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1.  Curcumin and 5-Fluorouracil-loaded, folate- and transferrin-decorated polymeric magnetic nanoformulation: a synergistic cancer therapeutic approach, accelerated by magnetic hyperthermia 
The efficient targeting and therapeutic efficacy of a combination of drugs (curcumin and 5-Fluorouracil [5FU]) and magnetic nanoparticles encapsulated poly(D,L-lactic-co-glycolic acid) nanoparticles, functionalized with two cancer-specific ligands are discussed in our work. This multifunctional, highly specific nanoconjugate resulted in the superior uptake of nanoparticles by cancer cells. Upon magnetic hyperthermia, we could harness the advantages of incorporating magnetic nanoparticles that synergistically acted with the drugs to destroy cancer cells within a very short period of time. The remarkable multimodal efficacy attained by this therapeutic nanoformulation offers the potential for targeting, imaging, and treatment of cancer within a short period of time (120 minutes) by initiating early and late apoptosis.
doi:10.2147/IJN.S49882
PMCID: PMC3891567  PMID: 24531392
nanotechnology; curcumin; 5FU; folate; transferrin; PLGA nanoparticle; magnetic hyperthermia
2.  Cholesterol Accumulation Regulates the Expression of Macrophage Proteins Implicated In Proteolysis and Complement Activation 
Objective
Cholesterol accumulation by macrophages plays a key role in atherogenesis. To begin to develop a global picture of this process, we used proteomics and transcriptomics to analyze foam cells generated with acetyl-LDL, a classic ligand for scavenger receptors.
Methods and Results
Tandem mass spectrometry and stringent statistical analysis revealed that foam cells differentially expressed 15 of the 542 proteins (2.8%) detected in macrophage-conditioned medium. Apolipoprotein E was one of the most up-regulated proteins, confirming that proteins involved in lipid metabolism are important targets for regulation by sterol accumulation. However, levels of proteins linked to complement activation and lysosomal proteolysis also changed markedly. Transcriptional analysis demonstrated that 698 of 19,700 genes (3.5%) were regulated in foam cells, including many genes important in sterol metabolism. We also found that cholesterol accumulation regulated genes implicated in complement activation, but failed to affect genes linked to proteolysis and macrophage polarization. Changes in protein levels in macrophage-conditioned medium were largely independent of changes in mRNA levels.
Conclusions
Loading sterol into macrophages regulates levels of complement proteins and lysosomal proteases—key players in the immune system and plaque rupture. Posttranscriptional mechanisms appear important for controlling levels of most of the proteins detected in macrophage medium.
doi:10.1161/ATVBAHA.112.300383
PMCID: PMC3501207  PMID: 23042816
cholesterol; proteolysis; complement; macrophage; atherosclerosis
3.  Molecular mechanisms of renal and extrarenal manifestations caused by inactivation of the electrogenic Na+-HCO3− cotransporter NBCe1 
The electrogenic Na+-HCO3− cotransporter NBCe1 plays an essential role in bicarbonate absorption from renal proximal tubules, but also mediates the other biological processes in extrarenal tissues such as bicarbonate secretion from pancreatic ducts, maintenance of tissue homeostasis in eye, enamel maturation in teeth, or local pH regulation in synapses. Homozygous mutation in NBCe1 cause proximal renal tubular acidosis (pRTA) associated with extrarenal manifestations such as short stature, ocular abnormalities, enamel abnormalities, and migraine. Functional analyses of NBCe1 mutants using different expression systems suggest that at least a 50% reduction of the transport activity may be required to induce severe pRTA. In addition to functional impairments, some NBCe1 mutants show trafficking defects. Some of the pRTA-related NBCe1 mutants showing the cytoplasmic retention have been shown to exert a dominant negative effect through hetero-oligomer complexes with wild-type NBCe1 that may explain the occurrence of extrarenal manifestations in the heterozygous carries of NBCe1 mutations. Both NBCe1 knockout (KO) and W516X knockin (KI) mice showed very severe pRTA and reproduced most of the clinical manifestations observed in human pRTA patients. Functional analysis on isolated renal proximal tubules from W516X KI mice directly confirmed the indispensable role of NBCe1 in bicarbonate absorption from this nephron segment. In this review, we will focus on the molecular mechanisms underling the renal and extrarenal manifestations caused by NBCe1 inactivation.
doi:10.3389/fphys.2013.00270
PMCID: PMC3787273  PMID: 24101904
NBCe1; pRTA; short stature; ocular abnormalities; migraine; enamel abnormalities; dominant negative effect
4.  Role of renal proximal tubule transport in thiazolidinedione-induced volume expansion 
World Journal of Nephrology  2012;1(5):146-150.
Thiazolidinediones (TZDs), pharmacological activators of peroxisome-proliferator-activated receptors γ (PPARγ), significantly improve insulin resistance and lower plasma glucose concentrations. However, the use of TZDs is associated with plasma volume expansion, the mechanism of which has been a matter of controversy. Originally, PPARγ-mediated enhanced transcription of the epithelial Na channel (ENaC) γ subunit was thought to play a central role in TZD-induced volume expansion. However, later studies suggested that the activation of ENaC alone could not explain TZD-induced volume expansion. We have recently shown that TZDs rapidly stimulate sodium-coupled bicarbonate absorption from renal proximal tubule (PT) in vitro and in vivo. TZD-induced transport stimulation was dependent on PPARγ/Src/EGFR/ERK, and observed in rat, rabbit and human. However, this stimulation was not observed in mouse PTs where Src/EGFR is constitutively activated. Analysis in mouse embryonic fibroblast cells confirmed the existence of PPARγ/Src-dependent non-genomic signaling, which requires the ligand binding ability but not the transcriptional activity of PPARγ. The TZD-induced enhancement of association between PPARγ and Src supports an obligatory role for Src in this signaling. These results support the view that TZD-induced volume expansion is multifactorial. In addition to the PPARγ-dependent enhanced expression of the sodium transport system(s) in distal nephrons, the PPARγ-dependent non-genomic stimulation of renal proximal transport may be also involved in TZD-induced volume expansion.
doi:10.5527/wjn.v1.i5.146
PMCID: PMC3782215  PMID: 24175252
Thiazolidinediones; Peroxisome-proliferator-activated receptors γ; Volume expansion; Edema; NBCe1; NHE3; Epithelial Na channel
5.  Bilateral Endoscopic Medial Maxillectomy for Bilateral Inverted Papilloma 
Case Reports in Otolaryngology  2012;2012:215847.
Inverted papilloma (IP) is a benign tumor of the nasal cavity and paranasal sinuses that is unilateral in most cases. Bilateral IP, involving both sides of the nasal cavity and sinuses, is extremely rare. This paper describes a large IP that filled in both sides of the nasal cavity and sinuses, mimicking association with malignancy. The tumor was successfully treated by bilateral endoscopic medial maxillectomy (EMM). The patient is without evidence of the disease 24 months after surgery. If preoperative diagnosis does not confirm the association with malignancy in IP, endoscopic sinus surgery (ESS) should be selected, and ESS, including EMM, is a good first choice of the treatment for IP.
doi:10.1155/2012/215847
PMCID: PMC3420519  PMID: 22953103
6.  Endoscopic Endonasal Transturbinate Approach to the Pterygopalatine Fossa in the Management of Juvenile Nasopharyngeal Angiofibromas 
Case Reports in Otolaryngology  2012;2012:786262.
Pterygopalatine fossa (PPF) is a difficult-to-access anatomic area located behind the posterior wall of the maxillary sinus. Juvenile nasopharyngeal angiofibroma (JNA) often affects this area, and the management of feeding artery to the tumor is important in the surgery. Endoscopic endonasal approach to the PPF without endangering all other nasal structures is useful in the management of JNA. We describe a new approach to the PPF, endoscopic transturbinate approach, which is effective in the management of JNA. Submucous inferior turbinoplasty was performed, and sphenopalatine artery, the feeder to the tumor, was identified at the sphenopalatine foramen. The posterior wall of maxillary sinus was removed. Internal maxillary artery was identified in the PPF and was ligated with a hemoclip. The tumor in the PPF was pushed into the nasal cavity. These procedures were all performed via submucous turbinate tunnel. Then, the tumor was successfully removed in en bloc from the nasal cavity by transnasal approach without ethmoidectomy. This approach improves accessibility and visualization in the PPF and potential to reduce intraoperative bleeding due to ligation of the feeder safely without touching the tumor. Endoscopic transturbinate approach is effective in the management of early stage of JNA.
doi:10.1155/2012/786262
PMCID: PMC3420430  PMID: 22953122
7.  Wegener's Granulomatosis with Extensive Bone Abnormalities Mimicking Fungal Sinusitis 
Case Reports in Otolaryngology  2012;2012:103403.
Abnormalities of the underlying bone of the paranasal sinuses have sometimes been shown in Wegener's granulomatosis (WG). We describe an interesting case of WG with extensive bone abnormalities in the sinuses mimicking fungal sinusitis. A 30-year-old woman presented with intermittent unilateral epistaxis. Biopsy was performed for the granulation tissue in the right nasal cavity, and she was diagnosed as having WG. Computed tomography (CT) revealed a ring-like calcification, mimicking a fungus ball, in the right maxillary sinus. Endoscopic sinus surgery was performed to confirm the diagnosis. A spherical bony structure, surrounded by granulation tissue, was identified in the maxillary sinus. The wall of the “bony ball” was fragile, like an egg shell. No fungus was found in the sinus. Thus, the extensive bone abnormalities were due to WG.
doi:10.1155/2012/103403
PMCID: PMC3420613  PMID: 22953096
8.  Functional Roles of Electrogenic Sodium Bicarbonate Cotransporter NBCe1 in Ocular Tissues 
Electrogenic Na+-HCO3- cotransporter NBCe1 is expressed in several tissues such as kidney, eye, and brain, where it may mediate distinct biological processes. In particular, NBCe1 in renal proximal tubules is essential for the regulation of systemic acid/base balance. On the other hand, NBCe1 in eye may be indispensable for the maintenance of tissue homeostasis. Consistent with this view, homozygous mutations in NBCe1 cause severe proximal renal tubular acidosis associated with ocular abnormalities such as band keratopathy, glaucoma, and cataract. The widespread expression of NBCe1 in eye suggests that the inactivation of NBCe1 per se may be responsible for the occurrence of these ocular abnormalities. In this review, we discuss about physiological and pathological roles of NBCe1 in eye.
doi:10.2174/1874364101206010036
PMCID: PMC3394102  PMID: 22798968
NBCe1; proximal renal tubular acidosis; band keratopathy; glaucoma; cataract.
9.  HDL suppresses the type I interferon response, a family of potent antiviral immunoregulators, in macrophages challenged with lipopolysaccharide 
Circulation  2010;122(19):1919-1927.
Background
High density lipoprotein (HDL) protects the artery wall by removing cholesterol from lipid-laden macrophages. However, recent evidence suggests that HDL might also inhibit atherogenesis by combating inflammation.
Methods and Results
To identify potential anti-inflammatory mechanisms, we challenged macrophages with lipopolysaccharide (LPS), an inflammatory microbial ligand for Toll-like receptor 4 (TLR4). HDL inhibited the expression of 30% (277 of 911) of the genes normally induced by LPS, microarray analysis revealed. One of its major targets was the type I interferon response pathway, a family of potent viral immunoregulators controlled by TLR4 and the TRAM/TRIF signaling pathway. Unexpectedly, HDL’s ability to inhibit gene expression was independent of macrophage cholesterol stores. Immunofluorescent studies suggested that HDL promoted TRAM translocation to intracellular compartments, which impaired subsequent signaling by TLR4 and TRIF. To examine the potential in vivo relevance of the pathway, we used mice deficient in apolipoprotein (apo) A-I, HDL’s major protein. After infection with Salmonella typhimurium, a Gram-negative bacterium that expresses LPS, apoA-I–deficient mice had 6-fold higher plasma levels of interferon-β –a key regulator of the type I interferon response– than did wild-type mice.
Conclusions
HDL inhibits a subset of LPS-stimulated macrophage genes that regulate the type I interferon response, and its action is independent of sterol metabolism. These findings raise the possibility that regulation of macrophage genes by HDL might link innate immunity and cardioprotection.
doi:10.1161/CIRCULATIONAHA.110.961193
PMCID: PMC2988582  PMID: 20974999
Lipid-raft; MyD88; chemokine; cytokine; interferon regulatory factor 7
10.  Insulin Resistance, Obesity, Hypertension, and Renal Sodium Transport 
Sodium transport through various nephron segments is quite important in regulating sodium reabsorption and blood pressure. Among several regulators of this process, insulin acts on almost all the nephron segments and is a strong enhancer of sodium reabsorption. Sodium-proton exchanger type 3 (NHE3) is a main regulator of sodium reabsorption in the luminal side of proximal tubule. In the basolateral side of the proximal tubule, sodium-bicarbonate cotransporter (NBCe1) mediates sodium and bicarbonate exit from tubular cells. In the distal nephron and the connecting tubule, epithelial sodium channel (ENaC) is of great importance to sodium reabsorption. NHE3, NBCe1, and ENaC are all regulated by insulin. Recently with-no-lysine (WNK) kinases, responsible for familial hypertension, stimulating sodium reabsorption in the distal nephron, have been found to be also regulated by insulin. We will discuss the regulation of renal sodium transport by insulin and its roles in the pathogenesis of hypertension in insulin resistance.
doi:10.4061/2011/391762
PMCID: PMC3095959  PMID: 21629870
11.  Expression of bone morphogenic protein in sinonasal inverted papilloma with new bone formation 
Allergy & Rhinology  2011;2(1):16-20.
Inverted papilloma (IP) is a common benign tumor in the nose and sinus. Osteogenesis in sinonasal IP is extremely rare; to date, only five cases of IP with new bone formation appear in the literature. In addition, the mechanism of osteogenesis in IP remains unclear. Here, we describe three cases of IP with new bone formation and an investigation into a possible role for bone morphogenic protein (BMP) in osteogenesis. Of three patients with sinonasal IP with new bone formation, two were treated by endoscopic sinus surgery and one was followed up with watchful waiting. Tumor tissues were subjected to immunohistochemistry to detect BMP expression. The patients were successfully treated surgically and showed no evidence of recurrence postoperatively. Follow-up examination is ongoing. Immunohistochemically, the tumors expressed BMP-4 but not BMP-2 or BMP-7. ESS could be successfully used to achieve complete removal of the sinonasal IPs with new bone formation. BMP-4 might be associated with new bone formation in the tumor.
doi:10.2500/ar.2011.2.0004
PMCID: PMC3390124  PMID: 22852110
Bone formation; bone morphogenic protein; endoscopic sinus surgery; inverted papilloma; sinonasal
12.  Sinonasal Schwannoma with New Bone Formation Expressing Bone Morphogenic Protein 
Schwannoma is a benign tumor that arises from the sheath of myelinated nerve fibers and may occur in any part of the body. Osteogenesis in schwannoma is extremely rare and, to date, new bone formation in sinonasal schwannoma has not yet been reported. Here, we describe the first reported case of sinonasal schwannoma with new bone formation. The tumor was successfully treated by endoscopic sinus surgery, and the patient showed no evidence of recurrence 24 months postoperatively. Immunohistochemically, the tumor expressed bone morphogenic protein 4, indicating a possible role of this protein in the new bone formation in schwannomas.
doi:10.1155/2010/154948
PMCID: PMC3010644  PMID: 21197441
13.  Function of the aux and rol genes of the Ri plasmid in plant cell division in vitro 
Plant Signaling & Behavior  2009;4(12):1145-1147.
Auxin-autonomous growth in vitro may be related to the integration and expression of the aux and rol genes from the root-inducing (Ri) plasmid in plant cells infected by agropine-type Agrobacterium rhizogenes. To elucidate the functions of the aux and rol genes in plant cell division, plant cell lines transformed with the aux1 and aux2 genes or with the rolABCD genes were established using tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells. The introduction of the aux1 and aux2 genes enabled the auxin-autonomous growth of BY-2 cells, but the introduction of the rolABCD genes did not affect the auxin requirement of the BY-2 cells. The results clearly show that the aux genes are necessary for auxinautotrophic cell division, and that the rolABCD genes are irrelevant in auxin autotrophy.
PMCID: PMC2819440  PMID: 20514230
Agrobacterium rhizogenes; auxin-autotrophic cell; auxin biosynthesis; hairy root; plant cell division; Ri plasmid; T-DNA; aux; rol; tobacco BY-2 cells
14.  Analysis of Nucleotide Sequences of Human Parvovirus B19 Genome Reveals Two Different Modes of Evolution, a Gradual Alteration and a Sudden Replacement: a Retrospective Study in Sapporo, Japan, from 1980 to 2008▿  
Journal of Virology  2009;83(21):10975-10980.
There have been no long-term systematic analyses of the molecular epidemiology of human parvovirus B19 (B19V). We investigated the variations of nucleotide sequences of B19V strains collected in Sapporo, Japan, from 1980 to 2008. In that period, six outbreaks of erythema infectiosum occurred regularly at 5-year intervals. The B19V strains collected successively, regardless of the outbreak, were analyzed for nucleotide variation in the subgenomic NS1-VP1u junction. The isolated strains can be classified into 10 subgroups. Two patterns of change of endemic strains were observed. One was a dynamic replacement of strains that occurred almost every 10 years, and the other was a gradual change consisting of an accumulation of point mutations.
doi:10.1128/JVI.00273-09
PMCID: PMC2772762  PMID: 19710152
15.  Expression of tumor necrosis factor-alpha converting enzyme and matrix metalloproteinase-3 in proliferated synovium in a patient with synovitis-acne-pustulosis-hyperostosis-osteitis syndrome: a case report 
Introduction
Synovitis-acne-pustulosis-hyperostosis-osteitis (SAPHO) syndrome is a rare disorder. The etiology remains unknown and the treatment is still empirical. Synovitis is one of the major manifestations, but information on histopathological features is still lacking. In this case, we investigated the histopathological features of SAPHO syndrome synovitis.
Case presentation
We present the case of a 53-year-old Japanese woman with SAPHO syndrome accompanied by marked knee synovitis and palmoplantar pustulosis. We found abundant sterile joint fluid in the right knee, and a blood test showed abnormally high values of C-reactive protein (17.26 mg/dl) and matrix metalloproteinase-3 (800 ng/ml). Arthroscopic surgery revealed marked proliferation of villous synovial tissues similar to rheumatoid arthritis and standard microscopic findings were also similar to rheumatoid arthritis. Furthermore, for the first time, we demonstrated by immunohistochemistry the expression of tumor necrosis factor-alpha (TNF-α) converting enzyme, TNF-α and matrix metalloproteinase-3 in the proliferated synovial lining cells. After arthroscopic synovectomy, her knee symptoms immediately diminished and laboratory data (matrix metalloproteinase-3 and C-reactive protein) normalized within 2 weeks of surgery.
Conclusion
We demonstrate the expression of TNF-α converting enzyme, TNF-α and matrix metalloproteinase-3 in SAPHO syndrome synovitis for the first time and also show, both macro- and microscopically, the similarity between SAPHO syndrome and rheumatoid arthritis synovitis. These new findings support the recently reported successful treatment of SAPHO syndrome with antirheumatic drugs, especially with anti-TNF-α agents.
doi:10.4076/1752-1947-3-9123
PMCID: PMC2827166
16.  The DNA-recognition mode shared by archaeal feast/famine-regulatory proteins revealed by the DNA-binding specificities of TvFL3, FL10, FL11 and Ss-LrpB 
Nucleic Acids Research  2009;37(13):4407-4419.
The DNA-binding mode of archaeal feast/famine-regulatory proteins (FFRPs), i.e. paralogs of the Esherichia coli leucine-responsive regulatory protein (Lrp), was studied. Using the method of systematic evolution of ligands by exponential enrichment (SELEX), optimal DNA duplexes for interacting with TvFL3, FL10, FL11 and Ss-LrpB were identified as TACGA[AAT/ATT]TCGTA, GTTCGA[AAT/ATT]TCGAAC, CCGAAA[AAT/ATT]TTTCGG and TTGCAA[AAT/ATT]TTGCAA, respectively, all fitting into the form abcdeWWWedcba. Here W is A or T, and e.g. a and a are bases complementary to each other. Apparent equilibrium binding constants of the FFRPs and various DNA duplexes were determined, thereby confirming the DNA-binding specificities of the FFRPs. It is likely that these FFRPs recognize DNA in essentially the same way, since their DNA-binding specificities were all explained by the same pattern of relationship between amino-acid positions and base positions to form chemical interactions. As predicted from this relationship, when Gly36 of TvFL3 was replaced by Thr, the b base in the optimal DNA duplex changed from A to T, and, when Thr36 of FL10 was replaced by Ser, the b base changed from T to G/A. DNA-binding characteristics of other archaeal FFRPs, Ptr1, Ptr2, Ss-Lrp and LysM, are also consistent with the relationship.
doi:10.1093/nar/gkp378
PMCID: PMC2715240  PMID: 19468044
17.  Albinism and cell viability in cycloartenol synthase deficient Arabidopsis 
Plant Signaling & Behavior  2008;3(11):978-980.
Phenotypes of Arabidopsis thaliana that carry mutations in CYCLOARTENOL SYNTHASE 1 (CAS1) which is required in sterol biosynthesis have been described. Knockout mutant alleles are responsible of a male-specific transmission defect. Plants carrying a weak mutant allele cas1-1 accumulate 2,3-oxidosqualene, the substrate of CAS1, in all analyzed organs. Mutant cas1-1 plants develop albino inflorescence shoots that contain low amount of carotenoids and chlorophylls. The extent of this albinism, which affects Arabidopsis stems late in development, may be modulated by the light/dark regime. The fact that chloroplast differentiation and pigment accumulation in inflorescence shoots are associated with a low CAS1 expression could suggest the involvement of 2,3-oxidosqualene in a yet unknown regulatory mechanism linking the sterol biosynthetic segment, located in the cytoplasm, and the chlorophyll and carotenoid biosynthetic segments, located in the plastids, in the highly complex terpenoid network. CAS1 loss of function in a mosaic analysis of seedlings further demonstrated that leaf albinism associated with an accumulation of 2,3-oxidosqualene is a novel phenotype for plant sterol deficient mutant.
PMCID: PMC2633748  PMID: 19704425
albinism; cell viability; sterol; terpenoid; light
18.  Complete blockage of the mevalonate pathway results in male gametophyte lethality 
Journal of Experimental Botany  2009;60(7):2055-2064.
Plants have two isoprenoid biosynthetic pathways: the cytosolic mevalonate (MVA) pathway and the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Since the discovery of the MEP pathway, possible metabolic cross-talk between these pathways has prompted intense research. Although many studies have shown the existence of such cross-talk using feeding experiments, it remains to be determined if native cross-talk, rather than exogenously applied metabolites, can compensate for complete blockage of the MVA pathway. Previously, Arabidopsis mutants for HMG1 and HMG2 encoding HMG-CoA reductase (HMGR) were isolated. Although it was shown that HMGR1 is a functional HMGR, the enzyme activity of HMGR2 has not been confirmed. It is demonstrated here that HMG2 encodes a functional reductase with similar activity to HMGR1, using enzyme assays and complementation experiments. To estimate the contribution of native cross-talk, an attempt was made to block the MVA pathway by making double mutants lacking both HMG1 and HMG2, but no double homozygotes were detected in the progeny of self-pollinated HMG1/hmg1 hmg2/hmg2 plants. hmg1 hmg2 male gametophytes appeared to be lethal based on crossing experiments, and microscopy indicated that ∼50% of the microspores from the HMG1/hmg1 hmg2/hmg2 plant appeared shrunken and exhibited poorly defined endoplasmic reticulum membranes. In situ hybridization showed that HMG1 transcripts were expressed in both the tapetum and microspores, while HMG2 mRNA appeared only in microspores. It is concluded that native cross-talk from the plastid cannot compensate for complete blockage of the MVA pathway, at least during male gametophyte development, because either HMG1 or HMG2 is required for male gametophyte development.
doi:10.1093/jxb/erp073
PMCID: PMC2682496  PMID: 19363204
Anther; cross-talk; HMG-CoA reductase; isoprenoid; male gametophyte; MEP pathway; MVA pathway; pollen; sterol; tapetum
19.  Dilated Cardiomyopathy Caused by Aberrant Endoplasmic Reticulum Quality Control in Mutant KDEL Receptor Transgenic Mice 
Molecular and Cellular Biology  2004;24(18):8007-8017.
Aberrant protein folding beyond the capacity of endoplasmic reticulum (ER) quality control leads to stress response in the ER. The Lys-Asp-Glu-Leu (KDEL) receptor, a retrieval receptor for ER chaperones in the early secretory pathway, contributes to ER quality control. To elucidate the function of the KDEL receptor in vivo, we established transgenic mice expressing a mutant KDEL receptor. We found that the mutant KDEL receptor sensitized cells to ER stress and that the mutant mice developed dilated cardiomyopathy. Ultrastructural analyses revealed expanded sarcoplasmic reticulums and protein aggregates that obstructed the adjacent transverse tubules of the mutant cardiomyocytes. Cardiomyocytes from the mutant mice were sensitive to ER stress when treated with tunicamycin and showed a functional defect in the L-type Ca2+ current. We observed ubiquitinated protein aggregates, enhanced expression of CHOP (a death-related transcriptional factor expressed upon ER stress), and apoptosis in the mutant hearts. These findings suggest that impairment of the KDEL receptor disturbs ER quality control, resulting in accumulation of misfolded proteins in the ER in an in vivo system, and that the dilated cardiomyopathy found in the mutant KDEL receptor transgenic mice is associated with ER stress.
doi:10.1128/MCB.24.18.8007-8017.2004
PMCID: PMC515036  PMID: 15340063
20.  Altered DNA binding specificity of Arnt by selection of partner bHLH–PAS proteins 
Nucleic Acids Research  2004;32(10):3169-3179.
The Ah receptor (AhR) and HLF are transcription factors involved in xenobiotic metabolism and hypoxic response, respectively. AhR and HLF heterodimerize with Arnt as the common partner, and bind to asymmetric E-boxes termed XRE and HRE, respectively. In order to investigate nucleotide preference of the heterodimers, reporter plasmids with oligonucleotides for XREs or HREs with systematic mutations were constructed and their activity was determined. Comparison of the activity revealed that DNA length and nucleotide preference recognized by Arnt subunit in the two heterodimers were largely different between XRE and HRE. We expressed AhR–Arnt and HLF–Arnt in Escherichia coli and used them for DNA binding. The dissociation constant of HLF–Arnt–HRE was 10.4 ± 1.6 nM. Competition activity of mutated XREs or HREs with wild type was consistent with their transcription activity. Bending of XRE and HRE induced by binding of the relevant heterodimers was observed with stronger bending of XRE than of HRE. By deletional and mutational analyses, an alanine and three arginine (Ala 8, Arg 9, Arg 11 and Arg 12) residues in the basic sequence of HLF were found to be indispensable for the transcriptional activity.
doi:10.1093/nar/gkh637
PMCID: PMC434443  PMID: 15190133
21.  Role of sarcolemmal KATP channels in cardioprotection against ischemia/reperfusion injury in mice 
Recently it has been postulated that mitochondrial ATP-sensitive K+ (mitoKATP) channels rather than sarcolemmal KATP (sarcKATP) channels are important as end effectors and/or triggers of ischemic preconditioning (IPC). To define the pathophysiological significance of sarcKATP channels, we conducted functional experiments using Kir6.2-deficient (KO) mice. Metabolic inhibition with glucose-free, dinitrophenol-containing solution activated sarcKATP current and shortened the action potential duration in ventricular cells isolated from wild-type (WT) but not KO mice. MitoKATP channel function was preserved in KO ventricular cells. In anesthetized mice, IPC reduced the infarct size in WT but not KO mice. Following global ischemia/reperfusion, the increase of left ventricular end-diastolic pressure during ischemia was more marked, and the recovery of contractile function was worse, in KO hearts than in WT hearts. Treatment with HMR1098, a sarcKATP channel blocker, but not 5-hydroxydecanoate, a mitoKATP channel blocker, produced a deterioration of contractile function in WT hearts comparable to that of KO hearts. These findings suggest that sarcKATP channels figures prominently in modulating ischemia/reperfusion injury in the mouse. The rapid heart rate of the mouse (>600 beats per minute) may magnify the relative importance of sarcKATP channels during ischemia, prompting caution in the extrapolation of the conclusions to larger mammals.
doi:10.1172/JCI14270
PMCID: PMC150878  PMID: 11854323
22.  Intranasal Immunization Enhances Clearance of Nontypeable Haemophilus influenzae and Reduces Stimulation of Tumor Necrosis Factor Alpha Production in the Murine Model of Otitis Media 
Infection and Immunity  2001;69(5):2964-2971.
Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing otitis media (OM). One of the outer membrane proteins of NTHi, P6, is a common antigen to all strains and is considered a candidate for mucosal vaccine. We have previously reported that intranasal immunization with P6 and cholera toxin (CT) could induce P6-specific immunoglobulin A (IgA) antibodies in the middle ear. In the present study, we assessed the effect of intranasal immunization for the protection against NTHi-induced OM. Mice were immunized intranasally with P6 and CT as an adjuvant on days 0, 7, and 14. Control mice were given phosphate-buffered saline (PBS) without antigen. One week after the final immunization, a suspension of live NTHi (107 CFU) was injected into the tympanic cavity to induce experimental OM. On days 3 and 7 after bacterial challenge, mice were killed and middle ear effusions (MEEs) were collected. All immunized mice showed elevated titers of P6-specific antibodies in MEEs. The rank order of specific antibody included, from highest to lowest levels, IgG, IgA, and IgM. In addition, immunized mice showed enhanced clearance of NTHi from the middle ear and the number of NTHi in MEEs of immunized mice was reduced by 97% on day 3 and by 92% on day 7 after bacterial challenge relative the number in the MEEs of control mice. The protective effect of intranasal immunization on the incidence of NTHi-induced experimental OM was evident on day 7 after challenge. By day 7, the number of MEEs in immunized mice was 64% less than that in control mice and the incidence of NTHi culture-positive MEEs in immunized mice was 56% less than that in control mice. Less stimulation of tumor necrosis factor alpha (TNF-α) production in the middle ear was evident on day 3 after challenge. Immunized mice showed lower concentrations of TNF-α in MEEs. These results indicate that intranasal immunization affords protection against experimental OM as evidenced by enhanced clearance of NTHi and less stimulation of TNF-α production in the middle ear. These findings suggest that a nasal vaccine might be useful for preventing OM.
doi:10.1128/IAI.69.5.2964-2971.2001
PMCID: PMC98249  PMID: 11292713
23.  Induction of Specific Immunoglobulin A and Th2 Immune Responses to P6 Outer Membrane Protein of Nontypeable Haemophilus influenzae in Middle Ear Mucosa by Intranasal Immunization 
Infection and Immunity  2000;68(4):2294-2300.
Nontypeable Haemophilus influenzae (NTHI) is a major pathogen of otitis media. One of the outer membrane proteins of NTHI, P6, is an antigen common to all strains and is considered as a candidate for mucosal vaccine. To elucidate the possibility of developing a nasal vaccine against nontypeable Haemophilus influenzae (NTHI) and to investigate mucosal immune responses in the middle ear, mice were immunized intranasally with the P6 outer membrane protein of NTHI, and P6-specific immune responses in the middle ear mucosa were examined. Mice were given with P6 and cholera toxin intranasally as an adjuvant on days 0, 7, and 14 and were killed on day 21. The P6-specific immunoglobulin A (IgA) antibody titer in ear wash was significantly elevated. Mononuclear cells were isolated from middle ear mucosa, and an increase in P6-specific IgA-producing cells was shown with an enzyme-linked immunospot assay. In addition, an increase in memory T cells in middle ear mucosa was detected with flow cytometric analysis after intranasal immunization. Moreover, in vitro stimulation with P6 resulted in proliferation of purified CD4+ T cells from immunized mice, and these T cells expressed Th2 cytokine mRNA. These results indicate that P6-specific IgA–B-cell immune responses and selected Th2 cytokine expressing Th cells were induced in middle ear mucosa by intranasal immunization. These findings suggest that a nasal vaccine is useful for preventing otitis media with effusion.
PMCID: PMC97416  PMID: 10722632

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