RESUMO
Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by defects in the ß-subunit of ß-N-acetylhexosaminidase (Hex), the enzyme that catabolizes GM2 ganglioside. Hex deficiency causes neuronal storage of GM2 and related glycoconjugates, resulting in progressive neurodegeneration and death, typically in infancy. No effective treatment exists for human patients. Adeno-associated virus (AAV) gene therapy led to improved clinical outcome and survival of SD cats treated before the onset of disease symptoms. Most human patients are diagnosed after clinical disease onset, so it is imperative to test AAV-gene therapy in symptomatic SD cats to provide a realistic indication of therapeutic benefits that can be expected in humans. In this study, AAVrh8 vectors injected into the thalamus and deep cerebellar nuclei of symptomatic SD cats resulted in widespread central nervous system enzyme distribution, although a substantial burden of storage material remained. Cats treated in the early symptomatic phase showed delayed disease progression and a significant survival increase versus untreated cats. Treatment was less effective when administered later in the disease course, although therapeutic benefit was still possible. Results are encouraging for the treatment of human patients and provide support for the development AAV-gene therapy for human SD.
Assuntos
Doença de Sandhoff , Animais , Gatos , Dependovirus/genética , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos/genética , Humanos , Doença de Sandhoff/genética , Doença de Sandhoff/terapia , beta-N-Acetil-Hexosaminidases/genéticaRESUMO
PURPOSE: Cystic fibrosis (CF), caused by pathogenic variants in the CF transmembrane conductance regulator (CFTR), affects multiple organs including the exocrine pancreas, which is a causal contributor to cystic fibrosis-related diabetes (CFRD). Untreated CFRD causes increased CF-related mortality whereas early detection can improve outcomes. METHODS: Using genetic and easily accessible clinical measures available at birth, we constructed a CFRD prediction model using the Canadian CF Gene Modifier Study (CGS; n = 1,958) and validated it in the French CF Gene Modifier Study (FGMS; n = 1,003). We investigated genetic variants shown to associate with CF disease severity across multiple organs in genome-wide association studies. RESULTS: The strongest predictors included sex, CFTR severity score, and several genetic variants including one annotated to PRSS1, which encodes cationic trypsinogen. The final model defined in the CGS shows excellent agreement when validated on the FGMS, and the risk classifier shows slightly better performance at predicting CFRD risk later in life in both studies. CONCLUSION: We demonstrated clinical utility by comparing CFRD prevalence rates between the top 10% of individuals with the highest risk and the bottom 10% with the lowest risk. A web-based application was developed to provide practitioners with patient-specific CFRD risk to guide CFRD monitoring and treatment.
Assuntos
Fibrose Cística , Diabetes Mellitus , Biomarcadores , Canadá , Fibrose Cística/complicações , Fibrose Cística/diagnóstico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Diabetes Mellitus/epidemiologia , Diabetes Mellitus/genética , Estudo de Associação Genômica Ampla , Humanos , Recém-NascidoRESUMO
Salutary responses to adeno-associated viral (AAV) gene therapy have been reported in the mouse model of Sandhoff disease (SD), a neurodegenerative lysosomal storage disease caused by deficiency of ß-N-acetylhexosaminidase (Hex). While untreated mice reach the humane endpoint by 4.1 months of age, mice treated by a single intracranial injection of vectors expressing human hexosaminidase may live a normal life span of 2 years. When treated with the same therapeutic vectors used in mice, two cats with SD lived to 7.0 and 8.2 months of age, compared with an untreated life span of 4.5 ± 0.5 months (n = 11). Because a pronounced humoral immune response to both the AAV1 vectors and human hexosaminidase was documented, feline cDNAs for the hexosaminidase α- and ß-subunits were cloned into AAVrh8 vectors. Cats treated with vectors expressing feline hexosaminidase produced enzymatic activity >75-fold normal at the brain injection site with little evidence of an immune infiltrate. Affected cats treated with feline-specific vectors by bilateral injection of the thalamus lived to 10.4 ± 3.7 months of age (n = 3), or 2.3 times as long as untreated cats. These studies support the therapeutic potential of AAV vectors for SD and underscore the importance of species-specific cDNAs for translational research.
Assuntos
Doenças do Gato/enzimologia , Doenças do Gato/terapia , Doença de Sandhoff/enzimologia , Doença de Sandhoff/terapia , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Doenças do Gato/genética , Gatos , Dependovirus/genética , Modelos Animais de Doenças , Terapia Genética/métodos , Vetores Genéticos/genética , Doença de Sandhoff/genética , beta-N-Acetil-Hexosaminidases/genéticaRESUMO
Mary Wollstonecraft Shelley's modern Prometheus shows us the eternal punishment of unrepaired shame--eternal entrapment within the shame triangle of victim, perpetrator and rescuer. This paper describes how Shelley's insight--that lack of love creates a monster living in shame--is being confirmed by neuroscience and how this is exemplified in two characters--the creature and Victor Frankenstein. Additionally, it delineates how pastoral counselors can help those suffering from unrepaired shame
Assuntos
Clero/métodos , Comportamento de Ajuda , Relações Profissional-Paciente , Autoimagem , Vergonha , Adaptação Psicológica , Revelação , Feminino , Humanos , Masculino , Medicina na LiteraturaRESUMO
Phasing of heterozygous alleles is critical for interpretation of cis-effects of disease-relevant variation. We sequenced 477 individuals with cystic fibrosis (CF) using linked-read sequencing, which display an average phase block N50 of 4.39 Mb. We use these samples to construct a graph representation of CFTR haplotypes, demonstrating its utility for understanding complex CF alleles. These are visualized in a Web app, CFTbaRcodes, that enables interactive exploration of CFTR haplotypes present in this cohort. We perform fine-mapping and phasing of the chr7q35 trypsinogen locus associated with CF meconium ileus, an intestinal obstruction at birth associated with more severe CF outcomes and pancreatic disease. A 20-kb deletion polymorphism and a PRSS2 missense variant p.Thr8Ile (rs62473563) are shown to independently contribute to meconium ileus risk (p = 0.0028, p = 0.011, respectively) and are PRSS2 pancreas eQTLs (p = 9.5 × 10-7 and p = 1.4 × 10-4, respectively), suggesting the mechanism by which these polymorphisms contribute to CF. The phase information from linked reads provides a putative causal explanation for variation at a CF-relevant locus, which also has implications for the genetic basis of non-CF pancreatitis, to which this locus has been reported to contribute.
Assuntos
Fibrose Cística , Obstrução Intestinal , Íleo Meconial , Recém-Nascido , Humanos , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Íleo Meconial/complicações , Mecônio , Obstrução Intestinal/complicações , Tripsina , Tripsinogênio/genéticaRESUMO
Over 400 variants in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) are CF-causing. CFTR modulators target variants to improve lung function, but marked variability in response exists and current therapies do not address all CF-causing variants highlighting unmet needs. Alternative epithelial ion channel/transporters such as SLC26A9 could compensate for CFTR dysfunction, providing therapeutic targets that may benefit all individuals with CF. We investigate the relationship between rs7512462, a marker of SLC26A9 activity, and lung function pre- and post-treatment with CFTR modulators in Canadian and US CF cohorts, in the general population, and in those with chronic obstructive pulmonary disease (COPD). Rs7512462 CC genotype is associated with greater lung function in CF individuals with minimal function variants (for which there are currently no approved therapies; p = 0.008); and for gating (p = 0.033) and p.Phe508del/ p.Phe508del (p = 0.006) genotypes upon treatment with CFTR modulators. In parallel, human nasal epithelia with CC and p.Phe508del/p.Phe508del after Ussing chamber analysis of a combination of approved and experimental modulator treatments show greater CFTR function (p = 0.0022). Beyond CF, rs7512462 is associated with peak expiratory flow in a meta-analysis of the UK Biobank and Spirometa Consortium (p = 2.74 × 10-44) and provides p = 0.0891 in an analysis of COPD case-control status in the UK Biobank defined by spirometry. These findings support SLC26A9 as a therapeutic target to improve lung function for all people with CF and in individuals with other obstructive lung diseases.
RESUMO
RATIONALE: We developed an evidence-based decision aid for patients with advanced cystic fibrosis considering referral for lung transplantation. OBJECTIVES: To prospectively evaluate whether use of the decision aid increased knowledge about the options, improved realistic expectations, and decreased decisional conflict in adult patients. METHODS: We performed a single-blind randomized controlled trial involving 149 adult patients with cystic fibrosis with an FEV(1) Assuntos
Fibrose Cística/cirurgia
, Tomada de Decisões Assistida por Computador
, Conhecimentos, Atitudes e Prática em Saúde
, Transplante de Pulmão/psicologia
, Participação do Paciente
, Adulto
, Fibrose Cística/psicologia
, Técnicas de Apoio para a Decisão
, Feminino
, Humanos
, Masculino
, Educação de Pacientes como Assunto
, Adulto Jovem
RESUMO
GM2 gangliosidosis is a fatal, progressive neuronopathic lysosomal storage disease resulting from a deficiency of beta-N-acetylhexosaminidase (EC 3.2.1.52) activity. GM2 gangliosidosis occurs with varying degrees of severity in humans and in a variety of animals, including cats. In the current research, European Burmese cats presented with clinical neurological signs and histopathological features typical of a lysosomal storage disease. Thin layer chromatography revealed substantial storage of GM2 ganglioside in brain tissue of affected cats, and assays with a synthetic fluorogenic substrate confirmed the absence of hexosaminidase activity. When the hexosaminidase beta-subunit cDNA was sequenced from affected cats, a 91 base pair deletion constituting the entirety of exon 12 was documented. Subsequent sequencing of introns 11 and 12 revealed a 15 base pair deletion at the 3' end of intron 11 that included the preferred splice acceptor site, generating two minor transcripts from cryptic splice acceptor sites in affected Burmese cats. In the cerebral cortex of affected cats, hexosaminidase beta-subunit mRNA levels were approximately 1.5 times higher than normal (P<0.001), while beta-subunit protein levels were substantially reduced on Western blots.
Assuntos
Doenças do Gato/enzimologia , Doenças por Armazenamento dos Lisossomos/veterinária , Degeneração Neural/complicações , Degeneração Neural/enzimologia , Cadeia beta da beta-Hexosaminidase/metabolismo , Animais , Sequência de Bases , Western Blotting , Gatos , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Cromatografia em Camada Fina , Análise Mutacional de DNA , Europa (Continente) , Gangliosidoses GM2/enzimologia , Gangliosidoses GM2/patologia , Lipídeos/análise , Doenças por Armazenamento dos Lisossomos/complicações , Doenças por Armazenamento dos Lisossomos/enzimologia , Dados de Sequência Molecular , MianmarRESUMO
BACKGROUND: Some people with chronic spinal cord injury (SCI) have low vitamin D levels and secondary hyperparathyroidism. OBJECTIVE: To determine whether, and to what extent, an acute calcium infusion decreased levels of N-telopeptide (NTx), a marker of osteoclastic activity, in individuals with chronic SCI. STUDY DESIGN: Case series. SUBJECTS: Eight men with chronic SCI. A relatively low serum 25 hydroxyvitamin D concentration (25[OH]D < or =20 ng/mL) and/or a high parathyroid hormone (PTH) (>55 pg/mL) was a prerequisite for study inclusion. METHODS: Calcium gluconate bolus 0.025 mmol elemental calcium/kg over 20 minutes followed by a constant infusion of 0.025 mmol/kg per hour for 6 hours was infused; blood samples were collected every 2 hours for measurement of serum total calcium, creatinine, NTx, and PTH. RESULTS: All results are expressed as means (+/- SDs). Baseline serum 25-hydroxyvitamin D level was 14.5 +/- 3.5 ng/mL (range: 10.2-19.6 ng/mL); PTH, 70 +/- 25 pg/mL (range: 37-100 pg/mL); and NTx, 21 +/- 7 nM bone collagen equivalents (BCE) (range: 14-34 nM). At 2, 4, and 6 hours after the calcium infusion, serum calcium rose from 9.3 +/- 0.2 to 10.8 +/- 0.9, 10.5 +/- 0.8, and 10.6 +/- 0.6 mg/d; PTH was suppressed from 70 +/- 25 pg/mL to 18 +/- 12, 16 +/- 9, and 15 +/- 9 pg/mL, respectively; NTx fell from 21 +/- 8 nM BCE to 17 +/- 5, 12 +/- 4, and 12 +/- 3 nM BCE, respectively. CONCLUSIONS: Serum NTx is a marker for bone collagen catabolism, and its reduction suggests that bone turnover was decreased. A relative deficiency of vitamin D associated with chronically elevated levels of PTH would be expected to increase bone turnover and to worsen the bone loss associated with immobilization.
Assuntos
Compostos Organometálicos/administração & dosagem , Traumatismos da Medula Espinal/sangue , Traumatismos da Medula Espinal/tratamento farmacológico , Trissacarídeos/administração & dosagem , Adulto , Análise de Variância , Cálcio/sangue , Colágeno Tipo I/sangue , Humanos , Masculino , Hormônio Paratireóideo/sangue , Peptídeos/sangue , Fatores de Tempo , Vitamina D/análogos & derivados , Vitamina D/sangue , Adulto JovemRESUMO
Overpopulation of free-roaming and wildlife animals negatively affects economy and public health in many parts of the world. Contraceptive vaccines are viewed as a valuable option for reducing numbers of unwanted animals. This study develops vaccines for potential use in animal contraception exploiting a DNA platform. Objectives of the study were to generate DNA constructs directed against gonadotropin-releasing hormone receptor (GnRHR), a crucial molecular player in animal reproduction, and characterize them for ability to promote immune responses and suppression of reproductive parameters in vivo. DNA constructs were created to encode for a recombinant protein composed of two domains: GnRHR, the target antigen, and ubiquitin (Ub), a support protein. Ub-GnRHR constructs administered intramuscularly or intradermally or containing different promoters were compared. CMV and EF1α promoters were shown to be superior to CAG. In fertility trials, mice immunized intradermally with Ub-GnRHR construct driven by EF1α had a significantly lower number of fetuses. Importantly, the impaired fertility was achieved with a single DNA immunization and without the use of adjuvants. The study demonstrated for the first time that targeting the GnRH receptor with DNA-based vaccines could be a viable option for animal contraception.
Assuntos
Anticoncepção/veterinária , Receptores LHRH/genética , Vacinas Anticoncepcionais/administração & dosagem , Vacinas de DNA/administração & dosagem , Animais , Anticorpos/sangue , Células CHO , Gatos , Cricetulus , Feminino , Fertilidade , Expressão Gênica , Imunização , Masculino , Camundongos , Regiões Promotoras Genéticas/genética , Receptores LHRH/imunologia , Testosterona/sangue , Ubiquitina/genéticaRESUMO
Patients with chronic spinal cord injury (SCI), a condition associated with reduced physical function, have been reported to have lower plasma insulin-like growth factor-I (IGF-I) levels than able-bodied persons. We evaluated the potential for daily low-dose baclofen administered over several weeks to increase plasma IGF-I levels. Ten healthy male outpatients with chronic SCI were studied prospectively. Patients received escalating doses of baclofen for 4 weeks at each dose level (5, 10, and 20 mg/d). At each dose of baclofen, an increase in the plasma IGF-I was noted; significant increases in plasma IGF-I occurred at 2 weeks after administration of drug at doses of 10 and 20 mg/d, with a subsequent rise to peak levels on baclofen 20 mg/d [baseline, 205+/-74; peak, 218+/-76 (not significant), 239+/-83 (P<.05), 263+/-87 microg/L (P<.05), at baclofen 5, 10, and 20 mg/d, respectively]. In conclusion, low-dose baclofen administration for 4 weeks stimulated the growth hormone-IGF-I axis in persons with SCI, with the potential for beneficial effects on body composition.
Assuntos
Baclofeno/administração & dosagem , Baclofeno/farmacologia , Fator de Crescimento Insulin-Like I/metabolismo , Relaxantes Musculares Centrais/administração & dosagem , Relaxantes Musculares Centrais/farmacologia , Traumatismos da Medula Espinal/tratamento farmacológico , Adulto , Baclofeno/uso terapêutico , Doença Crônica , Relação Dose-Resposta a Droga , Humanos , Masculino , Pessoa de Meia-Idade , Relaxantes Musculares Centrais/uso terapêutico , Paraplegia/tratamento farmacológico , Quadriplegia/tratamento farmacológicoRESUMO
Eleven subjects participated in a prospective placebo-controlled trial to address the efficacy of pamidronate in reducing bone loss in persons with acute spinal cord injury (SCI). We administered pamidronate (treatment) or normal saline (placebo) intravenously at baseline (22 to 65 days after injury) and sequentially over 12 months, with follow-up at 18 and 24 months. Regional bone mineral density (BMD) was lost over time, regardless of group. In the treatment group compared with the placebo group, we noted a mild early reduction in loss of total leg BMD. Significant bone loss from baseline occurred earlier in the placebo group at the regional sites than in the treatment group. However, by the end of the treatment and follow-up phases, both groups demonstrated a similar percent bone loss from baseline. Despite an early reduction in bone loss, pamidronate failed to prevent major, long-term bone loss in persons with acute neurologically complete SCI.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Densidade Óssea/efeitos dos fármacos , Ossos da Extremidade Inferior/efeitos dos fármacos , Difosfonatos/farmacologia , Osteoporose/prevenção & controle , Traumatismos da Medula Espinal/complicações , Doença Aguda , Adulto , Biomarcadores/sangue , Biomarcadores/urina , Conservadores da Densidade Óssea/uso terapêutico , Cálcio/sangue , Cálcio/urina , Colágeno/urina , Difosfonatos/uso terapêutico , Método Duplo-Cego , Esquema de Medicação , Feminino , Humanos , Imobilização/efeitos adversos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Osteoporose/etiologia , Pamidronato , Paraplegia/complicações , Estudos Prospectivos , Quadriplegia/complicaçõesRESUMO
BACKGROUND/OBJECTIVE: An increased prevalence of vitamin D deficiency has been reported in persons with chronic spinal cord injury (SCI), but treatment guidelines for replacement are not available. The purpose of this study was to evaluate two types of vitamin D therapy on calcium metabolism and vitamin D status in persons with SCI. METHODS: Ten subjects with chronic SCI who were vitamin D deficient received 25 hydroxyvitamin D3 [25(OH)D], 50 microg twice a week, for 14 days (Study 1). Regardless of vitamin D status, 40 subjects received vitamin D3 800 IU (20 microg) daily for 12 months (Study 2). Supplemental calcium was administered. The response to therapy was determined by the effect upon serum 25(OH)D levels. Results are expressed as mean +/- standard deviation. RESULTS: In Study 1, serum 25(OH)D levels increased by day 14 (8.7 +/- 2.1 vs 14.7 +/- 3.6 ng/mL; P < 0.0005). However, in 8 of 10 subjects, 25(OH)D levels were still below the absolute lower limit of normal. Serum calcium levels were not significantly different, but urinary calcium excretion increased (103 +/- 81 vs 184 +/- 145 mg/d; P < 0.01). Serum parathyroid hormone (PTH) levels decreased (35 +/- 26 vs 1 7 +/- 12 pg/ mL; P < 0.01). In Study 2, after 12 months of vitamin D supplementation, 9 subjects had an absolute and 23 had a relative vitamin D deficiency, compared with 33 and 6 subjects, respectively, at baseline. By 12 months, the 25(OH)D level increased (10.7 +/- 7.1 to 22.5 +/- 7.5 ng/mL; P < 0.0001) and the serum PTH level decreased (37 +/- 16 vs 25 +/- 11 pg/mL; P < 0.0001). CONCLUSIONS: Although 25(OH)D levels significantly increased in both studies, the replacement therapies employed were not sufficient to recommend for adoption for clinical use, indicating the need for higher doses and/or for longer periods of administration.
Assuntos
Calcifediol/uso terapêutico , Traumatismos da Medula Espinal/complicações , Deficiência de Vitamina D/complicações , Deficiência de Vitamina D/tratamento farmacológico , Adulto , Idoso , Calcifediol/sangue , Cálcio/sangue , Cálcio/uso terapêutico , Cálcio/urina , Humanos , Pessoa de Meia-Idade , Hormônio Paratireóideo/sangue , Traumatismos da Medula Espinal/sangue , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/urina , Falha de Tratamento , Deficiência de Vitamina D/sangueRESUMO
Although previous literature focusing on perceptions of victims of rape has examined how gender, race, and culture influence the attitudes one holds toward victims, these studies have yielded mixed results. This study compared perceptions of victims of rape across a wide range of ages, educational backgrounds, religions, and income levels, while focusing on gender and racial differences. Results indicate (N = 220) that victims of rape are generally viewed more sympathetically by females than by males and by Whites than by African Americans. However, the effect of race disappears when socioeconomic variables are controlled, suggesting a more complex relationship. Also, a hierarchical regression indicates that age, sex, education, and income are significant predictors of attitudes toward victims. This study builds on existing research that examines such attitudes from a cultural perspective and extends this literature by examining the interactive effects of several demographic variables within a community sample.
Assuntos
Negro ou Afro-Americano/psicologia , Vítimas de Crime , Estupro/psicologia , Religião , Classe Social , População Branca/psicologia , Adulto , Negro ou Afro-Americano/estatística & dados numéricos , Idoso , Idoso de 80 Anos ou mais , Características Culturais , Feminino , Humanos , Relações Interpessoais , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Missouri , Estupro/estatística & dados numéricos , Estudos de Amostragem , Fatores Sexuais , Inquéritos e Questionários , População Branca/estatística & dados numéricosRESUMO
Early diagnosis and effective treatment of malignant gliomas, which are heterogeneous brain tumors with variable expression of cell surface markers, are inhibited by the lack of means to characterize and target tumor-selective molecules. To create molecular profiles for RG2 rat glioma cells, we used phage display technology, an approach capable of producing valuable ligands to unknown cell surface targets. The ligands were selected from libraries of peptides displayed as fusion molecules on phage particles. Modifications of the selection conditions resulted in identification of three distinctive families of peptide ligands for malignant glioma cells. The first family with V (D)/(G) L P (E)/(T) H(3) binding motif appeared to target a marker that is common for glioma cells, normal brain cells, and cells of non-brain origin. The second group of peptide-presented phage displayed D (T)/S/(L) T K consensus sequence and contained peptides with pronounced glioma-selective properties. Phage clones expressing peptides with E (L)/V/(S) R G D S motif were found in cell lysates and represented the third family of glioma-specific ligands. All peptides within this family contain the RGD amino acid sequence, which is known to bind to a number of integrins. Phage clones that belong to this family were internalized by RG2 glioma cells about 63-fold more efficiently than by astrocytes. The approach described could be applicable for accurate detection of glioma expression patterns in individual tumors. Such patterns could be beneficial in the design of effective combinations of drugs for anti-glioma treatments.
Assuntos
Bacteriófagos/genética , Glioma/diagnóstico , Neuroglia/patologia , Biblioteca de Peptídeos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/patologia , Ligantes , Neuroglia/metabolismo , RatosRESUMO
GnRH receptors play vital roles in mammalian reproduction via regulation of gonadotropin secretion, which is essential for gametogenesis and production of gonadal steroids. GnRH receptors for more than 20 mammalian species have been sequenced, including human, mouse, and dog. This study reports the molecular cloning and sequencing of GnRH receptor (GnRHR) cDNA from the pituitary gland of the domestic cat, an important species in biomedical research. Feline GnRHR cDNA is composed of 981 nucleotides and encodes a 327 amino acid protein. Unlike the majority of mammalian species sequenced so far, but similar to canine GnRHR, feline GnRHR protein lacks asparagine in position three of the extracellular domain of the protein. At the amino acid level, feline GnRHR exhibits 95.1% identity with canine, 93.8% with human, and 88.9% with mouse GnRHR. Comparative sequence analysis of GnRHRs for multiple mammalian species led to resequencing of canine GnRHR, which differed from that previously published by a single base change that translates to a different amino acid in position 193. This single base change was confirmed in dogs of multiple breeds. Reverse transcriptase PCR analysis of GnRHR messenger RNA in different tissues from four normal cats indicated the presence of amplicons of varying lengths, including full-length as well as shortened GnRHR amplicons, pointing to the existence of truncated GnRHR transcripts in the domestic cat. This study is the first insight into molecular composition and expression of feline GnRHR and promotes better understanding of receptor organization, and distribution in various tissues of this species.
Assuntos
Gatos/genética , Clonagem Molecular , Cães/genética , Receptores LHRH/genética , Análise de Sequência/veterinária , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/química , DNA Complementar/genética , Feminino , Humanos , Masculino , Camundongos , Especificidade de Órgãos , Hipófise/química , RNA Mensageiro/análise , Receptores LHRH/análise , Receptores LHRH/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Homologia de SequênciaRESUMO
We have developed a gene delivery system that utilizes a cell-binding helper phage preselected from a landscape phage display library, and a phagemid harboring a marker gene and all regulatory elements (origins of replication and promoter-enhancer cassettes) necessary for replication of the phagemid and expression of the marker gene in the targeted cell. All the proteins required for encapsulation of the phagemid DNA and cell targeting are provided by the phage helper and are separate from the phagemid. Therefore, the resultant Phagemid Infective Particles (PIPs) are able to bind and infect target cells and express the marker gene from within the cell. Our approach, shown here for glioma cells, differs from others in that a phagemid expressing a model marker or particular therapeutic gene can be easily exchanged for a phagemid expressing a different therapeutic gene. Also, a different helper phage, selected from a phage display library, such as the f8-8-mer landscape library used here, can target any cell type and direct the encapsulation of any therapeutic gene encoding phagemid. Because of its versatility, the PIPs system may be readily used for optimization of the gene-delivery strategies applied to specific cell and tissue targets.
Assuntos
Bacteriófagos/genética , Plasmídeos/genética , Animais , Bacteriófagos/ultraestrutura , Linhagem Celular Tumoral , Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Plasmídeos/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Screening with a 7-mer phage display peptide library, a panel of cell-targeting peptides for the murine microglial cell line, EOC 20, was recognized. A number of similar, but not identical, sets of sequences representing more than 75% of all the cell line-binding clones were identified. Comparative analysis indicated that motif S/(T) F T/(X) Y W is present in the vast majority of the binding sequences. The selectivity and specificity of the dominant peptide sequence identified for microglia was confirmed using both phage displaying the peptide and the synthetic peptide alone.
Assuntos
Microglia/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Animais , Técnicas Biossensoriais , Linhagem Celular , Camundongos , Microglia/citologia , Biblioteca de Peptídeos , Ligação Proteica/genética , Ligação Proteica/imunologia , Sensibilidade e EspecificidadeRESUMO
Cell-binding ligands for RG2 rat glioma were identified in our recent study from a library of peptides that are displayed as fusion molecules on phage particles. Here, one of the phage clones was used to affinity purify those cell membrane components to which the displayed peptides bind. This phage clone, displaying the ELRGDSLP peptide, was shown to recognize glioma cells specifically in comparison to control phage-expressing peptides of either similar or irrelevant sequences. Blocking experiments with synthetic RGDS peptide demonstrated that the phage-glioma cell recognition occurs via the RGD motif known to be present in many integrin-binding proteins. To form an affinity matrix that would bind to glioma cell membrane molecules, ELRGDSLP phage particles were cross-linked using dextran polymer. Whole cell lysate from RG2 rat glioma cells was passed through the matrix, resulting in the isolation of cell membrane components having strong affinity to the peptides on phage and molecules associated with those components. One of the isolated proteins was found to be CD44s, a cell surface adhesion molecule involved in glioma cell invasion and migration, which likely formed a complex with an RGD-binding integrin. Cell membrane proteins isolated with this innovative approach could be used for the design of cell-specific anticancer treatments.