Enzymatic Protein Carboxyl Methylation in Rat Posterior Pituitary: Neurophysins in Rapid-Turnover Pool Determine Methyl Accepting Capacity

In vitro stimulation of intact rat posterior pituitary by either veratridine or K+ depolarization results in the concomitant release of neurophysins and in a decrease (70-80%) in their carboxyl methylation as measured either with L-[methyl-3H]methionine in the intact lobes after stimulation or in their homogenates with [methyl-3H]S-adenosyl-L-methionine and purified protein carboxyl methyltransferase. A similar reduction in neurophysin methylation (60%) was observed when the arrival of newly synthesized neurophysins at the posterior pituitary was blocked by colchicine. Experimental data indicate that the reduction in neurophysin content of the lobes after 12 h of colchicine treatment (less than 7%) or after in vitro stimulation (about 10%) cannot account for the marked reduction in neurophysin methylation. The results suggest that the granule pool characterized by rapid turnover of neurophysins probably represents the major source of methyl acceptor proteins in the lobe. In spite of the marked reduction in neurophysin methyl accepting capacity observed after stimulation, there was no parallel increase in methyl accepting capacity of the released neurophysins. We propose that a neurophysin subfraction that might be associated with the membrane of releasable granules participates in the methylation reaction in situ.     

Coenzyme Q10 restores oocyte mitochondrial function and fertility during reproductive aging

Female reproductive capacity declines dramatically in the fourth decade of life as a result of an age‐related decrease in oocyte quality and quantity. The primary causes of reproductive aging and the molecular factors responsible for decreased oocyte quality remain elusive. Here, we show that aging of the female germ line is accompanied by mitochondrial dysfunction associated with decreased oxidative phosphorylation and reduced Adenosine tri‐phosphate (ATP) level. Diminished expression of the enzymes responsible for CoQ production, Pdss2 and Coq6, was observed in oocytes of older females in both mouse and human. The age‐related decline in oocyte quality and quantity could be reversed by the administration of CoQ10. Oocyte‐specific disruption of Pdss2 recapitulated many of the mitochondrial and reproductive phenotypes observed in the old females including reduced ATP production and increased meiotic spindle abnormalities, resulting in infertility. Ovarian reserve in the oocyte‐specific Pdss2‐deficient animals was diminished, leading to premature ovarian failure which could be prevented by maternal dietary administration of CoQ10. We conclude that impaired mitochondrial performance created by suboptimal CoQ10 availability can drive age‐associated oocyte deficits causing infertility.     

Massive alterations of the methylation patterns around DNA transposons in the first four generations of a newly formed wheat allohexaploid

Rapid and reproducible genomic changes can be induced during the early stages of the life of nascent allopolyploid species. In a previous study, it was shown that following allopolyploidization, cytosine methylation changes can affect up to 11% of the wheat genome. However, the methylation patterns around transposable elements (TEs) were never studied in detail. We used transposon methylation display (TMD) to assess the methylation patterns of CCGG sites flanking three TE families (Balduin, Apollo, and Thalos) in the first four generations of a newly formed wheat allohexaploid. In addition, transposon display (TD), using a methylation-insensitive restriction enzyme, was applied to search for genomic rearrangements at the TE insertion sites. We observed that up to 54% of CCGG sites flanking the three TE families showed changes in methylation patterns in the first four generations of a newly formed wheat allohexaploid, where hypermethylation was predominant. Over 70% of the changes in TMD patterns occurred in the first two generations of the newly formed allohexaploid. Furthermore, analysis of 555 TE insertion sites by TD and 18 cases by site-specific PCR revealed a full additive pattern in the allohexaploid, an indication for lack of massive rearrangements. These data indicate that following allopolyplodization, DNA-TE insertion sites can undergo a significantly high level of methylation changes compared with methylation changes of other genomic sequences.     

Leukomogenic factors downregulate heparanase expression in acute myeloid leukemia cells

Heparanase is a heparan sulfate-degrading endoglycosidase expressed by mature monocytes and myeloid cells, but not by immature hematopoietic progenitors. Heparanase gene expression is upregulated during differentiation of immature myeloid cells. PML-RARalpha and PLZF-RARalpha fusion gene products associated with acute promyelocytic leukemia abrogate myeloid differentiation and heparanase expression. AML-Eto, a translocation product associated with AML FAB M2, also downregulates heparanase gene expression. The common mechanism that underlines the activity of these three fusion gene products involves the recruitment of histone deacetylase complexes to specific locations within the DNA. We found that retinoic acid that dissociates PML-RARalpha from the DNA, and which is used to treat acute promyelocytic leukemia patients, restores heparanase expression to normal levels in an acute promyelocytic leukemia cell line. The retinoic acid effects were also observed in primary acute promyelocytic leukemia cells and in a retinoic acid-treated acute promyelocytic leukemia patient. Histone deacetylase inhibitor reverses the downregulation of heparanase expression induced by the AML-Eto fusion gene product in M2 type AML. In summary, we have characterized a link between leukomogenic factors and the downregulation of heparanase in myeloid leukemic cells.     

Overcoming residual frustration in domain-swapping: the roles of disulfide bonds in dimerization and aggregation

The prevalence of domain-swapping in nature is a manifestation of the principle of minimal frustration in that the interactions designed by evolution to stabilize the protein are also involved in this mode of binding. We previously demonstrated that the Symmetrized-Go potential accurately predicts the experimentally observed domain-swapped structure of Eps8 based solely on the structure of the monomer. There can be, however, multiple modes of domain-swapping, reflecting a higher level of frustration, which is a consequence of symmetry. The human prion and cyanovirin-N are too frustrated to form unique domain-swapped structures on the basis of the Symmetrized-Go potential. However, supplementing the completely symmetric model with intermolecular and intramolecular disulfide bonds in the prion and cyanovirin-N proteins, respectively, yielded unique domain-swapped structures with a remarkable similarity to the experimentally observed ones. These results suggest that the disulfide bonds may sometimes be critical in overcoming the intrinsic frustration of the symmetrized energy landscapes for domain-swapping. We also discuss the implications of intermolecular disulfide bonds in the formation of mammalian prion aggregates.     

Enhanced Levels of the Aroma and Flavor Compound S-Linalool by Metabolic Engineering of the Terpenoid Pathway in Tomato Fruits

The aromas of fruits, vegetables, and flowers are mixtures of volatile metabolites, often present in parts per billion levels or less. We show here that tomato (Lycopersicon esculentum Mill.) plants transgenic for a heterologous Clarkia breweri S-linalool synthase (LIS) gene, under the control of the tomato late-ripening-specific E8 promoter, synthesize and accumulate S-linalool and 8-hydroxylinalool in ripening fruits. Apart from the difference in volatiles, no other phenotypic alterations were noted, including the levels of other terpenoids such as gamma- and alpha-tocopherols, lycopene, beta-carotene, and lutein. Our studies indicate that it is possible to enhance the levels of monoterpenes in ripening fruits by metabolic engineering.