RESUMO
BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive skin cancer, whose tumour cells often express CD56. While immune checkpoint inhibitors constitute a major advance for treating patients with MCC with advanced disease, new therapeutic options are still urgently required. OBJECTIVES: To produce and evaluate the therapeutic performance of a new antibody-drug conjugate (Adcitmer® ) targeting CD56 in preclinical models of MCC. METHODS: CD56 expression was evaluated in a MCC cohort (immunohistochemistry on a tissue microarray of 90 tumour samples) and MCC cell lines. Interaction of an unconjugated CD56-targeting antibody with CD56+ MCC cell lines was investigated by immunohistochemistry and imaging flow cytometry. Adcitmer® product was generated by the bioconjugation of CD56-targeting antibody to a cytotoxic drug (monomethyl auristatin E) using the McSAF Inside® bioconjugation process. The chemical properties and homogeneity of Adcitmer® were characterized by hydrophobic interaction chromatography. Adcitmer® cytotoxicity was evaluated in vitro and in an MCC xenograft mice model. RESULTS: Similar to previous reports, CD56 was expressed by 66% of MCC tumours in our cohort, confirming its relevance as a therapeutic target. Specific binding and internalization of the unconjugated CD56-targeting antibody was validated in MCC cell lines. The high homogeneity of the newly generated Adcitmer® was confirmed by hydrophobic interaction chromatography. The CD56-mediated cytotoxicity of Adcitmer® was demonstrated in vitro in MCC cell lines. Moreover, Adcitmer® significantly reduced tumour growth in a MCC mouse model. CONCLUSIONS: Our study suggests that Adcitmer® should be further assessed as a therapeutic option in patients with MCC, as an alternative therapy or combined with immune checkpoint inhibitors.
Assuntos
Carcinoma de Célula de Merkel , Neoplasias Cutâneas , Animais , Carcinoma de Célula de Merkel/tratamento farmacológico , Carcinoma de Célula de Merkel/patologia , Humanos , Imuno-Histoquímica , Camundongos , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Neoplasias Cutâneas/patologiaRESUMO
In this work we show the first microdosimetry measurements on a low energy proton beam with therapeutic-equivalent fluence rates by using the second generation of 3D-cylindrical microdetectors. The sensors belong to an improved version of a novel silicon-based 3D-microdetector design with electrodes etched inside silicon, which were manufactured at the National Microelectronics Centre (IMB-CNM, CSIC) in Spain. A new microtechnology has been employed using quasi-toroid electrodes of 25µm diameter and a depth of 20µm within the silicon bulk, resulting in a well-defined cylindrical radiation sensitive volume. These detectors were tested at the 18 MeV proton beamline of the cyclotron at the National Accelerator Centre (CNA, Spain). They were assembled into an in-house low-noise readout electronics to assess their performance at a therapeutic-equivalent fluence rate. Microdosimetry spectra of lineal energy were recorded at several proton energies starting from 18 MeV by adding 50µm thick tungsten foils gradually at the exit-window of the cyclotron external beamline, which corresponds to different depths along the Bragg curve. The experimentalyF¯values in silicon cover from (5.7 ± 0.9) to (8.5 ± 0.4) keV µm-1in the entrance to (27.4 ± 2.3) keV µm-1in the distal edge. Pulse height energy spectra were crosschecked with Monte Carlo simulations and an excellent agreement was obtained. This work demonstrates the capability of the second generation 3D-microdetectors to assess accurate microdosimetric distributions at fluence rates as high as those used in clinical centers in proton therapy.
Assuntos
Terapia com Prótons , Silício , Método de Monte Carlo , Prótons , RadiometriaRESUMO
We present a new and advanced methodology, developed for surface functionalisation of gold and to study immobilisation of an immuno-specific system of proteins. A combination of electrochemical quartz crystal microbalance and Raman spectroscopy techniques allowed a complete understanding of the system starting from surface functionalisation and progressing to the functional structure analysis of immobilised proteins. A simple electrochemical procedure was formulated to prepare sulphonyl chloride terminated gold surfaces that form a strong sulphonamide bond with the receptor protein staphylococcal protein A (SpA). On the SpA grafted surfaces, the immobilisation of a human IgG and consecutive binding of an immuno-specific anti-human IgG was observed. The surface functional groups form a strong interaction with SpA without disturbing its functional properties. The native functional structure of SpA and also the IgGs was found to be retained in their immobilised state.
Assuntos
Proteína Estafilocócica A/química , Eletroquímica , Imunoglobulina G/química , Análise Espectral RamanRESUMO
We have previously mapped a locus controlling Plasmodium falciparum blood infection levels (PFBI) to chromosome 5q31-q33. We genotyped 19 microsatellite markers on chromosome 5q31-q33 in a new sample of 44 pedigrees comprising 84 nuclear families and 292 individuals living in a P. falciparum endemic area. Using a nonparametric multipoint variance-component approach (by GENEHUNTER), we evidenced a peak of linkage close to D5S636 (P=0.0069), with a heritability of 0.46. Using a variance-component method for linkage-disequilibrium mapping of quantitative traits (by QTDT) and the Bonferroni correction for multiple testing, we further detected allelic association in the presence of linkage between blood infection levels and D5S487 (P=6 x 10(-5); P(c)=0.0011), which is located on the distal part of the peak. These results confirm the importance of chromosome 5q31-q33 in the genetic control of PFBI levels.
Assuntos
Cromossomos Humanos Par 5/genética , Eritrócitos/parasitologia , Ligação Genética , Malária Falciparum/genética , Malária Falciparum/parasitologia , Plasmodium falciparum , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Repetições de Microssatélites/genética , Plasmodium falciparum/crescimento & desenvolvimento , Locos de Características Quantitativas/genéticaRESUMO
The complete sequence of the Bombyx mori fibroin gene has been determined by means of combining a shotgun sequencing strategy with physical map-based sequencing procedures. It consists of two exons (67 and 15 750 bp, respectively) and one intron (971 bp). The fibroin coding sequence presents a spectacular organization, with a highly repetitive and G-rich (approximately 45%) core flanked by non-repetitive 5' and 3' ends. This repetitive core is composed of alternate arrays of 12 repetitive and 11 amorphous domains. The sequences of the amorphous domains are evolutionarily conserved and the repetitive domains differ from each other in length by a variety of tandem repeats of subdomains of approximately 208 bp which are reminiscent of the repetitive nucleosome organization. A typical composition of a subdomain is a cluster of repetitive units, Ua, followed by a cluster of units, Ub, (with a Ua:Ub ratio of 2:1) flanked by conserved boundary elements at the 3' end. Moreover some repeats are also perfectly conserved at the peptide level indicating that the evolutionary pressure is not identical along the sequence. A tentative model for the constitution and evolution of this unusual gene is discussed.
Assuntos
Bombyx/genética , Fibroínas/genética , Genes , Animais , Sequência de Bases , Éxons , Proteínas de Insetos/química , Proteínas de Insetos/genética , Íntrons , Substâncias Macromoleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Sequências Repetitivas de Aminoácidos , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Seda , Difração de Raios XRESUMO
Long interspersed elements (LINEs) are endogenous mobile genetic elements that have dispersed and accumulated in the genomes of higher eukaryotes via germline transposition, with up to 100,000 copies in mammalian genomes. In humans, LINEs are the major source of insertional mutagenesis, being involved in both germinal and somatic mutant phenotypes. Here we show that the human LINE retrotransposons, which transpose through the reverse transcription of their own transcript, can also mobilize transcribed DNA not associated with a LINE sequence by a process involving the diversion of the LINE enzymatic machinery by the corresponding mRNA transcripts. This results in the 'retroposition' of the transcribed gene and the formation of new copies that disclose features characteristic of the widespread and naturally occurring processed pseudogenes: loss of intron and promoter, acquisition of a poly(A) 3' end and presence of target-site duplications of varying length. We further show-by introducing deletions within either coding sequence of the human LINE-that both ORFs are necessary for the formation of the processed pseudogenes, and that retroviral-like elements are not able to produce similar structures in the same assay. Our results strengthen the unique versatility of LINEs as genome modellers.
Assuntos
Elementos Nucleotídeos Longos e Dispersos/genética , Pseudogenes/genética , Retroelementos/genética , Animais , Gatos , Linhagem Celular , Expressão Gênica , Genes Reporter , Teste de Complementação Genética , Vetores Genéticos , Humanos , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética , Transcrição Gênica , TransfecçãoRESUMO
SCID mice were grafted with human PBL (hu-PBL-SCID) from healthy or haemophilia A donors. Those containing human and no murine Ig in their plasma, were injected with 100 U VIII:Ag of a plasma derived (pd) FVIII or recombinant deleted Factor VIII (FVIII deltaII) and with 10 microg of tetanus toxoid as control immunogen. The frequency and the intensity of the humoral specific responses were measured in 253 mice humanized with PBL from 13 different donors. There was no significant difference in the frequency or intensity of the anti-FVIII immune responses to pd FVIII and FVIII deltaII. Neutralizing antibodies were only detected in the plasma of mice humanized with cells from haemophiliacs having FVIII inhibitors in their blood. The immune responses observed in hu-PBL-SCID mice correlated with the immune status of the corresponding human donor.
Assuntos
Fator VIII/imunologia , Adulto , Animais , Formação de Anticorpos , Fator VIII/antagonistas & inibidores , Feminino , Hemofilia A/sangue , Hemofilia A/terapia , Humanos , Imunização , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Proteínas Recombinantes/imunologia , Toxoide Tetânico/imunologia , Transplante HeterólogoRESUMO
We have determined the structure and organization of a 630-kb extrachromosomal element (amplisome) containing the dihydrofolate reductase-encoding gene (DHFR) in a methotrexate (MTX)-resistant human cell line, HeLa-Bu25-10B3. The size and copy number of amplisomes have previously been found to remain remarkably stable with or without selection. Both linear and open circular 630-kb amplisomes are present in these cells. We have been able to isolate the linear amplisomes after pulsed-field gel electrophoresis (PFGE), and transfect the amplisomes into MTX-sensitive recipient cells by electroporation, thus demonstrating that DNA as large as 630 kb can be transfected into mammalian cells. The NotI restriction site immediately upstream from DHFR on the circular amplisome is devoid of methylation, suggesting that it is transcriptionally active. Restriction mapping by PFGE reveals that there is only one copy of DHFR per amplisome and no repetitive structure is observed. The small size of the amplisomes, their stability and our ability to transfect large DNA molecules provide the necessary ingredients for the development of mammalian cloning vectors for large DNA fragments.
Assuntos
Tetra-Hidrofolato Desidrogenase/genética , Azacitidina , DNA/química , Eletroforese em Gel de Campo Pulsado , Herança Extracromossômica , Amplificação de Genes , Células HeLa , Humanos , Metilação , Conformação de Ácido Nucleico , Mapeamento por Restrição , TransfecçãoRESUMO
Immortalization of rodent embryo fibroblasts by SV40 is dominantly maintained by the large T antigen. The aim of this work is to characterize some of the events associated with the loss of proliferative capacity in a rat cell line, called REtsAF, which is conditionally immortalized by the tsA58 allele of SV40 large T antigen. DNA replication is arrested less than 24 h after the shift to the restrictive temperature (39 degrees C). This arrest occurs without specificity relative to the cell cycle stage, which suggests that a function essential throughout the cell cycle is affected. A two-dimensional SDS polyacrylamide gel electrophoresis analysis of proteins shows that, although the global rate of protein synthesis is only slightly affected at 39 degrees C, the rate of accumulation of specific proteins is either increased or decreased. Finally we present biochemical and electron microscopy data showing that alterations of the mitochondria occur upon shift to 39 degrees C.
Assuntos
Divisão Celular , Transformação Celular Neoplásica , Macrófagos/citologia , Vírus 40 dos Símios/genética , Animais , Células da Medula Óssea , Ciclo Celular , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Fenótipo , Temperatura , TransfecçãoRESUMO
Ditercalinium (NSC 335153) was synthesized as a bifunctional DNA intercalator. It is made of two 7-H pyridocarbazole rings joined by a rigid bis-ethyl bispiperidine chain. It binds to DNA with high affinity and elicits anti-tumor activity on a variety of animal tumors. 1H n.m.r. studies of ditercalinium bis-intercalated into d(CpGpCpG)2 have shown that the intercalation process occurs from the large groove of the DNA helix while the two intercalated rings are separated by two base pairs. Because of the linking chain rigidity of ditercalinium, DNA conformation has to be altered to permit the intercalation of the two rings. DNA must be bent toward the minor groove. In E. coli, ditercalinium elicits a specific toxicity on polA strains which is suppressed by an additional uvrA mutation. In vitro, the purified UvrA and UvrB proteins bind to the DNA-ditercalinium complex in an ATP dependent manner. The UvrABC complex induces single-strand nicks, but only when ditercalinium is bound to negatively supercoiled DNA. The life-time of the UvrAB-DNA-ditercalinium complex is greater than 50 min when free ditercalinium concentration is maintained constant in the incubation medium. The cytotoxicity of ditercalinium in E. coli results from the induction of a futile and abortive DNA repair. The reversible ditercalinium-DNA complex mimics a bulky DNA lesion, yet the UvrABC endonuclease is unable to cope with a reversible lesion since it cannot eliminate the causative agent. The interaction of UvrA and UvrB proteins has also been studied with DNA and other DNA-binding drugs forming high-affinity complexes such as distamycin. The Uvr protein recognition process appears to be associated with specific DNA structural alterations. In eukaryotic cells, ditercalinium is concentrated in mitochondria. Mitochondrial DNA is rapidly and totally degraded. Mitochondrial DNA coded proteins being no longer synthesized, the respiratory chain is progressively inactivated. The stimulation of the glycolytic pathway allows the cells to continue growth for several generations. Dihydro-orotate dehydrogenase is located in the inner membrane of mitochondria and its activity is dependent on mitochondria energization. It becomes inactive after ditercalinium treatment. A drop of the pyrimidine pool is then observed. Complementation of treated cells with uridine decreases 10-fold the ditercalinium toxicity. The cellular delayed toxicity of ditercalinium results from the slow induction of a pyrimidineless state associated with the progressive inactivation of mitochondria. The results show that DNA structural alterations induced by reversible drug-DNA complexes can be recognized by DNA repair enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Reparo do DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Proteínas de Escherichia coli , Animais , Carbazóis/farmacologia , Carbazóis/toxicidade , DNA/metabolismo , Interações Medicamentosas , Ensaios de Seleção de Medicamentos Antitumorais , Endodesoxirribonucleases/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Substâncias Intercalantes/farmacologia , Substâncias Intercalantes/toxicidade , Leucemia L1210/tratamento farmacológico , Mutação , Conformação de Ácido Nucleico/efeitos dos fármacos , Relação Estrutura-Atividade , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The bifunctional intercalator Ditercalinium (NSC 335153) demonstrates an anti-tumoral cytotoxicity markedly different from other intercalating agents. A delayed toxicity is observed in eucaryotic cells, both in vitro and in vivo, at drug concentrations far below those required to observe immediate toxic effects. Fluorescence microscopy demonstrates that Ditercalinium and the mitochondrial-staining fluorophore DiOC2(5) are concentrated in the same cellular organelles of L1210 cells. Electron microscopy of Ditercalinium-treated cells reveals extensive and progressive swelling of mitochondria, with no other ultrastructural changes observed. Ditercalinium uptake and toxicity are in part related to mitochondrial membrane potential. However, drug accumulation itself does not immediately alter the mitochondrial membrane potential. Cellular ATP pool levels and the rate of respiration fall progressively after drug treatment. Nucleotide pools in DC3F cells, measured between drug treatment and death, show marked drops in pyrimidine levels while purine nucleotide levels decline more slowly. Addition of uridine or cytidine partially rescues Ditercalinium-treated cells, while toxicity is increased in the presence of 2-deoxyglucose. The combined evidence indicates that the toxicity of Ditercalinium to murine leukemia cells (L1210) and Chinese Hamster lung cells (DC3F) is due to disruption of mitochondrial function.
Assuntos
Antineoplásicos/farmacologia , Carbazóis/farmacologia , Substâncias Intercalantes/farmacologia , Mitocôndrias/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Carbazóis/metabolismo , Carbazóis/uso terapêutico , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , DNA/metabolismo , Desoxiglucose/farmacologia , Membranas Intracelulares/fisiologia , Leucemia L1210/tratamento farmacológico , Potenciais da Membrana/efeitos dos fármacos , Microscopia Eletrônica , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Dilatação Mitocondrial/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Ditercalinium (NSC 366241), a dimer of 10-methoxy-7H-pyrido[4,3-c]carbazole quaternarized on the pyridine nitrogen by a rigid bis(1,1'-ethyl)-4,4'-bipiperidine linking chain, is endowed with antitumour properties and bis-intercalates with high affinity into DNA. New dimers have been designed in the same series to evaluate the importance of the rigidity of the linking chain for pharmacological activity. The dimers, characterized by one and two additional methylene groups between the two piperidine rings of the linking chain, remain as active as ditercalinium. However, a third additional CH2 group between the two piperidine rings leads to an inactive dimer. Relationships between the different pharmacological activities of the drugs and their intercalation complexes with DNA were investigated using viscosimetry, absorption spectroscopy and NMR analyses.
Assuntos
Antineoplásicos/síntese química , Substâncias Intercalantes/síntese química , Animais , Carbazóis/síntese química , Fenômenos Químicos , Química , Substâncias Intercalantes/farmacologia , Camundongos , Conformação MolecularRESUMO
Ditercalinium, a 7H-pyridocarbazole dimer (bisintercalator) belongs to a new class of antineoplastic intercalating agents. To investigate its mechanism of cytotoxicity, the effects of ditercalinium on DNA were assessed using normal (L1210) and drug-resistant (L1210/PyDi1) mouse leukemia cells. Alkaline elution assays demonstrated that ditercalinium produced no DNA strand breaks, DNA-protein cross-links, or DNA-DNA cross-links, eliminating these effects as cytotoxic lesions. This result sets ditercalinium apart from other intercalating agents with respect to its interaction with DNA. Nucleoids (histone-depleted chromatin) from ditercalinium-treated L1210 cells were considerably more compact than those from untreated cells, as determined by sedimentation in neutral sucrose gradients. In contrast, nucleoids from ditercalinium-treated L1210/PyDi1 (resistant) cells were similar in compactness to those from control cells. Thus, ditercalinium altered chromatin structure in vivo. The effect of the bisintercalator on purified DNA topoisomerase II, an intracellular target of monointercalators, was measured in vitro. Ditercalinium (5 X 10(-7) M) completely inhibited both the formation of covalent complexes between this enzyme and simian virus 40 DNA and the enzyme-induced DNA cleavage. In addition, ditercalinium induced DNA catenation in the presence of topoisomerase II and adenosine triphosphate. Thus, the cytotoxicity of ditercalinium may derive from a mechanism that, although involving topoisomerase II, is manifested by condensation of DNA rather than by the induction of protein-associated DNA strand breaks.
Assuntos
Carbazóis/farmacologia , Dano ao DNA , DNA de Neoplasias/efeitos dos fármacos , Substâncias Intercalantes/farmacologia , Inibidores da Topoisomerase II , Amsacrina/farmacologia , Animais , Reagentes de Ligações Cruzadas/farmacologia , Etoposídeo/farmacologia , Leucemia L1210/enzimologia , Camundongos , Conformação de Ácido Nucleico/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
The antitumor properties of 7H-pyridocarbazole dimers, a new series of bifunctional intercalators, have recently been described (Pelaprat, D. Delbarre, A., Le Guen, I., Roques, B. P., and Le Pecq, J. B. J. Med. Chem., 23: 1336-1343, 1980; and Roques, B. P., Pelaprat, D., Le Guen, I., Porcher, G., Gosse, C., and Le Pecq, J. B. Biochem. Pharmacol., 28: 1811-1815, 1979). In order to study the mechanism of action of these compounds, an L1210 subline was made resistant to one dimer (NSC 335153; ditercalinium). Selection of resistant cells was based on an in vitro-in vivo procedure as follows. Ascitic cells were taken from a leukemic mouse and incubated in vitro with the dimer for 1 hr. They were then injected into mice. After the development of the ascites, L1210 cells were collected and the process was repeated 13 times, until establishment of the resistance. Cloned resistant cells have maintained their resistance for 18 months of in vitro culture. The effects of two dimers (NSC 335153 and NSC 335154) on cell viability, growth, colony formation, and cell cycle progression were investigated on parental and resistant L1210 cells. The cross-resistance of these two L1210 cell lines to several cytotoxic agents was estimated. Several observations indicate that the mechanism of action of these dimers might be different from that of monointercalating agents: (a) these drugs induce a delayed toxicity (growth arrest occurring five generations after drug exposure) in sensitive but not in resistant cells; (b) cells exposed to the dimers arrested almost randomly in all phases of the cell cycle, whereas the corresponding monomer provokes a block in the G2 + M phase. Resistant cells were cross-resistant to 7H-pyridocarbazole monomer, Adriamycin, and vincristine but not to 6H-pyridocarbazole monomer derivatives, actinomycin D, and methotrexate.
Assuntos
Carbazóis/toxicidade , Leucemia L1210/patologia , Animais , Antineoplásicos/toxicidade , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Resistência a Medicamentos , Cinética , Leucemia L1210/tratamento farmacológico , Camundongos , Relação Estrutura-AtividadeRESUMO
The first chemical synthesis of 3 alpha, 7 alpha-dihydroxy-5 beta-androstan-17-one and 3 alpha-hydroxy-5 beta-androstane-7, 17-dione is reported. In this method, the 17 beta-side chain of commercial chenodesoxycholic acid was degraded in 6 steps after selective protection of the hydroxyl groups: 3 alpha-OH by a tert-butyldimethylsilyl group and 7 alpha-OH by an acetoxy group. The capacity of 3 alpha, 7 alpha-dihydroxy-5 beta-androstan-17-one and 3 alpha-hydroxy-5 beta-androstane-7, 17-dione to release a pyrogen by human leukocytes was investigated by two independent methods: supernatants from leukocytes incubated with a steroid are injected to rabbits whose fever is measured, or tested by the Limulus Test (a pyrogen detection technique). The 7-keto substituted etiocholanolone still possessed pyrogenic activity, while the 7 alpha-hydroxyl substituted one did not.
Assuntos
Etiocolanolona/análogos & derivados , Pirogênios/farmacologia , Animais , Fenômenos Químicos , Química , Etiocolanolona/síntese química , Etiocolanolona/farmacologia , Humanos , Técnicas In Vitro , Leucócitos/efeitos dos fármacos , Teste do Limulus , Métodos , Pirogênios/análise , CoelhosRESUMO
Consistent with changes in ploidy during germ cell maturation, the quantity of Feulgen DNA in round spermatids of voles is approximately a quarter of that in primary spermatocytes. The amount increases slightly in elongated spermatids, and then declines in spermatozoa from the testis, epididymis and vas deferens, below the level of round spermatids. Simultaneously nuclear area decreases so that in spermatozoa it is about one tenth that of primary spermatocytes. The rise in Feulgen DNA in elongated spermatids above the level found in round spermatids, and the decline in spermatozoa below this level, is probably caused by altered affinity of the desoxyribose for the leucofuchsin. This may be brought about by the complex physico-chemical changes occurring in chromatin and elsewhere in the nucleus and cell as they acquire their highly specialised final form.
Assuntos
Arvicolinae/anatomia & histologia , DNA/análise , Espermátides/análise , Espermatócitos/análise , Espermatozoides/análise , Animais , Arvicolinae/crescimento & desenvolvimento , Masculino , Maturidade SexualRESUMO
Oral treatment of bulls with ethylene dibromide caused a temporary reduction of the DNA and protein content and head area of epididymal and ejaculated spermatozoa.
Assuntos
DNA/análise , Dibrometo de Etileno/farmacologia , Hidrocarbonetos Bromados/farmacologia , Proteínas/análise , Espermatozoides/efeitos dos fármacos , Animais , Bovinos , Masculino , Espermatozoides/análise , Espermatozoides/metabolismoRESUMO
Polyploidy level of experimentally induced deciduomata was determined by cytophotometric measures of DNA-Feulgen, with the "Ultramikrospektrophotometer UMSP Zeiss" by scanning. The antimesometrial tissue reached 32 n, 72 H after the uterine trauma, whereas the mesometrial part did not exceed a 4 n polyploidy level. With the same technics we studied the ovarian hormone, its action upon the blastocyst cellular cycle. In delayed blastocysts, synthesis was never found. OEstradiol removed the inhibition and DNA synthesis then starts again.
Assuntos
Blastocisto/análise , DNA de Neoplasias/análise , Decídua/análise , Neoplasias Uterinas/análise , Animais , Implantação do Embrião , Feminino , Neoplasias Experimentais , Gravidez , Ratos , Fatores de TempoRESUMO
Cytophotometrics measures of DNA-Feulgen were realized in normal and delayed implantation blastocysts of the rat. During afternoon of the 5th day of progestation, blastocysts were of two types: those nuclei of which were in G1 gap and those nuclei of which were synthetising DNA. In delayed implantation, blastocyst nuclei were in G1 or G2 gap. Mitosis were rarely seen. After oestrogen injection DNA-synthesis started again. Cell kinetics before ovoimplantation is discussed.
Assuntos
Blastocisto/análise , DNA/análise , Implantação Tardia do Embrião , Implantação do Embrião , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/ultraestrutura , Divisão Celular/efeitos dos fármacos , Núcleo Celular/análise , DNA/biossíntese , Estrogênios/farmacologia , Feminino , Histocitoquímica , Gravidez , RatosRESUMO
Ploidy level of mesometral cells in experimentaly induced deciduomata is determinated by DNA-Feulgen cytophotometric measures. This tissue shown the first evolutive stages leading to polyploïdy (mono-binucleated cells) it was unable to reach a ploïdy level higher than 4 n.