RESUMO
Steroid-thyroid hormone receptors typically bind as dimers to DNA sequences that contain repeated elements termed half-sites. NGFI-B, an early response protein and orphan member of this receptor superfamily, binds to a DNA sequence that contains only one half-site (5'-AAAGGTCA-3'). A domain separate from the NGFI-B zinc fingers, termed the A box, was identified and is required for recognition of the two adenine-thymidine (A-T) base pairs at the 5' end of the NGFI-B DNA binding element. In addition, a domain downstream of the zinc fingers of the orphan receptor H-2 region II binding protein, termed the T box, determined binding to tandem repeats of the estrogen receptor half-site (5'-AGGTCA-3').
Assuntos
Núcleo Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Células CHO , Cricetinae , Proteínas de Ligação a DNA/genética , Cinética , Camundongos , Dados de Sequência Molecular , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Oligodesoxirribonucleotídeos/metabolismo , Reação em Cadeia da Polimerase , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência do Ácido Nucleico , Especificidade por Substrato , Fatores de Transcrição/genética , Transfecção , Dedos de Zinco/genéticaRESUMO
Prescribed fire was investigated as a method for controlling ixodid and argasid ticks in chaparral habitats in northern California. Two experimental and two adjacent control plots within a wildlife preserve were monitored for 1 yr postburn. Ticks were collected by flagging vegetation, by CO2-baited pitfall trap, and by live-trapping rodents. Twice as many rodents were caught at control sites compared with burn sites and no dusky-footed woodrats, Neotoma fuscipes Baird, were found in the treatment sites postburn. This species is known to be a reservoir of the agents of Lyme disease, Borrelia burgdorferi sensu stricto Johnson, Schmid, Hyde, Steigerwalt & Brenner, and human granulocytic anaplasmosis, Anaplasma phagocytophilum Dumler, Barbet, Bekker, Dasch, Palmer, Ray, Rikihisa, Rurangirwa. Six ixodid tick species were removed from rodents (Ixodes pacificus Cooley & Kohls, Ixodes jellisoni Cooley & Kohls, Ixodes spinipalpis Hadwen & Nuttall, Ixodes woodi Bishopp, Dermacentor occidentalis Marx, and Dermacentor parumapertus Neumann), two of which transmit bacterial zoonotic agents to people in the far-western United States. There was no decrease in number of ticks per animal trapped at either burn site compared with controls; in fact, the mean number of immature I. pacificus per rodent was significantly higher at one burn site than its control site. Soil refugia may protect ticks from fire-induced mortality; the argasid tick Ornithodoros coriaceus Koch, which lives in soil, was unaffected by the prescribed fire as were I. pacificus and D. occidentalis buried in packets 2.5 cm below ground. We conclude that although prescribed fires in chaparral habitats may diminish local rodent abundance, it does not decrease tick loads on rodents. Furthermore, burning chaparral does not result in a decreased abundance of adult ixodid ticks on vegetation and apparently does not affect argasid or ixodid ticks that are sheltered within soil refugia.
Assuntos
Dermacentor , Incêndios , Ixodes , Peromyscus/parasitologia , Controle de Ácaros e Carrapatos , Animais , Argasidae , California , Dipodomys/parasitologia , Ecossistema , Feminino , Longevidade , Masculino , Densidade Demográfica , Estações do AnoRESUMO
A method is described for the efficient cloning of any given DNA sequence into any desired location without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction (PCR) combined with the capacity of the type-IIS restriction endonuclease (ENase) Eam1104I to cut outside its recognition sequence. Primers that contain the Eam1104I recognition site (5'-CTCTTC) are used to amplify the DNA fragments being manipulated. Because the ENase is inhibited by site-specific methylation in the recognition sequence, all internal Eam1104I sites present in the DNA can be protected by performing the PCR amplification in the presence of 5-methyldeoxycytosine (m5dCTP). The primer-encoded Eam1104I sites are not affected by the modified nucleotides (nt) since the newly synthesized strand does not contain any cytosine residues in the recognition sequence. In addition, the ENase's ability to cleave several bases downstream from its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA fragments, resulting in 5' overhangs that are defined by the nt present within the cleavage site. Thus, the elimination of extraneous nt and the generation of unique, non-palindromic sticky ends permits the formation of seamless junctions in a directional fashion during the subsequent ligation event.
Assuntos
Clonagem Molecular/métodos , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Reação em Cadeia da Polimerase , Sequência de Bases , Primers do DNA/química , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos/genética , Metilação , Dados de Sequência MolecularRESUMO
The developmental timing of Ixodes pacficus Cooley & Kohls, the primary vector of the Lyme disease spirochete and the agent of human granulocytic ehrlichiosis in the far-western United States, was determined under field and laboratory conditions. During their seasonal peaks of abundance, each of the three parasitic stages of L. pacificus, both fed and unfed, was placed inside silk-screen packets. These packets were apportioned between four topographic exposures of two hilltop sites in northwestern California. The sites differed in vegetational composition and elevation: the first (elevation 390 m) was dominated by woodland-grass, the second (elevation 914 m) by chaparral. The timing of oviposition, larval eclosion, molting, and mortality were recorded in the field every 2-3 wk for 2.5 yr. Microenvironmental temperatures were measured on all four exposures at both sites. Accelerated developmental rates of all three stages were correlated with warmer soil temperatures and the time of placement in the field. In the laboratory, replete female I. pacificus maintained under uniform environmental conditions sustained constant preovipositional and prehatch periods independent of date-of-feeding. In the field, all unfed stages survived through one active feeding season with most larvae and nymphs remaining in behavioral diapause between late summer and early spring. No life stage survived through two active feeding periods which suggests that cohorts do not overlap. We concluded that I. pacificus takes a minimum of 3 yr to complete its life cycle in northwestern California.
Assuntos
Ixodes/crescimento & desenvolvimento , Animais , Feminino , Ixodes/fisiologia , Laboratórios , Larva , Estágios do Ciclo de Vida , Longevidade , Masculino , Oviposição , Coelhos , Estações do Ano , Fatores de TempoRESUMO
Ixodes (Ixodes) jellisoni Cooley & Kohls, a nonhuman biting and little known tick, is one of 4 members of the I. ricinus complex in the United States. A localized population of I. jellisoni inhabiting a grassland biotope in Mendocino County, CA, was studied from 1993 to 1997. Rodent trapping in all seasons revealed that the only host of both immature and adult I. jellisoni was the heteromyid rodent Dipodomys californicus Merriam. Field investigations suggested that I. jellisoni is nidicolous in habit, and laboratory findings demonstrated that it reproduces parthenogenetically. Known parthenogenetic females (n = 4) produced an average of 530 eggs of which 74% hatched, which was comparable to the fecundity and fertility of wild-caught females (n = 8). After the transstadial molt, 57 F1 or F2 nymphs derived from 2 wild-caught or 4 laboratory-reared, unmated females produced only females. Ixodes jellisoni males were not found on 112 wild-caught D. californicus individuals that were captured an average of 2 times. Collectively, these findings suggest that I. jellisoni may be obligatorily parthenogenetic. Borrelial isolates were obtained from 85% of 58 D. californicus and 33% of 21 I. jellisoni females removed from this rodent. None of the 7 infected female ticks passed borreliae ovarially to its F1 larval progeny. Eight D. californicus and 5 I. jellisoni-derived isolates that were genetically characterized belonged to 2 restriction pattern groups of Borrelia burgdorferi s.l. Neither restriction pattern group has been assigned to a particular genospecies yet. After placement on naturally infected D. californicus, noninfected larval ticks acquired and transstadially passed spirochetes as efficiently as (group 1 borreliae) or 6 times more efficiently (group 2 borreliae) than Ixodes pacificus Cooley & Kohls. As few as 1-4 infected I. jellisoni nymphs were capable of transmitting group 1 or group 2 borreliae to naive D. californicus. We conclude that I. jellisoni is a competent vector of both restriction fragment groups when D. californicus is used as the animal model.
Assuntos
Vetores Aracnídeos/microbiologia , Grupo Borrelia Burgdorferi , Ixodes/microbiologia , Animais , Sequência de Bases , Grupo Borrelia Burgdorferi/genética , DNA Bacteriano , Feminino , Doença de Lyme/transmissão , Masculino , Dados de Sequência Molecular , Doenças dos Roedores/microbiologia , Doenças dos Roedores/parasitologia , Infestações por Carrapato/parasitologia , Infestações por Carrapato/veterináriaRESUMO
REASONS FOR PERFORMING STUDY: Intra-arterial (i.a.) and intravenous (i.v.) regional limb perfusions (RLP) through the median artery and cephalic vein, respectively, have been previously investigated for administration of mesenchymal stem cells (MSCs) to the equine distal limb. Limitations due to thrombosis of the arteries after i.a. RLP and poor distribution of MSCs to the foot with i.v. RLP were observed. These techniques need to be modified for clinical use. OBJECTIVES: Evaluate the distribution, uptake and persistence of radiolabelled MSCs after i.a. injection through the median artery without a tourniquet and after i.v. RLP through the lateral palmar digital vein. STUDY DESIGN: In vivo experimental study. METHODS: (99m) Tc-HMPAO-labelled MSCs were injected through the median artery of one limb and the lateral palmar digital vein of the other limb of 6 horses under general anaesthesia. No tourniquet was used for the i.a. injection. A pneumatic tourniquet was placed on the metacarpus for i.v. injection. Scintigraphic images were obtained up to 24 h after injection. RESULTS: Intra-arterial injection resulted in MSCs retention within the limb despite the absence of a tourniquet and no thrombosis was observed. Both i.a. injection and i.v. RLP led to distribution of MSCs to the foot. The i.a. injection resulted in a more homogeneous distribution. The MSC uptake was higher with i.v. RLP at the initial timepoints, but no significant difference was present at 24 h. CONCLUSIONS: Both i.a. injection through the median artery without a tourniquet and i.v. RLP performed through the lateral palmar digital vein under general anaesthesia are safe and reliable methods for administration of MSCs to the equine foot. The i.a. technique is preferred owing to the better distribution, but is technically more challenging. The feasibility of performing these techniques on standing horses remains to be investigated.
Assuntos
Pé/irrigação sanguínea , Cavalos , Infusões Intra-Arteriais/veterinária , Infusões Intravenosas/veterinária , Transplante de Células-Tronco Mesenquimais/veterinária , Cintilografia/veterinária , Animais , Infusões Intra-Arteriais/métodos , Infusões Intravenosas/métodos , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Compostos Radiofarmacêuticos/farmacologia , Tecnécio Tc 99m Exametazima/farmacologiaRESUMO
REASONS FOR PERFORMING STUDY: Intralesional (i.l.) injection is currently the most commonly used technique for stem cell therapy in equine tendon injury. A comparison of different techniques of injection of mesenchymal stem cells for the treatment of tendon lesions is required. OBJECTIVES: We hypothesised that vascular perfusion of the equine distal limb with mesenchymal stem cells (MSCs) would result in preferential distribution of MSCs to acute tendon injuries. STUDY DESIGN: In vivo experimental study. METHODS: Lesions were surgically induced in forelimb superficial digital flexor tendons of 8 horses. Three or 10 days after lesion induction, technetium-99 hexamethyl propylene amine oxime-labelled MSCs were injected via i.v. or intra-arterial (i.a.) regional limb perfusion (RLP) at the level of the distal antebrachium and compared to i.l. injection. Mesenchymal stem cell persistence and distribution within the forelimb and tendon lesions was assessed with scintigraphy for 24 h. RESULTS: Lesion uptake was higher with i.l. injection than with RLP, but MSC persistence decreased similarly over time in all 3 techniques. Intra-arterial RLP resulted in a better distribution of MSCs and a higher uptake at the lesion site than i.v. RLP. Limbs perfused i.a. on Day 10 showed greater accumulation of MSCs in the lesion than limbs perfused on Day 3. Arterial thrombosis occurred in 50% of the i.v. RLP limbs and in 100% of the i.a. RLP limbs, which led to clinical complications in one horse. CONCLUSIONS AND POTENTIAL RELEVANCE: Compared with i.l. injection, RLP results in lower uptake but similar persistence of MSCs at the site of tendon lesions. A time dependent accumulation of MSCs was identified with i.a. RLP. The i.a. RLP appears more advantageous than the i.v. RLP in terms of distribution and uptake. However, the described i.a. technique produced arterial thrombosis and thus cannot currently be recommended for clinical use.
Assuntos
Doenças dos Cavalos/terapia , Macrófagos/metabolismo , Transplante de Células-Tronco Mesenquimais/veterinária , Tecnécio Tc 99m Exametazima/farmacologia , Traumatismos dos Tendões/terapia , Animais , Feminino , Membro Anterior , Doenças dos Cavalos/diagnóstico por imagem , Cavalos , Masculino , Cintilografia/veterinária , Traumatismos dos Tendões/diagnóstico por imagem , UltrassonografiaRESUMO
REASONS FOR PERFORMING STUDY: Mesenchymal stem cells (MSCs) are commonly injected intralesionally for treatment of soft tissue injuries in the horse. Alternative routes of administration would be beneficial for treatment of lesions that cannot be accessed directly or to limit needle-induced iatrogenic damage to the surrounding tissue. OBJECTIVES: The purpose of our study was to evaluate MSC distribution after intra-arterial (IA) and intravenous (IV) regional limb perfusions (RLP) using scintigraphy. We hypothesised that MSCs would persist in the distal limb after tourniquet removal and that both techniques would lead to diffuse MSC distribution. METHODS: Six horses were used in the study. MSCs were labelled with hexamethyl propylene amine oxime (HMPAO) and technetium-99m. RLP was performed through the median artery of one forelimb and the cephalic vein of the opposite limb under general anaesthesia. The tourniquet was left in place for 45 min. Scintigraphic images were obtained at 0, 45, 75 min, 6 h and 24 h post injection. RESULTS: Distribution of labelled MSCs through the entire distal limb was achieved with all 6 IA RLP, but 3 out of 6 IV RLP showed poor or absent uptake distal to the metacarpus. Mesenchymal stem cell persistence was 39% (30-60%) and 28% (14-50%) (median [minimum-maximum]) at 6 h for IA and IV RLP, respectively. Severe arterial thrombosis occurred in one horse after IA RLP. CONCLUSIONS: Both IA and IV RLP of the distal limb result in MSC persistence in perfused tissues. The IA perfusion resulted in more reliable cell distribution to the pastern and foot area. POTENTIAL RELEVANCE: Regional limb perfusion of MSCs might be used in cases where intralesional injection is not possible or in order to avoid iatrogenic needle damage. Further work is needed to assess the safety of IA RLP before its clinical use.
Assuntos
Células da Medula Óssea/fisiologia , Membro Anterior/irrigação sanguínea , Transplante de Células-Tronco Mesenquimais/veterinária , Cintilografia/veterinária , Tecnécio Tc 99m Exametazima/farmacologia , Animais , Feminino , Infusões Intra-Arteriais , Injeções Intravenosas , Masculino , Cintilografia/métodos , Compostos Radiofarmacêuticos/farmacologia , Transplante HomólogoRESUMO
In response to an outbreak of Rocky Mountain spotted fever (RMSF) in Baja California in early 2009, dogs at two shelters in neighbouring Imperial County, California, were evaluated for ectoparasites. Brown dog ticks (Rhipicephalus sanguineus), a recognized vector for RMSF, were found on 35 (30%) of 116 dogs but all ticks tested negative for Rickettsia rickettsii by PCR.
Assuntos
Vetores Aracnídeos/microbiologia , Doenças do Cão/epidemiologia , Rhipicephalus sanguineus/microbiologia , Rickettsia rickettsii/fisiologia , Febre Maculosa das Montanhas Rochosas/veterinária , Infestações por Carrapato/veterinária , Animais , California/epidemiologia , DNA Bacteriano/genética , Surtos de Doenças/veterinária , Doenças do Cão/microbiologia , Doenças do Cão/parasitologia , Cães , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase/veterinária , Rhipicephalus sanguineus/fisiologia , Febre Maculosa das Montanhas Rochosas/epidemiologia , Febre Maculosa das Montanhas Rochosas/microbiologia , Infestações por Carrapato/epidemiologia , Infestações por Carrapato/parasitologiaRESUMO
This study tested the hypotheses that ants (Formicidae) function as a first intermediate host of Mesocestoides (Cestoda: Mesocestoididae) and that deer mice (Peromyscus maniculatus) develop metacestode infections after ingesting cysticercoid or procercoid-infected ants. Field studies were conducted at an island fox (Urocyon littoralis littoralis) breeding facility located on San Miguel Island, California Channel Islands National Park, USA, where > 40% of captive foxes were infected with adult Mesocestoides. Eight percent (8%) of deer mice at the fox pen site were infected with Mesocestoides metacestodes while none were infected at a distant site where foxes were absent (campground), thereby indicating the potential localized presence of a first intermediate host. To test whether ants from San Miguel Island contained Mesocestoides DNA, a polymerase chain reaction (PCR)-based diagnostic assay was developed using nested primers that could detect a single hexacanth larva within pooled samples of ten ants. Ants (Lasius niger and Tapinoma sessile) collected near the fox breeding facility were tested using the nested-PCR assay. Seven of 223 pooled samples of L. niger (3.1%) and 2 of 84 pooled samples of T. sessile (2.4%) tested positive for Mesocestoides DNA, while none of the ants were positive at the campground site. Positive samples were sequenced and found to match DNA sequences from Mesocestoides obtained from island fox and deer mice. Finally, to determine whether ants function as a first intermediate host for Mesocestoides, colony-raised deer mice (n = 47) were fed L. niger (n = 3860) or T. sessile (n = 339) collected from the San Miguel Island fox breeding facility. No mouse became infected with Mesocestoides metacestodes after ingesting ants. While both L. niger and T. sessile from SMI were positive for Mesocestoides DNA, they were not infective to deer mice in the laboratory.
Assuntos
Formigas/parasitologia , Infecções por Cestoides/parasitologia , Mesocestoides/fisiologia , Peromyscus/parasitologia , Animais , California , DNA de Helmintos/análise , Vetores de Doenças , Comportamento Alimentar , Raposas/parasitologia , Interações Hospedeiro-Parasita , Mesocestoides/genética , Camundongos , Reação em Cadeia da Polimerase/métodosRESUMO
The murine MHC provides a unique genetic system for studying meiotic recombination. A large number of murine H-2 recombinants cross over within a stretch of the E beta gene referred to as the E beta hot spot. The crossing over of eight such recombinants, derived from the s and k haplotypes, was studied at the nucleotide level. A 3-kb stretch of DNA, 3' to the beta 1 exon of the E beta gene, was sequenced after amplification of the genomic DNA from B10.S (one of the parental strains) by polymerase chain reaction. A number of sequence variations were identified with respect to B10.A (the other parental strain). Examination of these sequence variations by RFLP, simple sequence length polymorphism, as well as direct sequencing after polymerase chain reaction-amplification of genomic DNA from the recombinants led to unambiguous identification of the cross-over sites. Although all eight recombinants crossed over within the beta 1-beta 2 intron, two discrete nonoverlapping sites were involved. Five of the recombinants B10.BASR1, B10.ASR1, B10.ASR12, B10.HTT, and B10.S(9R) crossed over within a maximum of 395 bp of DNA 3' to the beta 1 exon. The remaining three recombinants B10.ASR7, B10.ASR11, and B10.S(8R) crossed over within 950 bp of DNA, adjacent to the cross-over site noted above. Each of these stretches of DNA was completely identical in the two parental haplotypes precluding further dissection of the cross-over sites. These cross-over sites are within those reported for the b and k recombination.
Assuntos
Troca Genética , Antígenos H-2/genética , Íntrons , Recombinação Genética , Alelos , Animais , Sequência de Bases , Camundongos , Dados de Sequência Molecular , Sequências Repetitivas de Ácido NucleicoRESUMO
A method is described that allows for rapid and efficient generation of functional mutations in DNA-binding domains of proteins. The target DNA-binding domain is attached to the Gal4p transcriptional-activating domain and expressed in yeast. The binding site recognized by the target domain is placed upstream of a gene that produces a protein toxic to yeast cells, so that the chimeric protein activates its expression, providing a selection against DNA-binding domain function. The chimeric protein also activates expression of a gene necessary for histidine prototrophy, using a second DNA-binding domain included in the chimera (lexA), providing a selection against general activator mutations. Therefore, requiring growth in the absence of histidine focuses mutations to the target DNA-binding domain. This method was applied to the DNA-binding domain of the nuclear receptor NGFI-B. Nearly all mutations obtained concurred with previous studies of NGFI-B and other nuclear receptors, verifying the functional validity of the mutational profile obtained. In addition, by coupling this selection scheme with the two-hybrid system [Chien, C.-t., Bartel, P. L., Sternglanz, R. & Fields, S. (1991) Proc. Natl. Acad. Sci. USA 88, 9578-9582], mutations that alter protein interaction domains could also be obtained.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Receptores de Superfície Celular/metabolismo , Saccharomyces cerevisiae/genética , Serina Endopeptidases , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sítios de Ligação , Primers do DNA , Proteínas de Ligação a DNA/biossíntese , Galactose/farmacologia , Genes Fúngicos , Glutationa Transferase/biossíntese , Glutationa Transferase/isolamento & purificação , Glutationa Transferase/metabolismo , Dados de Sequência Molecular , Mutagênese , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Plasmídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Estrutura Secundária de Proteína , Receptores de Superfície Celular/genética , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , TATA Box , Fatores de Transcrição/biossínteseRESUMO
NGFI-A is a mammalian transcription factor that contains zinc fingers similar to those observed in several other proteins, including NGFI-C and Krox-20. To define precisely the DNA binding domain of NGFI-A, we selected mutants using a chimeric transcriptional activator that contains the NGFI-A zinc finger domain sandwiched between the lexA DNA binding domain and the GAL4 transcriptional activating domain. Expression of this lexA-NGFI-A-GAL4 (LAG) trimeric protein in yeast significantly retarded their growth, unlike an activator containing only the lexA and GAL4 components. This suggested that LAG inappropriately regulates genes in yeast that contain NGFI-A binding sites. Yeast that contained LAG reverted to wild-type growth at high frequency by inactivation of LAG. The mutations recovered from these revertants were specifically limited to the 83-residue NGFI-A zinc finger domain by requiring that the lexA and GAL4 portions of the LAG chimera remain functional. Nearly all of the 93 mutants obtained contained single missense mutations that mapped within the zinc fingers to residues thought to be important for zinc finger function. Deletion analysis of native NGFI-A verified that residues distant from the zinc fingers do not influence DNA binding, thus establishing the minimal functional DNA binding domain. Interestingly, many zinc finger residues ascribed specific functions by x-ray crystallography were never mutated in yeast, implying that the identity of these residues is not critical. Surprisingly, not all of the mutations tested significantly impaired NGFI-A-specific DNA binding, suggesting that the function of these zinc fingers is more diverse than previously recognized.