Susceptibility of Human Head and Neck Cancer Cells to Combined Inhibition of Glutathione and Thioredoxin Metabolism

Increased glutathione (GSH) and thioredoxin (Trx) metabolism are mechanisms that are widely implicated in resistance of cancer cells to chemotherapy. The current study determined if simultaneous inhibition of GSH and Trx metabolism enhanced cell killing of human head and neck squamous cell carcinoma (HNSCC) cells by a mechanism involving oxidative stress. Inhibition of GSH and Trx metabolism with buthionine sulfoximine (BSO) and auranofin (AUR), respectively, induced significant decreases in clonogenic survival compared to either drug alone in FaDu, Cal-27 and SCC-25 HNSCC cells in vitro and in vivo in Cal-27 xenografts. BSO+AUR significantly increased glutathione and thioredoxin oxidation and suppressed peroxiredoxin activity in vitro. Pre-treatment with N-acetylcysteine completely reversed BSO+AUR-induced cell killing in FaDu and Cal-27 cells, while catalase and selenium supplementation only inhibited BSO+AUR-induced cell killing in FaDu cells. BSO+AUR decreased caspase 3/7 activity in HNSCC cells and significantly reduced the viability of both Bax/Bak double knockout (DKO) and DKO-Bax reconstituted hematopoietic cells suggesting that necrosis was involved. BSO+AUR also significantly sensitized FaDu, Cal-27, SCC-25 and SQ20B cells to cell killing induced by the EGFR inhibitor Erlotinib in vitro. These results support the conclusion that simultaneous inhibition of GSH and Trx metabolism pathways induces oxidative stress and clonogenic killing in HNSCCs and this strategy may be useful in sensitizing HNSCCs to EGFR inhibitors.     

A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation

tRNAs are synthesized as immature precursors, and on their way to functional maturity, extra nucleotides at their 5' ends are removed by an endonuclease called RNase P. All RNase P enzymes characterized so far are composed of an RNA plus one or more proteins, and tRNA 5' end maturation is considered a universal ribozyme-catalyzed process. Using a combinatorial purification/proteomics approach, we identified the components of human mitochondrial RNase P and reconstituted the enzymatic activity from three recombinant proteins. We thereby demonstrate that human mitochondrial RNase P is a protein enzyme that does not require a trans-acting RNA component for catalysis. Moreover, the mitochondrial enzyme turns out to be an unexpected type of patchwork enzyme, composed of a tRNA methyltransferase, a short-chain dehydrogenase/reductase-family member, and a protein of hitherto unknown functional and evolutionary origin, possibly representing the enzyme's metallonuclease moiety. Apparently, animal mitochondria lost the seemingly ubiquitous RNA world remnant after reinventing RNase P from preexisting components.     

Evaluation of the energetic position of the lowest excited singlet state of beta-carotene by NEXAFS and photoemission spectroscopy.

In carotenoids the lowest energetic optical transition belonging to the pi-electron system is forbidden by symmetry, therefore the energetic position of the S(1) (2(1)A(g)) level can hardly be assessed by optical spectroscopy. We introduce a novel experimental approach: For molecules with pi-electron systems the transition C1s-->2p(pi*) from inner-atomic to the lowest unoccupied molecular orbital (LUMO) appears in X-ray absorption near edge spectra (NEXAFS) as an intense, sharp peak a few eV below the carbon K-edge. Whereas the peak position reflects the energy of the first excited singlet state in relation to the ionization potential of the molecule, intensity and width of the transition depend on hybridization and bonding partners of the selected atom. Complementary information can be obtained from ultraviolet photoelectron spectroscopy (UPS): At the low binding energy site of the spectrum a peak related to the highest occupied molecular orbital (HOMO) appears. We have measured NEXAFS and UPS of beta-carotene. Based on these measurements and quantum chemical calculations the HOMO and LUMO energies can be derived.     

172 Flexible Elemental Stoichiometry in Trichodesmium IMS101: Implications for Buoyancy-Mediated Vertical Migration

The cyanobacterium Trichodesmium is considered the most abundant and active nitrogen fixing plankton genus in tropical and subtropical marine waters. In the northern subtropical gyres of the Pacific and Atlantic Oceans, this organism is notable as a source of new production and as a potential biological shuttle for phosphorus to surface waters. We have conducted recent laboratory experiments revealing the remarkable stoichiometric flexibility exhibited by this diazotroph. These results indicate that Trichodesmium spp. are capable of viable growth with carbon to phosphorus ratios approximately 16 times Redfield stoichiometry. Such P-sparing is clearly an adaptation to the oligotrophic environments from which these cultures were isolated. Building on this research, additional work was performed to determine the maximal temporal scale of vertical migration and the implicit dark period physiology. An experiment was designed to assess the physiological changes that would occur as cells of varying growth stages migrated below the euphotic zone into more phosphorus replete waters. At different stages of growth sub-samples of phosphorus replete or phosphorus limited cells were transferred to dark conditions. For each dark bottle, chl-a, particulate carbon, phosphorus and nitrogen, trichome count and fluorescence parameters were measured daily. Our experimental results indicate that the maximum duration of dark period survival (and hence vertical migration) is approximately three days during which period cells were able to fully recover from light depravation and resume typical growth rates. The knowledge of Trichodesmium bioenergetics gained from this research will serve to further constrain the potential role of Trichodesmium spp. in biogeochemical cycling.     

Cyclic Adenosine 3′,5′-Monophosphate in Escherichia coli

The concentration of cyclic adenosine 3',5'-monophosphate (c-AMP) in Escherichia coli growing on different sources of carbon was studied. Cultures utilizing a source of carbon that supported growth relatively poorly had consistently higher concentrations of c-AMP than did cultures utilizing sugars that supported rapid growth. This relationship was also observed in strains defective in c-AMP phosphodiesterase and simultaneously resistant to catabolite repression; in such strains the c-AMP concentration was slightly higher for several sources of carbon tested. Cultures continued to synthesize c-AMP and secreted it into the medium, under conditions that brought about an inhibition of the intracellular accumulation of the cyclic nucleotide. Transient repression of the synthesis of beta-galactosidase was not associated with an abrupt decrease in the cellular concentration of c-AMP.     

Developmental regulation of the aldolase A muscle-specific promoter during in vivo muscle maturation is controlled by a nuclear receptor binding element.

During the post-natal period, skeletal muscles undergo important modifications leading to the appearance of different types of myofibers which exhibit distinct contractile and metabolic properties. This maturation process results from the activation of the expression of different sets of contractile proteins and metabolic enzymes, which are specific to the different types of myofibers. The muscle-specific promoter of the aldolase A gene (pM) is expressed mainly in fast-twitch glycolytic fibers in adult body muscles. We investigate here how pM is regulated during the post-natal development of different types of skeletal muscles (slow or fast-twitch muscles, head or body muscles). We show that pM is expressed preferentially in prospective fast-twitch muscles soon after birth; pM is up-regulated specifically in body muscles only later in development. This activation pattern is mimicked by a transgene which comprises only the 355 most proximal sequences of pM. Within this region, we identify a DNA element which is required for the up-regulation of the transgene during post-natal development in body muscles. Comparison of nuclear M1-binding proteins from young or adult body muscles show no qualitative differences. Distinct M1-binding proteins are present in both young and adult tongue nuclear extracts, compared to that present in gastrocnemius extracts.