RESUMO
INTRODUCTION: Sexual dysfunction and infertility are prevalent in the spina bifida (SB) population; however, the mechanism of how they affect a person with spina bifida is poorly understood. Additionally, the management of children with spina bifida becomes more difficult as they exit from pediatric institutes. OBJECTIVE: The present study sought to evaluate sexual health (using validated questionnaires) and fertility in adults with spina bifida and to correlate spinal cord level and ambulatory status with degree of sexual function. STUDY DESIGN: After institutional board review approval, 199 adult patients with SB, aged 18 and older and who were followed in one pediatric institution, were identified. Patients who were non-English speaking, cognitively and/or developmentally delayed, or unable to be contacted were excluded. Surveys regarding demographics, sexual health and infertility were mailed to the patients and administered in the clinic with the option to opt-out of the survey. Survey questions regarding sexual health were constructed using validated questionnaires: Female Sexual Function Index (FSFI) for females, and International Index of Erectile Function (IIEF) and Sexual Health Inventory for Men (SHIM) for males. Sexual dysfunction scores were correlated to the patients' spinal level and ambulatory status. RESULTS: Of the 121 eligible patients, 45 replied, with a response rate of 39%. For females, using a cut-off value of 26.5 for FSFI scoring, 25 out of 28 (89%) had sexual dysfunction. No association was seen between spinal level or ambulatory status and overall FSFI, satisfaction, or desire scores. For males, 10 out of 17 (59%) had severe erectile dysfunction (ED), and one out of 17 (6%) had no ED. No association was seen between ambulatory status and sexual function scores for the males. However, SHIM, satisfaction, and ED scores were higher in males with lower spinal lesions. People with spina bifida of both genders tended to have more severe dysfunction compared to those with sexual dysfunction of other etiologies, except with similar sexual desire scores. Regarding questions on fertility, no participant attempted to have children; thus, there was no infertility reported. DISCUSSION: Few studies have been conducted on sexual health and fertility in adults with SB. Three studies have utilized validated questionnaires and found varying degrees of sexual dysfunction in this subset of patients; however, only one study found sexual activity to be more likely in patients with more caudal levels of neurologic impairment. The present study also showed that SHIM, satisfaction, and ED scores were higher in males with lower spinal lesions. Limitations to this study primarily included the small sample size and low survey response rate. CONCLUSION: Limited information is known about adults with SB, and sexual function and fertility. While expressing sexual desire, adults with SB appear to experience high rates of sexual dysfunction. Fertility rates were inadequately assessed; this was possibly due to the high rate of sexual dysfunction. Sexual health in the SB population is an important component of the myriad of urologic care issues for these people. Due to the disparity in their care after reaching adulthood, it is prudent to follow these patients and understand their pathophysiology as they continue to mature through life.
Assuntos
Comportamento Sexual , Disfunções Sexuais Fisiológicas/epidemiologia , Disrafismo Espinal/complicações , Disrafismo Espinal/patologia , Adolescente , Adulto , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Infertilidade/epidemiologia , Masculino , Fatores Sexuais , Disrafismo Espinal/psicologia , Inquéritos e Questionários , Adulto JovemRESUMO
AIMS: To describe previously unreported retinal findings in patients with Alport Syndrome (AS), as well as review the range of ophthalmic manifestations. METHODS: Retrospective review of clinical records of patients with AS. RESULTS: Nine patients with AS were identified, of whom three had no eye findings, four showed classic features of AS, and two had new findings, bull's eye and vitelliform maculopathy. The genetic mutation responsible for the disease in the patient with vitelliform subretinal deposits was identified. CONCLUSIONS: Patients with AS can present with a variety of ophthalmic manifestations. Bull's eye maculopathy and vitelliform deposits can be features of AS. The mechanism of these new macular findings remains unknown. Possible pathophysiological overlap with other maculopathies including age-related macular degeneration is discussed.
Assuntos
Oftalmopatias Hereditárias/diagnóstico , Nefrite Hereditária/diagnóstico , Doenças Retinianas/diagnóstico , Adulto , Feminino , Fluorescência , Humanos , Hiperpigmentação/diagnóstico , Hiperpigmentação/genética , Masculino , Doenças Retinianas/genética , Estudos Retrospectivos , Tomografia de Coerência Óptica , Adulto JovemAssuntos
Imperícia/legislação & jurisprudência , Erros de Medicação/legislação & jurisprudência , Erros de Medicação/enfermagem , Cuidados de Enfermagem/normas , Humanos , Imperícia/estatística & dados numéricos , Erros de Medicação/efeitos adversos , Erros de Medicação/estatística & dados numéricos , National Academies of Science, Engineering, and Medicine, U.S., Health and Medicine Division , Gestão de Riscos/métodos , Estados Unidos/epidemiologiaAssuntos
Serviço Hospitalar de Emergência/economia , Serviço Hospitalar de Emergência/legislação & jurisprudência , Trabalho de Parto , Programas de Assistência Gerenciada/economia , Programas de Assistência Gerenciada/legislação & jurisprudência , Feminino , Humanos , Recursos Humanos de Enfermagem Hospitalar/organização & administração , Transferência de Pacientes/legislação & jurisprudência , Gravidez , Estados UnidosRESUMO
The aim of this study was to determine whether the antibodies raised in burn patients by active immunization with a Pseudomonas aeruginosa OMPs vaccine have a protective efficacy against infection with P. aeruginosa. The binding patterns with P. aeruginosa OMPs of immunized burn patient sera were similar to the sera of immunized healthy humans as determined by immunoblot and immunoprecipitation analyses. The sera pooled from immunized burn patients after three immunizations showed a significantly higher opsonophagocytic-killing activity than the corresponding pre-immune sera, while the sera from unimmunized patients collected at the same day did not. Passive immunization of mice with post-immune sera of burn patients significantly enhanced the survival rate upon a lethal challenge with P. aeruginosa compared to the pre-immune sera, indicating the protective ability of the antibodies induced in burn patients by immunization. These results suggest that anti-P. aeruginosa OMPs antibodies elicited in burn patients by active immunization are protective against infection with P. aeruginosa, and provide a rational for further development of the vaccine for prevention against P. aeruginosa infection in burn patients.
Assuntos
Anticorpos Antibacterianos/biossíntese , Proteínas da Membrana Bacteriana Externa/uso terapêutico , Vacinas Bacterianas/uso terapêutico , Queimaduras/imunologia , Queimaduras/microbiologia , Infecções por Pseudomonas/prevenção & controle , Pseudomonas aeruginosa/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/uso terapêutico , Reações Antígeno-Anticorpo , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Vacinas Bacterianas/imunologia , Humanos , Imunização Passiva/métodos , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas Opsonizantes/imunologia , Fagocitose/imunologia , Coelhos , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/uso terapêuticoRESUMO
In order to develop an effective means to treat Pseudomonas aeruginosa infections, we designed a large-scale process for purification of human IgG specific to P. aeruginosa outer membrane proteins (Oprs) from normal human sera. The process we developed includes affinity column chromatography using P. aeruginosa Oprs as ligands, protein A column chromatography and ultrafiltration, which enriched P. aeruginosa Oprs-specific IgG antibody by 500-fold. The purified anti-Oprs IgG was specific to the Oprs as confirmed by an ELISA competition assay and retained opsonophagocytic-killing capacity. In vivo protective efficacy of anti-Oprs IgG was evaluated by passive protection assays in mice where the 50% protective dose of anti-Oprs IgG against P. aeruginosa infections was 41 microg/kg, which was 20 times lower than that of normal serum IgG. When administered to mice 3 h after bacterial challenge, only anti-Oprs IgG afforded protection. These data demonstrate the feasibility of use of the purification process in producing functionally active target-specific human antibodies for clinical use and provide a rationale for use of anti-Oprs IgG as a valuable adjunct to treat P. aeruginosa infections.
Assuntos
Especificidade de Anticorpos , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/uso terapêutico , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/uso terapêutico , Infecções por Pseudomonas/prevenção & controle , Pseudomonas aeruginosa/imunologia , Animais , Afinidade de Anticorpos , Vacinas Bacterianas/isolamento & purificação , Humanos , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Endogâmicos ICR , Infecções por Pseudomonas/sangue , Infecções por Pseudomonas/imunologia , CoelhosRESUMO
In order to develop an effective means to treat and prevent Pseudomonas aeruginosa infections, we have purified P. aeruginosa outer membrane protein (Oprs)-specific human IgG antibody using a large-scale affinity column. In this study, we investigated the cross-protective activity of the purified anti-Oprs IgG against various immunotype strains of P. aeruginosa. The anti-Oprs IgG reacted with Oprs isolated from seven Fisher-Devlin immunotype strains of P. aeruginosa and was able to promote opsonophagocytic killing of all seven immunotype strains by human phagocytic cells. Administration of 500 microg anti-Oprs IgG to mice raised the LD50 of the P. aeruginosa strains by 8-250-fold, indicating the protective capacity against heterologous P. aeruginosa strains as well as homologous strains. In contrast, despite high titers against P. (aeruginosa Oprs, total serum IgG isolated from burn patient sera was no better than normal serum IgG in protecting mice from infection with P. aeruginosa. These data demonstrate that the affinity-purified human anti-Oprs IgG could afford protection against heterologous immunotype P. aeruginosa strains and provide a rationale to use anti-Oprs IgG as an adjunct for treatment of P. aeruginosa infections in humans.
Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Imunoglobulina G/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Reações Cruzadas , Modelos Animais de Doenças , Humanos , Imunização Passiva , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fagocitose/imunologia , Especificidade da EspécieRESUMO
In order to evaluate in humans the safety and immunogenicity of a Pseudomonas aeruginosa vaccine composed of outer membrane proteins (OMPs), CFC-101, we carried out a phase I/IIa clinical trial in healthy male volunteers. Groups of six volunteers were immunized either subcutaneously (s.c.) or intramuscularly (i.m.) with three dosages of the vaccine three times at 7-day intervals. The vaccine was well tolerated by volunteers. Local reactions in the injection sites were generally mild and transient. Significant increases in OMP-specific antibody were observed in both route groups after vaccinations but was higher in the i.m.-immunized group, where vaccination with 0.5 or 1.0 mg doses yielded 100% seroconversion. The specificity of the induced antibodies to P. aeruginosa OMP was demonstrated by western blot analysis and immunoprecipitation assay. An increase in Clq-binding capacity and ability to confer mice protection from lethal challenges with P. aeruginosa indicated the protective efficacy of the elicited antibodies. Based on these data, we concluded that the P. aeruginosa OMP vaccine is safe and effective in humans with an optimal dose of 0.5 and 1.0 mg and that i.m. is the better route than s.c. for this vaccine.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/farmacologia , Pseudomonas aeruginosa/imunologia , Adulto , Animais , Anticorpos Antibacterianos/sangue , Especificidade de Anticorpos , Vacinas Bacterianas/efeitos adversos , Vacinas Bacterianas/imunologia , Ativação do Complemento , Humanos , Imunização Passiva , Técnicas In Vitro , Leucócitos Mononucleares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Pessoa de Meia-Idade , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/prevenção & controle , Segurança , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Haemophilus influenzae is an important human pathogen. The lipooligosaccharide (LOS) of H. influenzae has been implicated as a virulence determinant. To better understand the assembly of LOS in nontypeable H. influenzae (NtHi), we have cloned and characterized the rfaD and rfaF genes of NtHi 2019, which encode the ADP-L-glycero-D-manno-heptose-6-epimerase and heptosyltransferase II enzymes, respectively. This cloning was accomplished by the complementation of Salmonella typhimurium lipopolysaccharide (LPS) biosynthesis gene mutants. These deep rough mutants are novobiocin susceptible until complemented with the appropriate gene. In this manner, we are able to use novobiocin resistance to select for specific NtHi LOS inner core biosynthesis genes. Such a screening system yielded a plasmid with a 4.8-kb insert. This plasmid was able to complement both rfaD and rfaF mutants of S. typhimurium. The LPS of these complemented strains appeared identical to the wild-type Salmonella LPS. The genes encoding the rfaD and rfaF genes from NtHi 2019 were sequenced and found to be similar to the analogous genes from S. typhimurium and Escherichia coli. The rfaD gene encodes a polypeptide of 35 kDa and the rfaF encodes a protein of 39 kDa, as demonstrated by in vitro transcription-translation studies. Isogenic mutants which demonstrated truncated LOS consistent with inner core biosynthesis mutants were constructed in the NtHi strain 2019. Primer extension analysis demonstrated the presence of a strong promoter upstream of rfaD but suggested only a very weak promoter upstream of rfaF. Complementation studies, however, suggest that the rfaF gene does have an independent promoter. Mass spectrometric analysis shows that the LOS molecules expressed by H. influenzae rfaD and rfaF mutant strains have identical molecular masses. Additional studies verified that in the rfaD mutant strain, D-glycero-D-manno-heptose is added to the LOS molecule in place of the usual L-glycero-D-manno-heptose. Finally, the genetic organizations of the inner core biosynthesis genes of S. typhimurium, E. coli, and several strains of H. influenzae were examined, and substantial differences were uncovered.
Assuntos
Carboidratos Epimerases/genética , Genes Bacterianos , Glicosiltransferases/genética , Haemophilus influenzae/genética , Sequência de Bases , Clonagem Molecular , Humanos , Dados de Sequência MolecularRESUMO
The HtrB protein was first identified in Escherichia coli as a protein required for cell viability at high temperature, but its expression was not regulated by temperature. We isolated an htrB homologue from non-typable Haemophilus influenzae strain (NTHi) 2019, which was able to functionally complement the E. coli htrB mutation. The promoter for the NTHi 2019 htrB gene overlaps the promoter for the rfaE gene, and the two genes are divergently transcribed. The deduced amino acid sequence of NTHi 2019 HtrB had 56% homology to E. coli HtrB. In vitro transcription-translation analysis confirmed production of a protein with an apparent molecular mass of 32-33 kDa. Primer extension analysis revealed that htrB was transcribed from a sigma 70-dependent consensus promoter and its expression was not affected by temperature. The expression of htrB and rfaE was 2.5-4 times higher in the NTHi htrB mutant B29 than in the parental strain. In order to study the function of the HtrB protein in Haemophilus, we generated two isogenic htrB mutants by shuttle mutagenesis using a mini-Tn3. The htrB mutants initially showed temperature sensitivity, but they lost the sensitivity after a few passages at 30 degrees C and were able to grow at 37 degrees C. They also showed hypersensitivity to deoxycholate and kanamycin, which persisted on passage. SDS-polyacrylamide gel electrophoresis analysis revealed that the lipo-oligosaccharide (LOS) isolated from these mutants migrated faster than the wild type LOS and its color changed from black to brown as has been described for E. coli htrB mutants. Immunoblotting analysis also showed that the LOS from the htrB mutants lost reactivity to a monoclonal antibody, 6E4, which binds to the wild type NTHi 2019 LOS. Electrospray ionization-mass spectrometry analysis of the O-deacylated LOS oligosaccharide indicated a modification of the core structure characterized in part by a net loss in phosphoethanolamine. Mass spectrometric analysis of the lipid A of the htrB mutant indicated a loss of one or both myristic acid substitutions. These data suggest that HtrB is a multifunctional protein and may play a controlling role in regulating cell responses to various environmental changes.
Assuntos
Haemophilus influenzae/genética , Haemophilus influenzae/metabolismo , Lipídeo A/metabolismo , Lipopolissacarídeos/metabolismo , Mutação , Proteínas de Bactérias/genética , Sequência de Bases , Sequência de Carboidratos , Clonagem Molecular , DNA Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos , Haemophilus influenzae/classificação , Lipídeo A/química , Lipopolissacarídeos/química , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese , Fosforilação , RNA Mensageiro/genética , Mapeamento por RestriçãoRESUMO
The lipooligosaccharide (LOS) of nontypeable Haemophilus influenzae (NTHi) is an important factor in pathogenesis and virulence. In an attempt to elucidate the genes involved in LOS biosynthesis, we have cloned the rfaE gene from NTHi 2019 by complementing a Salmonella typhimurium rfaE mutant strain with an NTHi 2019 plasmid library. The rfaE mutant synthesizes lipopolysaccharide (LPS) lacking heptose, and the rfaE gene is postulated to be involved in ADP-heptose synthesis. Retransformation with the plasmid containing 4 kb of NTHi DNA isolated from a reconstituted mutant into rfaE mutants gave wild-type LPS phenotypes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis confirmed the conversion of the rfaE mutant LPS to a wild-type LPS phenotype. Sequence analysis of a 2.4-kb BglII fragment revealed two open reading frames. One open reading frame encodes the RfaE protein with a molecular weight of 37.6 kDa, which was confirmed by in vitro transcription and translation, and the other encodes a polypeptide highly homologous to the Escherichia coli HtrB protein. These two genes are transcribed from the same promoter region into opposite directions. Primer extension analysis of the rfaE gene revealed a single transcription start site at 37 bp upstream of the predicted translation start site. The upstream promoter region contained a sequence (TA AAAT) homologous to the -10 region of the bacterial sigma 70-dependent promoters at an appropriate distance (7 bp), but not sequence resembling the consensus sequence of the -35 region was found. These studies demonstrate the ability to use complementation of defined LPS defects in members of the family Enterobacteriaceae to identify LOS synthesis genes in NTHi.
Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos/genética , Glicosiltransferases , Haemophilus influenzae/genética , Lipopolissacarídeos/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Sequência de Carboidratos , Clonagem Molecular , Teste de Complementação Genética , Biblioteca Genômica , Haemophilus influenzae/classificação , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Mapeamento por Restrição , Salmonella typhimurium , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The identical partial deep-core structure of Hep alpha 1-3Hep alpha 1-5KDO in Salmonella typhimurium LT2 LPS and Neisseria gonorrhoeae LOS enabled us to isolate a DNA fragment from N. gonorrhoeae that was able to complement the alpha 1,5 LOS heptosyltransferase defect in the S. typhimurium rfaC630 (SA1377) mutant. SDS-PAGE analysis confirmed the production of wild-type LPS in the transformant. Subcloning revealed that complementation was due to a 1.2 kb fragment. Sequence analysis revealed a complete open reading frame capable of encoding a 36-37 kDa peptide. In vitro transcription-translation analysis of the 1.2 kb clone confirmed that a 37 kDa protein was encoded by this DNA fragment. The DNA sequence-deduced protein had 36% identity and 58% similarity to S. typhimurium heptosyltransferase I (RfaC). Primer extension analysis indicated that transcription of the cloned gene in N. gonorrhoeae strain 1291 begins 144 bp upstream of the start codon at a G nucleotide. An isogenic mutant of N. gonorrhoeae strain 1291 with an m-Tn3 insertion inside the coding sequence expressed a single truncated LOS with a similar molecular mass to S. typhimurium rfaC LPS. We conclude that the 1.2 kb fragment encodes the alpha 1,5 LOS heptosyltransferase I (RfaC) in N. gonorrhoeae. Our studies also provide further evidence that the third KDO residue in S. typhimurium LPS is added after the core synthesis is completed.
Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos , Glicosiltransferases , Lipopolissacarídeos/biossíntese , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , Sequência de Carboidratos , Clonagem Molecular , Primers do DNA/genética , Lipopolissacarídeos/química , Dados de Sequência Molecular , Mutagênese Insercional , Neisseria gonorrhoeae/enzimologia , Biossíntese de Proteínas , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Transcrição GênicaRESUMO
The peribacteroid membrane (PBM) in legume root nodules is derived from plasma membrane following endocytosis of Rhizobium by fusion of newly synthesized vesicles. We studied the roles of plant Rab1p and Rab7p homologs, the small GTP-binding proteins involved in vesicular transport, in the biogenesis of the PBM. Three cDNAs encoding legume homologs of mammalian Rab1p and Rab7p were isolated from soybean (sRab1p, sRab7p) and Vigna aconitifolia (vRab7p). sRab1p was confirmed to be a functional counterpart of yeast Ypt1p (Rab1p) by complementation of a yeast ypt1-1 mutant. Both srab1 and vrab7 genes are induced during nodulation with the level of vrab7 mRNA being 12 times higher than that in root meristem and leaves. This induction directly correlates with membrane proliferation in nodules. Antisense constructs of srab1 and vrab7, under a nodule-specific promoter (leghemoglobin, Lbc3), were made in a binary vector and transgenic nodules were developed on soybean hairy roots obtained through Agrobacterium rhizogenes-mediated transformation. Both antisense srab1 and vrab7 nodules were smaller in size and showed lower nitrogenase activity than controls. The antisense srab1 nodules showed lack of expansion of infected cells, fewer bacteroids per cell and their frequent release into vacuoles. In contrast, antisense vrab7 expressing nodules showed accumulation of late endosomal structure and multivesicular bodies in the perinuclear region. These data suggest that both Rab1p and Rab7p are essential for the development of the PBM compartment in effective symbiosis.
Assuntos
Fabaceae/microbiologia , Proteínas de Ligação ao GTP/genética , Proteínas de Plantas/genética , Plantas Medicinais , Rhizobiaceae/fisiologia , Proteínas de Saccharomyces cerevisiae , Simbiose/fisiologia , Proteínas rab de Ligação ao GTP , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar , Fabaceae/genética , Teste de Complementação Genética , Membranas Intracelulares/metabolismo , Dados de Sequência Molecular , Morfogênese , Nitrogenase/análise , Plantas Geneticamente Modificadas , RNA Antissenso , Rhizobiaceae/ultraestrutura , Homologia de Sequência de Aminoácidos , Glycine max/genética , Glycine max/microbiologia , Glycine max/ultraestruturaRESUMO
A nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3.) was isolated from a Vigna aconitifolia (mothbean) root nodule cDNA library. Sequence analysis of Vigna uricase (VN-35) cDNA revealed 90% homology to that of soybean. The VN-35 cDNA was inserted in the antisense orientation downstream of the caMV-35S promoter, and transgenic hairy roots were formed on Vigna plants using Agrobacterium rhizogenes. Infection with Bradyrhizobium (cowpea) gave rise to root nodules on transgenic hairy roots supported by the wild-type shoot. Expression of antisense VN-35 RNA was detected in transgenic nodules on individual roots using polymerase chain reaction (PCR). The nodules expressing antisense VN-35 RNA were smaller in size and showed lower uricase activity than nodules formed on the hairy roots transformed with a binary vector containing beta-glucuronidase (GUS) gene (used as control), and the plants exhibited nitrogen deficiency symptoms. Ultrastructural analysis and immunogold labeling with antibody against soybean N-35 revealed that the growth of peroxisomes was retarded in transgenic nodules expressing antisense VN-35 RNA. These data suggest that a reduction in ureide biosynthesis limits the availability of symbiotically reduced nitrogen to the plant. The nodules of tropical legumes appear to be specialized in nitrogen assimilation and are developmentally controlled to produce and transport ureides.
Assuntos
Fabaceae/genética , Proteínas de Membrana , Microcorpos/fisiologia , Proteínas de Plantas/genética , Plantas Medicinais , RNA Antissenso/biossíntese , Urato Oxidase/genética , Sequência de Bases , DNA Complementar/genética , Fabaceae/enzimologia , Dados de Sequência Molecular , Morfogênese , Nitrogênio/metabolismo , Plantas Geneticamente Modificadas , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Using test plasmids containing the SV40 origin, we found a wide spectrum of permissiveness to their replication in different human cell lines. N-myc overexpressing neuroblastoma cells were highly permissive. LA-N-1 neuroblastoma cells were the most permissive of all the cell lines that we tested including the homologous CV-1 or COS-1 monkey kidney cells. Other human cell lines expressing various amounts of c-myc, and the 293 cell line expressing adenovirus E1A and E1B exhibited intermediate levels of permissiveness. T24 and EJ bladder carcinoma cells, which do not express the myc genes, were nonpermissive. Transient expression of c-myc or N-myc from plasmid vectors resulted in a modest stimulation of replication. Replication of test plasmids containing different configurations of the SV40 origin region was activated by the myc proteins. The high efficiency of replication in LA-N-1 cells is due to a combination of reasons including the overproduction of N-myc, high efficiency of expression of the SV40 replication initiator protein large T antigen from a cotransfected expression plasmid (containing the T antigen gene under the RSV LTR control), and other unknown host cell replication stimulatory factors. Replication of test plasmids was not detected in N-myc or c-myc overexpressing cells when the T antigen expression plasmid was not provided, showing that the myc proteins cannot substitute for T antigen in SV40 DNA replication.