Recurrent mutation in the human phenylalanine hydroxylase gene.

We report the identification of a missense mutation of Glu280 to Lys280 in the phenylalanine hydroxylase (PAH) gene of a phenylketonuria (PKU) patient in Denmark. The mutation is associated with haplotype 1 of the PAH gene in this population. This mutation has previously been found in North Africa, where it is in linkage disequilibrium with haplotype 38. While it is conceivable that this mutation could have been transferred from one haplotype background to another by a double crossover or gene conversion event, the fact that the mutation is exclusively associated with the two different haplotypes in the two distinct populations supports the hypothesis that these two PKU alleles are the result of recurrent mutations in the human PAH gene. Furthermore, since the site of mutation involves a CpG dinucleotide, they may represent hot spots for mutation in the human PAH locus.     

Evidence for the Glutamine Synthetase/Glutamate Synthase Pathway during the Photorespiratory Nitrogen Cycle in Spinach Leaves

Spinach leaf (Spinacia oleracea L.) discs infiltrated with [¹⁵N]glycine were incubated at 25°C in the light and in darkness for 0, 30, 60 and 90 minutes. The kinetics of ¹⁵N-incorporation into glutamine, glutamate, asparagine, aspartate, and serine from [¹⁵N]glycine was determined. At the beginning of the experiment, just after infiltration (0 min incubation) serine, and the amido-N of glutamine and asparagine were the only compounds significantly labeled in both light- and dark-treated leaf discs. Incorporation of ¹⁵N-label into the other amino acids was observed at longer incubation time. The per cent ¹⁵N-enrichment in all amino acids was found to increase with incubation. However, serine and the amido-N of glutamine remained the most highly labeled products in all treatments. The above pattern of ¹⁵N-labeling suggests that glutamine synthetase was involved in the initial refixation of ¹⁵NH₃ derived from [¹⁵N]glycine oxidation in spinach leaf discs.

The ¹⁵N-enrichment of the amino-N of glutamine was found to increase rapidly from 0 to 19% during incubation in the light. There was a comparatively smaller increase (4-9%) in the ¹⁵N-label of the amino-N of glutamine in tissue incubated in darkness. Furthermore the total flux of ¹⁵N-label into each of the amino acids examined was found to be greater in tissue incubated in the light than those in the dark. The above evidence indicates the involvement of the glutamine synthetase/glutamate synthase pathway in the recycling of photorespiratory NH₃ during glycine oxidation in spinach leaves.

     

Variation of cell kinetic parameters in relation to the growth rate of the Ehrlich ascites tumor.

A multicompartmental model of the cell cycle and proliferation kinetics was used to analyse the time-course behavior of the cell cycle time, the growth fraction, and the cell loss rate during Ehrlich ascites tumor growth. The growth rate of Ehrlich ascites tumor cells as the tumor aged was significantly influenced by change in the cell cycle time.     

Regulation of casein messenger RNA during the development of the rat mammary gland.

Casein mRNA was isolated and partially purified from RNA extracts of rat lactating mammary glands and translated in a teterologous cell-free protein synthesizing system derived from wheat germ. Casein mRNA activity was assayed by immunoprecipitation using a specific antiserum prepared against a mixture of the purified rat caseins. Properties of rat casein mRNA were examined using a variety of sizing techniques, including chromatography on Sepharose 4B, sedimentation on sucrose gradients after heat denaturation, and electrophoresis on 2.5% agarose gels in 6 M urea. Casein mRNA activity was found in an 8-16S region after gradient centrifugation with the peak occurring at 10.5 S. In addition, the binding of rat casein mRNA to dT-cellulose was examined. Only 40% of the total casein mRNA activity was selectively retained. A partial purification of casein mRNA was accomplished by a combination of these sizing and affinity chromatography techniques. In the purified preparations casein mRNA activity comprises approximately 90% of the total mRNA activity. Characterization of this material by agarose gel electrophoresis revealed two main bands of RNA at approximately 12 and 16 S, both containing casein mRNA activity. These mRNAs were of the correct size to code for two of the principal rat caseins of approximately 25,000 and 42,000 molecular weights. Casein mRNA and total mRNA activities were then compared in total RNA extracts at various stages of normal mammary gland development in the rat, i.e. during pregnancy, lactation, and involution following weaning. A selective induction of casein mRNA activity compared to total mRNA activity was found to occur during pregnancy and lactation. Moreover, a selective loss of activity was also observed during mammary gland involution. A surprisingly high level of casein mRNA activity was found in RNA extracts from early and midpregnant mammary glands.     

Induction of Apoptosis and Effect on the FAK/AKT/mTOR Signal Pathway by Evodiamine in Gastric Cancer Cells

Evodiamine isolated from Evodia rutaecarpa has been known to have anti-tumor activity against various cancer cell types. Although there have been reports showing the inhibitory effect of evodiamine on cell survival of gastric cancer cell, it is not clearly explained how evodiamine affects the expression and modification of proteins associated with apoptosis and upstream signal pathways. We confirmed the cytotoxic activity of evodiamine against AGS and MKN45 cells by a WST assay, cell morphological change, and clonogenic assay. The apoptotic cells were evaluated by Annexin V/PI analysis and Western blot and the expressions of apoptosis-related molecules were confirmed by Western blot. Evodiamine promoted apoptosis of AGS gastric cancer cells through both intrinsic and extrinsic signal pathways in a time- and dose-dependent manner. Evodiamine attenuated the expression of anti-apoptotic proteins, including Bcl-2, XIAP, and survivin, and elevated that of the pro-apoptotic protein Bax. Evodiamine also suppressed the FAK/AKT/mTOR signal pathway. Based on these results, we expect that the results from this study will further elucidate our understanding of evodiamine as an anti-cancer drug.