Small-molecule inhibitors of FABP4/5 ameliorate dyslipidemia but not insulin resistance in mice with diet-induced obesity

Fatty acid binding protein-4 (FABP4) and FABP5 are two closely related FA binding proteins expressed primarily in adipose tissue and/or macrophages. The small-molecule FABP4 inhibitor BMS309403 was previously reported to improve insulin sensitivity in leptin-deficient Lep(ob)/Lep(ob) (ob/ob) mice. However, this compound was not extensively characterized in the more physiologically relevant animal model of mice with diet-induced obesity (DIO). Here, we report the discovery and characterization of a novel series of FABP4/5 dual inhibitors represented by Compounds 1-3. Compared with BMS309403, the compounds had significant in vitro potency toward both FABP4 and FABP5. In cell-based assays, Compounds 2 and 3 were more potent than BMS309403 to inhibit lipolysis in 3T3-L1 adipocytes and in primary human adipocytes. They also inhibited MCP-1 release from THP-1 macrophages as well as from primary human macrophages. When chronically administered to DIO mice, BMS309403 and Compound 3 reduced plasma triglyceride and free FA levels. Compound 3 reduced plasma free FAs at a lower dose level than BMS309403. However, no significant change was observed in insulin, glucose, or glucose tolerance. Our results indicate that the FABP4/5 inhibitors ameliorate dyslipidemia but not insulin resistance in DIO mice.     

Pro-inflammatory effect of fibrinogen and FDP on vascular smooth muscle cells by IL-6, TNF-α and iNOS

AimsAtherosclerosis is a chronic inflammatory response of the arterial wall to multiple endothelial injuries. As one of the inflammatory markers, fibrinogen has been implicated in pathogenesis of atherosclerosis. But, it is not completely understood whether atherogenesis of fibrinogen is related to its pro-inflammatory effect on vascular smooth muscle cells (VSMCs). The purpose of the present study was to observe effects of fibrinogen and fibrin degradation products (FDP) on interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and inducible nitric oxide synthase (iNOS) generation in rat VSMCs.Main methodsRat VSMCs were cultured, and fibrinogen and FDP were used as stimulants for IL-6, TNF-α, and iNOS. IL-6 and TNF-α level in the supernatant were measured by ELISA, mRNA expression of IL-6, TNF-α, and iNOS were assayed with RT-PCR, and protein expression of iNOS was detected with western blot and immunocytochemistry.Key findingsFibrinogen and FDP both significantly stimulated mRNA and protein expressions of IL-6, TNF-α and iNOS in VSMCs in time- and concentration-dependent ways. The pro-inflammatory potency of FDP is higher than fibrinogen, which seems to mean that smaller fragments of the protein have greater pro-inflammatory activity. Fibrinogen and FDP promote more protein expressions of IL-6 and TNF-α compared to iNOS, suggesting that fibrinogen and FDP produce a pro-inflammatory effect on VSMCs mainly by IL-6 and TNF-α.SignificanceThese findings are helpful to better understand pro-inflammatory effect of fibrinogen on VSMCs involved in atherogenesis, and imply a therapeutic strategy targeting hyperfibrinogenemia in atherosclerosis.     

Genetic control of a transition from black to straw-white seed hull in rice domestication

The genetic mechanism involved in a transition from the black-colored seed hull of the ancestral wild rice (Oryza rufipogon and Oryza nivara) to the straw-white seed hull of cultivated rice (Oryza sativa) during grain ripening remains unknown. We report that the black hull of O. rufipogon was controlled by the Black hull4 (Bh4) gene, which was fine-mapped to an 8.8-kb region on rice chromosome 4 using a cross between O. rufipogon W1943 (black hull) and O. sativa indica cv Guangluai 4 (straw-white hull). Bh4 encodes an amino acid transporter. A 22-bp deletion within exon 3 of the bh4 variant disrupted the Bh4 function, leading to the straw-white hull in cultivated rice. Transgenic study indicated that Bh4 could restore the black pigment on hulls in cv Guangluai 4 and Kasalath. Bh4 sequence alignment of all taxa with the outgroup Oryza barthii showed that the wild rice maintained comparable levels of nucleotide diversity that were about 70 times higher than those in the cultivated rice. The results from the maximum likelihood Hudson-Kreitman-Aguade test suggested that the significant reduction in nucleotide diversity in rice cultivars could be caused by artificial selection. We propose that the straw-white hull was selected as an important visual phenotype of nonshattered grains during rice domestication.     

Axon regeneration mechanisms: insights from C. elegans

Understanding the mechanisms of axon regeneration is of great importance to the development of therapeutic treatments for spinal cord injury or stroke. Axon regeneration has long been studied in diverse vertebrate and invertebrate models, but until recently had not been analyzed in the genetically tractable model organism Caenorhabditis elegans. The small size, simple neuroanatomy, and transparency of C. elegans allows single fluorescently labeled axons to be severed in live animals using laser microsurgery. Many neurons in C. elegans are capable of regenerative regrowth, and can in some cases re-establish functional connections. Large-scale genetic screens have begun to elucidate the genetic basis of axon regrowth.     

Processes of organic carbon in mangrove ecosystems

In addition to carbon accumulation in plants, processes of organic carbon in mangrove ecosystems include origins of sediment organic carbon, carbon fluxes between mangroves and their adjacent systems (coastal waters and atmosphere), and cycling processes. Sediment organic carbon originates from suspending solids in coastal waters, mangrove plants and benthic algae. In mangroves with low organic carbon content in sediments, tidal seawater is the main origin of sediment organic carbon, while in mangroves with high sediment organic carbon contents, sediment organic carbon mainly originates from mangrove plants. Due to tidal flush, there is large material exchange between mangrove ecosystems and their adjacent coastal waters. In China, exports of organic carbon in litter falls and dissolved organic carbon from mangroves to their adjacent coastal waters have not been documented. Processes of mangrove litter falls, including production, decomposition, export and animal consumption, determine linkages among organic carbon among mangrove plants, secondary production and coastal ocean. Consumers especially benthic animals may influence organic carbon in mangrove ecosystems, because (1) their consumption rates are high, and their selective feeding on some food sources will change the relative quantities of export, bury and mineralization of organic carbon from different origins; (2) their consumption is much more than assimilation, resulting in the changes in sizes, forms and qualities of non-assimilated organic matters, and then the changes in availability of export, consumption or mineralization of organic carbon. Respiration and sulfate reduction are important mineralization processes of organic carbon in mangrove sediments. Mineralization rates of organic carbon in mangrove sediments are influenced by quantities, activities and particle sizes of organic matters, and other factors such as forest ages, root activities and animal burrowing activities. Researches on processes of mangrove organic carbon should be based on open systems, and ecological processes of organic carbon should be coupled with vegetation restoration.     

Cytoplasmic linker protein-170 enhances spreading and phagocytosis in activated macrophages by stabilizing microtubules

Activation of macrophages causes increased cell spreading, increased secretion of cytokines and matrix metalloproteinases, and enhanced phagocytosis. The intracellular mechanisms driving the up-regulation of these activities have not been completely clarified. We observe that classical activation of murine resident peritoneal or RAW 264.7 macrophages with a combination of IFN-gamma and LPS induces an increase in stabilized cytoplasmic microtubules (MTs), measured with an anti-acetylated alpha-tubulin Ab. We examined the mechanism of this MT stabilization and find that macrophage activation causes redistribution of the MT plus-end tracking protein, cytoplasmic linker protein-170 (CLIP-170). CLIP-170 is localized at the distal plus-ends of MTs in resting macrophages, but accumulates along the length of MTs in IFN-gamma/LPS-activated cells. A direct involvement of CLIP-170 in MT stabilization has not been thoroughly established. In this study, we show that expression of a mutant CLIP-170 chimeric protein (dominant-negative CLIP-170-GFP), lacking the MT-binding domain, prevents MT stabilization in activated RAW 264.7 macrophages. Furthermore, we find enhanced CLIP-170 association with MTs and MT stabilization by treating resting macrophages with okadaic acid, implicating the protein phosphatase 2A in CLIP-170 binding and MT stabilization in RAW 264.7 cells. Finally, we observed enhanced cell spreading and phagocytosis in both IFN-gamma/LPS-activated and okadaic acid-treated resting RAW 264.7 cells, which are markedly reduced in activated cells expressing dominant-negative CLIP-170-GFP. These results identify CLIP-170 as a key regulator of MT stabilization and establish a prominent role for stabilized MTs in cell spreading and phagocytosis in activated macrophages.     

Molecular and functional dissection of a putative RNA-binding region in yeast mitochondrial leucyl-tRNA synthetase

Aminoacylation and editing by leucyl-tRNA synthetases (LeuRS) require migration of the tRNA acceptor stem end between the canonical aminoacylation core and a separate domain called CP1 that is responsible for amino acid editing. The LeuRS CP1 domain can also support group I intron RNA splicing in the yeast mitochondria, although splicing-sensitive sites have been identified on the main body. The RDW peptide, a highly conserved peptide within an RDW-containing motif, resides near one of the beta-strand linkers that connects the main body to the CP1 domain. We hypothesized that the RDW peptide was important for interactions of one or more of the LeuRS-RNA complexes. An assortment of X-ray crystallography structures suggests that the RDW peptide is dynamic and forms unique sets of interactions with the aminoacylation and editing complexes. Mutational analysis identified specific sites within the RDW peptide that failed to support protein synthesis activity in complementation experiments. In vitro enzymatic investigations of mutations at Trp445, Arg449, and Arg451 in yeast mitochondrial LeuRS suggested that these sites within the RDW peptide are critical to the aminoacylation complex, but impacted amino acid editing activity to a much less extent. We propose that these highly conserved sites primarily influence productive tRNA interactions in the aminoacylation complex.     

Necrotic death of neurons following an excitotoxic insult is prevented by a peptide inhibitor of c-jun N-terminal kinase

Peptide inhibitors of c-Jun N-terminal kinase (JNK) have been shown to potently protect against cerebral ischemia. The protective effect has been ascribed to prevention of apoptosis, but cell death following cerebral ischemia is a consequence of both apoptotic and necrotic cell death. We evaluated whether a peptide inhibitor (TAT-TIJIP) of JNK could prevent necrotic cell death in an in vitro model of excitotoxic neuronal death. We find that TAT-TIJIP effectively prevented cell death by interfering with several processes which have been identified as leading to cell death by necrosis. In particular, reactive oxygen species production was reduced, as indicated by an 88% decrease in the rate of dihydroethidium fluorescence in the presence of TAT-TIJIP. Furthermore, TAT-TIJIP attenuated the increase in cytosolic calcium following the excitotoxic insult. The potent neuroprotective properties of JNK peptide inhibitors likely reflects their abilities to prevent cell death by necrosis as well as apoptosis.     

Neutralization of human papillomavirus with monoclonal antibodies reveals different mechanisms of inhibition

The mechanisms of human papillomavirus (HPV) neutralization by antibodies are incompletely understood. We have used HPV16 pseudovirus infection of HaCaT cells to analyze how several neutralizing monoclonal antibodies (MAbs) generated against HPV16 L1 interfere with the process of keratinocyte infection. HPV16 capsids normally bind to both the cell surface and extracellular matrix (ECM) of HaCaT cells. Surprisingly, two strongly neutralizing MAbs, V5 and E70, did not prevent attachment of capsids to the cell surface. However, they did block association with the ECM and prevented internalization of cell surface-bound capsids. In contrast, MAb U4 prevented binding to the cell surface but not to the ECM. The epitope recognized by U4 was inaccessible when virions were bound to the cell surface but became accessible after endocytosis, presumably coinciding with receptor detachment. Treatment of capsids with heparin, which is known to interfere with binding to cell surface heparan sulfate proteoglycans (HSPGs), also resulted in HPV16 localization to the ECM. These results suggest that the U4 epitope on the intercapsomeric C-terminal arm is likely to encompass the critical HSPG interaction residues for HPV16, while the V5 and E70 epitopes at the apex of the capsomer overlap the ECM-binding sites. We conclude that neutralizing antibodies can inhibit HPV infection by multiple distinct mechanisms, and understanding these mechanisms can add insight to the HPV entry processes.     

Conserved alpha-helix acts as autoinhibitory sequence in AMP-activated protein kinase alpha subunits

AMP-activated protein kinase (AMPK) acts as an energy sensor, being activated by metabolic stresses and regulating cellular metabolism. AMPK is a heterotrimer consisting of a catalytic alpha subunit and two regulatory subunits, beta and gamma. It had been reported that the mammalian AMPK alpha subunit contained an autoinhibitory domain (alpha1: residues 313-392) and had little kinase activity. We have found that a conserved short segment of the alpha subunit (alpha1-(313-335)), which includes a predicted alpha-helix, is responsible for alpha subunit autoinhibition. The role of the residues in this segment for autoinhibition was further investigated by systematic site-directed mutation. Several hydrophobic and charged residues, in particular Leu-328, were found to be critical for alpha1 autoinhibition. An autoinhibitory structural model of human AMPK alpha1-(1-335) was constructed and revealed that Val-298 interacts with Leu-328 through hydrophobic bonding at a distance of about 4 A and may stabilize the autoinhibitory conformation. Further mutation analysis showed that V298G mutation significantly activated the kinase activity. Moreover, the phosphorylation level of acetyl-CoA carboxylase, the AMPK downstream substrate, was significantly increased in COS7 cells overexpressing AMPK alpha1-(1-394) with deletion of residues 313-335 (Deltaalpha394) and a V298G or L328Q mutation, and the glucose uptake was also significantly enhanced in HepG2 cells transiently transfected with Deltaalpha394, V298G, or L328Q mutants, which indicated that these AMPK alpha1 mutants are constitutively active in mammalian cells and that interaction between Leu-328 and Val-298 plays an important role in AMPK alpha autoinhibitory function.