Homocysteine stimulates monocyte chemoattractant protein-1 expression in mesangial cells via NF-kappaB activation

Hyperhomocysteinemia is regarded as an independent risk factor for cardiovascular disorders. Although renal dysfunction or failure is one of the important factors causing hyperhomocysteinemia, the role of homocysteine (Hcy) in the development of glomerulosclerosis is largely unknown. One of the key events in the pathogenesis of glomerulosclerosis is the infiltration of circulating monocytes into affected glomeruli. The objective of the present study was to investigate the effect of Hcy on the expression of monocyte chemoattractant protein-1 (MCP-1) in kidney mesangial cells and the mechanisms involved. Levels of MCP-1 and mRNA were significantly elevated in Hcy-treated rat mesangial cells. This increase was associated with activation of NF-kappaB as a result of increased phosphorylation of the inhibitor protein IkappaBalpha. Monocyte chemotactic activity in these cells was also enhanced. In addition, there was a significant elevation of superoxide anion produced by Hcy-treated cells, which preceded the increased phosphorylation of IkappaBalpha. Addition of superoxide dismutase or NF-kappaB inhibitors to the culture medium abolished Hcy-induced NF-kappaB activation and MCP-1 expression. Taken together, these results indicate that Hcy induced MCP-1 expression in mesangial cells. Such a process was mediated by oxidative stress and NF-kappaB activation. This may further aggravate renal function in patients with hyperhomocysteinemia.     

Seeded Growth Route to Noble Calcium Carbonate Nanocrystal

A solution-phase route has been considered as the most promising route to synthesize noble nanostructures. A majority of their synthesis approaches of calcium carbonate (CaCO₃) are based on either using fungi or the CO₂ bubbling methods. Here, we approached the preparation of nano-precipitated calcium carbonate single crystal from salmacis sphaeroides in the presence of zwitterionic or cationic biosurfactants without external source of CO₂. The calcium carbonate crystals were rhombohedron structure and regularly shaped with side dimension ranging from 33–41 nm. The high degree of morphological control of CaCO₃ nanocrystals suggested that surfactants are capable of strongly interacting with the CaCO₃ surface and control the nucleation and growth direction of calcium carbonate nanocrystals. Finally, the mechanism of formation of nanocrystals in light of proposed routes was also discussed.     

Nuclear Targeting of 6-Phosphofructo-2-kinase (PFKFB3) Increases Proliferation via Cyclin-dependent Kinases

The regulation of metabolism and growth must be tightly coupled to guarantee the efficient use of energy and anabolic substrates throughout the cell cycle. Fructose 2,6-bisphosphate (Fru-2,6-BP) is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in glycolysis. The concentration of Fru-2,6-BP in mammalian cells is set by four 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1–4), which interconvert fructose 6-phosphate and Fru-2,6-BP. The relative functions of the PFKFB3 and PFKFB4 enzymes are of particular interest because they are activated in human cancers and increased by mitogens and low oxygen. We examined the cellular localization of PFKFB3 and PFKFB4 and unexpectedly found that whereas PFKFB4 localized to the cytoplasm (i.e. the site of glycolysis), PFKFB3 localized to the nucleus. We then overexpressed PFKFB3 and observed no change in glucose metabolism but rather a marked increase in cell proliferation. These effects on proliferation were completely abrogated by mutating either the active site or nuclear localization residues of PFKFB3, demonstrating a requirement for nuclear delivery of Fru-2,6-BP. Using protein array analyses, we then found that ectopic expression of PFKFB3 increased the expression of several key cell cycle proteins, including cyclin-dependent kinase (Cdk)-1, Cdc25C, and cyclin D3 and decreased the expression of the cell cycle inhibitor p27, a universal inhibitor of Cdk-1 and the cell cycle. We also observed that the addition of Fru-2,6-BP to HeLa cell lysates increased the phosphorylation of the Cdk-specific Thr-187 site of p27. Taken together, these observations demonstrate an unexpected role for PFKFB3 in nuclear signaling and indicate that Fru-2,6-BP may couple the activation of glucose metabolism with cell proliferation.Neoplastic transformation and growth require a massive increase in glucose uptake and glycolytic flux not only for energy production but also for the synthesis of nucleic acids, amino acids, and fatty acids. A central control point of glycolysis is the negative allosteric regulation of a rate-limiting enzyme, phosphofructokinase-1 (PFK-1),² by ATP (i.e. the Pasteur effect) (1, 2). When intracellular ATP production exceeds usage, ATP inhibits PFK-1 and glycolytic flux. Fructose 2,6-bisphosphate (Fru-2,6-BP) is a potent allosteric activator of PFK-1 that overrides this inhibitory influence of ATP on PFK-1, allowing forward flux of the entire pathway (3–5).The steady-state cellular concentration of Fru-2,6-BP is dependent on the activities of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB), which are encoded by four independent genes (PFKFB1–4) (6, 7). The PFKFB3 mRNA is distinguished by the presence of multiple copies of an AUUUA instability motif in its 3′-untranslated region and the PFKFB3 protein product has a high kinase:phosphatase activity ratio (740:1) (8). PFKFB3 mRNA is overexpressed by rapidly proliferating transformed cells and the PFKFB3 protein is highly expressed in solid tumors and leukemias (8–11). PFKFB3 expression is increased in response to several mitogenic stimuli, including progesterone, serum, and insulin (12–14). These studies indicate that the PFKFB3 enzyme may serve an essential function in the regulation of glucose metabolism during cell proliferation.The PFKFB3 mRNA is spliced into several variants that encode distinct carboxyl-terminal domains (9, 15). Importantly, the functional consequences of the disparate carboxyl-terminal variants of PFKFB3 are unknown. The mRNA splice variant 5 is the dominant PFKFB3 mRNA in human brain, several transformed cells, and colon adenocarcinoma tissues (9, 10). In the following series of experiments, we present data that the carboxyl-terminal domain of PFKFB3 variant 5 localizes the enzyme to the nucleus where its product, Fru-2,6-BP, increases the expression and activity of cyclin-dependent kinase-1. These data demonstrate a heretofore unidentified function of the PFKFB3 enzyme that is distinct from glycolysis, and provide a potential mechanism for the coupling of metabolism and proliferation.