Alternatively spliced products CC3 and TC3 have opposing effects on apoptosis

The human gene CC3 is a metastasis suppressor for small cell lung carcinoma (SCLC) in vivo. The ability of CC3 to impair the apoptotic resistance of tumor cells is likely to contribute to metastasis suppression. We describe here an alternatively spliced RNA of CC3, designated TC3, that encodes an unstable protein with antiapoptotic activity. TC3 and CC3 proteins share amino-terminal sequences, but TC3 has a unique short hydrophobic carboxyl terminus. Overexpression of CC3 results in massive death of rodent fibroblasts, but TC3 protects cells from CC3-induced death and from other death stimuli such as treatment with tumor necrosis factor or overexpression of Bax protein. The death-inducing activity of CC3 resides within its amino-terminal domain, which is conserved in TC3. The carboxyl terminus of TC3 is responsible for the antiapoptotic function of TC3; mutations in this domain abolish the ability of TC3 to protect cells from apoptosis. TC3 protein is short-lived due to its rapid degradation by proteasome, and it forms complexes with a regulatory subunit of proteasome known as s5alpha. The signal for the rapid degradation of TC3 resides within its carboxyl terminus, which is capable of conferring instability on a heterologous protein. The proapoptotic activity of CC3 in SCLC cells is induced by a wide variety of signals and involves disruption of the mitochondrial membrane potential (Deltapsim). The CC3 protein has sequence similarity to bacterial short-chain dehydrogenases/reductases and might represent a phylogenetically old effector of cell death similar to the recently identified apoptosis-inducing factor. CC3 and TC3 have opposing functions in apoptosis and represent a novel dual regulator of cell death.     

Macrophage colony-stimulating factor rapidly enhances beta-migrating very low density lipoprotein metabolism in macrophages through activation of a Gi/o protein signaling pathway

Previous studies have examined lipoprotein metabolism by macrophages following prolonged exposure (>24 h) to macrophage colony-stimulating factor (M-CSF). Because M-CSF activates several signaling pathways that could rapidly affect lipoprotein metabolism, we examined whether acute exposure of macrophages to M-CSF alters the metabolism of either native or modified lipoproteins. Acute incubation of cultured J774 macrophages and resident mouse peritoneal macrophages with M-CSF markedly enhanced low density lipoproteins (LDL) and beta-migrating very low density lipoproteins (beta-VLDL) stimulated cholesteryl [(3)H]oleate deposition. In parallel, M-CSF treatment increased the association and degradation of (125)I-labeled LDL or beta-VLDL without altering the amount of lipoprotein bound to the cell surface. The increase in LDL and beta-VLDL metabolism did not reflect a generalized effect on lipoprotein endocytosis and metabolism because M-CSF did not alter cholesterol deposition during incubation with acetylated LDL. Moreover, M-CSF did not augment beta-VLDL cholesterol deposition in macrophages from LDL receptor (-/-) mice, indicating that the effect of M-CSF was mediated by the LDL receptor. Incubation of macrophages with pertussis toxin, a specific inhibitor of G(i/o) protein signaling, had no effect on cholesterol deposition during incubation with beta-VLDL alone, but completely blocked the augmented response promoted by M-CSF. In addition, incubation of macrophages with the direct G(i/o) protein activator, mastoparan, mimicked the effect of M-CSF by enhancing cholesterol deposition in cells incubated with beta-VLDL, but not acetylated LDL. In summary, M-CSF rapidly enhances LDL receptor-mediated metabolism of native lipoproteins by macrophages through activation of a G(i/o) protein signaling pathway. Together, these findings describe a novel pathway for regulating lipoprotein metabolism.     

Naphthalene uptake by a Pseudomonas fluorescens isolate

The uptake of naphthalene has been investigated in the metabolizing cells of Pseudomonas fluorescens utilizing [1-14C]naphthalene. The uptake displayed an affinity constant (Kt) of 11 microM and a maximal velocity (Vmax) of 17 nmol.h-1.mg-1 cellular dry weight. Naphthalene uptake was not observed in a mutant strain, TG-5, which was unable to utilize naphthalene as a sole source of carbon for growth. Uptake was significantly inhibited (approximately 90%) by the presence of growth-inhibiting levels of either azide or 2,4-dinitrophenol and was sensitive to the presence of structural analogues of naphthalene. The intracellular levels of ATP were not significantly reduced by the presence of either azide or 2,4-dinitrophenol. The presence of alpha-naphthol was found to noncompetitively inhibit naphthalene uptake, displaying a Ki of 0.041 microM. It is concluded that the first step in the utilization of naphthalene by Pseudomonas fluorescens is its transport into the cell by a specific energy-linked transport system.     

Kinetic and structural characterization of DmpI from Helicobacter pylori and Archaeoglobus fulgidus, two 4-oxalocrotonate tautomerase family members

The tautomerase superfamily consists of structurally homologous proteins that are characterized by a β-α-β fold and a catalytic amino-terminal proline. 4-Oxalocrotonate tautomerase (4-OT) family members have been identified and categorized into five subfamilies on the basis of multiple sequence alignments and the conservation of key catalytic and structural residues. Representative members from two subfamilies have been cloned, expressed, purified, and subjected to kinetic and structural characterization. The crystal structure of DmpI from Helicobacter pylori (HpDmpI), a 4-OT homolog in subfamily 3, has been determined to high resolution (1.8 Å and 2.1 Å) in two different space groups. HpDmpI is a homohexamer with an active site cavity that includes Pro-1, but lacks the equivalent of Arg-11 and Arg-39 found in 4-OT. Instead, the side chain of Lys-36 replaces that of Arg-11 in a manner similar to that observed in the trimeric macrophage migration inhibitory factor (MIF), which is the title protein of another family in the superfamily. The electrostatic surface of the active site is also quite different and suggests that HpDmpI might prefer small, monoacid substrates. A kinetic analysis of the enzyme is consistent with the structural analysis, but a biological role for the enzyme remains elusive. The crystal structure of DmpI from Archaeoglobus fulgidus (AfDmpI), a 4-OT homolog in subfamily-4, has been determined to 2.4 Å resolution. AfDmpI is also a homohexamer, with a proposed active site cavity that includes Pro-1, but lacks any other residues that are readily identified as catalytic ones related to 4-OT activity. Indeed, the electrostatic potential of the active site differs significantly in that it is mostly neutral, in contrast to the usual electropositive features found in other 4-OT family members, suggesting that AfDmpI might accommodate hydrophobic substrates. A kinetic analysis has been carried out, but does not provide any clues about the type of reaction the enzyme might catalyze.     

Discovery of platelet-type 12-human lipoxygenase selective inhibitors by high-throughput screening of structurally diverse libraries

Human lipoxygenases (hLO) have been implicated in a variety of diseases and cancers and each hLO isozyme appears to have distinct roles in cellular biology. This fact emphasizes the need for discovering selective hLO inhibitors for both understanding the role of specific lipoxygenases in the cell and developing pharmaceutical therapeutics. To this end, we have modified a known lipoxygenase assay for high-throughput (HTP) screening of both the National Cancer Institute (NCI) and the UC Santa Cruz marine extract library (UCSC-MEL) in search of platelet-type 12-hLO (12-hLO) selective inhibitors. The HTP screen led to the characterization of five novel 12-hLO inhibitors from the NCI repository. One is the potent but non-selective michellamine B, a natural product, anti-viral agent. The other four compounds were selective inhibitors against 12-hLO, with three being synthetic compounds and one being alpha-mangostin, a natural product, caspase-3 pathway inhibitor. In addition, a selective inhibitor was isolated from the UCSC-MEL (neodysidenin), which has a unique chemical scaffold for a hLO inhibitor. Due to the unique structure of neodysidenin, steady-state inhibition kinetics were performed and its mode of inhibition against 12-hLO was determined to be competitive (K(i)=17microM) and selective over reticulocyte 15-hLO-1 (K(i) 15-hLO-1/12-hLO>30).     

Novel chemolithotrophic,thermophilic, anaerobic bacteria <Emphasis Type="Italic">Thermolithobacter ferrireducens</Emphasis> gen. nov., sp. nov. and <Emphasis Type="Italic">Thermolithobacter carboxydivorans</Emphasis> sp. nov.

Three thermophilic strains of chemolithoautotrophic Fe(III)-reducers were isolated from mixed sediment and water samples (JW/KA-1 and JW/KA-2(T): Calcite Spring, Yellowstone N.P., WY, USA; JW/JH-Fiji-2: Savusavu, Vanu Levu, Fiji). All were Gram stain positive rods (approximately 0.5 x 1.8 microm). Cells occurred singly or in V-shaped pairs, and they formed long chains in complex media. All utilized H(2) to reduce amorphous iron (III) oxide/hydroxide to magnetite at temperatures from 50 to 75 degrees C (opt. approximately 73 degrees C). Growth occurred within the pH(60C) range of 6.5-8.5 (opt. pH(60C) 7.1-7.3). Magnetite production by resting cells occurred at pH(60C) 5.5-10.3 (opt. 7.3). The iron (III) reduction rate was 1.3 mumol Fe(II) produced x h(-1) x ml(-1) in a culture with 3 x 10(7) cells, one of the highest rates reported. In the presence or absence of H(2), JW/KA-2(T) did not utilize CO. The G + C content of the genomic DNA of the type strain is 52.7 +/- 0.3 mol%. Strains JW/KA-1 and JW/KA-2(T) each contain two different 16S rRNA gene sequences. The 16S rRNA gene sequences from JW/KA-1, JW/KA-2(T), or JW/JH-Fiji-2 possessed >99% similarity to each other but also 99% similarity to the 16S rRNA gene sequence from the anaerobic, thermophilic, hydrogenogenic CO-oxidizing bacterium 'Carboxydothermus restrictus' R1. DNA-DNA hybridization between strain JW/KA-2(T) and strain R1(T) yielded 35% similarity. Physiological characteristics and the 16S rRNA gene sequence analysis indicated that the strains represent two novel species and are placed into the novel genus Thermolithobacter within the phylum 'Firmicutes'. In addition, the levels of 16S rRNA gene sequence similarity between the lineage containing the Thermolithobacter and well-established members of the three existing classes of the 'Firmicutes' is less than 85%. Therefore, Thermolithobacter is proposed to constitute the first genus within a novel class of the 'Firmicutes', Thermolithobacteria. The Fe(III)-reducing Thermolithobacter ferrireducens gen. nov., sp. nov. is designated as the type species with strain JW/KA-2(T) (ATCC 700985(T), DSM 13639(T)) as its type strain. Strain R1(T) is the type strain for the hydrogenogenic, CO-oxidizing Thermolithobacter carboxydivorans sp. nov. (DSM 7242(T), VKM 2359(T)).