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1.  β2-Adrenergic Receptor-Dependent Attenuation of Hypoxic Pulmonary Vasoconstriction Prevents Progression of Pulmonary Arterial Hypertension in Intermittent Hypoxic Rats 
PLoS ONE  2014;9(10):e110693.
In sleep apnea syndrome (SAS), intermittent hypoxia (IH) induces repeated episodes of hypoxic pulmonary vasoconstriction (HPV) during sleep, which presumably contribute to pulmonary arterial hypertension (PAH). However, the prevalence of PAH was low and severity is mostly mild in SAS patients, and mild or no right ventricular hypertrophy (RVH) was reported in IH-exposed animals. The question then arises as to why PAH is not a universal finding in SAS if repeated hypoxia of sufficient duration causes cycling HPV. In the present study, rats underwent IH at a rate of 3 min cycles of 4–21% O2 for 8 h/d for 6w. Assessment of diameter changes in small pulmonary arteries in response to acute hypoxia and drugs were performed using synchrotron radiation microangiography on anesthetized rats. In IH-rats, neither PAH nor RVH was observed and HPV was strongly reversed. Nadolol (a hydrophilic β1, 2-blocker) augmented the attenuated HPV to almost the same level as that in N-rats, but atenolol (a hydrophilic β1-blocker) had no effect on the HPV in IH. These β-blockers had almost no effect on the HPV in N-rats. Chronic administration of nadolol during 6 weeks of IH exposure induced PAH and RVH in IH-rats, but did not in N-rats. Meanwhile, atenolol had no effect on morphometric and hemodynamic changes in N and IH-rats. Protein expression of the β1-adrenergic receptor (AR) was down-regulated while that of β2AR was preserved in pulmonary arteries of IH-rats. Phosphorylation of p85 (chief component of phosphoinositide 3-kinase (PI3K)), protein kinase B (Akt), and endothelial nitric oxide synthase (eNOS) were abrogated by chronic administration of nadolol in the lung tissue of IH-rats. We conclude that IH-derived activation of β2AR in the pulmonary arteries attenuates the HPV, thereby preventing progression of IH-induced PAH. This protective effect may depend on the β2AR-Gi mediated PI3K/Akt/eNOS signaling pathway.
doi:10.1371/journal.pone.0110693
PMCID: PMC4211686  PMID: 25350545
2.  Genome Sequences of Two Nondomesticated Bacillus subtilis Strains Able To Form Thick Biofilms on Submerged Surfaces 
Genome Announcements  2014;2(5):e00946-14.
Genomes of two nondomesticated strains of Bacillus subtilis subspecies subtilis, NDmed and NDfood, have been sequenced. Both strains form very thick and spatially complex biofilms on submerged surfaces. Moreover, biofilms of the NDmed isolate were shown to be highly resistant to antimicrobials action.
doi:10.1128/genomeA.00946-14
PMCID: PMC4175202  PMID: 25291767
3.  Transitioning from conventional radiotherapy to intensity-modulated radiotherapy for localized prostate cancer: changing focus from rectal bleeding to detailed quality of life analysis 
Journal of Radiation Research  2014;55(6):1033-1047.
With the advent of modern radiation techniques, we have been able to deliver a higher prescribed radiotherapy dose for localized prostate cancer without severe adverse reactions. We reviewed and analyzed the change of toxicity profiles of external beam radiation therapy (EBRT) from the literature. Late rectal bleeding is the main adverse effect, and an incidence of >20% of Grade ≥2 adverse events was reported for 2D conventional radiotherapy of up to 70 Gy. 3D conformal radiation therapy (3D-CRT) was found to reduce the incidence to ∼10%. Furthermore, intensity-modulated radiation therapy (IMRT) reduced it further to a few percentage points. However, simultaneously, urological toxicities were enhanced by dose escalation using highly precise external radiotherapy. We should pay more attention to detailed quality of life (QOL) analysis, not only with respect to rectal bleeding but also other specific symptoms (such as urinary incontinence and impotence), for two reasons: (i) because of the increasing number of patients aged >80 years, and (ii) because of improved survival with elevated doses of radiotherapy and/or hormonal therapy; age is an important prognostic factor not only for prostate-specific antigen (PSA) control but also for adverse reactions. Those factors shift the main focus of treatment purpose from survival and avoidance of PSA failure to maintaining good QOL, particularly in older patients. In conclusion, the focus of toxicity analysis after radiotherapy for prostate cancer patients is changing from rectal bleeding to total elaborate quality of life assessment.
doi:10.1093/jrr/rru061
PMCID: PMC4229926  PMID: 25204643
prostate cancer; radiotherapy; rectal bleeding; incontinence; genitourinary symptom; erectile dysfunction
4.  Molecular Characterization of a Novel N-Acetyltransferase from Chryseobacterium sp. 
N-Acetyltransferase from Chryseobacterium sp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an acetyl group from acetyl-CoA to the amino group of l-2-phenylglycine to produce (2S)-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence. The gene, designated natA, was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid sequence of NatA showed high levels of identity to sequences of putative N-acetyltransferases of Chryseobacterium spp. but not to other known arylamine and arylalkylamine N-acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from known N-acetyltransferases. We heterologously expressed recombinant NatA (rNatA) in Escherichia coli and purified it. rNatA showed high activity for l-2-phenylglycine and its chloro- and hydroxyl-derivatives. The Km and Vmax values for l-2-phenylglycine were 0.145 ± 0.026 mM and 43.6 ± 2.39 μmol · min−1 · mg protein−1, respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good substrates of a known arylamine N-acetyltransferase and an arylalkylamine N-acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups to l-2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10:<1).
doi:10.1128/AEM.03449-13
PMCID: PMC3957623  PMID: 24375143
5.  Clinical Significance of Serum Soluble T Cell Regulatory Molecules in Clear Cell Renal Cell Carcinoma 
BioMed Research International  2014;2014:396064.
To clarify the role of serum soluble T cell regulatory molecules in clear cell renal cell carcinoma (CCRCC), we measured the serum levels of soluble interleukin-2 receptor (sIL-2R), soluble B7-H3 (sB7-H3), and soluble cytotoxic T lymphocyte associated antigen-4 (sCTLA-4) in 70 CCRCC patients and 35 healthy controls. We investigated correlations between the serum levels of these soluble T cell regulatory molecules and the pathological grade, clinical stage, and prognosis of CCRCC. We also assessed the relations among each of these soluble molecules. As a result, the serum level of sIL-2R was significantly higher in CCRCC patients than in healthy controls (P < 0.05). In addition, elevation of serum sIL-2R was significantly correlated with the clinical stage (P < 0.001), and the survival of patients with high sIL-2R levels was shorter than that of patients with low sIL-2R levels (P < 0.05). Furthermore, the serum level of sB7-H3 was also significantly correlated with the clinical stage (P < 0.05), while the sIL-2R and sB7-H3 levels showed a positive correlation with each other (R = 0.550, P < 0.0001). These results indicate that the serum level of sIL-2R reflects tumor progression in CCRCC patients. In addition, the possibility was suggested that the IL-2/IL-2R and B7-H3 pathways may be involved in the progression of CCRCC.
doi:10.1155/2014/396064
PMCID: PMC4095742  PMID: 25089268
6.  The Rho-kinase inhibitor HA-1077 suppresses proliferation/migration and induces apoptosis of urothelial cancer cells 
BMC Cancer  2014;14:412.
Background
Activation of Rho, one of the small GTPases, and its major downstream target Rho-kinase (ROCK) promotes the development and metastasis of cancer. We previously showed that elevation of Rho and ROCK expression was associated with tumor invasion, metastasis, and an unfavorable prognosis in patients with urothelial cancer of the bladder or upper urinary tract.
Methods
We investigated the effects of a ROCK inhibitor on the growth, migration, and apoptosis of bladder cancer cells. We also examined phosphorylation of RhoA (RhoA activity) by measuring its GTP-bound active form and assessed the expression of ROCK to explore the underlying molecular mechanisms.
Results
Lysophosphatidic acid (LPA) and geranylgeraniol (GGOH) induced an increase of cell proliferation and migration in association with promotion of RhoA activity and upregulation of ROCK expression. The ROCK inhibitor fasudil (HA-1077) suppressed cell proliferation and migration, and also induced apoptosis in a dose-dependent manner. HA-1077 dramatically suppressed the expression of ROCK-I and ROCK-II, but did not affect RhoA activity.
Conclusions
These findings suggest that ROCK could be a potential molecular target for the treatment of urothelial cancer.
doi:10.1186/1471-2407-14-412
PMCID: PMC4081468  PMID: 24908363
7.  Potential risk of alpha-glucosidase inhibitor administration in prostate cancer external radiotherapy by exceptional rectal gas production: a case report 
Introduction
Radiotherapy is a standard treatment for prostate cancer, and image-guided radiotherapy is increasingly being used to aid precision of dose delivery to targeted tissues. However, precision during radiotherapy cannot be maintained when unexpected intrafraction organ motion occurs.
Case presentation
We report our experience of internal organ motion caused by persistent gas production in a patient taking an alpha-glucosidase inhibitor. A 68-year-old Japanese man with prostate cancer visited our institution for treatment with helical tomotherapy. He suffered from diabetes mellitus and took an alpha-glucosidase inhibitor. Routine treatment planning computed tomography showed a large volume of rectal gas; an enema was given to void the rectum. Subsequent treatment planning computed tomography again showed a large volume of gas. After exercise (walking) to remove the intestinal gas, a third scan was performed as a test scan without tight fixation, which showed a sufficiently empty rectum for planning. However, after only a few minutes, treatment planning computed tomography again showed extreme accumulation of gas. Therefore, we postponed treatment planning computed tomography and consulted his doctor to suspend the alpha-glucosidase inhibitor, which was the expected cause of his persistent gas. Four days after the alpha-glucosidase inhibitor regimen was suspended, we took a fourth treatment planning computed tomography and made a treatment plan without gas accumulation. Thereafter, the absence of rectal gas accumulation was confirmed using daily megavolt computed tomography before treatment, and the patient received 37 fractions of intensity-modified radiotherapy at 74Gy without rectal gas complications. In this case study, the alpha-glucosidase inhibitor induced the accumulation of intestinal gas, which may have caused unexpected organ motion, untoward reactions, and insufficient doses to clinical targets.
Conclusions
We suggest that patients who are taking an alpha-glucosidase inhibitor for diabetes should discontinue use of that particular medicine prior to beginning radiotherapy.
doi:10.1186/1752-1947-8-136
PMCID: PMC4046525  PMID: 24886457
Tomotherapy; Alpha-glucosidase inhibitor; Prostate cancer; Internal organ motion
8.  Comparison of three tannases cloned from closely related lactobacillus species: L. Plantarum, L. Paraplantarum, and L. Pentosus 
BMC Microbiology  2014;14:87.
Background
Tannase (tannin acyl hydrolase, EC 3.1.1.20) specifically catalyzes the hydrolysis of the galloyl ester bonds in hydrolyzable tannins to release gallic acid. The enzyme was found not only in fungal species but also many bacterial species including Lactobacillus plantarum, L. paraplantarum, and L. pentosus. Recently, we identified and expressed a tannase gene of L. plantarum, tanLpl, to show remarkable differences to characterized fungal tannases. However, little is known about genes responsible for tannase activities of L. paraplantarum and L. pentosus. We here identify the tannase genes (i.e. tanLpa and tanLpe) of the above lactobacilli species, and describe their molecular diversity among the strains as well as enzymological difference between species inclusive of L. plantarum.
Results
The genes encoding tannase, designated tanLpa and tanLpe, were cloned from Lactobacillus paraplantarum NSO120 and Lactobacillus pentosus 21A-3, which shared 88% and 72% amino acid identity with TanLpl, cloned from Lactobacillus plantarum ATCC 14917T, respectively. These three enzymes could comprise a novel tannase subfamily of independent lineage, because no other tannases in the databases share significant sequence similarity with them. Each of tanLpl, tanLpa, and tanLpe was expressed in Bacillus subtilis RIK 1285 and recombinant enzymes were secreted and purified. The Km values of the enzymes on each galloyl ester were comparable; however, the kcat/Km values of TanLpa for EGCg, ECg, Cg, and GCg were markedly higher than those for TanLpl and TanLpe. Their enzymological properties were compared to reveal differences at least in substrate specificity.
Conclusion
Two tannase genes responsible for tannase activities of L. paraplantarum and L. pentosus were identified and characterized. TanLpl, TanLpa and TanLpe forming a phylogenetic cluster in the known bacterial tannase genes and had a limited diversity in each other. Their enzymological properties were compared to reveal differences at least in substrate specificity. This is the first comparative study of closely related bacterial tannases.
doi:10.1186/1471-2180-14-87
PMCID: PMC4233993  PMID: 24708557
9.  Polysaccharide-Degrading Thermophiles Generated by Heterologous Gene Expression in Geobacillus kaustophilus HTA426 
Applied and Environmental Microbiology  2013;79(17):5151-5158.
Thermophiles have important advantages over mesophiles as host organisms for high-temperature bioprocesses, functional production of thermostable enzymes, and efficient expression of enzymatic activities in vivo. To capitalize on these advantages of thermophiles, we describe here a new inducible gene expression system in the thermophile Geobacillus kaustophilus HTA426. Six promoter regions in the HTA426 genome were identified and analyzed for expression profiles using β-galactosidase reporter assay. This analysis identified a promoter region upstream of a putative amylose-metabolizing gene cluster that directed high-level expression of the reporter gene. The expression was >280-fold that without a promoter and was further enhanced 12-fold by maltose addition. In association with a multicopy plasmid, this promoter region was used to express heterologous genes. Several genes, including a gene whose product was insoluble when expressed in Escherichia coli, were successfully expressed as soluble proteins, with yields of 0.16 to 59 mg/liter, and conferred new functions to G. kaustophilus strains. Remarkably, cellulase and α-amylase genes conferred the ability to degrade cellulose paper and insoluble starch at high temperatures, respectively, generating thermophiles with the potential to degrade plant biomass. Our results demonstrate that this novel expression system expands the potential applications of G. kaustophilus.
doi:10.1128/AEM.01506-13
PMCID: PMC3753961  PMID: 23793634
10.  High-dose-rate interstitial brachytherapy for mobile tongue cancer: preliminary results of a dose reduction trial 
Purpose
To compare the outcome of our facility with another about the shortened schedule (60 Gy in 10 fractions to 54 Gy in 9 fractions) of high-dose-rate interstitial brachytherapy (HDR ISBT) for mobile tongue cancer.
Material and methods
Eighteen patients were treated with HDR ISBT as a monotherapy in dose reduction schedule with some unique technique to determine the border of tumor accuracy (lugol's staining and metal marker), and to minimize adverse effect (lead-lined silicon block) at our facility.
Results
The 2-year local and regional control rates and cause-specific survival rate were 82%, 80%, and 83% and moderate to severe late complications occurred in five patients (28%), which were almost the same treatment results achieved by another facility.
Conclusions
We recommend 54 Gy in 9 fractions over 7 days as a feasible treatment to reduce patient discomfort in mobile tongue cancer patients.
doi:10.5114/jcb.2014.40726
PMCID: PMC4003422  PMID: 24790616
dose reduction; high-dose-rate brachytherapy; tongue cancer
11.  High-dose-rate brachytherapy as monotherapy for prostate cancer: technique, rationale and perspective 
High-dose-rate (HDR) brachytherapy as monotherapy is a comparatively new brachytherapy procedure for prostate cancer. Although clinical results are not yet mature enough, it is a highly promising approach in terms of potential benefits for both radiation physics and radiobiology. In this article, we describe our technique for monotherapeutic HDR prostate brachytherapy, as well as the rationale and theoretical background, with educational intent.
doi:10.5114/jcb.2014.42026
PMCID: PMC4003433  PMID: 24790627
prostate cancer; radiotherapy; high-dose-rate (HDR) brachytherapy; monotherapy; hypofractionation
12.  Axitinib for preoperative downstaging of renal cell carcinoma with sarcomatoid differentiation and direct invasion of the duodenum and inferior vena cava: a case report 
OncoTargets and therapy  2014;7:289-295.
Background
Renal cell carcinoma (RCC) with sarcomatoid differentiation is invasive, refractory to treatment, and has a higher mortality. Therefore, systemic therapy is still challenging, and the curative resection of localized or locally advanced RCC with sarcomatoid differentiation is very important. Axitinib is a potent and selective second-generation vascular endothelial growth factor receptor tyrosine kinase inhibitor with improved safety and tolerability. Axitinib is generally recommended as second-line therapy for advanced RCC because the phase III axitinib versus sorafenib in advanced RCC (AXIS) trial demonstrated that it achieved longer progression-free survival than sorafenib in patients with metastatic RCC after failure of an approved first-line regimen.
Methods
We present a 73-year-old man who had a large (13 cm in diameter) right RCC with sarcomatoid differentiation that directly invaded the duodenum and inferior vena cava. The patient presented with gastrointestinal bleeding, was unable to eat solid food, and had become emaciated. Thus, his classification was poor risk with anemia, hypercalcemia, and poor performance status, according to the Memorial Sloan-Kettering Cancer Center criteria. He seemed unlikely to survive if radical nephrectomy, cavotomy with thrombectomy, and pancreatoduodenectomy were performed. To reduce the tumor burden and potential operative complications, we administered axitinib as first-line neoadjuvant therapy.
Results
Six weeks of treatment reduced the tumor burden without causing severe toxicities. Subsequently, radical right nephrectomy, cavotomy with thrombectomy, and pancreatoduodenectomy were performed successfully. The pathological treatment effect of axitinib was grade 2 (two-thirds necrosis). The resected tumor showed a heterogeneous reaction for phosphorylated Akt (Ser-473) by Western blotting and immunohistochemistry, indicating that parts of the tumor were sensitive to axitinib and other parts were not.
Conclusion
Axitinib might be promising as preoperative or neoadjuvant therapy for locally advanced RCC (>cT3b or >cTanyN1).
doi:10.2147/OTT.S58089
PMCID: PMC3931632  PMID: 24627641
renal cell carcinoma; sarcomatoid differentiation; axitinib; tyrosine kinase inhibitors; phosphorylated Akt
13.  Death from axillary haemorrhage during haemodialysis in a patient with a history of microscopic polyangiitis 
BMJ Case Reports  2012;2012:bcr1120115194.
An older female with a history of microscopic polyangiitis underwent haemodialysis through an end-to-side anastomosis between the left basilica vein and brachial artery. During the last haemodialysis session, repeated punctures induced haemorrhage that required brachial compression. Twenty min posthaemodialysis, haemorrhage had expanded from the axilla to the left lateral thorax. Autopsy disclosed axillary haematoma. The haemorrhage was not derived from punctured vessels or the left axillary artery. Although neither an alveolar nor a glomerular microscopic polyangiitis lesion was detected, fragility of the axillary small vessels due to microscopic polyangiitis, ageing, atherosclerosis and steroid therapy were underlying factors in the haematoma. Aspirin and heparin may have promoted haemorrhage, while shunt vessel stenosis with disturbed flow may have increased the axillary vessel pressure when the shunt vessels were compressed for haemostasis. This is the first report of a death due to haemorrhage from ruptured axillary vessels related to haemodialysis or microscopic polyangiitis.
doi:10.1136/bcr.11.2011.5194
PMCID: PMC3263122  PMID: 22665880
14.  An improved Bacillus subtilis cell factory for producing scyllo-inositol, a promising therapeutic agent for Alzheimer’s disease 
Background
Bacillus subtilis 168 possesses an efficient pathway to metabolize some of the stereoisomers of inositol, including myo-inositol (MI) and scyllo-inositol (SI). Previously we reported a prototype of a B. subtilis cell factory with modified inositol metabolism that converts MI into SI in the culture medium. However, it wasted half of initial 1.0% (w/v) MI, and the conversion was limited to produce only 0.4% (w/v) SI. To achieve a more efficient SI production, we attempted additional modifications.
Results
All “useless” genes involved in MI and SI metabolism were deleted. Although no elevation in SI production was observed in the deletion strain, it did result in no wastage of MI anymore. Thus additionally, overexpression of the key enzymes, IolG and IolW, was appended to demonstrate that simultaneous overexpression of them enabled complete conversion of all MI into SI.
Conclusions
The B. subtilis cell factory was improved to yield an SI production rate of 10 g/L/48 h at least. The improved conversion was achieved only in the presence of enriched nutrition in the form of 2% (w/v) Bacto soytone in the medium, which may be due to the increasing demand for regeneration of cofactors.
doi:10.1186/1475-2859-12-124
PMCID: PMC3878828  PMID: 24325193
Bacillus subtilis; scyllo-inositol; myo-inositol; Bioconversion; Alzheimer’s disease
15.  PhaP phasins play a principal role in poly-β-hydroxybutyrate accumulation in free-living Bradyrhizobium japonicum 
BMC Microbiology  2013;13:290.
Background
Bradyrhizobium japonicum USDA110, a soybean symbiont, is capable of accumulating a large amount of poly-β-hydroxybutyrate (PHB) as an intracellular carbon storage polymer during free-living growth. Within the genome of USDA110, there are a number of genes annotated as paralogs of proteins involved in PHB metabolism, including its biosynthesis, degradation, and stabilization of its granules. They include two phbA paralogs encoding 3-ketoacyl-CoA thiolase, two phbB paralogs encoding acetoacetylCoA reductase, five phbC paralogs encoding PHB synthase, two phaZ paralogs encoding PHB depolymerase, at least four phaP phasin paralogs for stabilization of PHB granules, and one phaR encoding a putative transcriptional repressor to control phaP expression.
Results
Quantitative reverse-transcriptase PCR analyses of RNA samples prepared from cells grown using three different media revealed that PHB accumulation was related neither to redundancy nor expression levels of the phbA, phbB, phbC, and phaZ paralogs for PHB-synthesis and degradation. On the other hand, at least three of the phaP paralogs, involved in the growth and stabilization of PHB granules, were induced under PHB accumulating conditions. Moreover, the most prominently induced phasin exhibited the highest affinity to PHB in vitro; it was able to displace PhaR previously bound to PHB.
Conclusions
These results suggest that PHB accumulation in free-living B. japonicum USDA110 may not be achieved by controlling production and degradation of PHB. In contrast, it is achieved by stabilizing granules autonomously produced in an environment of excess carbon sources together with restricted nitrogen sources.
doi:10.1186/1471-2180-13-290
PMCID: PMC4029623  PMID: 24330393
Bradyrhizobium japonicum; Phasin; PHB
16.  Increased expression of system large amino acid transporter (LAT)-1 mRNA is associated with invasive potential and unfavorable prognosis of human clear cell renal cell carcinoma 
BMC Cancer  2013;13:509.
Background
The system L amino acid transporter (LAT) has an important role in the transport of various amino acids, and there have been reports about the relation of this system to cancer. Although LATs are highly expressed in the kidneys, little is known about their influence on human renal cancer.
Methods
To clarify the role of LATs in human clear cell renal cell carcinoma (RCC), we investigated the expression of mRNAs for LAT1, LAT2, LAT3, LAT4, and 4F2hc in clear cell RCC tissues. The mRNAs of these five genes were analyzed by the real-time reverse transcription polymerase chain reaction in matched sets of tumor and non-tumor tissues obtained at operation from 82 Japanese patients with clear cell RCC. We also measured phosphorylated S6 ribosomal protein (Ser-235/236) proteins levels in 18 paired tumor and non-tumor tissues of the patients by Western blotting.
Results
Expression of LAT1 mRNA was significantly increased in tumor tissue compared with non-tumor tissue, while expression of LAT2 and LAT3 mRNAs was reduced. There was no difference in the expression of LAT4 and 4F2hc mRNAs between tumor and non-tumor tissues. Increased expression of LAT1 mRNA was associated with less differentiated tumors, local invasion, microscopic vascular invasion, and metastasis. Kaplan-Meier survival analysis showed that a higher serum LAT1 mRNA level was associated with a shorter overall survival time. Phosphorylated S6 ribosomal protein levels were associated with metastatic potential. LAT1 mRNA levels positively correlated with phosphorylated S6 ribosomal protein proteins levels in primary tumors.
Conclusions
These findings suggest that LAT1 mRNA is related to the invasive and progressive potential of clear cell RCC.
doi:10.1186/1471-2407-13-509
PMCID: PMC3832879  PMID: 24168110
17.  Enantioselectivity in the cytochrome P450-dependent conversion of tegafur to 5-fluorouracil in human liver microsomes 
Tegafur (FT) is a prodrug of 5-fluorouracil (5-FU) used in cancer chemotherapy, and the bioactivation of FT to 5-FU is mainly catalyzed by cytochrome P450 (CYP) in hepatic microsomes. FT has a chiral center and is a racemate consisting of the enantiomers, R- and S-FT. In the present study, we clarified the enantioselectivity in the conversion of FT to 5-FU and identified human CYP isoforms involved in the metabolism of its enantiomers using human hepatic preparations and recombinant CYP isoforms. Although 5-FU was generated from both FT enantiomers, R-FT was a preferred substrate than S-FT, because of the considerably higher intrinsic clearance for 5-FU formation from R-FT in liver. Eadie–Hofstee plots in microsomes showed that the conversions of R- and S-FT to 5-FU followed biphasic and monophasic kinetics, respectively. Based on the evaluation using cDNA-expressed enzymes, CYP2A6 showed the highest activity for 5-FU formation from R-FT with the Km value similar to that of the high-affinity component in microsomes. Also, CYP2A6 was the most effective catalyst for S-FT. Inhibition studies using CYP-selective inhibitors and anti-CYP antibodies demonstrated that CYP2A6 mainly contributed to the enantioselective metabolism of FT, and were almost in accordance with the relative percentage contribution of each CYP isoform to the metabolism of FT estimated using relative activity factor methods. These results suggest that the enantioselectivity in the bioactivation of FT to 5-FU in humans is mainly due to the large difference of the catalytic activity of CYP2A6 between R- and S-FT.
doi:10.1002/prp2.9
PMCID: PMC4184574  PMID: 25505563
5-fluorouracil; cytochrome P450 2A6; enantioselectivity; microsomes; tegafur; thymidine phosphorylase
18.  A novel angiomatoid epithelioid sarcoma cell line, Asra-EPS, forming tumors with large cysts containing hemorrhagic fluid in vivo 
BMC Research Notes  2013;6:305.
Background
Whereas we can use several human epithelioid sarcoma (ES) cell lines for basic and preclinical research, an angiomatoid ES cell line has not been reported to date. We have treated a case of an angiomatoid ES developing in the right upper extremity of a 67-year-old man.
Methods
An angiomatoid ES cell line, Asra-EPS was newly established and characterized for its morphology, growth rate and chromosomal analysis. Tumorigenicity of Asra-EPS cells was also analyzed in athymic nude mice.
Results
Asra-EPS cells were round, polygonal or spindle-shaped with an abundant cytoplasm and have been maintained continuously in vitro for over 150 passages during more than 15 months. These cells secreted cancer antigen 125 (CA 125), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) into the culture medium. Asra-EPS cells were tumorigenic when implanted in nude mice with tumors reaching a volume of 1000 mm3 at around 50 days. Histological features of tumors formed in mice were essentially the same as those of the original tumor, exhibiting a multinodular proliferation of eosinophilic epithelioid and spindle-shaped cells with prominent areas of hemorrhage and blood-filled cystic spaces strikingly corresponding to the potential of hemorrhagic cyst formation in the original tumor. They showed immunopositive staining for cytokeratins (AE1/AE3 and CAM5.2), epithelial membrane antigen (EMA), vimentin, CD31, CD34 and CA 125, but negative for integrase interactor 1 (INI-1) and factor VIII-related antigen.
Conclusions
The established cell line represents a biologically relevant new tool to investigate the molecular pathology of human angiomatoid ES and to evaluate the efficacy of novel therapeutics both in vitro and in vivo.
doi:10.1186/1756-0500-6-305
PMCID: PMC3734118  PMID: 23915498
Epithelioid sarcoma; Asra-EPS; VEGF; CA 125; INI-1; Cystogenesis
19.  Three-dimensional image-based high-dose-rate interstitial brachytherapy for mobile tongue cancer 
Journal of Radiation Research  2013;55(1):154-161.
To investigate the influence of a 3D image-based treatment-planning method for high-dose-rate interstitial brachytherapy (HDR-ISBT) for mobile tongue cancer, we analyzed dose–volume histogram results for the clinical target volume (CTV) and the mandible. Between October 2010 and November 2011, one and four patients having T2 and T3 tumors, respectively, were treated with HDR-ISBT. Multiplane implantation using 9–15 treatment applicators was performed. Lugol's iodine staining, metal markers, ultrasonography, and magnetic resonance imaging were used to identify the contours of the gross tumor volume (defined as the CTV). The results of the image-based treatment plan were compared with those of the conventional simulated plan on the basis of a reference point 5 mm from the applicator position. The mean D90(CTV) and V100(CTV) were 112% of the prescribed dose (PD) and 98.1%PD, respectively, for the image-based plan, and 113%PD and 97.2%PD, respectively, for the conventional plan. The median CTVref/Vref was 0.23 for the image-based plan and 0.16 for the conventional plan (P = 0.01). The mean D0.1 cm3 (mandible), D1 cm3 (mandible), and D2 cm3 (mandible) were 80.1%PD, 62.5%PD, and 55.7%PD, respectively, for the image-based plan, and 109.1%PD (P = 0.02), 82.4%PD (P = 0.005), and 74%PD (P = 0.004), respectively, for the conventional plan). Image-based treatment planning may achieve high-conformity radiotherapy for the CTV and decrease irradiated doses to the mandible.
doi:10.1093/jrr/rrt079
PMCID: PMC3885112  PMID: 23732769
high-dose-rate interstitial brachytherapy; mobile tongue cancer; image-based plan; dose–volume histogram
20.  Counterselection System for Geobacillus kaustophilus HTA426 through Disruption of pyrF and pyrR 
Applied and Environmental Microbiology  2012;78(20):7376-7383.
Counterselection systems facilitate marker-free genetic modifications in microbes by enabling positive selections for both the introduction of a marker gene into the microbe and elimination of the marker from the microbe. Here we report a counterselection system for Geobacillus kaustophilus HTA426, established through simultaneous disruption of the pyrF and pyrR genes. The pyrF gene, essential for pyrimidine biosynthesis and metabolization of 5-fluoroorotic acid (5-FOA) to toxic metabolites, was disrupted by homologous recombination. The resultant MK54 strain (ΔpyrF) was auxotrophic for uracil and resistant to 5-FOA. MK54 complemented with pyrF was prototrophic for uracil but insensitive to 5-FOA in the presence of uracil. To confer 5-FOA sensitivity, the pyrR gene encoding an attenuator to repress pyrimidine biosynthesis by sensing uracil derivatives was disrupted. The resultant MK72 strain (ΔpyrF ΔpyrR) was auxotrophic for uracil and resistant to 5-FOA. MK72 complemented with pyrF was prototrophic for uracil and 5-FOA sensitive even in the presence of uracil. The results suggested that pyrF could serve as a counterselection marker in MK72, which was demonstrated by efficient marker-free integrations of heterologous β-galactosidase and α-amylase genes. The integrated genes were functionally expressed in G. kaustophilus and conferred new functions on the thermophile. This report describes the first establishment of a pyrF-based counterselection system in a Bacillus-related bacterium, along with the first demonstration of homologous recombination and heterologous gene expression in G. kaustophilus. Our results also suggest a new strategy for establishment of counterselection systems.
doi:10.1128/AEM.01669-12
PMCID: PMC3457123  PMID: 22885745
21.  The emerging role of high-dose-rate (HDR) brachytherapy as monotherapy for prostate cancer 
Journal of Radiation Research  2013;54(5):781-788.
High-dose-rate (HDR) brachytherapy as monotherapy is a comparatively new brachytherapy procedure for prostate cancer. In addition to the intrinsic advantages of brachytherapy, including radiation dose concentration to the tumor and rapid dose fall-off at the surrounding normal tissue, HDR brachytherapy can yield a more homogeneous and conformal dose distribution through image-based decisions for source dwell positions and by optimization of individual source dwell times. Indication can be extended even to T3a/b or a part of T4 tumors because the applicators can be positioned at the extracapsular lesion, into the seminal vesicles, and/or into the bladder, without any risk of source migration or dropping out. Unlike external beam radiotherapy, with HDR brachytherapy inter-/intra-fraction organ motion is not problematic. However, HDR monotherapy requires patients to stay in bed for 1–4 days during hospitalization, even though the actual overall treatment time is short. Recent findings that the α/β value for prostate cancer is less than that for the surrounding late-responding normal tissue has made hypofractionation attractive, and HDR monotherapy can maximize this advantage of hypofractionation. Research on HDR monotherapy is accelerating, with a growing number of publications reporting excellent preliminary clinical results due to the high ‘biologically effective dose (BED)’ of >200 Gy. Moreover, the findings obtained for HDR monotherapy as an early model of extreme hypofractionation tend to be applied to other radiotherapy techniques such as stereotactic radiotherapy. All these developments point to the emerging role of HDR brachytherapy as monotherapy for prostate cancer.
doi:10.1093/jrr/rrt027
PMCID: PMC3766299  PMID: 23543798
prostate cancer; high-dose-rate (HDR); brachytherapy; monotherapy; hypofractionation
22.  Enrichment and characterization of a bacterial culture that can degrade 4-aminopyridine 
BMC Microbiology  2013;13:62.
Background
The agrichemical 4-aminopyridine is used as a bird repellent in crop fields and has an epileptogenic action in a variety of animals, including man and mouse. 4-Aminopyridine is biodegraded in the environment through an unknown mechanism.
Results
A 4-aminopyridine-degrading enrichment culture utilized 4-aminopyridine as a carbon, nitrogen, and energy source, generating 4-amino-3-hydroxypyridine, 3,4-dihydroxypyridine, and formate as intermediates. 4-Amino-3-hydroxypyridine could not be further metabolized and probably accumulated as a dead-end product in the culture. Biodegradability tests and partial sequence analysis of the enrichment culture indicated that 4-aminopyridine was mainly degraded via 3,4-dihydroxypyridine and that the metabolite is probably cleaved by 3-hydroxy-4-pyridone dioxygenase. Seven culturable predominant bacterial strains (strains 4AP-A to 4AP-G) were isolated on nutrient agar plates. Changes in the bacterial populations of 4-aminopyridine, 3,4-dihydroxypyridine, or formate/ammonium chloride enrichment cultures were monitored by denaturing gradient gel electrophoresis (DGGE) profiling of PCR-amplified 16S rRNA gene fragments. Sequence analysis of the 16S rRNA gene fragments derived from predominant DGGE bands indicated that Pseudomonas nitroreducens 4AP-A and Enterobacter sp. 4AP-G were predominant in the three tested enrichment cultures and that the unculturable strains Hyphomicrobium sp. 4AP-Y and Elizabethkingia sp. 4AP-Z were predominant in 4-aminopyridine and formate/ammonium chloride enrichment cultures and in the 3,4-dihydroxypyridine enrichment culture, respectively. Among the culturable strains, strain 4AP-A could utilize 3,4-dihydroxypyridine as a growth substrate. Although we could not isolate strain 4AP-Y on several media, PCR-DGGE analysis and microscopy indicated that the unique bi-polar filamentous bacterial cells gradually became more dominant with increasing 4-aminopyridine concentration in the medium.
Conclusions
Hyphomicrobium sp. 4AP-Y, P. nitroreducens 4AP-A, and Elizabethkingia sp. 4AP-Z probably play important roles in 4-aminopyridine degradation in crop fields. In the enrichment culture, 3,4-dihydroxypyridine and its metabolites including formate might be shared as growth substrates and maintain the enrichment culture, including these indispensable strains.
doi:10.1186/1471-2180-13-62
PMCID: PMC3637104  PMID: 23517195
4-aminopyridine; 4-amino-3-hydroxypyridine; 3,4-dihydroxypyridine; Hyphomicrobium; Pseudomonas nitroreducens; Elizabethkingia
23.  Cobalt Protoporphyrin Accelerates TFEB Activation and Lysosome Reformation during LPS-Induced Septic Insults in the Rat Heart 
PLoS ONE  2013;8(2):e56526.
Lipopolysaccharide (LPS)-induced myocardial dysfunction is caused, at least in part, by mitochondrial dysfunction. Mitochondrial dysfunction and the oxidative damage associated with it are scavenged through various cellular defense systems such as autophagy to prevent harmful effects. Our recent study has demonstrated that cobalt protoporphyrin IX (CoPPIX), a potent inducer of heme oxygenase-1 (HO-1), ameliorates septic liver injuries by enhancing mitochondrial autophagy in rats. In our current study, we show that CoPPIX (5 mg/kg s.c.) not only accelerates the autophagic response but also promotes lysosome reformation in the rat heart treated with LPS (15 mg/kg i.p.). Lysosomal membrane-associated protein-2 (LAMP2), which is essential to the maintenance of lysosomal functions in the heart, is depleted transiently but restored rapidly during LPS administration in the rat. Activation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, was also observed, indicating a hyper consumption and subsequent reformation of the lysosome to meet the increased demand for autophagosome cleaning. CoPPIX was found to promote these processes and tended to restore the LPS-induced suppression of cardiac performances whilst chloroquine (CQ; 20 mg/kg i.p.), an inhibitor of lysosomes and autophagic protein degradation, abrogates these beneficial effects. The cardioprotective effect of CoPPIX against LPS toxicity was also observed via decreased levels of cardiac releasing enzymes in the plasma. Taken together, our current data indicate that lysosome reformation mediated by TFEB may be involved in cardioprotection against LPS-induced septic insults, and serve as a novel mechanism by which CoPPIX protects the heart against oxidative stress.
doi:10.1371/journal.pone.0056526
PMCID: PMC3574118  PMID: 23457579
24.  Usefulness of Running Wheel for Detection of Congestive Heart Failure in Dilated Cardiomyopathy Mouse Model 
PLoS ONE  2013;8(1):e55514.
Background
Inherited dilated cardiomyopathy (DCM) is a progressive disease that often results in death from congestive heart failure (CHF) or sudden cardiac death (SCD). Mouse models with human DCM mutation are useful to investigate the developmental mechanisms of CHF and SCD, but knowledge of the severity of CHF in live mice is necessary. We aimed to diagnose CHF in live DCM model mice by measuring voluntary exercise using a running wheel and to determine causes of death in these mice.
Methodology/Principal Findings
A knock-in mouse with a mutation in cardiac troponin T (ΔK210) (DCM mouse), which results in frequent death with a t1/2 of 70 to 90 days, was used as a DCM model. Until 2 months of age, average wheel-running activity was similar between wild-type and DCM mice (approximately 7 km/day). At approximately 3 months, some DCM mice demonstrated low running activity (LO: <1 km/day) while others maintained high running activity (HI: >5 km/day). In the LO group, the lung weight/body weight ratio was much higher than that in the other groups, and the lungs were infiltrated with hemosiderin-loaded alveolar macrophages. Furthermore, echocardiography showed more severe ventricular dilation and a lower ejection fraction, whereas Electrocardiography (ECG) revealed QRS widening. There were two patterns in the time courses of running activity before death in DCM mice: deaths with maintained activity and deaths with decreased activity.
Conclusions/Significance
Our results indicate that DCM mice with low running activity developed severe CHF and that running wheels are useful for detection of CHF in mouse models. We found that approximately half of ΔK210 DCM mice die suddenly before onset of CHF, whereas others develop CHF, deteriorate within 10 to 20 days, and die.
doi:10.1371/journal.pone.0055514
PMCID: PMC3561288  PMID: 23383212
25.  High dose rate brachytherapy for oral cancer 
Journal of Radiation Research  2012;54(1):1-17.
Brachytherapy results in better dose distribution compared with other treatments because of steep dose reduction in the surrounding normal tissues. Excellent local control rates and acceptable side effects have been demonstrated with brachytherapy as a sole treatment modality, a postoperative method, and a method of reirradiation. Low-dose-rate (LDR) brachytherapy has been employed worldwide for its superior outcome. With the advent of technology, high-dose-rate (HDR) brachytherapy has enabled health care providers to avoid radiation exposure. This therapy has been used for treating many types of cancer such as gynecological cancer, breast cancer, and prostate cancer. However, LDR and pulsed-dose-rate interstitial brachytherapies have been mainstays for head and neck cancer. HDR brachytherapy has not become widely used in the radiotherapy community for treating head and neck cancer because of lack of experience and biological concerns. On the other hand, because HDR brachytherapy is less time-consuming, treatment can occasionally be administered on an outpatient basis. For the convenience and safety of patients and medical staff, HDR brachytherapy should be explored. To enhance the role of this therapy in treatment of head and neck lesions, we have reviewed its outcomes with oral cancer, including Phase I/II to Phase III studies, evaluating this technique in terms of safety and efficacy. In particular, our studies have shown that superficial tumors can be treated using a non-invasive mold technique on an outpatient basis without adverse reactions. The next generation of image-guided brachytherapy using HDR has been discussed. In conclusion, although concrete evidence is yet to be produced with a sophisticated study in a reproducible manner, HDR brachytherapy remains an important option for treatment of oral cancer.
doi:10.1093/jrr/rrs103
PMCID: PMC3534285  PMID: 23179377
brachytherapy; oral cancer; high dose rate

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