Quantification of hexokinase mRNA in mouse blastocysts by competitive reverse transcriptase polymerase chain reaction.

Hexokinase (HX), the enzyme that catalyses the initial reaction in glycolysis, is an important enzyme in glucose metabolism during human and mouse embryonic development. In our previous investigations of the genetic activities of HX, we observed an increased incidence of HX gene expression in blastocysts in comparison with morulae, and variability in the incidence of HX gene expression between embryos at the same developmental stages. These observations prompted us to quantify HX mRNA in mouse blastocysts to define the biological significance of the variable gene transcription. We modified our qualitative reverse transcription-nested polymerase chain reaction (RT-nPCR) assay for HX mRNA in single or groups of embryos to quantify HX mRNA by competitive RT-nPCR. HX mRNA was quantified in cohorts of mouse blastocysts cultured in glucose/phosphate-containing human tubal fluid (HTF) media. These blastocysts expressed HX in minute amounts, averaging 1.95 x 10(-18) g of mRNA. This is the first attempt at quantification of single gene mRNA in preimplantation embryos. Further investigations using similar techniques will enable comparative analyses between embryos to be performed to determine the correlation between specific levels of HX mRNA transcripts in individual embryos and embryonic viability and competence for further development and implantation.

Genetic expression of hexokinase and glucose phosphate isomerase in late-stage mouse preimplantation embryos: transcription activities in glucose/phosphate-containing HTF and glucose/phosphate-free P1 media.

In mouse and human preimplantation development, pyruvate is consumed preferentially during early embryogenesis; however, during the morula and blastocyst stages, glucose is the preferred energy substrate. Studies have suggested that the glycolytic enzymes, hexokinase and glucose phosphate isomerase, are important enzymes in glucose metabolism during these later stages of human and mouse preimplantation development. In order to investigate the genetic activities of these enzymes in late-stage mouse embryos developing in vitro, we analysed hexokinase and glucose phosphate isomerase transcription activities by qualitative RNA assays using reverse transcriptase-nested polymerase chain reaction amplification of individual mouse morulae and early blastocysts incubated in glucose/phosphate-free preimplantation stage one (P1) medium and glucose/phosphate-containing human tubal fluid (HTF) medium. We observed an increased incidence of hexokinase transcripts in the population of blastocysts compared with morulae, and differences in transcript incidence between early blastocysts developing in HTF medium and in P1 medium. In contrast, glucose phosphate isomerase transcripts were consistantly present in all embryos analysed, and appear to be constitutively expressed during late-stage mouse embryogenesis. The different activity patterns of the two glycolytic genes may reflect different mechanisms of gene regulation or differential transcript stability during the later stages of mouse preimplantation development.

Do products of the myc proto-oncogene play a role in transcriptional regulation of the prothymosin alpha gene?

The Myc protein has been reported to activate transcription of the rat prothymosin alpha gene by binding to an enhancer element or E box (CACGTG) located in the first intron (S. Gaubatz et al., Mol. Cell. Biol. 14:3853-3862, 1994). The human prothymosin alpha gene contains two such motifs: in the promoter region at kb -1.2 and in intron 1, approximately 2 kb downstream of the transcriptional start site in a region which otherwise bears little homology to the rat gene. Using chloramphenicol acetyltransferase (CAT) reporter constructs driven either by the 5-kb human prothymosin alpha promoter or by a series of truncated promoters, we showed that removal of the E-box sequence had no effect on transient expression of CAT activity in mouse L cells. When intron 1 of the prothymosin alpha gene was inserted into the most extensive promoter construct downstream of the CAT coding region, a diminution in transcription, which remained virtually unchanged upon disruption of the E boxes, was observed. CAT constructs driven by the native prothymosin alpha promoter or the native promoter and intron were indifferent to Myc; equivalent CAT activity was observed in the presence of ectopic normal or mutant Myc genes. Similarly, expression of a transiently transfected wild-type prothymosin alpha gene as the reporter was not affected by a repertoire of myc-derived genes, including myc itself and dominant or recessive negative myc mutants. In COS-1 cells, equivalent amounts of the protein were produced from transfected prothymosin alpha genes regardless of whether genomic E boxes were disrupted, intron 1 was removed, or a repertoire of myc-derived genes was included in the transfection cocktail. More importantly, cotransfection of a dominant negative Max gene failed to reduce transcription of the endogenous prothymosin alpha gene in COS cells or the wild-type transfected gene in COS or L cells. Taken together, the data do not support the idea that Myc activates transcription of the intact human prothymosin alpha gene or reporter constructs that mimic its structure. Rather, they suggest that the human prothymosin alpha promoter and downstream elements are buffered so as to respond poorly, if at all, to transient fluctuations in transcription factors which regulate other genes.

Phosphorylation of human and bovine prothymosin alpha in vivo.

Prothymosin alpha is post-translationally modified. When human myeloma cells were metabolically labeled with [32P]orthophosphoric acid, they synthesized [32P]prothymosin alpha. The incorporated radioactivity was resistant to DNase and RNases A, T1, and T2, but could be completely removed by alkaline phosphatase. No evidence was found for an RNA adduct as postulated by Vartapetian et al. [Vartapetian, A., Makarova, T., Koonin, E. V., Agol, V. I., & Bogdanov, A. (1988) FEBS Lett. 232, 35-38]. Thin-layer electrophoresis of partially hydrolyzed [32P]prothymosin alpha indicated that serine residues were phosphorylated. Analysis of peptides derived from bovine prothymosin alpha and human [32P]prothymosin alpha by treatment with endoproteinase Lys-C revealed that the amino-terminal 14-mer, with serine residues at positions 1, 8, and 9, was phosphorylated at a single position. Approximately 2% of the peptide in each case contained phosphate. Further digestion of the phosphopeptide with Asp-N followed by C18 reversed-phase column chromatography produced two peptides: a phosphate-free 9-mer containing amino acids 6-14 and a labeled peptide migrating slightly faster than the N-terminal 5-mer derived from the unmodified 14-mer. Positive identification of the phosphorylated amino acid was obtained by colliding the 14-residue phosphopeptide with helium in the mass spectrometer and finding phosphate only in a nested set of phosphorylated fragments composed of the first three, four, and five amino acids. The results prove that prothymosin alpha contains N-terminal acetylserine phosphate. In a synchronized population of human myeloma cells, phosphorylation occurred throughout the cell cycle. Furthermore, prothymosin alpha appeared to be stable, with a half-life slightly shorter than the generation time. Although prothymosin alpha is known to be essential for cell division, the constancy of both the amount of the protein and the degree of its phosphorylation suggests that prothymosin alpha does not directly govern mitosis.

Flavin levels in the rat retina.

Exposure of riboflavin and its coenzymes adenine dinucleotide (FAD) and riboflavin-5'-phosphate (FMN) to UV and visible light results in the generation of radicals and photodegradative products that can damage surrounding macromolecules. Vertebrates and invertebrates have lost the ability to synthesize riboflavin and must obtain it or its coenzymes from food. The present study evaluated the relationship between FAD, FMN, and riboflavin concentrations in retina and blood of male Sprague-Dawley rats. Rations were provided in the form of purified diets containing 0, 3, 6, 30, and 300 mg riboflavin kg-1 diet. Analysis of flavins by HPLC showed that saturation levels of FAD, FMN and riboflavin in the retina and blood were achieved with diets containing 3 mg riboflavin kg-1. Retinal flavins were not significantly elevated by further increases in dietary riboflavin concentration, but an unidentified flavin appeared in the blood of rats given rations containing concentrations above 3 mg kg-1. The concentration of this unknown flavin varied in proportion to the level of dietary riboflavin.

Analysis of flavins in ocular tissues of the rabbit.

Riboflavin is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), coenzymes required for the activity of flavoenzymes involved in the transfer of electrons in oxidation-reduction reactions. Flavins are light sensitive and rapidly degrade when exposed to light in the near ultraviolet and visible wavelengths. Some of the byproducts of flavin photodegradation are toxic. A quantitative survey of flavins in rabbit ocular tissues is reported. Adult male Dutch-Belt Rabbits were fed purified diets containing 3, 30, or 300 mg riboflavin/kg for 1 month. A method of aqueous extraction and high-performance liquid chromatography with fluorescence detection was used to measure riboflavin, FMN, and FAD in cornea, lens cortex, lens nucleus, retina, and blood. The retina contained the highest flavin concentration. In all tissues, the primary flavin was FAD followed by FMN and riboflavin. The highest concentration of riboflavin occurred in the cornea followed by the retina, lens cortex, and lens nucleus. A trend toward increasing concentrations of riboflavin occurred in the retina and blood in response to excess dietary riboflavin, but the concentration changes were not statistically significant. The highest concentration of FAD and FMN occurred in the retina followed by the cornea and the lens cortex and nucleus. The relative contribution of riboflavin, FMN, and FAD to the total flavin pool was markedly different in the various tissues of the eye. The proportion of tissue flavins present as riboflavin decreased from anterior to posterior. It was highest in the cornea followed by lens and retina. The pattern of distribution for FMN was: cornea greater than retina greater than lens cortex and nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of FAD, FMN, and riboflavin in the retina by microextraction and high-performance liquid chromatography.

The presence of flavins in the retina has been known for some time. However, the small size of the tissue has made it difficult to quantify the levels of the individual flavins, riboflavin (RB), FMN, and FAD without pooling large numbers of retinas. A procedure to extract and quantitate RB, FMN, and FAD in retinal tissue from as few as four rat retinas has been developed. The procedure resolves these three classes of flavins and provides a recovery near 100%. For the analysis, HPLC using a reverse-phase column with cyclohexyl functional groups was coupled to a fluorescence detector. The microextraction-HPLC procedure was reproducible for the quantitative analysis of flavins in the retina and equally applicable for analysis of flavins in liver and plasma.

Lipids of the retinal pigment epithelium in RCS dystrophic and normal rats.

The lipid composition of retinal pigment epithelial cells was determined for normal cells which have full phagocytic ability and for a genetic variant with impaired phagocytic function. Retinal pigment epithelial cells from 9-14-day-old congenic strains of normal (RCS-rdy+) and dystrophic (RCS-rdy/rdy) rats were separated from intact retinas and homogenized in 0.08 M Tris base, pH 7.4. The lipids were extracted using 2:1 chloroform--methanol. Fatty-acid methyl esters identified by gas chromatography were: 16:0, 17:0, 18:0, 18:1, 18:2 omega 6, 20:0, 20:2, 22:0, 20:4 omega 6, 22:4, 22:5, 22:6 omega 3. Major fatty acids for both normal and dystrophic cells were: 16:0, 18:0, 20:4 omega 6, 22:6 omega 3. One- and two-dimensional thin-layer chromatography was used to determine phospholipid composition of pigment epithelial cells at two different age groups. The relative amount of phosphatidylethanolamine was significantly higher in dystrophic RPE cells compared with normal cells (20.7% for 9-11-day-old and 17.3% for 12-14-day-old dystrophic rats). Cells from normal animals contained a higher level of phosphatidylethanolamine in the older age group whereas RPE cells from dystrophic animals contained a lower level of phosphatidylcholine in the older group. Anomalous phospholipid composition of dystrophic pigment epithelial cells may be associated with a change in cellular membranes and a defect in the cellular processes involved in phagocytic function.