Efeitos da eliminação de neurônios infragranulares sobre a especificação de neurônios supragranulares do córtex cerebral

The cerebral cortex of mammals is histologically organized into different layers of excitatory neurons that have distinct patterns of connections with cortical or subcortical targets. During development, these cortical layers are established through an intricate combination of neuronal specification and migration in a radial pattern known as "insideout": deep-layer neurons are generated prior to upper-layer neurons. In the last few decades, several genes encoding transcription factors involved in the sequential specification of neurons destined to different cortical layers have been identified. However, the influence of early-generated neurons in the specification of subsequent neuronal cohorts remains unclear. To investigate this possible influence, we induced the selective death of cortical neurons from layer V and VI before the generation of layer II, III and IV neurons. Thus, we can evaluate the effects of ablation of early born neurons on the phenotype of late born neurons. Our data shows that one-day after ablation, layer VI neurons expressing the transcription factor TBR1 are newly generated while virtually no neuron expressing TBR1 was generated in the same age in control animals. This suggests that progenitors involved in the generation of neurons destined for superficial layers suffer interference from the selective death of neurons in deep layers, changing their specification. We also observed that while TBR1-positive neurons are located exclusively in deep cortical layers of control animals, many TBR1-positive neurons are misplaced in superficial layers of ablated animals, suggesting that the migration of cortical neurons could be controlled independently of neuronal phenotypes. Furthermore, we observed an increase in layer V neurons expressing CTIP2 and neurons expressing SATB2 and that these cells have changed their distributions. As a conclusion, our data indicate the existence of a mechanism of control exercised by the early-generated neurons in the cerebral cortex on the fate of the progenitors involved in the generation of the following cortical neurons. This mechanism could help to control the number of neurons in different layers and contribute to the establishment of different cortical areas

GM-CSF receptor targeted treatment of primary AML in SCID mice using Diphtheria toxin fused to huGM-CSF

The severe combined immunodeficient (SCID) mouse model may be used to evaluate new approaches for the treatment of acute myeloid leukemia (AML). We have previously demonstrated the killing of SCID mouse leukemia initiating cells by in vitro incubation with human GM-CSF fused to Diphtheria toxin (DT-huGM-CSF). In this report, we show that in vivo treatment with DT-huGM-CSF eliminates AML growth in SCID mice. Seven cases of AML were studied. SCID mice were treated intraperitoneally with the maximally tolerated dose of 75 microg/kg/day for 7 days. Antileukemic efficacy was determined at days 40 and 80 after transplantation, by enumerating the percentages of human cells in SCID bone marrow using flow cytometry and short tandem repeat polymerase chain reaction (STR-PCR) analysis. Four out of seven AML cases were sensitive to in vivo treatment with DT-huGM-CSF at both evaluation time points. In three of these cases, elimination of human cells was demonstrated by flow cytometry and STR-PCR. One AML case showed moderate sensitivity for DT-huGM-CSF, and growth of the two remaining AML cases was not influenced by DT-huGM-CSF. Sensitivity was correlated with GM-CSFR expression. Our data show that DT-huGM-CSF can be used in vivo to reduce growth of AML and warrant further development of DT-huGM-CSF for the treatment of human AML.

Effect of electroporation-mediated diphtheria toxin A expression on PSA positive human prostate xenograft tumors in SCID mice

Current therapies to treat prostate cancer are often limited. Since it has been shown that very low concentrations of diphtheria toxin A (DT-A) result in abrogation of protein synthesis and apoptosis of cells, DT-A might serve as an efficient killer in cancer gene therapy. For this purpose we investigated in a quantitative manner using a stereological approach the apoptotic effect of DT-A in androgen receptor (AR) and prostate specific antigen (PSA) expressing cells after tumor formation in both flanks of SCID mice.

Isolation and characterization of iron-independent positive dominant mutants of the diphtheria toxin repressor DtxR

It is well known that the functional activity of the diphtheria toxin repressor DtxR is controlled by iron, which serves as an essential cofactor necessary for activation of target DNA binding by this regulatory element. In this communication, we describe the isolation and characterization of a unique series of DtxR mutants that are constitutively active and repress the expression of β-galactosidase from a diphtheria tox promoter/operator–lacZ transcriptional fusion, even in the absence of iron. These self-activating mutants of DtxR (SAD) were isolated through the use of a positive selection system for the cloning of functional dtxR alleles and target DNA operator sites. Of the four independently isolated SAD mutants that were characterized, two (SAD2 and SAD11) were found to carry a single missense mutation (E175K) in their respective C-terminal SH3-like domains. In contrast, the mutant allele encoding SAD3 was found to carry a total of six missense mutations distributed throughout the N- and C-terminal domains of the repressor. Partial diploid analysis of strains carrying both native dtxR and alleles encoding either SAD2 or SAD3 demonstrate that these iron-independent mutants possess a positive dominant phenotype in the regulation of β-galactosidase expression from a diphtheria tox promoter/operator–lacZ transcriptional fusion.

Regulated expression of the diphtheria toxin A chain by a tumor-specific chimeric transcription factor results in selective toxicity for alveolar rhabdomyosarcoma cells

Alveolar rhabdomyosarcoma (ARMS) cells often harbor one of two unique chromosomal translocations, either t(2;13)(q35;q14) or t(1;13)(p36;q14). The chimeric proteins expressed from these rearrangements, PAX3-FKHR and PAX7-FKHR, respectively, are potent transcriptional activators. In an effort to exploit these unique cancer-specific molecules to achieve ARMS-specific expression of therapeutic genes, we have studied the expression of a minimal promoter linked to six copies of a PAX3 DNA binding site, prs-9. In transient transfections, expression of the prs-9-regulated reporter genes was ≈250-fold higher than expression of genes lacking the prs-9 sequences in cell lines derived from ARMS, but remained at or below baseline levels in other cells. High expression of these prs-9-regulated genes was also observed in a cancer cell line that lacks t(2;13) but was stably transfected with a plasmid expressing PAX3-FKHR. Transfection of a plasmid containing the diphtheria toxin A chain gene regulated by prs-9 sequences (pA3–6PED) was selectively cytotoxic for PAX3-FKHR-expressing cells. This was shown by inhibition of gene expression from cotransfected plasmids and by direct cytotoxicity after transfected cells were isolated by cell sorting. Gene transfer of pA3–6PED may thus be useful as a cancer-specific treatment strategy for t(2;13)- or t(1;13)-positive ARMS. Furthermore, gene transfer of fusion protein-regulated toxin genes might also be applied to the treatment of other cancers that harbor cancer-specific chromosomal translocations involving transcription factors.

Solution structure and peptide binding studies of the C-terminal Src homology 3-like domain of the diphtheria toxin repressor protein

The diphtheria toxin repressor (DtxR) is the best-characterized member of a family of homologous proteins that regulate iron uptake and virulence gene expression in the Gram-positive bacteria. DtxR contains two domains that are separated by a short, unstructured linker. The N-terminal domain is structurally well-defined and is responsible for Fe2+ binding, dimerization, and DNA binding. The C-terminal domain adopts a fold similar to eukaryotic Src homology 3 domains, but the functional role of the C-terminal domain in repressor activity is unknown. The solution structure of the C-terminal domain, consisting of residues N130-L226 plus a 13-residue N-terminal extension, has been determined by using NMR spectroscopy. Residues before A147 are highly mobile and adopt a random coil conformation, but residues A147-L226 form a single structured domain consisting of five β-strands and three helices arranged into a partially orthogonal, two-sheet β-barrel, similar to the structure observed in the crystalline Co2+ complex of full-length DtxR. Chemical shift perturbation studies demonstrate that a proline-rich peptide corresponding to residues R125-G139 of intact DtxR binds to the C-terminal domain in a pocket formed by residues in β-strands 2, 3, and 5, and helix 3. Binding of the proline-rich peptide by the C-terminal domain of DtxR presents an example of peptide binding by a prokaryotic Src homology 3-like protein. The results of this study, combined with previous x-ray studies of intact DtxR, provide insights into a possible biological function of the C-terminal domain in regulating repressor activity.

Fused polycationic peptide mediates delivery of diphtheria toxin A chain to the cytosol in the presence of anthrax protective antigen.

The lethal factor (LF) and edema factor (EF) of anthrax toxin bind by means of their amino-terminal domains to protective antigen (PA) on the surface of toxin-sensitive cells and are translocated to the cytosol, where they act on intracellular targets. Genetically fusing the amino-terminal domain of LF (LFN; residues 1-255) to certain heterologous proteins has been shown to potentiate these proteins for PA-dependent delivery to the cytosol. We report here that short tracts of lysine, arginine, or histidine residues can also potentiate a protein for such PA-dependent delivery. Fusion of these polycationic tracts to the amino terminus of the enzymic A chain of diphtheria toxin (DTA; residues 1-193) enabled it to be translocated to the cytosol by PA and inhibit protein synthesis. The efficiency of translocation was dependent on tract length: (LFN > Lys8 > Lys6 > Lys3). Lys6 was approximately 100-fold more active than Arg6 or His6, whereas Glu6 and (SerSerGly)2 were inactive. Arg6DTA was partially degraded in cell culture, which may explain its low activity relative to that of Lys6DTA. The polycationic tracts may bind to anionic sites at the cell surface (possibly on PA), allowing the fusion proteins to be coendocytosed with PA and delivered to the endosome, where translocation to the cytosol occurs. Excess free LFN blocked the action of LFNDTA, but not of Lys6DTA. This implies that binding to the LF/EF site is not an obligatory step in translocation and suggests that the polycationic tag binds to a different site. Besides elucidating the process of translocation in anthrax toxin, these findings may aid in developing systems to deliver heterologous proteins and peptides to the cytoplasm of mammalian cells.

Genetic construction and properties of a diphtheria toxin-related substance P fusion protein: in vitro destruction of cells bearing substance P receptors.

We have genetically replaced the native receptor binding domain of diphtheria toxin with an extended form of substance P (SP): SP-glycine (SP-Gly). The resulting fusion protein, DAB389SP-Gly, is composed of the catalytic and transmembrane domains of diphtheria toxin genetically coupled to SP-Gly. Because native SP requires a C-terminal amide moiety to bind with high affinity to the SP receptor, the precursor form of the fusion toxin, DAB389SP-Gly, was converted to DAB389SP by treatment with peptidylglycine-alpha-amidating monooxygenase. We demonstrate that following conversion, DAB389SP is selectively cytotoxic for cell lines that express either the rat or the human SP receptor. We also demonstrate that the cytotoxic action of DAB389SP is mediated via the SP receptor and dependent upon passage through an acidic compartment. To our knowledge, this is the first reported use of a neuropeptide as the targeting ligand for a fusion toxin; and the first instance in which an inactive precursor form of a fusion toxin is converted to the active form by a posttranslational modification.

Transgenic mice carrying the diphtheria toxin A chain gene under the control of the granzyme A promoter: expected depletion of cytotoxic cells and unexpected depletion of CD8 T cells.

We have generated transgenic mice bearing the diphtheria toxin A chain (DTA) gene under the control of granzyme A (GrA) promoter sequences (GrA-DTA). GrA is expressed in activated cytotoxic cells but not in their immediate progenitors. These GrA-DTA mice are deficient in cytotoxic functions, indicating that most cytotoxic cells express GrA in vivo. Surprisingly, one founder strain containing a multicopy GrA-DTA insert show a marked and selective deficiency in CD8+ cells in peripheral lymphoid organs. This depletion was not observed in thymus, where the distribution of CD4+ and CD8+ cells is normal. Moreover, the emigration of T cells from thymus is normal, indicating that the depletion occurs in the periphery. GrA-DTA mice should be useful as models to dissect the role of cytotoxic cells in immune responses and as recipients of normal and neoplastic hematopoietic cells. The selective depletion of CD8+ cells in one founder strain could have implications for postthymic T-cell development.

In vivo konditionale Depletion von latentem murinen Cytomegalovirus

Die Lunge stellt einen Hauptort der CMV-Latenz dar. Die akute CMV-Infektion wird durch infiltrierende antivirale CD8 T-Zellen terminiert. Das virale Genom verbleibt jedoch im Lungengewebe in einem nicht replikativen Zustand, der Latenz, erhalten. Es konnte bereits gezeigt werden, dass während der Latenz die Major Immediate Early- (MIE) Gene ie1- und ie2 sporadisch transkribiert werden. Bisher konnte diese beginnende Reaktivierung latenter CMV-Genome nur in einer Momentaufnahme gezeigt werden (Kurz et al., 1999; Grzimek et al., 2001; Simon et al., 2005; zur Übersicht: Reddehase et al., 2008). Die sporadische Expression der MIE-Gene führt jedoch zur Präsentation eines antigenen IE1-Peptids und somit zur Stimulation antiviraler IE1-Peptid-spezifischer CD8 T-Zellen, die durch ihre Effektorfunktion die beginnende Reaktivierung wieder beenden. Dies führte uns zu der Hypothese, dass MIE-Genexpression über einen Zeitraum betrachtet (period prevalence) häufiger stattfindet als es in einer Momentaufnahme (point prevalence) beobachtet werden kann.rnrnUm die Häufigkeit der MIE-Genexpression in der Dynamik in einem definierten Zeitraum zu erfassen, sollte eine Methode entwickelt werden, welche es erstmals ermöglicht, selektiv und konditional transkriptionell aktive Zellen sowohl während der akuten Infektion als auch während der Latenz auszulöschen. Dazu wurde mit Hilfe der Zwei-Schritt BAC-Mutagenese ein rekombinantes death-tagged Virus hergestellt, welches das Gen für den Diphtherie Toxin Rezeptor (DTR) unter Kontrolle des ie2-Promotors (P2) enthält. Ist der P2 transkriptionell aktiv, wird der DTR an der Zelloberfläche präsentiert und die Zelle wird suszeptibel für den Liganden Diphtherie Toxin (DT). Durch Gabe von DT werden somit alle Zellen ausgelöscht, in denen virale Genome transkriptionell aktiv sind. Mit zunehmender Dauer der DT-Behandlung sollte also die Menge an latenten viralen Genomen abnehmen.rnrnIn Western Blot-Analysen konnte das DTR-Protein bereits 2h nach der Infektion nachgewiesen werden. Die Präsentation des DTR an der Zelloberfläche wurde indirekt durch dessen Funktionalität bewiesen. Das rekombinante Virus konnte in Fibroblasten in Gegenwart von DT nicht mehr replizieren. In akut infizierten Tieren konnte die virale DNA-Menge durch eine einmalige intravenöse (i.v.) DT-Gabe signifikant reduziert werden. Verstärkt wurde dieser Effekt durch eine repetitive i.v. DT-Gabe. Auch während der Latenz gelang es, die Zahl der latenten viralen Genome durch repetitive i.v. und anschließende intraperitoneale (i.p.) DT-Gabe zu reduzieren, wobei wir abhängig von der Dauer der DT-Gabe eine Reduktion um 60\% erreichen konnten. Korrespondierend zu der Reduktion der DNA-Menge sank auch die Reaktivierungshäufigkeit des rekombinanten Virus in Lungenexplantatkulturen. rnrnrnUm die Reaktivierungshäufigkeit während der Latenz berechnen zu können, wurde durch eine Grenzverdünnungsanalyse die Anzahl an latenten viralen Genomen pro Zelle bestimmt. Dabei ergab sich eine Kopienzahl von 9 (6 bis 13). Ausgehend von diesen Ergebnissen lässt sich berechnen, dass, bezogen auf die gesamte Lunge, in dem getesteten Zeitraum von 184h durch die DT-Behandlung 1.000 bis 2.500 Genome pro Stunde ausgelöscht wurden. Dies entspricht einer Auslöschung von 110 bis 280 MIE-Gen-exprimierenden Lungenzellen pro Stunde. Damit konnte in dieser Arbeit erstmals die Latenz-assoziierte Genexpression in ihrer Dynamik dargestellt werden.rn

Granulocyte death regulation by naturally occurring autoantibodies

Programmed cell death (PCD) plays a central role in the regulation of granulocytes that are key effector cells of the innate immune system. Granulocytes are produced in high amounts in the bone marrow. A safe elimination of granulocytes by cell death (apoptosis) is essential to maintain the numbers of these cells balanced. In many acute and chronic inflammatory diseases, delayed apoptosis is one mechanism that contributes to accumulation of neutrophil and eosinophil granulocytes at the site of inflammation. On the other hand, a safe elimination of granulocytes by cell death is required to avoid unwanted tissue damage for instance by secretion of toxic products from these cells. Recent evidence shows that humans produce an array of naturally occurring autoantibodies (NAbs) with the capacity to regulate granulocyte death, including agonistic and antagonistic NAbs that bind to the receptors Fas, Siglec-8, and Siglec-9. Together with other factors, these various NAbs exhibit different properties in terms of the form of cell death they induce, the molecular signaling pathways they engage, as well as the efficacy or potency by which they induce cell death. Moreover, several regulatory mechanisms seem to exist that control their biological activity. Novel insights support the concept of granulocyte death regulation by NAbs, which might have important implications for our understanding of the pathogenesis and treatment of inflammatory diseases, including many autoimmune and allergic disorders.

Estimates of life expectancy gains from reducing/eliminating major causes of death in the USA: An update analysis for population in 2001--2008

Evaluation of the impact of a disease on life expectancy is an important part of public health. Potential gains in life expectancy (PGLE) that can properly take into account the competing risks are an effective indicator for measuring the impact of the multiple causes of death. This study aimed to measure the PGLEs from reducing/eliminating the major causes of death in the USA from 2001 to 2008. To calculate the PGLEs due to the elimination of specific causes of death, the age-specific mortality rates for heart disease, malignant neoplasms, Alzheimer disease, kidney diseases and HIV/AIDS and life table constructing data were obtained from the National Center for Health Statistics, and the multiple decremental life tables were constructed. The PGLEs by elimination of heart disease, malignant neoplasms or HIV/AIDS continued decreasing from 2001 to 2008, but the PGLE by elimination of Alzheimer's disease or kidney diseases revealed increased trends. The PGLEs (by years) for all race, male, female, white, white male, white female, black, black male and black female at birth by complete elimination of heart disease 2001–2008 were 0.336–0.299, 0.327–0.301, 0.344–0.295, 0.360–0.315, 0.349–0.317, 0.371–0.316,0.278–0.251, 0.272–0.255, and 0.282–0.246 respectively. Similarly, the PGLEs (by years) for all race, male, female, white, white male, white female, black, black male and black female at birth by complete elimination of malignant neoplasms, Alzheimer's disease, kidney disease or HIV/AIDS 2001–2008 were also uncovered, respectively. Most diseases affect specific population, such as, HIV/AIDS tends to have a greater impact on people of working age, heart disease and malignant neoplasms have a greater impact on people over 65 years of age, but Alzheimer's disease and kidney diseases have a greater impact on people over 75 years of age. To measure the impact of these diseases on life expectancy in people of working age, partial multiple decremental life tables were constructed and the PGLEs were computed by partial or complete elimination of various causes of death during the working years. Thus, the results of the study outlined a picture of how each single disease could affect the life expectancy in age-, race-, or sex-specific population in USA. Therefore, the findings would not only assist to evaluate current public health improvements, but also provide useful information for future research and disease control programs.^

Channel formation by antiapoptotic protein Bcl-2

Bcl-2 is the prototypical member of a large family of apoptosis-regulating proteins, consisting of blockers and promoters of cell death. The three-dimensional structure of a Bcl-2 homologue, Bcl-XL, suggests striking similarity to the pore-forming domains of diphtheria toxin and the bacterial colicins, prompting exploration of whether Bcl-2 is capable of forming pores in lipid membranes. Using chloride efflux from KCl-loaded unilamellar lipid vesicles as an assay, purified recombinant Bcl-2 protein exhibited pore-forming activity with properties similar to those of the bacterial toxins, diphtheria toxin, and colicins, i.e., dependence on low pH and acidic lipid membranes. In contrast, a mutant of Bcl-2 lacking the two core hydrophobic α-helices (helices 5 and 6), predicted to be required for membrane insertion and channel formation, produced only nonspecific effects. In planar lipid bilayers, where detection of single channels is possible, Bcl-2 formed discrete ion-conducting, cation-selective channels, whereas the Bcl-2 (Δh5, 6) mutant did not. The most frequent conductance observed (18 ± 2 pS in 0.5 M KCl at pH 7.4) is consistent with a four-helix bundle structure arising from Bcl-2 dimers. However, larger channel conductances (41 ± 2 pS and 90 ± 10 pS) also were detected with progressively lower occurrence, implying the step-wise formation of larger oligomers of Bcl-2 in membranes. These findings thus provide biophysical evidence that Bcl-2 forms channels in lipid membranes, suggesting a novel function for this antiapoptotic protein.

Cloning and characterization of a cellular apoptosis susceptibility gene, the human homologue to the yeast chromosome segregation gene CSE1.

We recently isolated human cDNA fragments that render MCF-7 breast cancer cells resistant to cell death caused by Pseudomonas exotoxin, Pseudomonas exotoxin-derived immunotoxins, diphtheria toxin, and tumor necrosis factor. We report here that one of these fragments is an antisense fragment of a gene homologous to the essential yeast chromosome segregation gene CSE1. Cloning and analysis of the full-length cDNA of the human CSE1 homologue, which we name CAS for cellular apoptosis susceptibility gene, reveals a protein coding region with similar length (971 amino acids for CAS, 960 amino acids for CSE1) and 59% overall protein homology to the yeast CSE1 protein. The conservation of this gene indicates it has an important function in human cells consistent with the essential role of CSE1 in yeast. CAS is highly expressed in human tumor cell lines and in human testis and fetal liver, tissues that contain actively dividing cells. Furthermore, CAS expression increases when resting human fibroblasts are induced to proliferate and decreases when they are growth-arrested. Thus, CAS appears to play an important role in both toxin and tumor necrosis factor-mediated cell death, as well as in cell proliferation.

Structures of the apo- and the metal ion-activated forms of the diphtheria tox repressor from Corynebacterium diphtheriae.

The diphtheria tox repressor (DtxR) of Corynebacterium diphtheriae plays a critical role in the regulation of diphtheria toxin expression and the control of other iron-sensitive genes. The crystal structures of apo-DtxR and of the metal ion-activated form of the repressor have been solved and used to identify motifs involved in DNA and metal ion binding. Residues involved in binding of the activated repressor to the diphtheria tox operator, glutamine 43, arginine 47, and arginine 50, were located and confirmed by site-directed mutagenesis. Previous biochemical and genetic data can be explained in terms of these structures. Conformational differences between apo- and Ni-DtxR are discussed with regard to the mechanism of action of this repressor.