ABSTRACT
Cell-permeable small molecules are powerful tools for unraveling complex cellular pathways. We demonstrate that nuclear hormone receptors can be engineered through mutagenesis to create orthogonal ligand-receptor pairs to control transcription. Mutated residues in the retinoid X receptor (RXR) were chosen from structural analysis of RXR and the retinoic acid receptor (RAR) ligand binding domains. The potential ligands screened for activation of variant receptors are "near drugs"--compounds synthesized during structure-activity studies that are structurally similar to an approved drug yet inactive on the wild-type receptor. One variant, Q275C;I310M;F313I, is poorly activated by ligands for the wild-type receptor but is activated by a "near drug", fulfilling the criteria of an orthogonal ligand-receptor pair. These experiments demonstrate that nuclear hormone receptors are well suited to supply orthogonal ligand-receptor pairs for experimental biology, biotechnology, and gene therapy. Our findings also demonstrate the general principle that inactive compounds synthesized during drug discovery can be combined with mutant proteins to rapidly create new tools for controlling cellular processes.
Subject(s)
Mutagenesis, Site-Directed , Receptors, Retinoic Acid/genetics , Transcription Factors/genetics , Tretinoin/metabolism , Alitretinoin , Amino Acid Substitution , Animals , Cell Line , Ligands , Plasmids/genetics , Receptors, Retinoic Acid/agonists , Receptors, Retinoic Acid/metabolism , Retinoid X Receptors , Structure-Activity Relationship , Substrate Specificity , Transcription Factors/agonists , Transcription Factors/metabolism , Transcriptional Activation , Transfection , Tretinoin/chemistry , Tretinoin/pharmacologyABSTRACT
Autovaccines are therapeutic vaccines manufactured from a disease causing micro-organism for individual treatment of patients, animals, or sometimes herds to treat chronic or recurrent infections. Despite the common use of autovaccines in veterinary medicine, their mechanism of action, i.e. the immunologic effector mechanism activated after administration, has never been investigated. Here we present data concerning the use of autovaccines to treat metritis infection in a group of dairy cows. Following autovaccination we observed a significant decrease in CD4+ cells paralleled by an increase in T-cells expressing the gammadelta-T-cell receptor (gammadelta-TCR) in the peripheral blood of the treated animals. Lymphocyte proliferation assays showed an initial increase in antigen-specific responsiveness followed by a decrease in this responsiveness during autovaccination treatment. We therefore conclude that administration of an autovaccine leads to the activation of immunologic effector mechanisms which contribute to recovery of the diseased animals.
Subject(s)
Actinomycosis/veterinary , Bacterial Vaccines/therapeutic use , Cattle Diseases/therapy , Puerperal Infection/veterinary , Uterine Diseases/veterinary , Actinomyces/immunology , Actinomycosis/immunology , Actinomycosis/therapy , Animals , B-Lymphocytes/immunology , Bacterial Vaccines/isolation & purification , Cattle , Cattle Diseases/immunology , Chronic Disease , Female , Flow Cytometry , In Vitro Techniques , Lymphocyte Activation , Puerperal Infection/immunology , Puerperal Infection/therapy , T-Lymphocytes, Helper-Inducer/immunology , Uterine Diseases/immunology , Uterine Diseases/therapyABSTRACT
In an attempt to determine cranial ultrasonographic features of preterm infants with intraventricular hemorrhage (IVH) and/or periventricular white matter abnormalities (PVWMA) that could serve as more specific predictors of cerebral palsy (CP), we reviewed the cranial sonograms of 34 infants with IVH and/or PVWMA. Fourteen of the 34 infants studied (41%) developed CP. One of five infants with grade III IVH alone developed CP. Eleven infants with PVWMA did not develop cysts and only two (18%) developed CP (p = 0.04). Of the 18 infants who went on to develop cysts, four had a small, discrete solitary cyst and 14 had large cystic areas. Three of the four with small cysts were neurologically normal, whereas only three of the 14 with large cysts were neurologically normal (p = 0.04). Preterm infants with grade III IVH in the absence of any parenchymal lesion had a more favorable neurologic outcome than those with IVH and concomitant PVWMA. Infants with PVWMA in the presence or absence of IVH had much poorer neurologic prognoses. In infants with PVWM abnormalities, both the presence and extent of cystic lesions, though not their location, are the strongest predictors of long-term neurologic outcome.
Subject(s)
Cerebral Palsy/diagnostic imaging , Cerebral Ventricles/blood supply , Infant, Premature, Diseases/diagnostic imaging , Cerebral Ventricles/diagnostic imaging , Cysts/diagnostic imaging , Diagnosis, Differential , Humans , Infant, Newborn , Nerve Fibers, Myelinated/diagnostic imaging , Skull/diagnostic imaging , UltrasonographyABSTRACT
Clostridium botulinum toxin, type C, could be demonstrated by means of temperature induced microcomplement fixation in blood serum and in aspirated rumen fluid of cattle suffering from botulism. The results were already available after seven hours. Botulinum toxin likewise could be identified from hair of a suspicious carcass. The investigations confirm the high sensitivity of this method.
Subject(s)
Botulinum Toxins/analysis , Botulism/veterinary , Cattle Diseases/diagnosis , Complement Fixation Tests , Animals , Botulism/diagnosis , Cattle , Female , Hair/chemistry , Hot Temperature , Male , Rumen/chemistryABSTRACT
Na-hylan, a chemically modified sodium hyaluronate jelly, was studied mechanically and histologically as a surgical device to diminish tendon adhesion in the rabbit long toe extensor three weeks after surgical abrasion. This device was found to be highly effective (55% of treated tendons formed no adhesions compared to 5% of controls, and only 18% formed severe adhesions compared to 62% of controls). No gross or histologic evidence of significant acute or chronic inflammatory reaction to the Na-hylan was found.
