Satoru Oshiro

Homocysteine Induces Programmed Cell Death in Human Vascular Endothelial Cells through Activation of the Unfolded Protein Response

Severe hyperhomocysteinemia is associated with endothelial cell injury that may contribute to an increased incidence of thromboembolic disease. In this study, homocysteine induced programmed cell death in human umbilical vein endothelial cells as measured by TdT-mediated dUTP nick end labeling assay, DNA ladder formation, induction of caspase 3-like activity, and cleavage of procaspase 3. Homocysteine-induced cell death was specific to homocysteine, was not mediated by oxidative stress, and was mimicked by inducers of the unfolded protein response (UPR), a signal transduction pathway activated by the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum. Dominant negative forms of the endoplasmic reticulum-resident protein kinases IRE1α and -β, which function as signal transducers of the UPR, prevented the activation of glucose-regulated protein 78/immunoglobulin chain-binding protein and C/EBP homologous protein/growth arrest and DNA damage-inducible protein 153 in response to homocysteine. Furthermore, overexpression of the point mutants of IRE1 with defective RNase more effectively suppressed the cell death than the kinase-defective mutant. These results indicate that homocysteine induces apoptosis in human umbilical vein endothelial cells by activation of the UPR and is signaled through IRE1. The studies implicate that the UPR may cause endothelial cell injury associated with severe hyperhomocysteinemia.

Glial cells contribute more to iron and aluminum accumulation but are more resistant to oxidative stress than neuronal cells

Iron (Fe) and aluminum (Al) have been implicated in the pathogenesis of Alzheimers disease (AD). In this study, we examined neuronal and glial cells to clarify which contributes most to metal accumulation after internalization through the transferrin-independent iron uptake (Tf-IU) systems in primary neuronal and glial predominant (NP and GP) cells from rat cerebral cortex, which affect the accumulation of transition metals in a variety of cultured cells. Al more significantly upregulated the Tf-IU activity in GP cells than in NP cells. GP cells were more resistant to Fe and Al exposure than NP cells. However, a chemiluminescence analysis specific for reactive oxygen species (ROS) showed that ROS levels in Fe- or Al-loaded NP cells were twice as high as in Fe- or Al-loaded GP cells. Northern blot analysis and gel retardation assay showed that the Al and Fe exposure taken up by the cells suppress Tf receptor mRNA expression to a greater extent in GP than NP cells, indicating that Al and Fe more markedly accumulate in glial than in neuronal cells. These results suggest that glial cells rather than neuronal cells contribute to the metal accumulation and are more resistant to oxidative stress caused by metals than neuronal cells. The present study may help to explain the pathogenesis of neurodegeneration in AD disorders caused by metal-generated oxidative stress.

Transcriptional activation of heme oxygenase-1 gene in mouse spleen, liver and kidney cells after treatment with lipopolysaccharide or hemoglobin.

Heme oxygenase (HO)−1 catalyzes the conversion of heme to biliverdin, iron and carbon monoxide. HO−1 is induced by many reagents including heme, Hb and lipopolysaccharide (LPS). LPS is known to activate the HO−1 gene in cultured mouse liver and macrophage cells through oxidative activation of NF‐κB. But little is known about the effect of LPS and Hb on the HO−1 gene in living organisms. To study this issue, we examined the HO−1 and its mRNA levels in mouse liver, spleen and kidney after intravenous administration of LPS and Hb. On LPS treatment, the amount of HO−1 and its mRNA increased markedly mainly in mouse spleen, but on Hb treatment the amounts of HO‐1 and its mRNA increased slightly only in liver. Run‐off transcription assay supported the above results and band shift assays also revealed that LPS significantly activates an NF‐κB‐like factor in spleen cells, while Hb slightly activates it in liver cells. According to our previous study, a small amount of Hb injected to mouse is selectively taken up by liver as Hb—haptoglobin complex.

Structure of the human transcription factor TFIIF revealed by limited proteolysis with trypsin

In this study, the human general transcription factor IIF (TFIIF), a heteromeric complex of RAP74 and RAP30 subunits, was subjected to limited proteolysis with trypsin. The central region of RAP74 was demonstrated to be highly sensitive to trypsin while both the N‐ and C‐terminal regions contained trypsin‐resistant structures. In contrast, RAP30 digestion occurred after proteolysis of RAP74. The digestion pattern of RAP74 recruited into the preinitiation complex showed no marked difference from that of IIF, while RAP30 in the complex was protected from trypsin. These results indicate that RAP74 apparently contains three structural domains, the central one of which is externally surfaced and unstructured, but RAP30 is internally wrapped by RAP74. Furthermore, the accessibility of the central region of RAP74 is unaltered in the minimal preinitiation complex, while RAP30 is involved in promoter recognition through its DNA binding activity.

A New Effect of Aluminum on Iron Metabolism in Mammalian Cells

There is an increasing number of reports about new effects of aluminum on iron metabolism in mammalian cells. In this review, based on our recent work and that by others, we describe how aluminum disturbs iron homeostasis. The effects of aluminum may help to explain the pathogenesis of neurodegeneration in Alzheimer’s disease.