The application of a quantitative videographic technique has provided an opportunity to compare the quantitative volumetric expansion of cultured oocyte complexes (COCs) to quantitative changes in gap junction down-regulation and hyaluronic acid synthesis and to investigate the effects of physiological agents that influence these processes. Results of these experiments support the idea that the down-regulation of cumulus gap junctions is required for the initial phase of cumulus cell disaggregation and confirm earlier reports that hyaluronic acid synthesis plays a major role in additional expansion of the cumulus. These studies also provide evidence that the degree of expansion observed in culture lacking substrates of hyaluronic synthesis is significantly attentuated when compared with expansion occurring in vivo and that the failure of cultured complexes to expand maximally can be overcome by the addition of substrates of hyaluronic acid synthesis to the culture medium.

Modulation of connexin 43 (cx43) in the myometrium of timed pregnant rats was studied using enzyme-linked immunosorbent assay (ELISA), immunocytochemical localization, and immunoblot. These techniques utilized site-specific antibodies directed against a portion of the carboxyl tail of cx43. We found that cx43 is synthesized several days prior to labor but accumulates within the cytoplasm until parturition, when it is rapidly transported to the plasma membrane and assembled into gap junction plaques at the cell surface. These cx43-positive gap junctions begin to disappear from the plasma membrane within hours of delivery of the last pup and are completely absent within 24 hr following delivery. These structures are apparently internalized and degraded within the cytoplasm. ELISA documents a reduction of total cellular cx43 to baseline levels within 5 days following parturition. While the timing of synthesis, cytoplasmic storage, concentration in apparent Golgi vesicles, and transport to and assembly in the plasma membrane are accelerated in three models of preterm labor, the sequence of these events and the correlation of parturition with the formation of gap junctions are identical to those documented in normal labor. These results support the hypothesis that effective labor requires the synthesis and assembly of cx43 into functional gap junctions at the myometrial cell surface.

Hydroxylation is a novel protein modification catalyzed by a family of oxygenases that depend on fundamental nutrients and metabolites for activity. Protein hydroxylases have been implicated in a variety of key cellular processes that play important roles in both normal homeostasis and pathogenesis. Here, in this review, we summarize the current literature on a highly conserved sub-family of oxygenases that catalyze protein histidyl hydroxylation. We discuss the evidence supporting the biochemical assignment of these emerging enzymes as ribosomal protein hydroxylases, and provide an overview of their role in immunology, bone development, and cancer.

MINA53 is a JmjC domain 2-oxoglutarate-dependent oxygenase that catalyzes ribosomal hydroxylation and is a target of the oncogenic transcription factor c -MYC. Despite its anticancer target potential, no small-molecule MINA53 inhibitors are reported. Using ribosomal substrate fragments, we developed mass spectrometry assays for MINA53 and the related oxygenase NO66. These assays enabled the identification of 2-(aryl)alkylthio-3,4-dihydro-4-oxoypyrimidine-5-carboxylic acids as potent MINA53 inhibitors, with selectivity over NO66 and other JmjC oxygenases. Crystallographic studies with the JmjC demethylase KDM5B revealed active site binding but without direct metal chelation; however, molecular modeling investigations indicated that the inhibitors bind to MINA53 by directly interacting with the iron cofactor. The MINA53 inhibitors manifest evidence for target engagement and selectivity for MINA53 over KDM4–6. The MINA53 inhibitors show antiproliferative activity with solid cancer lines and sensitize cancer cells to conventional chemotherapy, suggesting that further work investigating their potential in combination therapies is warranted.

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