We have previously demonstrated that systemic administration of testosterone increases the rate of axonal regeneration following facial nerve crush in adult male hamsters. In the present study, the molecular mechanisms by which androgens could enhance axonal regeneration were examined at a cellular level. Specifically, the following question was addressed using quantitative in situ hybridization with cDNA probes complementary to betaII, and alpha1 tubulin mRNAs: Does exogenous testosterone augment axotomy-induced changes in tubulin mRNA expression in hamster facial motoneurons (FMN)? Castrated adult male hamsters were subjected to right facial nerve severance, with the left side serving as internal control. One-half of the animals received testosterone replacement in the form of subcutaneously implanted silastic capsules containing crystalline testosterone propionate, and the other half were implanted with blank capsules immediately following the axotomy. Postoperative survival times from 2-14 days were examined. Axotomy alone resulted in a significant increase in the levels of both betaII and alpha1 tubulin mRNAs in facial motor neurons between 2-14 days after injury. Administration of testosterone selectively augmented the axotomy-induced increases in betaII-tubulin, but not alpha1 tubulin, mRNA, levels at 7 and 14 days post axotomy. These results demonstrating an effect of testosterone in altering the neuronal cytoskeletal response to axotomy suggest that testosterone may enhance the regenerative properties of motor neurons via molecular mechanisms that involve selective alterations of the neuronal cytoskeleton.

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A key step in the ability of neurons to survive injury and successfully regenerate involves ribosomal RNA production. Testosterone propionate (TP), augments facial nerve regeneration in the adult hamster. TP modulates the nucleolar reaction in injured facial motoneurons, such that mature ribosome levels increase more rapidly and in greater magnitude than with injury only. In this study, molecular and electron microscopic stereologic approaches were used to determine the effects of axotomy and steroid treatment on ribosomal transcription and processing in facial motoneurons. Castrated adult male hamsters were subjected to right facial nerve transection at the stylomastoid foramen. Half the animals were subcutaneously implanted with one Silastic TP capsule, with the remainder sham implanted. For the in situ hybridization experiments, postoperative survival times were 0.5, 2, or 6 hours. In situ hybridization with a ribosomal DNA probe specific to the external transcribed spacer region located at the 5' end of the ribosomal gene was accomplished. Transcriptional activation of the rRNA gene occurred rapidly, within 2 hours, after injury only. Unexpectedly, TP treatment did not alter the time course or magnitude of rRNA transcriptional activity. For the electron microscope experiments, the postoperative time of 12 hours was selected. Stereologic analysis of 3 nucleolar subcomponents, fibrillar centers (site of rRNA transcription), nucleolonema (site of rRNA processing), and granular material (site of preribosome storage), was accomplished. TP decreased the nucleolonemal strands and the granular material, relative to injury only. These results suggest that, although rRNA transcription is rapidly activated by axotomy, rRNA processing is temporarily stalled. TP does not affect the early, axotomy-induced transcriptional activation of the ribosomal gene, but may, instead, prevent the subsequent disruption in rRNA processing. An hypothesis for the molecular mechanism by which steroids augment the regenerative capabilities of injured facial motoneurons is presented.

Background: Organic solvents used for extraction of tacrolimus from whole blood samples lower the apparent affinity of the antibody used in a diagnostic immunoassay, thereby affecting the detection limit. Methods: We used in vitro recombinant antibody engineering to screen and isolate clones from diverse libraries with mutagenic complementarity regions (CDRs) from tacrolimus 1-60-46 hybridoma cell line, with improved binding to tacrolimus in the presence of 10% methanol organic solvent solution. Results: We isolated a number of clones with mutations in variable heavy (VH) CDR 2, variable light (VL) CDR 1, and VL CDR 3 with improved binding. Various combinatorial pairings constructed from these individual mutations contained >10-fold improvements in both the dissociation rate and overall equilibrium affinity constants. Selected clones produced as IgG have increased functional sensitivity, with a 3- to 6-fold reduction in the limit of detection relative to the parental tacrolimus 1-60-46 monoclonal antibody in the Architect® Tacrolimus immunodiagnostic assay. Conclusions: The recent advent of recombinant in vitro antibody display technologies in general, and yeast surface display in particular, allows the flexibility to engineer new or augment specific analytical characteristics, such as affinity, specificity, or stability, into previously isolated and otherwise desirable antibodies to enhance assay performance. These in vitro selections can also be performed under conditions meant to mimic the assay in which the reagent will ultimately be used, to increase the likelihood of successful assay development.