Rapamycin induces binding activity to the terminal oligopyrimidine tract of ribosomal protein mRNA in rats

Cathepsin G, elastase, and proteinase 3 are serine proteinases released by activated neutrophils. Cathepsin G can cleave angiotensinogen to release angiotensin II, but this activity has not been previously reported for elastase or proteinase 3. In this study we show that elastase and proteinase 3 can release angiotensin I from angiotensinogen and release angiotensin II from angiotensin I and angiotensinogen. The relative order of potency in releasing angiotensin II by the three proteinases at equivalent concentrations is cathepsin G > elastase > proteinase 3. When all three proteinases are used together, the release of angiotensin II is greater than the sum of the release when each proteinase is used individually. Cathepsin G and elastase can also degrade angiotensin II, reactions which might be important in regulating the activity of angiotensin II. The release and degradation of angiotensin II by the neutrophil proteinases are reactions which could play a role in the local inflammatory response and wound healing.     

Insulin activates CCAAT/enhancer binding proteins and proinflammatory gene expression through the phosphatidylinositol 3-kinase pathway in vascular smooth muscle cells

Phosphatidylinositol 3-kinase (PI3K) is a key molecule mediating signals of insulin in vascular smooth muscle cells (VSMCs). To examine the effect of chronic activation of PI3K on the gene expression of VSMCs, membrane-targeted p110CAAX, a catalytic subunit of PI3K, was overexpressed in rat VSMCs by adenovirus-mediated gene transfer. Similar to insulin's effects, cells overexpressing p110CAAX exhibited a 10- to 15-fold increase in monocyte chemoattractant protein-1 (MCP-1) mRNA expression as compared with the control cells. Electrophoretic mobility shift assay analysis showed that the overexpression of p110CAAX activated neither the NF-kappaB binding nor the activator protein (AP-1) binding activities. We found that two CCAAT/enhancer binding protein (C/EBP) binding sites located between 2.6 and 3.6 kb upstream of the MCP-1 gene were responsible for the induction by p110CAAX. The overexpression of C/EBP-beta and C/EBP-delta but not C/EBP-alpha caused 6- to 8-fold induction of MCP-1 promoter activity. Consistently, the overexpression of p110CAAX as well as insulin induced mRNA expression and nuclear expression of C/EBP-beta and C/EBP-delta in VSMCs. These results clearly indicate that the activation of PI3K induced proinflammatory gene expression through activating C/EBP-beta and C/EBP-delta but not NF-kappaB, which may explain the proinflammatory effect of insulin in the insulin-resistant state.     

Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis

The specific biochemical steps required for glucose-regulated insulin exocytosis from beta-cells are not well defined. Elevation of glucose leads to increases in cytosolic [Ca2+]i and biphasic release of insulin from both a readily releasable and a storage pool of beta-granules. The effect of elevated [Ca2+]i on phosphorylation of isolated beta-granule membrane proteins was evaluated, and the phosphorylation of four proteins was found to be altered by [Ca2+]i. One (a 18/20-kDa doublet) was a Ca2+-dependent increase in phosphorylation, and, surprisingly, three others (138, 42, and 36 kDa) were Ca2+-dependent dephosphorylations. The 138-kDa beta-granule phosphoprotein was found to be kinesin heavy chain (KHC). At low levels of [Ca2+]i KHC was phosphorylated by casein kinase 2, but KHC was rapidly dephosphorylated by protein phosphatase 2B beta (PP2Bbeta) as [Ca2+]i increased. Inhibitors of PP2B specifically reduced the second, microtubule-dependent, phase of insulin secretion, suggesting that dephosphorylation of KHC was required for transport of beta-granules from the storage pool to replenish the readily releasable pool of beta-granules. This is distinct from synaptic vesicle exocytosis, because neurotransmitter release from synaptosomes did not require a Ca2+-dependent KHC dephosphorylation. These results suggest a novel mechanism for regulating KHC function and beta-granule transport in beta-cells that is mediated by casein kinase 2 and PP2B. They also implicate a novel regulatory role for PP2B/calcineurin in the control of insulin secretion downstream of a rise in [Ca2+]i.     

F(0) of ATP synthase is a rotary proton channel. Obligatory coupling of proton translocation with rotation of c-subunit ring

Coupling of proton flow and rotation in the F(0) motor of ATP synthase was investigated using the thermophilic Bacillus PS3 enzyme expressed functionally in Escherichia coli cells. Cysteine residues introduced into the N-terminal regions of subunits b and c of ATP synthase (bL2C/cS2C) were readily oxidized by treating the expressing cells with CuCl(2) to form predominantly a b-c cross-link with b-b and c-c cross-links being minor products. The oxidized ATP synthases, either in the inverted membrane vesicles or in the reconstituted proteoliposomes, showed drastically decreased proton pumping and ATPase activities compared with the reduced ones. Also, the oxidized F(0), either in the F(1)-stripped inverted vesicles or in the reconstituted F(0)-proteoliposomes, hardly mediated passive proton translocation through F(0). Careful analysis using single mutants (bL2C or cS2C) as controls indicated that the b-c cross-link was responsible for these defects. Thus, rotation of the c-oligomer ring relative to subunit b is obligatory for proton translocation; if there is no rotation of the c-ring there is no proton flow through F(0).     

Mature glycosylation and trafficking of nicastrin modulate its binding to presenilins

Nicastrin is an integral component of the high molecular weight presenilin complexes that control proteolytic processing of the amyloid precursor protein and Notch. We report here that nicastrin is most probably a type 1 transmembrane glycoprotein that is expressed at moderate levels in the brain and in cultured neurons. Immunofluorescence studies demonstrate that nicastrin is localized in the endoplasmic reticulum, Golgi, and a discrete population of vesicles. Glycosidase analyses reveal that endogenous nicastrin undergoes a conventional, trafficking-dependent maturation process. However, when highly expressed in transfected cells, there is a disproportionate accumulation of the endo-beta-N-acetylglucosaminidase H-sensitive, immature form, with no significant increase in the levels of the fully mature species. Immunoprecipitation revealed that presenilin-1 interacts preferentially with mature nicastrin, suggesting that correct trafficking and co-localization of the presenilin complex components are essential for activity. These findings demonstrate that trafficking and post-translational modifications of nicastrin are tightly regulated processes that accompany the assembly of the active presenilin complexes that execute gamma-secretase cleavage. These results also underscore the caveat that simple overexpression of nicastrin in transfected cells may result in the accumulation of large amounts of the immature protein, which is apparently unable to assemble into the active complexes capable of processing amyloid precursor protein and Notch.     

Sequence analysis of 193.4 and 83.9 kbp of mouse and chicken genomic DNAs containing the entire Prkdc (DNA-PKcs) gene

The catalytic subunit of DNA-dependent protein kinase plays critical roles in nonhomologous end joining in repair of DNA double-strand breaks and V(D)J recombination. In addition to the SCID phenotype, it has been suggested that the molecule contributes to the polymorphic variations in radiosensitivity and susceptibility to cancer in mouse strains. Here we show the nucleotide sequence of approximately 193-kbp and 84-kbp genomic regions encoding the entire Prkdc gene (also known as DNA-PKcs) in the mouse and chicken, respectively. A large retroposon was found in intron 51 in the mouse but not in the human or chicken. Comparative analyses of the genome strongly suggested that the region contains only two genes for Prkdc and Mcm4; however, several conserved sequences and cis elements were also predicted.     

Causal relationship between the loss of RUNX3 expression and gastric cancer

Runx3/Pebp2alphaC null mouse gastric mucosa exhibits hyperplasias due to stimulated proliferation and suppressed apoptosis in epithelial cells, and the cells are resistant to growth-inhibitory and apoptosis-inducing action of TGF-beta, indicating that Runx3 is a major growth regulator of gastric epithelial cells. Between 45% and 60% of human gastric cancer cells do not significantly express RUNX3 due to hemizygous deletion and hypermethylation of the RUNX3 promoter region. Tumorigenicity of human gastric cancer cell lines in nude mice was inversely related to their level of RUNX3 expression, and a mutation (R122C) occurring within the conserved Runt domain abolished the tumor-suppressive effect of RUNX3, suggesting that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer.