Inhibition of glyoxalase I by the enediol mimic S-(N-hydroxy-N-methylcarbamoyl)glutathione. The possible basis of a tumor-selective anticancer strategy.

In principle, competitive inhibitors of glyoxalase I that also serve as substrates for the thioester hydrolase glyoxalase II might function as tumor-selective anti-cancer agents, given the role of these enzymes in removing cytotoxic methylglyoxal from cells and the observation that glyoxalase II activity is abnormally low in some types of cancer cells. In support of the feasibility of this anticancer strategy, an inhibitor of this type has been synthesized by a thioester-interchange reaction between glutathione and N-hydroxy-N-methylcarbamate 4-chlorophenyl ester to give S-(N-hydroxy-N-methylcarbamoyl)glutathione (1). This compound was designed to be a tight-binding inhibitor of glyoxalase I, on the basis of its stereoelectronic similarity to the enediol(ate) intermediate that forms along the reaction pathway of this enzyme. Indeed, 1 is a competitive inhibitor of yeast glyoxalase I, with an inhibition constant (Ki = 68 microM) that is approximately 30-fold lower than that reported for S-D-lactoylglutathione and approximately 7-fold lower than the Km for glutathione-methylglyoxal thiohemiacetal. In addition, 1 is a substrate for bovine liver glyoxalase II, with a Km (0.48 mM) approximately equal to that of the normal substrate S-D-lactoyglutathione and a kcat approximately 2 x 10(-5)-fold that of the normal substrate. Membrane transport studies show that 1 can be delivered into human erythrocytes (used here as a model cell) either by direct diffusion of 1 across the cell membrane or by more rapid diffusion of the glycylethyl ester of 1 across the cell membrane, followed by the catalyzed hydrolysis of the ester to give 1.     

Refolding of S-methylmethionyl basic pancreatic trypsin inhibitor

The normal CH₂CH₂SCH₃ side-chain of Met52 of bovine pancreatic trypsin inhibitor was converted to CH₂CH₂S⁺(CH₃)₂ with methyl iodide. After unfolding and breakage of the three disulphide bonds, the reduced protein refolded some three to six times more slowly than unmodified bovine pancreatic trypsin inhibitor. This was shown to be due to the decreased occurrence of the normal one- and two-disulphide initial intermediates. Modifications of the Met52 side-chain suggest that it normally has an important conformational role in the initial stages of folding, participating in extensive hydrophobic interactions with other parts of the protein. This role is different from that in the final folded state, where modification produced no detectable change in stability.     

An epigenomic approach to therapy for tamoxifen-resistant breast cancer

Tamoxifen has been a frontline treatment for estrogen receptor alpha (ERα)-positive breast tumors in premenopausal women. However, resistance to tamoxifen occurs in many patients. ER still plays a critical role in the growth of breast cancer cells with acquired tamoxifen resistance, suggesting that ERα remains a valid target for treatment of tamoxifen-resistant (Tam-R) breast cancer. In an effort to identify novel regulators of ERα signaling, through a small-scale siRNA screen against histone methyl modifiers, we found WHSC1, a histone H3K36 methyltransferase, as a positive regulator of ERα signaling in breast cancer cells. We demonstrated that WHSC1 is recruited to the ERα gene by the BET protein BRD3/4, and facilitates ERα gene expression. The small-molecule BET protein inhibitor JQ1 potently suppressed the classic ERα signaling pathway and the growth of Tam-R breast cancer cells in culture. Using a Tam-R breast cancer xenograft mouse model, we demonstrated in vivo anti-breast cancer activity by JQ1 and a strong long-lasting effect of combination therapy with JQ1 and the ER degrader fulvestrant. Taken together, we provide evidence that the epigenomic proteins BRD3/4 and WHSC1 are essential regulators of estrogen receptor signaling and are novel therapeutic targets for treatment of Tam-R breast cancer.     

Improved gold chloride staining method for anatomical analysis of sensory nerve endings in the shoulder capsule and labrum as examples of loose and dense fibrous tissues

Consistency in gold chloride staining is essential for anatomical analysis of sensory nerve endings. The gold chloride stain for this purpose has been modified by many investigators, but often yields inconsistent staining, which makes it difficult to differentiate structures and to determine nerve ending distribution in large tissue samples. We introduce additional steps and major changes to the modified Gairns’ protocol. We controlled the temperature and mixing rate during tissue staining to achieve consistent staining and complete solution penetration. We subjected samples to sucrose dehydration to improve cutting efficiency. We then exposed samples to a solution containing lemon juice, formic acid and paraformaldehyde to produce optimal tissue transparency with minimal tissue deformity. We extended the time for gold chloride impregnation 1.5 fold. Gold chloride was reduced in the labrum using 25% formic acid in water for 18 h and in the capsule using 25% formic acid in citrate phosphate buffer for 2 h. Citrate binds gold nanoparticles, which minimizes aggregation in the tissue. We stored samples in fresh ultrapure water at 4° C to slow reduction and to maintain color contrast in the tissue. Tissue samples were embedded in Tissue Tek and sectioned at 80 and 100 μm instead of using glycerin and teasing the tissue apart as in Gairns’ modified gold chloride method. We attached sections directly to gelatin subbed slides after sectioning with a cryostat. The slides then were processed and coverslipped with Permount. Staining consistency was demonstrated throughout the tissue sections and neural structures were clearly identifiable.     

Invasive feral pigs impact native tree ferns and woody seedlings in Hawaiian forest

Invasive mammals can fundamentally alter native plant communities, especially on isolated islands where plants evolved without them. The globally invasive feral pig (Sus scrofa) can be particularly destructive to native plant communities. Tree ferns are an important understory component in many forests facilitating the establishment of a variety of species. However, the extent and effects of feral pig damage to tree ferns, and associated impacts on plant community regeneration, are largely unknown. We quantified the effect that feral pig damage has on tree fern growth, survival, and epiphytic woody seedling abundance over 1 year on 438 randomly selected tree ferns of three endemic species (Cibotium chamissoi, Cibotium glaucum, and Cibotium menziesii) in a Hawaiian montane wet forest with high tree fern and feral pig densities. Across all tree fern species, feral pigs damaged 13 % of individuals over 1 year. Compared with undamaged tree ferns, moderately- to heavily-damaged individuals had decreases of 4 to 27 % in trunk length increment and lost tenfold more fronds. Tree fern angle (standing, leaning, prone, or semi-prone) and woody seedling abundance co-varied with feral pig damage. Specifically, damaged tree ferns were more often prone or semi-prone and supported more seedlings, but also had annual mortality up to 34 % higher than undamaged tree ferns. Overall, feral pig damage had substantial negative effects on tree ferns by reducing growth and survival. Given the importance of tree ferns as regeneration sites for a variety of native plants, feral pig damage to tree ferns will likely alter future forest composition and structure. Specifically, feral pig damage to tree ferns reduces potential establishment sites for species that either regenerate preferentially as epiphytes or are currently restricted to epiphytic establishment due to ground rooting by feral pigs.