RESUMEN
Many of the most contentious questions that concern the ecology of helminths could be resolved with data on helminth abundance over the past few decades or centuries, but unfortunately these data are rare. A new sub-discipline - the historical ecology of parasitism - is resurrecting long-term data on the abundance of parasites, an advancement facilitated by the use of biological natural history collections. Because the world's museums hold billions of suitable specimens collected over more than a century, these potential parasitological datasets are broad in scope and finely resolved in taxonomic, temporal and spatial dimensions. Here, we set out best practices for the extraction of parasitological information from natural history collections, including how to conceive of a project, how to select specimens, how to engage curators and receive permission for proposed projects, standard operating protocols for dissections and how to manage data. Our hope is that other helminthologists will use this paper as a reference to expand their own research programmes along the dimension of time.
Asunto(s)
Helmintos , Parásitos , Animales , Ecología , MuseosRESUMEN
The social-ecological system of the Lake Manyara basin (Northern Tanzania), a UNESCO Biosphere reserve (BR) suffers from social-economic and environmental problems due to decreasing water levels, erosion and land and water use conflicts. We propose an integrated assessment of the social-ecological interactions of the area to support future sustainable management. Within the Drivers-Pressures-State-Impact-Response (DPSIR) framework an integrated literature review and several methods of knowledge collection were combined to identify future management priorities and challenges. During focus groups with farmers and pastoralists, stakeholders confirmed the role played by land use changes as driver and pressure in the landscape, e.g. through increased erosion rates and siltation of the lake. Moreover, economic and social issues were identified as prominent factors being influenced by, or influencing these processes. These statements match the scientific literature. During participatory mapping exercises different spatial and resource allocation perceptions appeared amongst pastoralists and farmers. The multidisciplinary approach proved to be useful to acquire an integrated and comprehensive understanding of the state, challenges and opportunities of Lake Manyara BR, to feed into a decision support system in service of an integrated management plan. Our assessment suggests that improved water governance in a multi-actor approach (with a focus on distribution of benefits, rights, and a specific role of the water authorities) should be a priority for future integrated management strategies. Also, awareness raising amongst decision makers, scientists and local communities is needed to demonstrate the advantages of an integrated approach. And finally, visible and fair mechanisms to share conservation revenues should be designed in a way that local benefits can be obtained together with incentive mechanisms for co-management and conservation.
Asunto(s)
Conservación de los Recursos Naturales , Lagos , Ecosistema , TanzaníaRESUMEN
This study represents the first exploration of the parasite fauna of cichlid fishes in the Mweru-Luapula subregion (Central Africa). Twelve species of cichlids and 14 species of Monogenea from three genera (Cichlidogyrus, Gyrodactylus and Scutogyrus) were collected. We present a first record of the gill parasite fauna of eight host species, Oreochromis mweruensis, Orthochromis sp. 'Mambilima', Sargochromis mellandi, Serranochromis angusticeps, S. stappersi, S. thumbergi and Tylochromis mylodon. The host range of ten parasite species was expanded. The study further includes the description of Cichlidogyrus consobrini sp. n. from S. mellandi and Orthochromis sp. 'Mambilima'. A new morphotype of C. halli is characterized, and three species - C. papernastrema, C. quaestio and C. zambezensis - are redescribed. Furthermore, the biodiversity and host specificity of these parasites is compared with that of cichlid parasites from Lake Kariba and Cameroon. Two species, including C. consobrini sp. n. and a new morphotype of C. halli, are putative endemics. The parasite fauna in Bangweulu-Mweru is highly similar in species composition to Lake Kariba, but in Bangweulu-Mweru the same parasite species are more host-specific, probably because of hydrogeographical differences between the two regions.
Asunto(s)
Biodiversidad , Infecciones por Cestodos/veterinaria , Cíclidos/parasitología , Enfermedades de los Peces/parasitología , Especificidad del Huésped , Platelmintos/fisiología , África Central , Animales , Infecciones por Cestodos/parasitología , Cíclidos/clasificación , Branquias/parasitología , Platelmintos/clasificación , Platelmintos/genética , Platelmintos/aislamiento & purificaciónRESUMEN
Adaptive radiation occurs when species diversify rapidly to occupy an array of ecological niches. As opportunities for parasite infection and transmission may greatly vary among these niches, adaptive radiation is expected to be associated with a turnover of the parasite community. As major agents of natural and sexual selection, parasites may play a central role in host diversification. The study of parasite turnover may thus be of general relevance and could significantly improve our understanding of adaptive radiation. In this study, we examined the parasite faunas of eleven species belonging to the tribe Tropheini, one of several adaptive radiations of cichlid fishes in Lake Tanganyika. The most parsimonious ancestral foraging strategy among the Tropheini is relatively unselective substrate browsing of aufwuchs. Several lineages evolved more specialized foraging strategies, such as selective combing of microscopic diatoms or picking of macro-invertebrates. We found that representatives of these specialized lineages bear reduced infection with food-web-transmitted acanthocephalan helminths, but not with parasites with a direct life cycle. Possibly, the evolution of selective foraging strategies entailed reduced ingestion of intermediate invertebrate hosts of acanthocephalans. We conclude that some species belonging to the Tropheini virtually escape acanthocephalan infection as a by-product of trophic specialization.
Asunto(s)
Evolución Biológica , Cíclidos/parasitología , Helmintos/patogenicidad , Filogenia , Animales , Cadena Alimentaria , Parásitos , TanzaníaRESUMEN
Geographical isolation, habitat variation and trophic specialization have contributed to a large extent to the astonishing diversity of cichlid fishes in the Great East African lakes. Because parasite communities often vary across space and environments, parasites can accompany and potentially enhance cichlid species diversification. However, host dispersal may reduce opportunities for parasite-driven evolution by homogenizing parasite communities and allele frequencies of immunity genes. To test for the relationships between parasite community variation, host dispersal and parasite-induced host evolution, we studied two sympatric cichlid species with contrasting dispersal capacities along the shores of southern Lake Tanganyika. Whereas the philopatric Tropheus moorii evolved into several genetically differentiated colour morphs, Simochromis diagramma is phenotypically rather uniform across its distribution range and shows only weak population structure. Populations of both species were infected with divergent parasite communities and harbour differentiated variant pools of an important set of immune genes, the major histocompatibility complex (MHC). The overall extent of geographical variation of parasites and MHC genes was similar between host species. This indicates that immunogenetic divergence among populations of Lake Tanganyika cichlids can occur even in species that are strongly dispersing. However, because this also includes species that are phenotypically uniform, parasite-induced evolution may not represent a key factor underlying species diversification in this system.
Asunto(s)
Distribución Animal , Cíclidos/genética , Cíclidos/parasitología , Inmunogenética , Simpatría , Animales , Cíclidos/inmunología , Genética de Población , Complejo Mayor de Histocompatibilidad/genética , Parásitos , Fenotipo , TanzaníaRESUMEN
Studying the genetic basis of host-parasite interactions represents an outstanding opportunity to observe eco-evolutionary processes. Established candidates for such studies in vertebrates are immunogenes of the major histocompatibility complex (MHC). The MHC has been reported to reach high intra- and interindividual diversity, and a diverse MHC might be advantageous when facing infections from multiple parasites. However, other studies indicated that individuals with an intermediate number of MHC alleles are less infected with parasites or have other fitness advantages. In this study, we assessed the optimal number of MHC alleles in the blunt-head cichlid Tropheus moorii from Lake Tanganyika. We investigated the influence of the interindividual variation in number of MHC length variants on parasite infection and body condition, measured by the amount of perivisceral fat reserves. Surprisingly, there was no correlation between parasite infection and number of MHC length variants or perivisceral fat deposits. However, the individual number of MHC length variants significantly correlated with the amount of perivisceral fat deposits in males, suggesting that male individuals with an intermediate number of alleles might be able to use their fat reserves more efficiently.
Asunto(s)
Tejido Adiposo/anatomía & histología , Cíclidos/anatomía & histología , Cíclidos/genética , Cíclidos/parasitología , Genes MHC Clase II , Animales , Tamaño Corporal , Femenino , Genotipo , Modelos Lineales , Masculino , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Carga de ParásitosRESUMEN
Ocriplasmin, a truncated form of plasmin, is commercialized in the USA and in Europe under the trade name Jetrea(®), and indicated for the treatment of symptomatic vitreomacular adhesion and vitreomacular traction including when associated with macular hole ≤400 µm, respectively. We have shown in a previous study that ocriplasmin undergoes autolytic degradation when injected in eye vitreous, which leads to its rapid inactivation. In order to investigate this process further, we have introduced in ocriplasmin a variety of amino acid substitutions within or in the immediate vicinity of the three major autolytic cleavage sites. We demonstrate here that autolytic inactivation of ocriplasmin is a sequential process where initial cleavage occurs primarily between residues 156 and 157. Reduction or even blocking of autolysis can be achieved by mutating a limited number of key residues. In this study, we also report the identification of a series of ocriplasmin variants with improved resistance to autolysis and unimpaired catalytic activity. Such variants represent useful tools for the exploration of therapeutic approaches aiming at non-surgical resolution of vitreomacular adhesion.
Asunto(s)
Análisis Mutacional de ADN , Fibrinolisina/genética , Fibrinolisina/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Ingeniería de Proteínas , Proteolisis , Secuencia de Aminoácidos , Dominio Catalítico , Activación Enzimática , Fibrinolisina/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Mutación Puntual , Cuerpo Vítreo/enzimologíaRESUMEN
Jewel scarabs, beetles in the genus Chrysina Kirby (Coleoptera: Rutelinae: Scarabaeidae), receive their name from the bright, often gold, green elytra that reflect light like a precious stone. Jewel scarabs are commonly observed at light traps in Mesoamerican cloud forests, and their association with mountain forests makes them potentially interesting candidates for cloud forest conservation monitoring. The absence of survey protocols and identification tools, and the little ecological information available are barriers. In the present study, collection of Chrysina species assembled during biodiversity surveys by Operation Wallacea in Cusuco National Park (CNP), Honduras, were studied. The aim of this overview is to provide an easy to use identification tool for in the field, hopefully stimulating data collection on these beetles. Based on the data associated with the collection localities, elevation distribution of the species in the park was analyzed. The limited data points available were complemented with potential distribution areas generated with distribution models based on climate and elevation data. This study is aimed at initializing the development of a survey protocol for Chrysina species that can be used in cloud forest conservation monitoring throughout Central America. A list of Chrysina species recorded from Honduras so far is provided. The six identified and one unidentified species recorded from CNP are easy to identify in the field based on color and straightforward morphological characteristics. Literature research revealed ten species currently recorded from Honduras. This low species richness in comparison with surrounding Central American countries indicates the poor knowledge of this genus in Honduras. Chrysina species richness in CNP increases with elevation, thereby making the genus one of a few groups of organisms where this correlation is observed, and rendering it a suitable invertebrate representative for cloud forest habitats in Central America.
Asunto(s)
Escarabajos/clasificación , Ecosistema , Animales , Conservación de los Recursos Naturales , Monitoreo del Ambiente , Femenino , Honduras , MasculinoRESUMEN
1. The potential of the matrix metalloproteinase (MMP) inhibitor ABT-518 to affect pre-adipocyte differentiation in vitro and adipose tissue development in vivo was investigated using mouse models of adipogenesis and obesity. 2. Differentiation of 3T3-F442A pre-adipocytes into mature adipocytes was enhanced in a dose-dependent manner by the addition of ABT-518 (0-100 µmol/L). This was associated with increased expression of the adipogenic markers adipocyte fatty acid-binding protein 2 (AP2), peroxisome proliferator-activated receptor γ and adiponectin. 3. Feeding 5-week-old male wild-type mice with a high-fat diet, with or without ABT-518 (to achieve a dose of 100 mg/kg per day), for 16 weeks resulted in a significant reduction in bodyweight throughout the experimental period. Magnetic resonance spectroscopy revealed that the lipid : water ratio was significantly lower in ABT-518-treated mice. The total weight of isolated subcutaneous or gonadal fat depots did not differ significantly following ABT-518 treatment, but adipocyte and blood vessel size were significantly reduced in the gonadal fat. 4. Administration of ABT-518-2 (100 mg/kg per day for 10 weeks) to 5-week-old male wild-type mice with established obesity maintained on a high-fat diet had no effect on total bodyweight at the end of the experiment, but was associated with reduced blood vessel size in the fat depots. 5. Thus, the MMP inhibitor ABT-518 stimulates differentiation of 3T3-F442A pre-adipocytes in vitro. It mildly reduces bodyweight gain in a murine model of diet-induced obesity, but does not affect established obesity.
Asunto(s)
Adipocitos Blancos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Tejido Adiposo Blanco/efectos de los fármacos , Formamidas/uso terapéutico , Gelatinasas/antagonistas & inhibidores , Obesidad/prevención & control , Inhibidores de Proteasas/uso terapéutico , Células 3T3 , Adipocitos Blancos/citología , Adipocitos Blancos/metabolismo , Adiponectina/genética , Adiponectina/metabolismo , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Adiposidad/efectos de los fármacos , Animales , Vasos Sanguíneos/patología , Tamaño de la Célula/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Formamidas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Obesidad/patología , PPAR gamma/genética , PPAR gamma/metabolismo , Inhibidores de Proteasas/farmacología , ARN Mensajero/metabolismo , Aumento de Peso/efectos de los fármacosRESUMEN
The elucidation of the whole genome of the nematode Caenorhabditis elegans allowed for the identification of ortholog genes belonging to the pigment dispersing hormone/factor (PDH/PDF) peptide family. Members of this peptide family are known from crustaceans, insects and nematodes and seem to exist exclusively in ecdysozoans where they play a role in different processes, ranging from the dispersion of integumental and eye (retinal) pigments in decapod crustaceans to circadian rhythms in insects and locomotion in C. elegans. Two pdf genes (pdf-1 and pdf-2) encoding three different peptides: PDF-1a, PDF-1b and PDF-2 have been identified in C. elegans. These three C. elegans PDH-like peptides are similar but not identical in primary structure to PDHs from decapod crustaceans. We investigate whether this divergence has an influence on the pigment dispersing function of the peptides in a decapod crustacean, namely the shrimp Palaemon pacificus. We show that C. elegans PDF-1a and b peptides display cross-functional activity by dispersing pigments in the epithelium of P. pacificus at physiological doses. Moreover, by means of a comparative amino acid sequence analysis of nematode and crustacean PDH-like peptides, we can pinpoint several potentially important residues for eliciting pigment dispersing activity in decapod crustaceans. Although there is no sequence information on a receptor for PDH in decapod crustaceans, we postulate that there is general conservation of the PDH/PDF signaling system based on structural similarities of precursor proteins and receptors (including those from a branchiopod crustacean and from C. elegans).
Asunto(s)
Caenorhabditis elegans/metabolismo , Crustáceos/metabolismo , Péptidos/química , Secuencia de Aminoácidos , Animales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Datos de Secuencia Molecular , Péptidos/farmacologíaRESUMEN
In comparison with terrestrial and freshwater ecosystems, information about speciation modes and the role of selection in marine environments is scarce. Recent studies have indicated that spectral adaptation could play an important role in the diversification of marine species flocks. Natural selection influences specific amino acids (AAs) that are involved in the spectral tuning mechanism of visual pigment genes. To study the wider occurrence and the characteristics of spectral adaptation in marine radiations, a reinterpretation of the rhodopsin (RH1) data of American seven-spined gobies (genus Elacatinus; Gobiidae; Teleostei) was carried out. Reanalysis revealed that some AAs, which are well known in the literature as spectral tuning sites, are variable in Elacatinus. Those crucial AA substitutions originated polyphyletically, indicating convergent evolution within the genus Elacatinus. Moreover, statistical tests based on the d(N)/d(S) ratio detected selection in several phylogenetic lineages and at specific AAs. Many of these AAs were previously shown to be under selection in other marine radiations. Therefore, the current phylogenetic approach provided an extended list of AAs that are probably involved in spectral tuning, and which should be validated by mutagenic experiments.
Asunto(s)
Perciformes/genética , Rodopsina/genética , Selección Genética , Animales , Demografía , Regulación de la Expresión Génica , Rodopsina/metabolismoRESUMEN
The CphA metallo-beta-lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results in a drastic decrease in the hydrolytic activity. In this study, we analyzed the role of Asn116 and Cys221, two residues of the active site. These residues were replaced by site-directed mutagenesis and the different mutants were characterized. The C221S and C221A mutants were seriously impaired in their ability to bind the first, catalytic zinc ion and were nearly completely inactive, indicating a major role for Cys221 in the binding of the catalytic metal ion. By contrast, the binding of the second zinc ion was only slightly affected, at least for the C221S mutant. Mutation of Asn116 did not lead to a drastic decrease in the hydrolytic activity, indicating that this residue does not play a key role in the catalytic mechanism. However, the substitution of Asn116 by a Cys or His residue resulted in an approximately fivefold increase in the affinity for the second, inhibitory zinc ion. Together, these data suggested that the first zinc ion is located in the binding site involving the Cys221 and that the second zinc ion binds in the binding site involving Asn116 and, presumably, His118 and His196.
Asunto(s)
Aeromonas hydrophila/enzimología , Proteínas Bacterianas/química , Zinc/metabolismo , beta-Lactamasas/química , Asparagina , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Cisteína , Cinética , Datos de Secuencia Molecular , Relación Estructura-Actividad , beta-Lactamasas/metabolismoRESUMEN
Modalities that act through different mechanisms can often provide synergistic antitumor activity for the treatment of refractory tumors when used in combination. Here we report a gene therapy approach in which the genes for the angiogenesis inhibitor, endostatin, and the marker protein and potent immunogen, green fluorescent protein (GFP), were delivered to murine neuroblastoma cells prior to inoculation of the tumor cells into syngeneic immunocompetent mice. Although the effect of either angiogenesis inhibition or immunomodulation alone resulted in only a modest delay in tumor growth, when these approaches were used in combination, prevention of the formation of appreciable tumors was effected in 15 of 24 (63%) mice. The combination of endostatin and GFP expression elicited a strong immune response that was T cell-mediated and was reactive against both GFP and tumor cell line-specific antigens. This afforded treated mice protection against subsequent tumor challenge with unmodified tumor cells. These results suggest that antiangiogenic and immunotherapy strategies, when used in a gene therapy-mediated approach, can act synergistically in an effective multimodality anticancer approach.
Asunto(s)
Colágeno/biosíntesis , Colágeno/genética , Terapia Genética/métodos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Neuroblastoma/terapia , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Inhibidores de la Angiogénesis/farmacología , Animales , División Celular , Movimiento Celular , Separación Celular , Células Cultivadas , Clonación Molecular , Terapia Combinada , Endostatinas , Endotelio Vascular/citología , Citometría de Flujo , Proteínas Fluorescentes Verdes , Humanos , Inmunoterapia/métodos , Ratones , Ratones SCID , Plásmidos/metabolismo , Biosíntesis de Proteínas , Proteínas Recombinantes/metabolismo , Retroviridae/genética , Linfocitos T/metabolismo , Factores de Tiempo , Transcripción Genética , Transducción Genética , Células Tumorales Cultivadas , Venas Umbilicales/citologíaRESUMEN
BACKGROUND: The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme destroys the potential stabilizing agent during the course of the experiments. In this work, enzyme unfolding was followed by monitoring the progressive decrease of the rate of substrate utilization by the Staphylococcus aureus PC1 beta-lactamase, at temperatures above the melting point of the enzyme. RESULTS: Enzyme inactivation was directly followed by spectrophotometric measurements. In the presence of substrate concentrations above the K(m) values, significant stabilization was observed with all tested compounds. A combination of unfolding kinetic measurements and enzymatic studies, both under steady-state and non-steady-state regimes, allowed most of the parameters characteristic of the two concurrent phenomena (i.e. substrate hydrolysis and enzyme denaturation) to be evaluated. In addition, molecular modelling studies show a good correlation between the extent of stabilization, and the magnitude of the energies of interaction with the enzyme. CONCLUSIONS: Our analysis indicates that the enzyme is substantially stabilized towards heat-induced denaturation, independently of the relative proportions of non-covalent Henri-Michaelis complex (ES) and acyl-enzyme adduct (ES*). Thus, for those substrates with which the two catalytic intermediates are expected to be significantly populated, both species (ES and ES*) appear to be similarly stabilized. This analysis contributes a new quantitative approach to the problem.
Asunto(s)
Antibacterianos/metabolismo , Staphylococcus aureus/enzimología , beta-Lactamasas/química , beta-Lactamasas/metabolismo , Acilación , Estabilidad de Enzimas , Cinética , Modelos Moleculares , Unión Proteica , Desnaturalización Proteica , Pliegue de Proteína , Temperatura , Factores de Tiempo , beta-LactamasRESUMEN
Unassembled Ig heavy chains are retained in the ER via the binding of BiP to the C(H)1 domain, which remains unoxidized. Interestingly, this domain folds rapidly, albeit nonproductively, when heavy chains are released from BiP in vitro with ATP. The in vivo cycling of BiP from heavy chains was monitored using BiP ATPase mutants as kinetic traps. Our data suggest that BiP does not cycle from the C(H)1 domain of free heavy chains. However, heavy and light chain assembly occurs rapidly and requires the ATP-dependent release of BiP. We propose that BiP's ATPase cycle is stalled or nonproductive when it is bound to free heavy chains. The binding of light chains to the complex reactivates the cycle and releases BiP.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Choque Térmico , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/fisiología , Chaperonas Moleculares/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Células COS , Chaperón BiP del Retículo Endoplásmico , Regiones Constantes de Inmunoglobulina/metabolismo , Pliegue de ProteínaRESUMEN
Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-beta-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by the reaction products. As shown by electrospray mass spectrometry, the inactivation of CphA by cefoxitin and moxalactam is accompanied by the formation of stable adducts with mass increases of 445 and 111 Da, respectively. The single thiol group of the inactivated enzyme is no longer titrable, and dithiothreitol treatment of the complexes partially restores the catalytic activity. The mechanism of inactivation by moxalactam was studied in detail. Hydrolysis of moxalactam is followed by elimination of the 3' leaving group (5-mercapto-1-methyltetrazole), which forms a disulfide bond with the cysteine residue of CphA located in the active site. Interestingly, this reaction is catalyzed by cacodylate.
Asunto(s)
Aeromonas hydrophila/enzimología , Proteínas Bacterianas , Cefamicinas/metabolismo , Cefamicinas/farmacología , Moxalactam/metabolismo , Moxalactam/farmacología , beta-Lactamasas/metabolismo , Cefamicinas/química , Hidrólisis , Cinética , Estructura Molecular , Moxalactam/química , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-Actividad , Inhibidores de beta-LactamasasRESUMEN
Penicillin-binding protein 4a (PBP4a) from Bacillus subtilis was overproduced and purified to homogeneity. It clearly exhibits DD-carboxypeptidase and thiolesterase activities in vitro. Although highly isologous to the Actinomadura sp. strain R39 DD-peptidase (B. Granier, C. Duez, S. Lepage, S. Englebert, J. Dusart, O. Dideberg, J. van Beeumen, J. M. Frère, and J. M. Ghuysen, Biochem. J. 282:781-788, 1992), which is rapidly inactivated by many beta-lactams, PBP4a is only moderately sensitive to these compounds. The second-order rate constant (k(2)/K) for the acylation of the essential serine by benzylpenicillin is 300,000 M(-1) s(-1) for the Actinomadura sp. strain R39 peptidase, 1,400 M(-1) s(-1) for B. subtilis PBP4a, and 7,000 M(-1) s(-1) for Escherichia coli PBP4, the third member of this class of PBPs. Cephaloridine, however, efficiently inactivates PBP4a (k(2)/K = 46,000 M(-1) s(-1)). PBP4a is also much more thermostable than the R39 enzyme.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/aislamiento & purificación , Muramoilpentapéptido Carboxipeptidasa/metabolismo , Peptidil Transferasas , Bacillus subtilis/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Cinética , Lactamas/metabolismo , Muramoilpentapéptido Carboxipeptidasa/química , Muramoilpentapéptido Carboxipeptidasa/genética , Proteínas de Unión a las Penicilinas , Plásmidos , Tioléster Hidrolasas/metabolismoRESUMEN
The design of effective gene delivery systems for gene transfer in primary human blood cells is important both for fundamental hematopoiesis research and for cancer gene therapy strategies. Here, we evaluated electroporation as a nonviral means for transfection of activated human T lymphocytes and adult bone marrow (BM) CD34+ cells. We describe optimal culture and electroporation parameters for efficient gene delivery in prestimulated T lymphocytes (16.3 +/-1.3%), as well as 2-day cultured adult BM CD34+ cells (29.6+/-4.6%). PHA-stimulated T cells were most receptive for transfection after 48h of in vitro culture, while T cells stimulated by CD3 cross-linking and interleukin (IL)-2 achieved maximum transfection levels after 72 h of prestimulation. Kinetic analysis of EGFP expression revealed that activated T lymphocytes maintained transgene expression at high levels for a prolonged period. In addition, fresh unstimulated BM CD34+ cells were consistently transfected (5.2+/-0.4%) with minimal cytotoxicity (<5%), even without preliminary CD34+ cell purification. Both T cells and CD34+ cells retained their phenotype and functional capacity after electroporation. These results demonstrate that electroporation is a suitable nonviral transfection technique that may serve applications in gene therapy protocols using T lymphocytes or CD34+ cells.
Asunto(s)
Antígenos CD34 , Células de la Médula Ósea , Electroporación/métodos , Linfocitos T , Transfección/métodos , Adulto , Células de la Médula Ósea/inmunología , Citometría de Flujo , Colorantes Fluorescentes , Expresión Génica , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Activación de Linfocitos , TransgenesRESUMEN
Important functional differences exist between primitive CD34++ CD38- hematopoietic progenitor cells derived from human fetal liver (FL) and adult bone marrow (ABM). FL progenitors are known to have higher proliferative capacities and lower cytokine requirements than their ABM counterparts. In this study, we isolated FL and ABM CD34++ CD38- cells and used a two-stage culture system to investigate the effects of transforming growth factor-beta (TGF-beta) and blocking anti-TGF-beta antibodies (anti-TGF-beta) on these cells. First, we demonstrate that FL progenitors are significantly less sensitive to the inhibitory effects of TGF-beta than ABM cells. Second, whereas ABM cells are significantly stimulated by anti-TGF-beta, only very limited effects are seen on FL cells. Third, we show that the effect of anti-TGF-beta is mainly situated at the level of the initial cell cycles of very primitive progenitor cells with a high proliferation potential. Fourth, we demonstrate that blocking the effects of endogenous TGF-beta reduces the growth factor requirements of ABM cells in order to proliferate and differentiate. Based on these data, we hypothesize that at least part of the functional differences that exist between adult and fetal stem cells can be accounted for by a developmental different responsiveness to TGF-beta.
Asunto(s)
Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/fisiología , Factor de Crecimiento Transformador beta/farmacología , Adulto , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , Células Cultivadas , Femenino , Feto/citología , Feto/fisiología , Células Madre Hematopoyéticas/citología , Humanos , EmbarazoRESUMEN
Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 family of chaperones and one of the most abundant proteins in the ER lumen. It is known to interact transiently with many nascent proteins as they enter the ER and more stably with protein subunits produced in stoichiometric excess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To begin to understand what controls the likelihood that a nascent protein entering the ER will associate with BiP, we have examined the in vivo folding of a murine lambdaI immunoglobulin (Ig) light chain (LC). This LC is composed of two Ig domains that can fold independent of the other and that each possess multiple potential BiP-binding sequences. To detect BiP binding to the LC during folding, we used BiP ATPase mutants, which bind irreversibly to proteins, as "kinetic traps." Although both the wild-type and mutant BiP clearly associated with the unoxidized variable region domain, we were unable to detect binding of either BiP protein to the constant region domain. A combination of in vivo and in vitro folding studies revealed that the constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond. Thus, the simple presence of a BiP-binding site on a nascent chain does not ensure that BiP will bind and play a role in its folding. Instead, it appears that the rate and stability of protein folding determines whether or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.