Novel Transmembrane Receptor Involved in Phagosome Transport of Lysozymes and β-Hexosaminidase in the Enteric Protozoan Entamoeba histolytica

Lysozymes and hexosaminidases are ubiquitous hydrolases in bacteria and eukaryotes. In phagocytic lower eukaryotes and professional phagocytes from higher eukaryotes, they are involved in the degradation of ingested bacteria in phagosomes. In Entamoeba histolytica, which is the intestinal protozoan parasite that causes amoebiasis, phagocytosis plays a pivotal role in the nutrient acquisition and the evasion from the host defense systems. While the content of phagosomes and biochemical and physiological roles of the major phagosomal proteins have been established in E. histolytica, the mechanisms of trafficking of these phagosomal proteins, in general, remain largely unknown. In this study, we identified and characterized for the first time the putative receptor/carrier involved in the transport of the above-mentioned hydrolases to phagosomes. We have shown that the receptor, designated as cysteine protease binding protein family 8 (CPBF8), is localized in lysosomes and mediates transport of lysozymes and β-hexosaminidase α-subunit to phagosomes when the amoeba ingests mammalian cells or Gram-positive bacillus Clostridium perfringens. We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8. We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens. Repression of CPBF8 also resulted in decrease in the cytopathy against the mammalian cells, suggesting that CPBF8 may also be involved in, besides the degradation of ingested bacteria, the pathogenesis against the mammalian hosts. This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes

Sistema biométrico multimodal baseado em pupilometria dinâmica

Orientador : Prof. Dr. Alessandro ZimmerDissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduaçao em Engenharia Elétrica. Defesa: Curitiba, 20/12/2011Bibliografia: fls. 113-121Resumo: A identificação pessoal através de características biométricas, ou seja, de medidas e atributos relacionados ao corpo humano, comparada ao uso de senhas, chaves ou documentos, apresenta a vantagem de não depender da posse de algo ou de um conhecimento que pode ser perdido ou roubado. No entanto, a maioria dos traços individuais usados nestes tipos de sistemas está sujeita a fraudes, através da reprodução das características em meios artificiais, como fotografias, próteses e gravações. A identificação baseada em características dinâmicas e reflexos humanos, tais como os movimentos sacádicos do olho e a contração pupilar, é uma alternativa que pode resolver este tipo de problema, mas a quantidade de informação envolvida não é suficiente para discriminar indivíduos. O uso conjunto de mais de uma característica biométrica, na chamada biometria multimodal, também é uma forma de dificultar a ocorrência de evasão. Neste trabalho de pesquisa, foi estudada a possibilidade de desenvolvimento de um sistema biométrico multimodal menos propenso a fraudes, baseado tanto nas características do reflexo pupilar à luz quanto na textura da íris. Um método de geração do vetor de primitivas e comparação de amostras foi proposto e testado por 59 voluntários, apresentando resultados promissores para esta nova abordagem. No melhor caso, a taxa de erros iguais ficou em 2,44%.Abstract: Personal identification using biometric features, i.e. measurements and attributes of the human body, compared to the use of passwords, key or documents, has the advantage of being independent of some object or some knowledge that can be lost or stolen. However, most of the individual features used in these types of systems are subject to frauds, by reproducing the human characteristics by artificial means, such as pictures, prosthesis and recordings. Identification based on dynamic characteristics and human reflexes, like saccadic movements and the pupil constriction, is an alternative that can solve this kind of problem, but the amount of information is usually not enough to identify a person. The use of multiple biometric characteristics, which is named multimodal biometrics, is also a way to avoid the occurrence of fraud, by involving more than one individual feature. In this research work, the possibility of developing a multimodal biometrics system less subject to fraud was studied based on the characteristics of the pupillary light reflex and the iris texture. A method for the generation of a vector of features and for the comparison of samples has been proposed and tested by 59 volunteers, leading to promising results for this new approach. In the best case, the equal error rate was 2.44%

Hepatitis C virus quasispecies in cancerous and noncancerous hepatic lesions: the core protein-encoding region.

We have shown that highly proofreading DNA polymerase is required for the polymerase chain reaction in the genetic analysis of hepatitis C virus (HCV). To clarify the status of HCV quasispecies in hepatic tissue using proofreading DNA polymerase, we performed a genetic analysis of the HCV core protein-encoding region in cancerous and noncancerous lesions derived from 4 patients with hepatocellular carcinoma. In contrast to the previously published data, we observed neither deletions nor stop codons in the analyzed region and no significant difference in the complexity of HCV quasispecies between cancerous and noncancerous lesions. This result suggests that the HCV core gene is never structurally defective in hepatic tissues, including cancerous lesions. However, in 3 of the patients, the consensus HCV species differed between cancerous and noncancerous lesions, suggesting that the predominant replicating HCV species differs between these 2 types of lesions. Moreover, during the course of the study, we obtained several interesting variants possessing a substitution at codon 9 of the core gene, whose substitution has been shown to induce the production of the F protein synthesized by a - 2/+1 ribosomal frameshift.</p

Cysteine protease-binding protein family 6 mediates the trafficking of amylases to phagosomes in the enteric protozoan entamoeba histolytica

金沢大学医薬保健研究域薬学系Phagocytosis plays a pivotal role in nutrient acquisition and evasion from the host defense systems in Entamoeba histolytica, the intestinal protozoan parasite that causes amoebiasis. We previously reported that E. histolytica possesses a unique class of a hydrolase receptor family, designated the cysteine protease-binding protein family (CPBF), that is involved in trafficking of hydrolases to lysosomes and phagosomes, and we have also reported that CPBF1 and CPBF8 bind to cysteine proteases or α-hexosaminidase β-subunit and lysozymes, respectively. In this study, we showed by immunoprecipitation that CPBF6, one of the most highly expressed CPBF proteins, specifically binds to β-amylase and γ-amylase. We also found that CPBF6 is localized in lysosomes, based on immunofluorescence imaging. Immunoblot and proteome analyses of the isolated phagosomes showed that CPBF6 mediates transport of amylases to phagosomes. We also demonstrated that the carboxyl-terminal cytosolic region of CPBF6 is engaged in the regulation of the trafficking of CPBF6 to phagosomes. Our proteome analysis of phagosomes also revealed new potential phagosomal proteins. © 2013, American Society for Microbiology

Novel transmembrane receptor involved in phagosome transport of lysozymes and β-hexosaminidase in the enteric protozoan Entamoeba histolytica

金沢大学医薬保健研究域薬学系Lysozymes and hexosaminidases are ubiquitous hydrolases in bacteria and eukaryotes. In phagocytic lower eukaryotes and professional phagocytes from higher eukaryotes, they are involved in the degradation of ingested bacteria in phagosomes. In Entamoeba histolytica, which is the intestinal protozoan parasite that causes amoebiasis, phagocytosis plays a pivotal role in the nutrient acquisition and the evasion from the host defense systems. While the content of phagosomes and biochemical and physiological roles of the major phagosomal proteins have been established in E. histolytica, the mechanisms of trafficking of these phagosomal proteins, in general, remain largely unknown. In this study, we identified and characterized for the first time the putative receptor/carrier involved in the transport of the above-mentioned hydrolases to phagosomes. We have shown that the receptor, designated as cysteine protease binding protein family 8 (CPBF8), is localized in lysosomes and mediates transport of lysozymes and β-hexosaminidase α-subunit to phagosomes when the amoeba ingests mammalian cells or Gram-positive bacillus Clostridium perfringens. We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8. We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens. Repression of CPBF8 also resulted in decrease in the cytopathy against the mammalian cells, suggesting that CPBF8 may also be involved in, besides the degradation of ingested bacteria, the pathogenesis against the mammalian hosts. This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes. © 2012 Furukawa et al

A reliable internally controlled RT-nested PCR method for the detection of hepatitis C virus RNA.

We have developed a reliable internally controlled RT-nested PCR method for the detection of hepatitis C virus (HCV) RNA using in vitro synthesized Renilla luciferase (Rluc) RNA as an internal control. Using this method, the 5'-noncoding region of HCV RNA (144 nucleotides) and Rluc RNA (276 nucleotides) were efficiently amplified in a single tube, and the sensitivity and specificity of this method were comparable to standard RT-nested PCR. This method was successfully performed on RNA specimens obtained from in vitro HCV-infected human hepatocyte PH5CH8 cells, which support HCV replication. In addition, we demonstrated that this method was useful for the evaluation of antiviral reagents by confirming the anti-HCV activity of bovine lactoferrin, which we previously found to be a new inhibitor of HCV infection. Therefore, this method may be useful for the studies of not only HCV but also of other viruses.</p

Sex-specific post-translational regulation of the gamete fusogen GCS1 in the isogamous volvocine alga Gonium pectorale

Male and female, generally defined based on differences in gamete size and motility, likely have multiple independent origins, appearing to have evolved from isogamous organisms in various eukaryotic lineages. Recent studies of the gamete fusogen GCS1/HAP2 indicate that this protein is deeply conserved across eukaryotes, and its exclusive and/or functional expression generally resides in males or in male homologues. However, little is known regarding the conserved or primitive molecular traits of males and females within eukaryotes. Here, using morphologically indistinguishable isogametes of the colonial volvocine Gonium pectorale, we demonstrated that GCS1 is differently regulated between the sexes. G. pectorale GCS1 molecules in one sex (homologous to “male”) are transported from the gamete cytoplasm to the protruded fusion site, whereas those of the other sex (“females”) are quickly degraded within the cytoplasm upon gamete activation. This molecular trait difference might be conserved across various eukaryotic lineages and may represent male and female prototypes originating from a common eukaryotic ancestor

Hoxa13 regulates expression of common Hox target genes involved in cartilage development to coordinate the expansion of the autopodal anlage

To elucidate the role of Hox genes in limb cartilage development, we identified the target genes of HOXA11 and HOXA13 by ChIP‐Seq. The ChIP DNA fragment contained evolutionarily conserved sequences and multiple highly conserved HOX binding sites. A substantial portion of the HOXA11 ChIP fragment overlapped with the HOXA13 ChIP fragment indicating that both factors share common targets. Deletion of the target regions neighboring Bmp2 or Tshz2 reduced their expression in the autopod suggesting that they function as the limb bud‐specific enhancers. We identified the Hox downstream genes as exhibiting expression changes in the Hoxa13 knock out (KO) and Hoxd11‐13 deletion double mutant (Hox13 dKO) autopod by Genechip analysis. The Hox downstream genes neighboring the ChIP fragment were defined as the direct targets of Hox. We analyzed the spatial expression pattern of the Hox target genes that encode two different categories of transcription factors during autopod development and Hox13dKO limb bud. (a) Bcl11a, encoding a repressor of cartilage differentiation, was expressed in the E11.5 autopod and was substantially reduced in the Hox13dKO. (b) The transcription factors Aff3, Bnc2, Nfib and Runx1t1 were expressed in the zeugopodal cartilage but not in the autopod due to the repressive or relatively weak transcriptional activity of Hox13 at E11.5. Interestingly, the expression of these genes was later observed in the autopodal cartilage at E12.5. These results indicate that Hox13 transiently suspends the cartilage differentiation in the autopodal anlage via multiple pathways until establishing the paddle‐shaped structure required to generate five digits