The high efficiency of capillary electrophoresis (CE) coupled with universality of direct UV absorbance detection allowed the quantification of the following matrices: (1) oxalate in numerous biological systems such as: human albumin, urine, parental nutrition solutions (intralipid infusion, multi-vitamin infusion), fifteen premature baby formulas produced by the two leading baby food companies, commercial skim and 1% fat milk emulsions; (2) histidine-containing antioxidants, carnosine and anserine, in pork and chicken muscle tissue extracts; (3) melatonin in the presence of the products of tryptophan metabolism and (4) taxol in the presence of such interferents as cephalomannine and baccatine III. The importance of accurate oxalate quantification in various biological systems is directly connected to the formation of kidney and, possibly, gall bladder stones in premature babies and adults, since oxalate is a direct precursor in the formation of calcium oxalate, the final product of metabolism of oxalic acid. The method developed allowed quantification of underivatized samples with a 6 ppb detection limit for oxalate using direct photometric detection. The naturally occurring histidine-containing dipeptides, carnosine and anserine, are directly responsible for the following properties of muscle tissues of pork, chicken, fish, etc.: freshness, color, texture, and flavor. The present methods of antioxidant quantification based on HPLC with fluorescence detection lack sensitivity, specificity and reproducibility. A reliable CE method using direct UV absorbance detection was developed for fast, accurate, reproducible, sensitive and selective quantification of the analytes at ppm levels in the muscle extracts and at ppb levels in aqueous standards without derivatization of the muscle extracts. Much attention has been focused lately on the healing properties of the naturally occurring hormone melatonin. The analyte is currently quantified by HPLC with fluorescence and/or electrochemical detection, which, unfortunately, lacks specificity and sensitivity for melatonin in the presence of tryptophan and its products of metabolism. An attempt was made to develop a specific, sensitive, fast and simple method of analyte quantification without any kind of sample derivatization, solid phase and/or liquid phase extraction, and/or preconcentration. This allowed the separation and quantification of melatonin in the presence of major HPLC interferents such as tryptophan, hydroxytryptophan, and seratonin. Taxol, a highly functionalized taxane diterpene amide, has recently emerged as an effective chemotherapeutic agent for treatment of ovarian tumors, breast carcinomas and malignant melanoma. Micellar electrokinetic chromatography was successfully applied to separate several taxanes in about 11 minutes using minute amounts of sample and minimal amounts of buffer to decrease the solvent waste. (Abstract shortened by UMI.)

The program of a differentiated somatic cell can be altered by nuclear transplantation, cell fusion or co-culture. This work demonstrates that somatic gene function can be manipulated in vitro. We developed two novel strategies by which reprogramming of gene expression in a differentiated somatic cell can take place. Our first approach relies on the establishment of connections through gap junctions between somatic cells and blastomeres. We show that mouse epithelial cells are capable of forming gap junctions with blastomeres following injection into cleavage stage mouse embryos. In contrast, fibroblasts adhere but do not form gap junctions with blastomeres. Adhesion of the somatic cells to blastomeres was assessed by electron microscopy and immunological procedures using cell adhesion markers. Transfer of a fluorescent soluble dye from somatic cells to the adherent blastomeres demonstrated formation of functional gap junctions. Establishment of connections between epithelial cells and blastomeres correlated with induction of expression of the embryonic and ES cell-specific transcription factor, Oct-4, in the epithelial cells. We argue that communication between epithelial cells and blastomeres results in changes in somatic cell gene expression. Our second approach relies on the production of cell extracts to which permeabilized fibroblasts are exposed. The resulting fibroblasts are reprogrammed and thus take on characteristics and functions typical of the cell type from which the extract was derived, including the induction of Oct-4. The development of these reprogramming systems can be used to define the factors necessary for reprogramming somatic cells. Ultimately, these strategies for altering genome function could be applied to develop cell therapeutics.

Cloning of animals by nuclear transplantation has demonstrated that reprogramming of nuclear function is possible. However, low pregnancy rates, elevated pregnancy losses and lethal abnormalities in most cloned animals born argue that somatic cell reprogramming by nuclear transplantation is not always complete. Here, we report the identification of four nuclear markers of incomplete reprogramming in nuclear transplant mouse embryos. Nuclear transplant embryos exhibit (i) pronucleur assembly of A-type lamins, (ii) increased NuMA content, (iii) stronger anchoring of AKAP95 and (iv) a greater proportion of heterochromatin, compared to fertilized embryos. We propose that deficiencies in reprogramming through nuclear transplantation result from failure to morphologically remodel the somatic donor nucleus into a normal, fully functional pronucleus.

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