Drug-inducible and simultaneous regulation of endogenous genes by single-chain nuclear receptor-based zinc-finger transcription factor gene switches.

Chemically inducible gene switches that regulate expression of endogenous genes have multiple applications for basic gene expression research and gene therapy. Single-chain zinc-finger transcription factors that utilize either estrogen receptor homodimers or retinoid X receptor-alpha/ecdysone receptor heterodimers are shown here to be effective regulators of ICAM-1 and ErbB-2 transcription. Using activator (VP64) and repressor (Krüppel-associated box) domains to impart regulatory directionality, ICAM-1 was activated by 4.8-fold and repressed by 81% with the estrogen receptor-inducible transcription factors. ErbB-2 was activated by up to threefold and repressed by 84% with the retinoid X receptor-alpha/ecdysone receptor-inducible transcription factors. The dynamic range of these proteins was similar to the constitutive system and showed negligible basal regulation when ligand was not present. We have also demonstrated that the regulation imposed by these inducible transcription factors is dose dependent, sustainable for at least 11 days and reversible upon cessation of drug treatment. Importantly, these proteins can be used in conjunction with each other with no detectable overlap of activity enabling concurrent and temporal regulation of multiple genes within the same cell. Thus, these chemically inducible transcription factors are valuable tools for spatiotemporal control of gene expression that should prove valuable for research and gene therapy applications.

Developmentally regulated telomerase activity is correlated with chromosomal healing during chromatin diminution in Ascaris suum.

Telomerase is the ribonucleoprotein complex responsible for the maintenance of the physical ends, or telomeres, of most eukaryotic chromosomes. In this study, telomerase activity has been identified in cell extracts from the nematode Ascaris suum. This parasitic nematode is particularly suited as a model system for the study of telomerase, because it shows the phenomenon of chromatin diminution, consisting of developmentally programmed chromosomal breakage, DNA elimination, and new telomere formation. In vitro, the A. suum telomerase is capable of efficiently recognizing and elongating nontelomeric primers with nematode-specific telomere repeats by using limited homology at the 3' end of the DNA to anneal with the putative telomerase RNA template. The activity of this enzyme is developmentally regulated, and it correlates temporally with the phenomenon of chromatin diminution. It is up-regulated during the first two rounds of embryonic cell divisions, to reach a peak in 4-cell-stage embryos, when three presomatic blastomeres prepare for chromatin diminution. The activity remains high until the beginning of gastrulation, when the last of the presomatic cells undergoes chromatin diminution, and then constantly decreases during further development. In summary, our data strongly argue for a role of this enzyme in chromosome healing during the process of chromatin diminution.

Caenorhabditis elegans unc-51 gene required for axonal elongation encodes a novel serine/threonine kinase.

Mutations in the unc-51 gene of the nematode Caenorhabditis elegans result in various abnormalities in axonal elongation and axonal structures. We cloned the unc-51 gene by tagging with the transposon Tc1. The wild-type unc-51 gene, which rescued the mutant phenotypes, encodes a novel serine/threonine kinase of 856 amino acids. Mutation sites were identified in the unc-51 gene of six mutants. A Lys-->Met mutation created in vitro in the kinase domain led to the loss of rescuing activity and was dominant negative, indicating that the kinase domain of Unc-51 is essential for the function. Expression of an unc-51/lacZ fusion gene was observed in many neurons at all stages. We propose that protein phosphorylation by the unc-51 product is important for axonal elongation and possibly for axonal guidance.

Effects of pindolol therapy on the size of experimentally induced myocardial infarction in the pig.

The effect of pindolol on experimental myocardial infarction was studied in a pig model. Intravenous application of 0.05 mg pindolol per kg body weight was initiated one hour after coronary ligation and repeated at 12-hour intervals for five days. No significant difference in infarct size could be found between pindolol-treated animals (20.4 +/- 0.6% SEM of whole ventricular mass, n = 6) and untreated controls (20.5 +/- 1.2% SEM, n = 9). Hemodynamic data did not change significantly throughout the experiment. These results differ in part from those reported by other investigators: The disagreement may be due to the specific pharmacological properties of the applied drug, to variations in the dosages of beta blockers, as well as to differences in the study design.

Nervous control of migratory myoelectric complex of the small bowel.

The effects on the migratory myoelectric complex (MMC) of transplantation of small bowel loops were investigated in miniature swine with chronically implanted bipolar electrodes. Four experimental models (A--D) were studied. In groups A and B, jejunal loops were either interchanged in sequence or transplanted to the ileum with preservation of intact neurovascular pedicles. In groups C and D, bowel segments were totally isolated and replanted in situ (group C) or as modified Thiry-Vella loops (group D). Weekly records of fasting myoelectrical activity were then taken. Group A and B animals showed reintegration of the transplanted bowel segments into a normal MMC sequence after a remodeling period. The replanted bowel segments of groups C and D displayed MMCs throughout the study period. The in situ replanted segments of group C were reintegrated into the normal sequence after a delay of 3--4 wk. These results indicate that the organization of the MMC is not solely determined by the extrinsic nervous system.