RESUMO
Synthetic vectors have been considered as a safer and more versatile alternative to viral-based gene delivery systems. A variety of very simple synthetic vector systems, e.g., cationic lipid- and polymer-complexed plasmid DNA have activity in vivo but it appears to be mediated by non-specific electrostatic interactions limiting targeting. In order to avoid these problems, we designed a sterically stabilized layered colloidal system. The steric polymer coating reduces non-specific interactions. We have synthesized a PEG conjugate of PEI that complexes DNA to form small, stable colloids with a steric polymer coat on their surface. The polymer enhances colloidal stability and reduces non-specific binding and toxicity. It also renders the complex inactive presumably due to reduced binding. Ligands are then appended to the distal end of the steric polymer to restore cell binding and expression at target cells. We prepared conjugates with RGD peptide ligands appended to the distal end of the steric polymer. The resulting conjugates also form complexes but with ligands exposed on their surface restoring binding and activity. Labeled oligonucleotides and DNA were used to measure intracellular distribution. Oligonucleotides are found localized in the nucleus, whereas the labeled plasmid DNA remained in the cytoplasm. Import of plasmid DNA into the nucleus appears to be very inefficient yet sufficient for expression.
Assuntos
Vetores Genéticos/química , Fenômenos Químicos , Físico-Química , Vetores Genéticos/farmacologia , Ligantes , Luciferases/genética , Tamanho da Partícula , Plasmídeos , Polietilenoglicóis , Polietilenoimina , PolímerosRESUMO
A chemical affinity system exhibiting antibody-like properties is described. The system exploits bioconjugates with appended phenylboronic acid (PBA) moieties and a support-bound phenylboronic acid complexing reagent derived from salicylhydroxamic acid (SHA) for protein immobilization on a chromatographic support. The structure of the PBA.SHA complex was characterized by 11B NMR and mass spectrometry and compared with complexes derived from model compounds. Protein modification reagents were synthesized from 3-aminophenylboronic acid and utilized to prepare bioconjugates from alkaline phosphatase (AP) and horseradish peroxidase (HRP). AP obtained from one source afforded PBA bioconjugates exhibiting significant loss of enzymatic activity, whereas AP obtained from a second source afforded PBA bioconjugates exhibiting only a modest loss of enzymatic activity. Conversely, HRP afforded PBA bioconjugates exhibiting no loss of enzymatic activity. SHA-modified Sepharose was prepared by reaction of methyl 4-[(6-aminohexanoylamino)methyl]salicylate with CNBr-activated Sepharose 4B, followed by treatment with aqueous alkaline hydroxylamine. PBA-AP and PBA-HRP conjugates were efficiently immobilized on SHA-Sepharose at pH 8.3. PBA-AP conjugates were retained after washing with acidic buffers at pH 6.7, 4.2, and 2.5, whereas PBA-HRP conjugates were retained after washing with buffer at pH 6.7, but were eluted to some extent at and below pH 4.2. The results are interpreted in terms of multivalent interactions involving boronic acid complex formation between the enzyme bioconjugates and immobilized complexing reagent.
Assuntos
Ácidos Borônicos/química , Cromatografia/métodos , Enzimas Imobilizadas/química , Salicilamidas/química , Sefarose/química , Fosfatase Alcalina/química , Fosfatase Alcalina/metabolismo , Cromatografia/instrumentação , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Ligação ProteicaRESUMO
Short-chain fatty acids, such as butyrate and propionate, are under investigation as therapeutic stimulants of fetal hemoglobin production in the beta-hemoglobin disorders. Significant limitations to these fatty acids and derivatives as optimal therapeutics are their rapid metabolism in vivo and their induction of cell growth arrest in the G1 phase of the cell cycle. This antiproliferative activity is related to their inhibition of metabolic transport pumps which are essential for cell proliferation. Other small carbon compounds, the phenylalkyl acids, phenoxyacetic acids, and phenylacetic acids, which are structurally resistant to oxidative metabolism, are shown here to induce fetal globin production in human erythroid cultures at concentrations of 0.2 mM, lower than those required for most other fatty acids. Certain of these compounds were found not to inhibit cellular neutral amino acid transport function in erythroid cells, nor to inhibit erythroid colony (Bfu-e) growth. Certain of these compounds even stimulated human Bfu-e proliferation in vitro beyond that induced by optimal concentrations of hematopoietic growth factors. The combination of increased fetal globin chain production by these compounds and their stimulatory effects on erythropoiesis result in an increase in Hb F-expressing erythroid cells in culture several-fold greater than that achieved by the butyrates. These new compounds thus have the potential to provide superior therapy for the beta-hemoglobinopathies and other anemias.
Assuntos
Eritroblastos/citologia , Eritropoese/efeitos dos fármacos , Ácidos Graxos Voláteis/farmacologia , Transporte Biológico/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Globinas/biossíntese , Globinas/efeitos dos fármacos , HumanosRESUMO
An enzyme-linked immunosorbent assay (ELISA) for detection of Bordetella avium infection in turkey poults was developed. One-week-old poults challenged intratracheally with 10(12) colony-forming units of B. avium had detectable titers (greater than or equal to 11), with an average of 13.6% positive samples when the birds were 6 to 11 weeks old. The method was sensitive enough to detect maternal antibodies to B. avium in poults up to 3 weeks of age. The same poults challenged at 1 week of age had 100% tracheal infection up to 3 weeks of age, which dropped to 0% by 6 weeks. The method resulted in no false-positive samples (titer = 0) from birds not infected with B. avium and tested weekly between 4 and 11 weeks of age. Antibodies in turkey flocks infected with Newcastle disease virus, hemorrhagic enteritis virus, and Mycoplasma meleagridis, and birds infected with Escherichia coli had no apparent cross-reactivity to the B. avium antigens used in the ELISA. The percentages of B. avium-positive serum samples collected from different turkey flocks did not significantly differ (P greater than 0.05) when samples were tested by the developed ELISA at different times, an indication of the reproducibility of the method.
Assuntos
Infecções por Bordetella/veterinária , Ensaio de Imunoadsorção Enzimática/normas , Doenças das Aves Domésticas/diagnóstico , Perus , Animais , Anticorpos Antibacterianos/sangue , Bordetella/imunologia , Infecções por Bordetella/diagnóstico , Reações Cruzadas , Reações Falso-Positivas , Análise dos Mínimos Quadrados , Valor Preditivo dos Testes , Análise de Regressão , Reprodutibilidade dos TestesRESUMO
The synthesis of a pentadecamer of the 5'-phosphate of the antiviral nucleoside ribavirin (1'-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) has been achieved. This homoribopolymer is terminated at the 5'-position with an (6-aminohexyl)phosphate group to permit conjugation to a carrier and at the 3'-position by a thymidine-5'-phosphate. The synthesis was accomplished using the methyl phosphoramidite approach to oligoribonucleotides. The homoribopolymer was insensitive to ribonuclease A but was sensitive to ribonuclease T2 digestion.
Assuntos
Antivirais/síntese química , Polímeros/síntese química , Ribonucleotídeos/síntese química , Antivirais/metabolismo , Biopolímeros , Fenômenos Químicos , Química , Endorribonucleases/metabolismo , Substâncias Macromoleculares , Estrutura Molecular , Polímeros/metabolismo , Ribonuclease Pancreático/metabolismo , Ribonucleotídeos/metabolismo , TimidinaRESUMO
A series of 2-substituted octahydrobenzo[f]quinolines has been synthesized and assayed for dopamine agonist activity. Only the compounds corresponding to the beta-rotameric conformation of dopamine showed biphasic activity in competition binding studies with the radioligand [3H]spiroperidol. These findings suggest that the congeners possessing the beta-rotamer conformation show receptor-binding characteristics that resemble those of the ergolines more closely than do those of the corresponding alpha-rotamer congeners.
Assuntos
Dopaminérgicos/síntese química , Quinolinas/síntese química , Animais , Bovinos , Dopaminérgicos/farmacologia , Ergolinas/farmacologia , Técnicas In Vitro , Conformação Molecular , Quinolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Endometriosis of the sciatic nerve is rare but must be included in the differential diagnosis of sciatic pain. Patients present with typical signs and symptoms of sciatica, which are cyclic in nature. Electromyography and computed tomographic scanning are useful in diagnosis. At laparoscopy or laparotomy, a characteristic "pocket sign" is frequently seen, and may be the only clue to the presence of endometriosis. The patient often requires definitive surgery with total abdominal hysterectomy and bilateral salpingo-oophorectomy. However, conservative surgery with excision of the endometriosis from the nerve can be successful in selected patients who wish to preserve reproductive function.
Assuntos
Endometriose , Neoplasias do Sistema Nervoso Periférico , Nervo Isquiático , Ciática/etiologia , Adulto , Feminino , Humanos , GravidezRESUMO
The flexible dopamine (DA) molecule exists in one or the other of its two conformational extremes (alpha- or beta-rotamer) and its receptor in the anterior pituitary gland exists in a high and a low affinity state. A series of novel, rigid DA congeners (2-substituted octahydrobenzo[f]quinolines) was synthesized and used to investigate the conformation of DA preferred by its anterior pituitary receptor and the significance of recognition of the two affinity states to the inhibition of prolactin (PRL) secretion. Analysis of competition curves of congeners for [3H]spiperone binding to bovine anterior pituitary membranes was used to calculate affinity constants. Congeners in the beta-rotamer conformation showed a biphasic competition curve as observed for DA. The curves were resolved into high (nM) and low (microM) affinity binding sites. This biphasic binding could be converted to monophasic low affinity binding in the presence of a nonhydrolyzable GTP analog. The congeners in the alpha-rotameric conformation showed monophasic low affinity binding. The potency of congeners to suppress PRL release was evaluated in cell cultures of dispersed bovine anterior pituitary. Congeners recognizing the high affinity binding site were 100-fold more potent in suppressing PRL release than those recognizing only low affinity binding sites. Dihydroxy congeners versus monohydroxy congeners and cyanomethyl group substituted versus methylthiomethyl substituted congeners occupied greater proportions of high affinity binding sites. Increasing proportions of high affinity sites occupied increased the potency of the congener to suppress PRL release. These results suggest that the beta-rotameric conformational extreme of DA is preferred by its receptor in the anterior pituitary gland and that the high affinity state of this receptor is functionally important in mediating the inhibition of PRL secretion.
Assuntos
Dopamina/fisiologia , Adeno-Hipófise/fisiologia , Receptores Dopaminérgicos/fisiologia , Ligação Competitiva , Membrana Celular/metabolismo , Dopamina/análogos & derivados , Guanosina Trifosfato/metabolismo , Conformação Molecular , Movimento (Física) , Prolactina/metabolismo , Taxa Secretória/efeitos dos fármacos , Espiperona/metabolismo , Relação Estrutura-AtividadeRESUMO
Determinations of carbon monoxide binding curves for hemoglobin from Brevoortia tyrannus under equilibrium and photostationary conditions show that in the light, the curve is shifted to the right and altered in shape. The Bohr effect is much less in the light. The kinetics of the transition between equilibrium and photostationary states has been examined. All of the results are satisfactorily described using the two-state model of Monod, J. Wyman, J., and Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118 with the assumption that light produces an additive increase in the rate of dissociation of ligand from the R and T states.