Miniaturized analytical assays in biotechnology

Biotechnology today is a well-established paradigm in many areas of human endeavor, such as the pharmaceutical industry, agriculture, management of the environment and many others. Meanwhile, biology is undergoing a spectacular transition: whereas systematic biology was replaced gradually by molecular biology, the latter is rapidly being transformed into a new systematic era in which entire genomes are being charted by ever more sophisticated analytical techniques.In the wake of this onslaught of data, new fields are germinating, such as bioinformatics in an attempt to find answers to fundamental questions, answers that may be hidden in the massive amounts of data already available today.     

Nonphotic phase shifting in female Syrian hamsters: interactions with the estrous cycle

Nonphotic phase shifting of circadian rhythms was examined in female Syrian hamsters. Animals were stimulated at zeitgeber time 4.5 by either placing them in a novel running wheel or by transferring them to a clean home cage. Placement in a clean home cage was more effective than novel wheel treatment in stimulating large (> 1.5 h) phase shifts. Peak phase shifts (ca. 3.5 h) and the percentage of females showing large phase shifts were comparable to those found in male hamsters stimulated with novel wheels. The amount of activity induced by nonphotic stimulation and the amount of phase shifting varied slightly with respect to the 4-day estrous cycle. Animals tended to run less and shift less on the day of estrus. Nonphotic stimulation on proestrus often resulted in a 1-day delay of the estrous cycle reflected in animals' postovulatory vaginal discharge and the expression of sexual receptivity (lordosis). This delay of the estrous cycle was associated with large phase advances and high activity. These results extend the generality of nonphotic phase shifting to females for the first time and raise the possibility that resetting of circadian rhythms can induce changes in the estrous cycle.     

Arsenic trioxide enhances radiation response of 9L glioma in the rat brain

Arsenic trioxide (ATO) at low doses induces leukemia cells to undergo apoptosis and at higher doses causes blood flow to solid tumors to shut down. To determine whether a potential synergistic interaction exists between ATO at the non-toxic dose level in the rat and radiation, the present study was carried out with orthotopic 9L malignant gliomas growing in the brains of rats. Animals died within 50 days of treatment when 12-day-old 9L gliomas growing in the brain of Fischer rats were treated with either the drug alone (8 mg/kg) or radiation alone (25 Gy). In contrast, the overall tumor cure rate exceeded 50% at a follow-up time of 120 days after the combined treatment with radiation and ATO. Long-term surviving animals showed no clinical or disproportionately enhanced histopathological changes in the brain parenchyma. Early changes in tumor physiology showed that the vascular leakage of FITC-dextran conjugates was apparent within 8 h of drug administration. Last, the use of diffusion magnetic resonance imaging as an early surrogate marker of therapeutic efficacy corroborated the effects of drug with and without radiation on brain histology and animal survival.     

Evaluation of 17alpha-E-(trifluoromethylphenyl)vinyl estradiols as novel estrogen receptor ligands

As part of our program to develop novel ligands for the estrogen receptor, we synthesized the series of isomeric 17alpha-(trifluoromethyl)phenylvinyl estradiols using our solid-phase organic synthesis methodology. The compounds were evaluated for their relative binding affinity (RBA) using the ERalpha-LBD and in vivo potency using the immature rat uterotrophic growth assay. The ortho-isomer had the highest RBA values, 48-223, and the highest estrogenicity in vivo. The other isomers had significantly lower affinities and were weaker agonists in the uterotrophic assay. The results suggest that introduction of substituents at the 17alpha-position of estradiol is tolerated by the ER-LBD and permit agonist responses in the intact animal, however, the effect is sensitive to the position of groups on the phenyl ring. This study demonstrates that the 17alpha-position of estradiol is a reasonable site for modification but the position and physicochemical properties of such modifications may significantly affect the affinity and efficacy of the ligand.     

A single nucleotide polymorphism in the<Emphasis Type="Italic"> Emp3</Emphasis> gene defines the H4 minor histocompatibility antigen

Minor histocompatibility antigens (minor H antigen) elicit strong T-cell-mediated responses during both graft rejection and graft versus leukemia (GvL) among MHC-matched individuals (where MHC is major histocompatibility complex). Employing expression-cloning methodology, we have identified a cDNA clone, MI-35, encoding the immunodominant H4^b minor H antigen within the classical mouse H4 complex. The minimal antigenic epitope derived from H4^b presented on K^b class I MHC is SGIVYIHL (SYL8) and the polymorphism is due to CT nucleotide modification in p3 resulting in the change of threonine (ACT) to isoleucine (ATT). The results presented here demonstrate that amino acid variation in the allelic epitopes results in the low abundance of H4^a peptide. The differential peptide copy number resulted in an immunodominant cytotoxic T cells (CTL) response directed against H4^b while the anti-B6 response directed against H4^a was easily dominated. These results provide a molecular mechanism for the H4 minor H antigen and suggest a novel mechanism by which alloantigenic disparity caused by conservative amino acid changes can be augmented by posttranslational antigen processing events.     

Comparative analysis of expressed sequence tags from Sesamum indicum and Arabidopsis thaliana developing seeds

Sesame (Sesamum indicum) is an important oilseed crop which produces seeds with 50% oil that have a distinct flavor and contains antioxidant lignans. Because sesame lignans are known to have antioxidant and health-protecting properties, metabolic pathways for lignans have been of interest in developing sesame seeds. As an initial approach to identify genes involved in accumulation of storage products and in the biosynthesis of antioxidant lignans, 3328 expressed sequence tags (ESTs) were obtained from a cDNA library of immature seeds 5-25 days old. ESTs were clustered and analyzed by the BLASTX or FASTAX program against the GenBank NR and Arabidopsis proteome databases. To compare gene expression profiles during development of green and non-green seeds, a comparative analysis was carried out between developing sesame and Arabidopsis seed ESTs. Analyses of these two seed EST sets have helped to identify similar and different gene expression profiles during seed development, and to identify a large number of sesame seed-specific genes. In particular, we have identified EST candidates for genes possibly involved in biosynthesis of sesame lignans, sesamin and sesamolin, and also suggest a possible metabolic pathway for the generation of cofactors required for synthesis of storage lipid in non-green oilseeds. Seed-specific expression of several candidate genes has been confirmed by northern blot analysis.