Yeast cytochrome c is a sequence-specific DNA-binding protein

A protein binding to the alcohol oxidase 2 upstream activation sequence (AOX2UAS) of the methylotropic yeast, Pichia pastoris, has been purified and identified as cytochrome c (cyt c). Cyt c purified from P. pastoris or Saccharomyces cerevisiae binds to AOX2UAS. Specific point mutations in AOX2UAS abolish cyt c binding. We conclude that yeast cyt c is a sequence-specific DNA-binding protein and may have a regulatory role in the nucleus.     

Decreased expression of the Ets family transcription factor Fli-1 markedly prolongs survival and significantly reduces renal disease in MRL/lpr mice

Increased Fli-1 mRNA is present in PBLs from systemic lupus erythematosus patients, and transgenic overexpression of Fli-1 in normal mice leads to a lupus-like disease. We report in this study that MRL/lpr mice, an animal model of systemic lupus erythematosus, have increased splenic expression of Fli-1 protein compared with BALB/c mice. Using mice with targeted gene disruption, we examined the effect of reduced Fli-1 expression on disease development in MRL/lpr mice. Complete knockout of Fli-1 is lethal in utero. Fli-1 protein expression in heterozygous MRL/lpr (Fli-1(+/-)) mice was reduced by 50% compared with wild-type MRL/lpr (Fli-1(+/+)) mice. Fli-1(+/-) MRL/lpr mice had significantly decreased serum levels of total IgG and anti-dsDNA Abs as disease progressed. Fli-1(+/-) MRL/lpr mice had significantly increased splenic CD8(+) and naive T cells compared with Fli-1(+/+) MRL/lpr mice. Both in vivo and in vitro production of MCP-1 were significantly decreased in Fli-1(+/-) MRL/lpr mice. The Fli-1(+/-) mice had markedly decreased proteinuria and significantly lower pathologic renal scores. At 48 wk of age, survival was significantly increased in the Fli-1(+/-) MRL/lpr mice, as 100% of Fli-1(+/-) MRL/lpr mice were alive, in contrast to only 27% of Fli-1(+/+) mice. These findings indicate that Fli-1 expression is important in lupus-like disease development, and that modulation of Fli-1 expression profoundly decreases renal disease and improves survival in MRL/lpr mice.     

Potent antiproliferative effects of resveratrol on human osteosarcoma SJSA1 cells: Novel cellular mechanisms involving the ERKs/p53 cascade

The chemopreventive activity of resveratrol (RSVL) has been demonstrated in several types of cancer. However, its effects and the underling mechanisms remain poorly understood. In this study, we investigated the involvement of the mitogen activated protein kinase (MAPK)/p53 signal transduction mechanism in RSVL-induced growth inhibition using a human osteosarcoma cell line. We demonstrate that RSVL reduces cell viability and growth of SJSA1 osteosarcoma cells. Morphological profiles and 4,6-diamidino-2-phenylindole nuclear staining of RSVL-treated cells indicated marked nuclear fragmentation. Cleavage of the (116-kDa) poly(ADP-ribose) polymerase protein into an 89-kDa fragment (a proapoptotic marker system) was substantially augmented by RSVL treatment. RSVL-dependent growth impairment was preceded by enhanced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 (at Thr202/Tyr204). Likewise, RSVL increased the phosphorylation of p53 tumor suppressor protein (at Ser15). The effects of RSVL on ERKs and on p53 phosphorylation were abrogated by either the MAPK inhibitor PD98059 or the p53 inhibitor pifithrine-alpha. The present study indicates that RSVL antiproliferative effects on osteosarcoma cells are mediated by the activation of the ERKs/p53 signaling pathway and therefore identifies new targets for strategies to treat and/or prevent osteosarcoma.     

Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels

The voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane mediates metabolic flow, Ca(2+), and cell death signaling between the endoplasmic reticulum (ER) and mitochondrial networks. We demonstrate that VDAC1 is physically linked to the endoplasmic reticulum Ca(2+)-release channel inositol 1,4,5-trisphosphate receptor (IP(3)R) through the molecular chaperone glucose-regulated protein 75 (grp75). Functional interaction between the channels was shown by the recombinant expression of the ligand-binding domain of the IP(3)R on the ER or mitochondrial surface, which directly enhanced Ca(2+) accumulation in mitochondria. Knockdown of grp75 abolished the stimulatory effect, highlighting chaperone-mediated conformational coupling between the IP(3)R and the mitochondrial Ca(2+) uptake machinery. Because organelle Ca(2+) homeostasis influences fundamentally cellular functions and death signaling, the central location of grp75 may represent an important control point of cell fate and pathogenesis.     

MGOUN3: evidence for chromatin-mediated regulation of FLC expression

The MGOUN3(MGO3)/BRUSHY1(BRU1)/TONSOKU(TSK) gene of Arabidopsis thaliana encodes a nuclear leucine-glycine-asparagine (LGN) domain protein that may be implicated in chromatin dynamics and genome maintenance. Mutants with defects in MGO3 display a fasciated stem and disorganized meristem structures. The transition to flowering was examined in mgo3 mutants and it was found that, under short days, the mutants flowered significantly earlier than the wild-type plants. Study of flowering-time associated gene expression showed that the floral transition inhibitor gene FLC was under-expressed in the mutant background. Ectopic expression of the flower-specific genes AGAMOUS (AG), PISTILLATA (PI), and SEPALLATA3 (SEP3) in mgo3 vegetative organs was also detected. Western blot and chromatin immunoprecipitation experiments suggested that histone H3 acetylation may be altered in the mgo3 background. Together, these data suggest that MGO3 is required for the correct transition to flowering and that this may be mediated by histone acetylation and associated changes in FLC expression.     

The antifungal protein AFP secreted by Aspergillus giganteus does not cause detrimental effects on certain mammalian cells

The antifungal protein AFP is a small, cystein-rich protein secreted by the imperfect ascomycete Aspergillus giganteus. The protein efficiently inhibits the growth of filamentous fungi, including a variety of serious human and plant pathogens mainly of the genera Aspergillus and Fusarium, whereas AFP does not affect the growth of yeast and bacteria. This restricted susceptibility range makes it very attractive for medical or biotechnological use to combat fungal infection and contamination. We, therefore, analyzed whether AFP affects the growth or function of a number of mammalian cells. Here we show that the protein neither provokes any cytotoxic effects on human endothelial cells isolated from the umbilical vein nor activates the immune system. Moreover, potassium currents of neurons and astrocytes do not change in the presence of AFP and neither excitatory processes nor the intracellular calcium homeostasis of cultured skeletal muscle myotubes are affected by AFP. Our data, therefore, suggest that AFP is indeed a promising candidate for the therapeutic or biotechnological use as a potential antifungal agent.     

Fibronectin and laminin enhance engraftibility of cultured hematopoietic stem cells

To test the hypothesis that extracellular matrix (ECM) components maintain stem cell property, murine bone marrow (BM) cells were expanded in fibronectin and laminin coated plate in the presence of cytokines. We observed significant phenotypic and functional improvement of expanded cells. In 10 days, 800-fold expansion of colony-forming unit-granulocyte erythrocyte monocyte megakaryocyte (CFU-GEMM) was observed in the cultured cells. No apparent activation of cell cycle was observed, but CD29 and very late antigen-4 (VLA-4) expression was increased, as compared to the normal BM cells. A fraction of the expanded cells became verapamil sensitive, suggesting upregulation of multi-drug resistant gene(s), as found in the primitive hematopoietic stem cells (HSCs). Competitive repopulation assay confirmed that HSCs compartment was amplified during culture. Overall, our study clearly demonstrated that ex vivo culture of murine HSCs in the presence of fibronectin and laminin resulted in expansion of primitive stem cells and improvement in the marrow engraftibility.     

Synergistic and Antagonistic Effects of Combined Subzero Temperature and High Pressure on Inactivation of Escherichia coli

The combined effects of subzero temperature and high pressure on the inactivation of Escherichia coli K12TG1 were investigated. Cells of this bacterial strain were exposed to high pressure (50 to 450 MPa, 10-min holding time) at two temperatures (−20°C without freezing and 25°C) and three water activity levels (a_w) (0.850, 0.992, and ca. 1.000) achieved with the addition of glycerol. There was a synergistic interaction between subzero temperature and high pressure in their effects on microbial inactivation. Indeed, to achieve the same inactivation rate, the pressures required at −20°C (in the liquid state) were more than 100 MPa less than those required at 25°C, at pressures in the range of 100 to 300 MPa with an a_w of 0.992. However, at pressures greater than 300 MPa, this trend was reversed, and subzero temperature counteracted the inactivation effect of pressure. When the amount of water in the bacterial suspension was increased, the synergistic effect was enhanced. Conversely, when the a_w was decreased by the addition of solute to the bacterial suspension, the baroprotective effect of subzero temperature increased sharply. These results support the argument that water compression is involved in the antimicrobial effect of high pressure. From a thermodynamic point of view, the mechanical energy transferred to the cell during the pressure treatment can be characterized by the change in volume of the system. The amount of mechanical energy transferred to the cell system is strongly related to cell compressibility, which depends on the water quantity in the cytoplasm.     

Which diameter and angle rule provides optimal flow patterns in a coronary bifurcation?

The branching angle and diameter ratio in epicardial coronary artery bifurcations are two important determinants of atherogenesis. Murray's cubed diameter law and bifurcation angle have been assumed to yield optimal flows through a bifurcation. In contrast, we have recently shown a 7/3 diameter law (HK diameter model), based on minimum energy hypothesis in an entire tree structure. Here, we derive a bifurcation angle rule corresponding to the HK diameter model and critically evaluate the streamline flow through HK and Murray-type bifurcations. The bifurcations from coronary casts were found to obey the HK diameter model and angle rule much more than Murray's model. A finite element model was used to investigate flow patterns for coronary artery bifurcations of various types. The inlet velocity and pressure boundary conditions were measured by ComboWire. Y-bifurcation of Murray type decreased wall shear stress-WSS (10%-40%) and created an increased oscillatory shear index-OSI in atherosclerosis-prone regions as compared with HK-type bifurcations. The HK-type bifurcations were found to have more optimal flow patterns (i.e., higher WSS and lower OSI) than Murray-type bifurcations which have been traditionally believed to be optimized. This study has implications for changes in bifurcation angles and diameters in percutaneous coronary intervention.     

Angiotensin II Induces Vascular Endocannabinoid Release, Which Attenuates Its Vasoconstrictor Effect via CB1 Cannabinoid Receptors

In the vascular system angiotensin II (Ang II) causes vasoconstriction via the activation of type 1 angiotensin receptors. Earlier reports have shown that in cellular expression systems diacylglycerol produced during type 1 angiotensin receptor signaling can be converted to 2-arachidonoylglycerol, an important endocannabinoid. Because activation of CB(1) cannabinoid receptors (CB(1)R) induces vasodilation and reduces blood pressure, we have tested the hypothesis that Ang II-induced 2-arachidonoylglycerol release can modulate its vasoconstrictor action in vascular tissue. Rat and mouse skeletal muscle arterioles and mouse saphenous arteries were isolated, pressurized, and subjected to microangiometry. Vascular expression of CB(1)R was demonstrated using Western blot and RT-PCR. In accordance with the functional relevance of these receptors WIN55212, a CB(1)R agonist, caused vasodilation, which was absent in CB(1)R knock-out mice. Inhibition of CB(1)Rs using O2050, a neutral antagonist, enhanced the vasoconstrictor effect of Ang II in wild type but not in CB(1)R knock-out mice. Inverse agonists of CB(1)R (SR141716 and AM251) and inhibition of diacylglycerol lipase using tetrahydrolipstatin also augmented the Ang II-induced vasoconstriction, suggesting that endocannabinoid release modulates this process via CB(1)R activation. This effect was independent of nitric-oxide synthase activity and endothelial function. These data demonstrate that Ang II stimulates vascular endocannabinoid formation, which attenuates its vasoconstrictor effect, suggesting that endocannabinoid release from the vascular wall and CB(1)R activation reduces the vasoconstrictor and hypertensive effects of Ang II.