In the gut-associated lymphoid tissues, Th17 cells mediate mucosal homeostasis and inflammation. During HIV infection, Th17 cells become depleted and functionally impaired, which is implicated in the pathogenesis of chronic inflammation in patients treated with highly active antiretroviral therapy. IL-7 is a cytokine that mediates homeostatic responses in T lymphocytes, such as proliferation and survival, which are dysregulated during HIV infection. Whether similar dysregulation occurs in Th17 cells has yet to be reported. IL-7 receptor α (CD127) expression and IL-7 responses were therefore measured in blood-derived Th17 cells from uninfected individuals and effectively treated, HIV-infected individuals by flow cytometry. Th17 cells from uninfected individuals expressed CD127 and, in response to IL-7, exhibited phosphorylation of STAT5, upregulation of Bcl-2, and proliferation. During HIV infection, expression of CD127 and pSTAT5 in Th17 cells was comparable to that observed in cells from uninfected individuals. Interestingly, expression of Bcl-2 was upregulated while proliferation was dramatically impaired. These findings may provide further insight into the mechanisms by which Th17 cells fail to become restored during HIV infection.

<p>It has been long known that many types of cancers have high metabolic requirements and use reprogrammed metabolism to support cellular activities. The first identified metabolic alteration in cancer cells was elevated glucose uptake, glycolysis activity and lactate production even in the presence of oxygen. This metabolic program, termed aerobic glycolysis or the Warburg effect, provides cells with energy as well as biosynthetic substrates to sustain cell survival and rapid cell proliferation. Cancer metabolism is closely linked to genetic mutations and oncogenic signaling pathways, such as PI3K/Akt, cMyc and HIF pathways. These oncogenic signals can direct metabolic reprogramming while changes in metabolic status can regulate activities of these signaling pathways in turn. In addition to glucose, later studies also found utilization of alternate nutrients in cancer cells, including glutamine and lipids. Glutamine is the second major metabolic fuel and can be converted to various substrates to support cell bioenergetics needs and biosynthetic reactions. Usage of metabolic fuels in cancer cells, however, is variable. While certain cancers display addiction to one type of nutrient, others are capable of using multiple nutrients. </p><p>The unique metabolic features of cancer cells raise the possibility of targeting metabolism as a novel therapeutic approach for cancer treatment. Using pharmacological inhibitors, previous research has provided corroborating evidence that metabolic stress can impact survival and growth of proliferative cancer cells by regulating cell apoptotic machinery and cell cycle checkpoints. Due to lack of genetic tools and side effects from these inhibitors, however, mechanistic understanding of cell response to metabolic inhibition was limited in these studies. More importantly, how metabolic stress affects cancer progression in a physiological condition has not yet been well investigated. Lastly, current research has not examined metabolic program in indolent cancers and the metabolic requirements and activities in less proliferative cells also remain to be understood.</p><p>This work examines nutrients utilization in B cell derived acute and chronic leukemia (B-ALL and B-CLL). B-ALL is an aggressive form of leukemia. Using cell lines and primary patient samples, we found B-ALL cells primarily used glucose through aerobic glycolysis, similar to other proliferative cancer cells. B-ALL cells were also more sensitive to inhibition of glycolysis than normal B cells. Employing an untargeted metabolomics profiling in combination with isotope labeled glucose tracing approach, we show in a B-ALL model that genetic ablation of glucose transporter Glut1 partially reduced glucose uptake, sufficiently hindered anabolic pathways and promoted catabolic metabolism. This metabolic shift led to sharply curtailed B-ALL proliferation in vitro and reduced leukemic burden in vivo. Furthermore, this partial inhibition of glucose metabolism sensitized B-ALL cells to apoptotic stimuli and non-cytotoxic metabolic inhibition significantly enhanced efficacy of a tyrosine kinase inhibitor to eliminate B-ALL cells in vitro and in vivo. Thus, partial inhibition of glucose metabolism can provide a plausible adjuvant therapy to treat cancers that depend on glycolysis for survival and proliferation. </p><p>In contrast to B-ALL, B-CLL is an indolent form of cancer. Most B-CLL cells exhibited low glucose metabolic activities that were comparable with normal B cells at resting stage. Similar to chronically stimulated and anergic B cells, these B-CLL cells also failed to upregulate glucose metabolism in response to IgM stimulation. We also observed an altered amino acid and acyl-carnitine profile and increased glutaminase mRNA in B-CLL relative to normal B cells, suggesting the capability of using alternate nutrients such as glutamine in these cells. Finally, we explored the possibility of suppressing mitochondria metabolism to induce B-CLL cell death through inhibition of the nuclear hormone receptor and metabolic regulator ERRalpha. ERRalpha is known to regulate mitochondrial metabolism and was expressed higher in B-CLL than normal B cells. ERRalpha inhibition decreased viability of oncogene transformed pro-B cells, suggesting ERRalpha as a potential target for B-CLL treatment.</p><p> Collectively, this work investigates metabolic phenotype in two forms of leukemia derived from B cells. It reveals different metabolic requirements and activities in aggressive and indolent leukemia and explores different approaches to suppress metabolism in these cancers. Findings of this work shed light on how to potentially design metabolic approach to improve cancer treatment.</p> / Dissertation

The most common secondary structure of proteins is the alpha-helix. The alpha-helix can be involved in various protein-protein interactions (PPIs) through the recognition of three or more side chains along one face of the alpha-helix (Wells and McClendon, 2007). In recent years, there has been an increasing interest in the development of peptidic and non-peptidic compounds that bind to PPI surfaces. We focused on the design and synthesis of compounds that mimic the orientation of side chain residues of an alpha-helical protein domain. Although our scaffolds could potentially inhibit various PPIs, we focused mainly on the disruption of interactions among the Bcl-2-family of proteins and the Mdm-2-family of proteins to favor apoptosis in cancer cells. A summary of Bcl-2 and Mdm-2 structure and function relationships that focuses on the possibility of using peptidic and non-peptidic alpha-helical mimics as PPI inhibitors is described in Chapter One. Chapter Two discusses the design and synthesis of 3-substituted-2,6- and 2,5-piperazinedione oligomers as more hydrophilic scaffolds compared to previously reported alpha-helical mimetics (Yin, et al., 2005). A key feature of this design is the linkage of the units by a hydrazine bond. While we were able to prepare several monomers containing the hydrazine linkage, synthesis of the dimers and trimers is very challenging. Due to the difficulty of synthesizing oligomeric piperazine-diones in practical yields, we next focused on the design and synthesis of novel 2,5-terpyrimidinylene scaffolds as an alternative to obtain alpha-helical mimetics; this is discussed in Chapter Three. The main outcome of this project was the efficient preparation of a "first-generation" non-peptidic compound library via a facile iterative synthesis enabled by the key conversion of 5-cyanopyrimidine to 5-carboxamidine. Chapter Three also discusses our progress towards the synthesis of structurally similar substituted-2,5-terpyrimidinylenes, but with more drug-like properties as determined by QikProp calculations. Chapter Four describes an independent study on the synthesis of a guanidine derivative as an alkylating agent for the synthesis of cysteine peptide nucleic acids, CPNA, which is another current project in our lab.

No description available.

Aim: To explore the protective effects and involved mechanisms of two angiogenic growth factors, vascular endothelial growth factor (VEGF165) and angiopoietin-1 in cardiac myoblasts. Methods: Replication-deficient adenovirus encoding for human VEGF165 (Ad-VEGF165) or angiopoietin-1 (Ad-Ang1) were transfected into H9C2 cardiac myoblasts. Recombinant adenovirus encoding for green fluorescent protein (Ad-GFP) was used as vehicle control. Twenty-four hours later, cell apoptosis was induced by 300 μmmol of H2O2. Genomic DNA was extracted and DNA fragmentation was analyzed in 1.6% agarose gels. Phosphatidylinositol-3 kinase(PI-3 K) activity and bcl-2 expression level were investigated in H9C2 after gene transfection 24 hours later by an immol/Lunoprecipitated kinase assay and Western blot assay respectively. The effect of wartmannin, a specific inhibitor of PI-3 K, on DNA fragmentation, PI-3 K activity and bcl-2 expression was also analyzed by a pre-treatment of 30 minutes before transfection. Results: Apoptotic DNA fragmentation induced by H2O2 was significantly inhibited by the transfaction of Ad-VEGF165 and/or Ad-Ang1 but then aborted by the pretreatment of wartmannin. PI-3 K activity was significantly elevated after Ad-VEGF165 + Ad-Ang1 transfection as compared to Ad-GFP transfection group(2.60 vs 1.32, P < 0.01). Anti-apoptotic factor bcl-2 expression was upregulated in Ad-VEGF165 (2.1-fold), Ad-Ang1 (1.7-fold) and Ad-VEGF165 + Ad-Ang1 (1.7-fold) treated groups as compared to Ad-GFP transfection group. Wortmannin suppressed PI-3 K activiation induced by Ad-VEGF165 (from 1.83 to 0.69, P < 0.05). Ad-Ang1 (from 1.80 to 0.97, P = 0.07) or Ad-VEGF165a + Ad-Ang1 (from 2.60 to 0.42, P < 0.01). However, upregulation of bcl-2 induced by Ad-VEGF165 and/or Ad-Ang1 was not aborted by wortmannin pretreatment. Conclusions: VEGF165 and/or Ang1 can protect cardiac myoblasts from apoptosis induced by H2O2 throught PI-3 K and bcl-2 pathway. The anti-apoptotic function of either VEGF165 or Ang1 could be served as a now therapeutic target including their angiogenic benefits.

Our previous work in cultured cells has shown that the maintenance of mitochondrial Ca2+ homeostasis is essential for cell survival, and that the anti-apoptotic protein Bcl-2 is able to maintain a threshold level of mitochondrial Ca2+ by the inhibition of permeability transition. To test whether Bcl-2 also affects the mitochondrial Na+-Ca2+ exchange (NCE), a major efflux pathway for mitochondrial Ca2+, studies using transgenic mice that overexpress Bcl-2 in the heart have been performed. NCE activity was determined as the Na+-dependent Ca2+ efflux in the isolated mitochondria. Overexpression of Bcl-2 led to a significant reduction of NCE activity as well as increased resistance to permeability transition in the mitochondria of transgenic heart. This was accompanied by increased matrix Ca2+ level, enhanced formation of NADH and enhanced oxidation of pyruvate, an NAD+-linked substrate. Furthermore, there was induction of cellular Ca2+ transport proteins including the Na+-Ca2+ exchanger of the sarcolemma (NCX). Bcl-2 not only stimulates NCX expression in the sarcolemma but also attenuates the Na+-Ca2+ exchange in the mitochondria. These results are consistent with the protection by Bcl-2 against apoptosis in heart following ischemia/reperfusion.

京都大学 / 0048 / 新制・論文博士 / 博士(医科学) / 乙第12971号 / 論医科博第2号 / 新制||医科||5(附属図書館) / 32409 / 新制||医科||5 / 横浜市立大学大学院国際総合科学研究科バイオ科学専攻 / (主査)教授 長船 健二, 教授 中畑 龍俊, 教授 髙折 晃史 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

CLIC1 is an intracellular membrane protein that is unusual in that it can exist in both a soluble and an integral membrane form. The manner in which this protein inserts into membranes is unknown although it is proposed to undergo a change in structure whereby it initially experiences a degree of unfolding and then refolds into its new membrane-bound conformation. This study focuses on the characterisation of CLIC1 in terms of its secondary, tertiary and quaternary structure, the determination of its conformational stability at equilibrium and the establishment of its unfolding kinetics, all under conditions of varying pH, polarity, redox conditions, temperature and ionic strength. CLIC1 was found to be most stable at pH 7.0 / 20oC. The unfolding process is two-state and cooperative, producing a DG(H2O) of ~10 kcal/mol and a m-value of ~2 kcal/mol per molar urea. A decrease in pH to 5.5 or an increase in temperature to 37oC resulted in the stabilisation of an equilibrium intermediate species under mild denaturing conditions and a destabilisation of the native state. This was further evidenced by an increase in the rate of unfolding of CLIC1 from the native state to the denatured state under these conditions. A state with similar properties to the intermediate species was detected in the absence of urea at pH 5.5 / 37oC and under non-reducing conditions at both pH 7.0 / 20oC and pH 5.5 / 20oC. The intermediate species is more hydrophobic than either the native or denatured state; it is stabilised by salts, has a reduced secondary structure, increased flexibility and a buried Trp35 relative to the native state. The rate of formation of the intermediate species is a slow process which may involve an oligomerisation step. The results from this study provide an interpretation for the structure and mechanism of CLIC1 pore formation in vivo by comparing the effects of the environment on the structure and stability of the protein.

No description available.

The regulated cell death apoptosis enables redundant or compromised cells in ontogeny and homeostasis to remove themselves receptor-dependent after extrinsic signaling or after internal stress by BCL-2 proteins on the outer mitochondrial membrane (OMM). Mitochondrial BCL-2 proteins are also often needed for receptor-mediated signaling in apoptosis. Then, the truncated BH3-only protein BID (tBID) blocks retrotranslocation of the pro-apoptotic BCL-2 proteins BAX and BAK from the mitochondria into the cytosol. BAX and BAK in turn permeabilize the OMM. Although the BCL-2 proteins are controlled by a complex regulatory network, a specific mechanism for the inhibition of tBID remained unknown. Curiously, it was suggested that hexokinases, which channel glucose into the metabolism, have an intriguing function in the regulation of apoptosis. Recent analysis of transient hexokinase interactions with BAX revealed its participation in the inhibition of BAX and also BAK by retrotranslocation from mitochondria to the cytosol. In contrast to general apoptosis inhibition by anti-apoptotic BCL-2 proteins, hexokinase I and hexokinase 2 specifically inhibit tBID and thus the mitochondrial apoptosis pathway in response to death receptor signaling. Mitochondrial hexokinase localization and BH3 binding of cytosolic hexokinase domains are prerequisites for protection against receptor-mediated cell death, whereas glucose metabolism is not. This mechanism protects cells from apoptosis induced by cytotoxic T cells.