Michio Hayashi

Constitutive Activation of the pH-Responsive Rim101 Pathway in Yeast Mutants Defective in Late Steps of the MVB/ESCRT Pathway

ABSTRACT In many fungi, transcriptional responses to alkaline pH are mediated by conserved signal transduction machinery. In the homologous system in Saccharomyces cerevisiae, the zinc-finger transcription factor Rim101 is activated under alkaline conditions to regulate transcription of target genes. The activation of Rim101 is exerted through proteolytic processing of its C-terminal inhibitory domain. Regulated processing of Rim101 requires several proteins, including the calpain-like protease Rim13/Cpl1, a putative protease scaffold Rim20, putative transmembrane proteins Rim9, and Rim21/Pal2, and Rim8/Pal3 of unknown biochemical function. To identify new regulatory components and thereby determine the order of action among the components in the pathway, we screened for suppressors of rim9Δ and rim21Δ mutations. Three identified suppressors—did4/vps2, vps24, and vps4—all belonged to “class E” vps mutants, which are commonly defective in multivesicular body sorting. These mutations suppress rim8, rim9, and rim21 but not rim13 or rim20, indicating that Rim8, Rim9, and Rim21 act upstream of Rim13 and Rim20 in the pathway. Disruption of DID4, VPS24, or VPS4, by itself, uncouples pH sensing from Rim101 processing, leading to constitutive Rim101 activation. Based on extensive epistasis analysis between pathway-activating and -inactivating mutations, a model for architecture and regulation of the Rim101 pathway is proposed.

Plasma adrenomedullin in diabetes

Vol 350 • November 15, 1997 1449 had secondary DVT and six cancers were diagnosed among these patients during their hospital stay (1·2%). When thrombosis was idiopathic, the rate of occult cancer was significantly higher (3·9%, p<0·02). Among the 64 patients with bilateral DVT, 41 (64%) had secondary bilateral DVT and two cancers were diagnosed (4·9%). When bilateral DVT was idiopathic, the rate of cancer was significantly higher than when it was not (35%, p<0·001). To our knowledge, this is the first study to establish the increased risk of occult cancer in bilateral DVT. The low rate of bilateral DVT probably explains the lack of data in the published research. However, this was a retrospective study with possible bias. We cannot rule out the possibility that clinical examination was more meticulous in patients with bilateral DVT. It is also possible that screening tests were more extensive in patients with bilateral venous thrombosis. However, most cancers are diagnosed by clinical examination, chest radiography, or standard biological screening tests, which were routinely done in all our patients. Further followup will be required to determine the rate of undiagnosed cancers. The rate of known cancers in our series (13%) and the rate of occult cancers found during the hospital stay (3·2%) were similar to those in other studies. Symptomatic pulmonary embolism was more common in patients with bilateral DVT than in those with unilateral DVT. The bilateral thrombosis can explain an increased risk of PE. The presence of PE and the location of DVT (proximal versus inferior vena cava) were not associated with the risk of occult cancer. Our study shows an increased rate of occult cancers in bilateral DVT (mainly in idiopathic bilateral DVT), with malignant disease in one-third of the patients. These data call for an extensive search for occult cancer in this situation, that concerns less than 10% of patients with DVT.

Synergistic activation of NF-?b and inducible isoform of nitric oxide synthase induction by interferon-? and tumor necrosis factor-? in INS-1 cells

Interferon‐γ (IFN‐γ) is known to exert deleterious effects on pancreatic β‐cells and is implicated in the development of type 1 (autoimmune) diabetes mellitus. In this study, we investigated signaling mechanisms mediating the effects of IFN‐γ in pancreatic β‐cells using a differentiated rat insulin‐secreting cell line, INS‐1, with special reference to the activation of transcription factors STAT (signal transducers and activators of transcription)1 and NF‐κB. Exposure of INS‐1 cells to 100 IU/ml IFN‐γ for 24 h resulted in significant inhibition of nutrient‐induced insulin secretion associated with impaired metabolism. In combination with tumor necrosis factor‐α (TNF‐α) (50 ng/ml), IFN‐γ elicited severe cytotoxicity and induced the expression of the inducible isoform of nitric oxide synthase (iNOS) mRNA. IFN‐γ promoted tyrosine phosphorylation and DNA‐binding of STAT1 through Janus kinase (JAK)1 activation without apparent phosphorylation of JAK2. TNF‐α did not affect STAT1 activation, but stimulated DNA‐binding and transcriptional activity of NF‐κB, both of which were further increased by IFN‐γ. These effects of IFN‐γ and TNF‐α seem physiologically relevant, because either inhibition of STAT1 by the tyrosine kinase inhibitor herbimycin A or that of NF‐κB by sulfasalazine resulted in the reduction of iNOS mRNA expression. In conclusion, IFN‐γ activates STAT1 and potentiates TNF‐α–induced NF‐κB activation in INS‐1 cells, thereby inducing iNOS and cell destruction. J. Cell. Physiol. 184:46–57, 2000.

Pho85 Kinase, a Cyclin-Dependent Kinase, Regulates Nuclear Accumulation of the Rim101 Transcription Factor in the Stress Response of Saccharomyces cerevisiae

ABSTRACT The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to changing environmental conditions. The Pho85 kinase, one of the yeast cyclin-dependent kinases (CDK), is known to play an important role in the cellular response to alterations in parameters such as nutrient levels and salinity. Several genes whose expression is regulated, either directly or indirectly, by the Rim101 transcription factor become constitutively activated when Pho85 function is absent,. Because Rim101 is responsible for adaptation to alkaline conditions, this observation suggests an interaction between Pho85 and Rim101 in the response to alkaline stress. We have found that Pho85 affects neither RIM101 transcription, the proteolytic processing that is required for Rim101 activation, nor Rim101 stability. Rather, Pho85 regulates the nuclear accumulation of active Rim101, possibly via phosphorylation. Additionally, we report that Pho85 and the transcription factor Pho4 are necessary for adaptation to alkaline conditions and that PTK2 activation by Pho4 is involved in this process. These findings illustrate novel roles for the regulators of the PHO system when yeast cells cope with various environmental stresses potentially threatening their survival.

Roles of CTPL/Sfxn3 and Sfxn family members in pancreatic islet

Pancreatic AR42J cells have the feature of pluripotency of the precursor cells of the gut endoderm. Betacellulin (BTC) and activin A (Act) convert them into insulin‐secreting cells. Using mRNA differential display techniques, we have identified a novel mitochondrial transporter, which is highly expressed during the course of differentiation, and have designated it citrate transporter protein‐like protein (CTPL). Recently sideroflexin 1 (Sfxn1) was shown to be a susceptible gene of flexed‐tail (f/f) mice, and CTPL has turned out to be a rat orthologous protein of Sfxn3, a member of sideroflexin family. CTPL/Sfxn3 was targeted to mitochondrial membrane like Sfxn1. The expression levels of CTPL/Sfxn3, Sfxn2, and Sfxn5 were upregulated in the early phase of differentiation into insulin‐secreting cells but the expression levels of Sfxn1 and Sfxn3 did not change. All Sfxn family members were expressed in rat pancreatic islet. The expression levels of CTPL/Sfxn3, Sfxn2, and Sfxn5 were also upregulated in islets of streptozotocin‐induced diabetic rats compared to normal rats. The downregulation of CTPL/Sfxn3 in a rat insulinoma cell line, INS‐1, with the antisense oligonucleotide did not affect the insulin secretion. Taken together, CTPL/Sfxn3 and some other family members might be important in the differentiation of pancreatic β‐cells as a channel or a carrier molecule and be related to the regeneration of pancreatic endocrine cells.

The Role of Adrenomedullin in the Renal NADPH Oxidase and (Pro)renin in Diabetic Mice

Adrenomedullin has an antioxidative action and protects organs in various diseases. To clarify the role of adrenomedullin in diabetic nephropathy, we investigated the NADPH oxidase expression, renin-secreting granular cell (GC) hyperplasia, and glomerular matrix expansion in the streptozotocin (STZ)-induced diabetic adrenomedullin gene knockout (AMKO) mice compared with the STZ-diabetic wild mice at 10 weeks. The NADPH oxidase p47phox expression and lipid peroxidation products were enhanced in the glomeruli of the diabetic mice compared with that observed in the controls in both wild and AMKO mice. These changes were more obvious in the AMKO mice than in the wild mice. Glomerular mesangial matrix expansion was more severe in the diabetic AMKO mice than in the diabetic wild mice and exhibited a positive correlation with the degree of lipid peroxidation products in the glomeruli. Proteinuria was significantly higher in the diabetic AMKO mice than in the diabetic wild mice. The GC hyperplasia score and the renal prorenin expression were significantly increased in the diabetic AMKO mice than in the diabetic wild mice, and a positive correlation was observed with the NADPH oxidase expression in the macula densa. The endogenous adrenomedullin gene exhibits an antioxidant action via the inhibition of NADPH oxidase probably by suppressing the local renin-angiotensin system.

A Rare Case Report of Central Line-associated Bloodstream Infection Caused by Cryptococcus arboriformis

Cryptococcus arboriformis (C. arboriformis) is a novel Cryptococcus species belonging to the genus Trichosporonales. This novel species was identified definitively in 2007 using D1/D2 26S ribosomal DNA gene sequencing. In this article, we present a rare case of central line-associated bloodstream infection caused by C. arboriformis with successful treatment of this infection.