivery of an exogenous Ag into the conventional cytosolic pathway.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Bordatella pertussis adenylate cyclase: a toxin with multiple talents.

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin (CyaA) that is able to deliver its amino-terminal catalytic domain into the cytosol of eukaryotic cells. The novelty of the structural organization and conformational flexibility of the CyaA catalytic domain has opened up the way for exploiting this protein as a tool for several biological applications, including epitope delivery, protein targeting and characterization of protein-protein interactions.

Adenylate Cyclase Toxin↗
PubMed1998

Charge-dependent translocation of Bordetella pertussis adenylate cyclase toxin into eukaryotic cells: implication for the in vivo delivery of CD8(+) T cell epitopes into antigen-presenting cells.

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400-aa residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver T cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted CD8(+) cytotoxic T cell responses in vivo. Here, we constructed a series of recombinant toxins harboring at the same insertion site various peptide sequences of 11-25 amino acids, corresponding to defined CD8(+) T cell epitopes and differing in the charge of the inserted sequence. We show that inserted peptide sequences containing net negative charges (-1 or -2) decreased or completely blocked (charge of -4) the internalization of the toxin into target cells in vitro and abolished the induction of cytotoxic T cell responses in vivo. The blocking of translocation due to the inserted acidic sequences can be relieved by appropriate mutations in the flanking region of CyaA that counterbalance the inserted charges. Our data indicate that (i) the electrostatic charge of the peptides inserted within the catalytic domain of CyaA is critical for its translocation into eukaryotic cells and (ii) the delivery of T cell epitopes into the cytosol of antigen-presenting cells by recombinant CyaA toxins is essential for the in vivo stimulation of specific cytotoxic T cells. These findings will help to engineer improved recombinant CyaA vectors able to stimulate more efficiently cellular immunity.

Adenylate Cyclase Toxin↗
PubMed1998

Uncoupling of p21WAF1/CIP1/SDI1 mRNA and protein expression upon genotoxic stress.

The p21WAF1/CIP1/SDI1 gene is an important regulator of crucial cellular processes, including cell cycle control, cellular differentiation, and the response to genotoxic stress. Induction of p21 gene expression upon DNA damage is widely believed to be p53-dependent. In the present study we analysed the expression of p21 following genotoxic stress, using different DNA-damaging agents and cellular systems. We found that the p21 response markedly varied between different cell lines and also for different genotoxic agents within the same cell line. Genotoxic induction of p21 mRNA expression can occur in the presence of p53-antagonists, such as overexpressed mdm-2 or human papillomavirus (HPV) E6, and in cells harbouring mutated p53 genes. Moreover, upon genotoxic stress, p21 mRNA and protein expression were found to be uncoupled in several cell lines. Thus, transcriptional and postranscriptional changes in p21 expression following DNA damage are not necessarily linked to the intracellular p53 status but strongly depend on the individual cellular background and the type of DNA-damaging agent. Our findings indicate that p21 expression following genotoxic stress underlies a complex control and can be substantially modulated on the posttranscriptional level in a cell-specific manner.

Cells, Cultured↗
PubMed1998

A bacterial two-hybrid system based on a reconstituted signal transduction pathway.

We describe a bacterial two-hybrid system that allows an easy in vivo screening and selection of functional interactions between two proteins. This genetic test is based on the reconstitution, in an Escherichia coli cya strain, of a signal transduction pathway that takes advantage of the positive control exerted by cAMP. Two putative interacting proteins are genetically fused to two complementary fragments, T25 and T18, that constitute the catalytic domain of Bordetella pertussis adenylate cyclase. Association of the two-hybrid proteins results in functional complementation between T25 and T18 fragments and leads to cAMP synthesis. Cyclic AMP then triggers transcriptional activation of catabolic operons, such as lactose or maltose, that yield a characteristic phenotype. In this genetic test, the involvement of a signaling cascade offers the unique property that association between the hybrid proteins can be spatially separated from the transcriptional activation readout. This permits a versatile design of screening procedures either for ligands that bind to a given "bait," as in the classical yeast two-hybrid system, or for molecules or mutations that block a given interaction between two proteins of interest.

Adenylyl Cyclases↗
PubMed1998

Pleiotropic effects of cAMP on germination, antibiotic biosynthesis and morphological development in Streptomyces coelicolor.

In wild-type Streptomyces coelicolor MT1110 cultures, cyclic adenosine 3',5' monophosphate (cAMP) was synthesized throughout the developmental programme with peaks of accumulation both during germination and later when aerial mycelium and actinorhodin were being produced. Construction and characterization of an adenylate cyclase disruption mutant (BZ1) demonstrated that cAMP facilitated these developmental processes. Although pulse-labelling experiments showed that a similar germination process was initiated in BZ1 and MT1110, germ-tube emergence was severely delayed in BZ1 and never occurred in more than 85% of the spores. Studies of growth and development on solid glucose minimal medium (SMMS, buffered or unbuffered) showed that MT1110 and BZ1 produced acid during the first rapid growth phase, which generated substrate mycelium. Thereafter, on unbuffered SMMS, only MT1110 resumed growth and produced aerial mycelium by switching to an alternative metabolism that neutralized its medium, probably by reincorporating and metabolizing extracellular acids. BZ1 was not able to neutralize its medium or produce aerial mycelium on unbuffered SMMS; these defects were suppressed by high concentrations (>1 mM) of cAMP during early growth or on buffered medium. Other developmental mutants (bldA, bldB, bldC, bldD, bldG) also irreversibly acidified this medium. However, these bald mutants were not suppressed by exogenous cAMP or neutralizing buffer. BZ1 also differentiated when it was cultured in close proximity to MT1110, a property observed in cross-feeding experiments between bald mutants and commonly thought to reflect diffusion of a discrete positively acting signalling molecule. In this case, MT1110 generated a more neutral pH environment that allowed BZ1 to reinitiate growth and form aerial mycelium. The fact that actinorhodin synthesis could be induced by concentrations of cAMP (< 20 microM) found in the medium of MT1110 cultures, suggested that it may serve as a diffusible signalling molecule to co-ordinate antibiotic biosynthesis.

Adenylyl Cyclases↗
PubMed1998

Hybrid Bordetella pertussis-Escherichia coli RNA polymerases: selectivity of promoter activation.

We constructed hybrid Bordetella pertussis-Escherichia coli RNA polymerases and compared productive interactions between transcription activators and cognate RNA polymerase subunits in an in vitro transcription system. Virulence-associated genes of B. pertussis, in the presence of their activator BvgA, are transcribed by all variants of hybrid RNA polymerases, whereas transcription at the E. coli lac promoter regulated by the cyclic AMP-catabolite gene activator protein has an absolute requirement for the E. coli alpha subunit. This suggests that activator contact sites involve a high degree of selectivity.

Amino Acid Sequence↗