Effects of overexpression of PKAc genes on expressions of lignin-modifying enzymes by Pleurotus ostreatus.

We studied the role of genes encoding the cAMP-dependent protein kinase A catalytic subunit (PKAc) in the ligninolytic system in Pleurotus ostreatus. The wild-type P. ostreatus strain PC9 has two PKAc-encoding genes: PKAc1 and PKAc2 (protein ID 114122 and 85056). In the current study, PKAc1 and PKAc2 were fused with a ß-tubulin promoter and introduced into strain PC9 to produce the overexpression strains PKAc1-97 and PKAc2-69. These strains showed significantly higher transcription levels of isozyme genes encoding lignin-modifying enzymes than strain PC9, but the specific gene expression patterns differed between the two recombinant strains. Both recombinants showed 2.05-2.10-fold faster degradation of beechwood lignin than strain PC9. These results indicate that PKAc plays an important role in inducing the wood degradation system in P. ostreatus.

Effects of calmodulin on expression of lignin-modifying enzymes in Pleurotus ostreatus.

Previously, we suppressed the expression of genes encoding isozymes of lignin peroxidase (LiP) and manganese peroxidase (MnP) using a calmodulin (CaM) inhibitor, W7, in the white-rot fungus Phanerochaete chrysosporium; this suggested that CaM positively regulates their expression. Here, we studied the role of CaM in another white-rot fungus, Pleurotus ostreatus, which produces MnP and versatile peroxidase (VP), but not LiP. W7 upregulated Mn(2+)-dependent oxidation of guaiacol, suggesting that CaM negatively regulates the production of the enzymes. Suppression of CaM in P. ostreatus using RNAi also led to upregulation of enzyme activity, whereas overexpression of CaM in P. ostreatus caused downregulation. Real-time RT-PCR showed that MnP1-6 and VP3 levels in the CaM-knockdown strain were higher than those in the wild-type strain, while MnP-5 and -6 and VP1 and 2 levels in the CaM-overexpressing strain were lower than in the wild type. Moreover, we also found that another ligninolytic enzyme, laccase, which is not produced by P. chrysosporium, was negatively regulated by CaM in P. ostreatus similar to MnP and VP. Although overexpression of CaM did not reduce the ability of P. ostreatus to digest beech wood powder, the percentage of lignin remaining in the digest was slightly higher than in the wild-type strain digest.

Effect of solvent polarity on enantioselectivity in Candida antarctica lipase B catalyzed kinetic resolution of primary and secondary alcohols.

The Candida antarctica lipase B (CAL-B) catalyzed kinetic resolution of primary and secondary alcohols via acetylation is dependent on the permittivity (ε) of the reaction solvent. For example, the enantiomeric ratio (E) vs ε plot for the acetylation of 1-(naphth-2-yl)ethanol (1) exhibits a convex shape, taking the maximum E value at a medium ε value (11.2), whereas the same plot for the acetylation of benzyl 3-hydroxybutylate (3) exhibits a concave shape, taking the minimum E value at a similar ε value (11.6). Kinetic studies reveal that the difference in shape of the E vs ε plots originates from the relative reaction rate between the enantiomers with different Michaelis constants (Km). Thus, when the enantiomer with a larger Km value in the middle ε region reacts more slowly than its antipode, the ε dependence of E exhibits a convex shape. On the other hand, when the enantiomer reacts more quickly, it exhibits a concave shape. The E vs ε plot for the acetylation of 2-methoxy-2-phenylethanol (7) exhibits a convex shape with the maximum E value (20) at ε = 14.1. The E value can be further improved to almost reach the efficiency required for industrial applications (E ≈ 30) by the addition of a nitro compound.

Advantageous properties of a new fungicide, isofetamid.

The fungicidal properties of a new fungicide, isofetamid, were examined to assess its antifungal spectrum, mode of action, and effects on the infection process of Botrytis cinerea. Additionally, we investigated its fungicidal activity against isolates of B. cinerea resistant to existing fungicides. In mycelial growth inhibition tests, isofetamid exhibited excellent fungicidal activity against ascomycetes but showed no activity against basidiomycetes and oomycetes. Respiratory enzyme assay using mitochondria revealed that isofetamid inhibited succinate dehydrogenase activity prepared from B. cinerea and other ascomycetes fungi used in the study. On the other hand, the activity of mitochondria prepared from Pythium, potato and rat were not inhibited. Isofetamid inhibited also many stages of the infection processes in B. cinerea. Furthermore, it exhibited high fungicidal activity against B. cinerea isolates that were resistant to existing fungicides.

Switching of the diastereomer deposited during the crystallization of N-[(S)-1-phenylethyl]-2'-carbamoyl-1,1'-binaphthalene-2-carboxylic acid: investigation of the mechanism of dielectrically controlled resolution.

Dielectrically controlled resolution (DCR) has been achieved during the crystallization of (S)-1-phenylethylamides of racemic 1,1'-binaphthalene-2,2'-dicarboxylic acid (RS(a),S)-1. For example, a water well-shaped plot is obtained for the diastereomeric excess (de) of the deposited amide versus the solvent permittivity (ε) for the crystallization of (RS(a),S)-1 from three-component mixed solvents, consisting of 25 vol % of dichloromethane and 75 vol % of varying ratios of two solvents (i.e., an alcohol and either hexane or water). The de value drastically changes within two narrow ε ranges and diastereomerically pure crystals of either (R(a),S)-1 (13.9 ≤ ε ≤ 17.9) or (S(a),S)-1·CH(2)Cl(2) (ε ≤ 11.9 and ε ≥ 21.8) deposit, depending on the solvent permittivity. X-ray crystallographic analyses reveal that the major difference between the crystal structures of (S(a),S)-1 and (R(a),S)-1 is the presence of solvent molecules that fill the spatial voids in the (S(a),S)-1 crystals. The ε-dependence of the chemical shifts of (S(a),S)-1 and (R(a),S)-1 suggests that their aggregation states are similar in the same solvents and change discontinuously at two ε values. The ε-dependence of the C═O stretching vibrations suggests that the lower ε is a transition point where the amide molecules, which aggregate through intermolecular hydrogen bonds in low-permittivity solvents, begin to dissociate. An absorption experiment suggests that dichloromethane is easily incorporated into solvent-free (S(a),S)-1 crystals in high-permittivity solvents. On the basis of these observations, a feasible molecular mechanism is proposed for the present DCR phenomenon.

The white-rot fungus pleurotus ostreatus transformant overproduced intracellular cAMP and laccase.

Transformation of Pleurotus ostreatus PC9 with the mutated heterotrimeric G protein alpha subunit (Gα) gene resulted in higher laccase (Lac) activity and intracellular cyclic adenosine monophosphate (cAMP) concentrations as compared to those in wild-type PC9. The transformant also exhibited higher Lac activity than the wild type when cultured in a medium containing known Lac inducers CuSO4 and ferulic acid.

Fungicidal spectrum and biological properties of a new fungicide, pyriofenone.

Pyriofenone is a new fungicide developed by Ishihara Sangyo Kaisha, Ltd. To determine the fungicidal spectrum of pyriofenone, in vivo pot tests and in vitro mycelial growth-inhibition tests were conducted. Pyriofenone showed excellent activity against wheat and cucumber powdery mildew and moderate efficacy against rice blast in the pot tests. In the mycelial growth-inhibition tests, most fungi were not affected by pyriofenone except for Botrytis cinerea, Helminthosporium sacchari, Pseudocercosporella herpotrichoides, Pyricularia oryzae, Rosellinia necatrix, and Verticillium dahliae. The fungicidal properties of pyriofenone on powdery mildew in cucumber and wheat were evaluated precisely. Pyriofenone exhibited excellent preventive and residual activities. It had high rainfastness in the cucumber leaves against powdery mildew. Pyriofenone also showed inhibitory activity on lesion development upon application until 2 days after inoculation, and the lesion expansion and sporulation of the cucumber powdery mildew fungus were effectively controlled. Furthermore, pyriofenone showed translaminar and vapor activities.

Docetaxel with or without estramustine for estramustine refractory castration-resistant prostate cancer: a single institution experience.

BACKGROUND: The significance of combination of docetaxel (DTX) with estramustine phosphate (EMP) in castration-resistant prostate cancer (CRPC) patients remains unclear. In this study, we aimed to retrospectively evaluate the efficacy and toxicity of DTX with or without EMP and to elucidate the significance of DTX and EMP combination therapy in Japanese EMP-refractory CRPC patients. METHODS: To compare the efficacy and toxicity of DTX and EMP, we divided CRPC patients, who were confirmed to be resistant to EMP, into the following two groups: group D (n = 28), which included patients treated with DTX (60 mg/m2, once in every four weeks) alone, and group DE (n = 33), which included patients treated with a combination of DTX (60 mg/m2, once in every four weeks) and EMP (twice daily oral administration at 280 mg). RESULTS: Prostate specific antigen (PSA) response (> 50% decline in PSA) was observed in six patients (21%) in group D and eight patients (24%) in group DE. The median time to progression (TTP) was 12.0 months and 6.2 months and the median overall survival (OS) was 26.4 months and 24.3 months in group D and DE, respectively. There was no statistical difference between the two groups in terms of PSA response, TTP, and OS. The incidence of adverse events of grade 3/4 was low in both the groups, and there was no statistical difference between the two groups. CONCLUSIONS: Although treatment with DTX at 60 mg/m2 was effective and highly tolerated in EMP-refractory Japanese CRPC patients, the DTX and EMP combination therapy might not exhibit any survival benefit for CRPC patients.

Genetic basis for the hierarchical interaction between Tobamovirus spp. and L resistance gene alleles from different pepper species.

The pepper L gene conditions the plant's resistance to Tobamovirus spp. Alleles L(1), L(2), L(3), and L(4) confer a broadening spectra of resistance to different virus pathotypes. In this study, we report the genetic basis for the hierarchical interaction between L genes and Tobamovirus pathotypes. We cloned L(3) using map-based methods, and L(1), L(1a), L(1c), L(2), L(2b), and L(4) using a homology-based method. L gene alleles encode coiled-coil, nucleotide-binding, leucine-rich repeat (LRR)-type resistance proteins with the ability to induce resistance response to the viral coat protein (CP) avirulence effectors by themselves. Their different recognition spectra in original pepper species were reproduced in an Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana. Chimera analysis with L(1), which showed the narrowest recognition spectrum, indicates that the broader recognition spectra conferred by L(2), L(2b), L(3), and L(4) require different subregions of the LRR domain. We identified a critical amino acid residue for the determination of recognition spectra but other regions also influenced the L genes' resistance spectra. The results suggest that the hierarchical interactions between L genes and Tobamovirus spp. are determined by the interaction of multiple subregions of the LRR domain of L proteins with different viral CP themselves or some protein complexes including them.

Clinical efficacy of primary combined androgen blockade for Japanese men with clinically localized prostate cancer unsuitable for local definitive treatment: a single institution experience.

BACKGROUND: Primary hormonal therapy has been mostly used for patients with advanced prostate cancer, as international guidelines do not recommend its use for patients at earlier disease stages. However, there seems to be a discrepancy between the guideline recommendations and clinical practice on the use of primary androgen deprivation therapy for localized prostate cancer in Japan. Therefore, we retrospectively analyzed a single-institution experience in primary combined androgen blockade (CAB) for localized prostate cancer. PATIENTS AND METHODS: The study included 187 patients with T1c-T3a prostate cancer unsuitable for local definitive treatment and treated with primary CAB. Clinical outcomes, predictive factors of PSA relapse and adverse events were investigated. RESULTS: The progression-free, disease-specific, and overall survival rates of all patients at 5 years were 63.0, 99.4 and 95.9%, respectively. Of the several parameters isolated as predictors of prostate-specific antigen (PSA) progression, nadir PSA level and the percentage of positive biopsy cores (%PBC) remained as independent prognostic factors on multivariate analysis. Toxicities were mild to moderate and well tolerated. CONCLUSIONS: Primary CAB treatment brought initial disease control without relapse in the majority of our selected cases. The %PBC may help predict time to relapse in the pretreatment setting. The results implicate that CAB can be an option as a primary treatment for clinically localized prostate cancer unsuitable for local definitive treatment. To confirm the exact efficacy of primary CAB, these findings should be reviewed in a large cohort of patients with long-term follow-up from various viewpoints, including disease control, toxicities, quality-of-life and medical cost.

Transcriptional effect of a calmodulin inhibitor, W-7, on the ligninolytic enzyme genes in Phanerochaete chrysosporium.

We investigated the effects of a calmodulin (CaM) inhibitor, W-7, on the expression of lignin peroxidase (LiP) and manganese peroxidase (MnP) genes in Phanerochaete chrysosporium to consider the role of cam gene, which was upregulated in parallel with the total activities of LiP and MnP in our previous transcriptomic analysis. The addition of 100 µM W-7 to the fungal cultures repressed the total activities of LiP and MnP, whereas the addition of 100 µM W-5, which is a control drug of W-7, retained approximately half of them, indicating that the effect of W-7 was attributable to CaM inhibition. Real-time reverse transcription polymerase chain reaction analysis revealed that most of lip and mnp isozyme genes predicted from whole-genome data were significantly inhibited by W-7 at the transcription level (P ≤ 0.05). These results suggest that CaM has an important role for the expression of isozyme genes of LiP and MnP at the transcription level.

Outcome of adjuvant radiotherapy after radical prostatectomy for prostate cancer patients.

AIM: To examine the outcome and risk factors of biochemical failure (BCF) in Japanese prostate cancer (PCa) patients treated with adjuvant radiotherapy (RT) after radical prostatectomy (RP). PATIENTS AND METHOD: In this study we enrolled 83 Japanese patients having clinically organ-confined PCa without neoadjuvant treatments who received conventional RT (60 Gy) after RP. All patients had extracapsular extension (ECE) and/or positive surgical margin (PSM) of the RP specimens, but no lymph node metastasis. The disease-specific, clinical failure-free, and BCF-free survivals were analyzed. Furthermore, the risk factors affecting the BCF-free survival were examined in detail. RESULTS: The 5-year disease-specific, clinical failure-free, and BCF-free survival rates were 100, 99, and 87%, respectively. The clinicopathological factors associated with BCF were seminal vesicle invasion (SVI) (p = 0.024), perineural invasion (PNI) (p = 0.03), and pre-RT prostate-specific antigen (PSA) (p = 0.014). In the patients with PSM (n = 59), the entire surgical margin-positive patients had a significantly higher risk of BCF than the focal surgical margin-positive patients (p = 0.015). Multivariate analysis showed that SVI and pre-RT PSA were independent prognostic factors of BCF (p = 0.0142, p = 0.0225, respectively). 22% of our patients had only low-grade adverse effects. CONCLUSION: The outcome of adjuvant RT after RP in the Japanese patients with ECE and/or PSM was excellent, and the adverse effects were mild and tolerable. However, the patients with SVI, PNI, entire surgical margin-positive specimens, or high pre-RT PSA had poor biochemical control by only adjuvant RT after RP.

Single amino acid substitution in the methyltransferase domain of Paprika mild mottle virus replicase proteins confers the ability to overcome the high temperature-dependent Hk gene-mediated resistance in Capsicum plants.

Capsicum plants harboring the Hk gene (Hk) show resistance to Paprika mild mottle virus (PaMMV) at 32 degrees C but not 24 degrees C. To identify the viral elicitor that activates the Hk-mediated resistance, several chimeric viral genomes were constructed between PaMMV and Tobacco mosaic virus-L. Infection patterns of these chimeric viruses in Hk-harboring plants revealed responsibility of PaMMV replicase genes for activation of the Hk-mediated resistance. The comparison of nucleotide sequence of replicase genes between PaMMV and PaHk1, an Hk-resistance-breaking strain of PaMMV, revealed that the adenine-to-uracil substitution at the nucleotide position 721 causes an amino acid change from threonine to serine at the 241st residue in the methyltransferase domain. Introduction of the A721U mutation into the replicase genes of parental PaMMV overcame the Hk resistance at 32 degrees C. The results indicate that Hk-mediated resistance is induced by PaMMV replicase proteins and that methyltransferase domain has a role in this elicitation.

Changes in the gene expression of the white rot fungus Phanerochaete chrysosporium due to the addition of atropine.

We constructed a LongSAGE (Long Serial Analysis of Gene Expression) library from a 3-d culture of Phanerochaete chrysosporium supplemented with atropine, which inhibits the production of lignin-degrading enzymes. The library (the atropine library) contains 13,108 LongSAGE tags and 6,783 unique tags. The gene expression profile represented by the tags was compared with those of two previously constructed libraries, one of which was constructed using 2-d cultures in which the fungus had not yet produced ligninolytic enzymes (the 2-d library) and the other was constructed using 3-d cultures in which the fungus had just started to produce the enzymes (the 3-d library). We found a total of 595 genes that were at least twice more highly or at least twice less highly expressed in the 3-d library than in the 2-d library or the atropine library, and the fluctuations were statistically significant. The relationships among these 595 genes were considered using cluster analysis. Of the 595 genes, 164 showed expression patterns similar to those of four ligninolytic enzyme genes, which were more expressed on day 3 than under any other conditions. Many of these 164 genes comprised genes possibly involved in lignin degradation, lipid metabolism, xenobiotic degradation, stress response, or signal transduction pathways.

Significance of serum calcitonin gene-related peptide levels in prostate cancer patients receiving hormonal therapy.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is one of the neuroendocrine markers. The serum CGRP levels in untreated prostate cancer (PCa) patients reportedly reflect the tumor volume and aggressiveness. However, the detailed evaluation of the serum CGRP levels in the PCa patients treated with hormonal therapy (HT) has never been reported. We measured the serum CGRP levels in PCa patients receiving HT to elucidate its clinical significance. MATERIALS AND METHODS: We used serum samples from 129 PCa patients. Thirty-six patients had never received any treatment, and 93 had been treated with HT. The serum CGRP was measured by immunoradiometric assay, and we analyzed the correlation between the serum CGRP level and the clinicopathological factors. RESULTS: The serum CGRP levels in the patients with higher clinical stages and histological grades were significantly higher than in those with lower stages and grades. Although in the patients treated with HT, the serum CGRP levels significantly correlated with clinical stage, these levels did not correlate with histological grade and the kind of HT. The highest serum CGRP level was observed in the stage D1 and stage D2 patients, not in the stage D3 patients treated with HT. CONCLUSION: The serum CGRP levels were significantly elevated in the PCa patients with high grade or high stage. However, in PCa patients receiving HT, various clinical factors influence the CGRP levels. Thus, evaluation of the levels should be careful.

[Retroperitoneal solitary fibrous tumor: a case report].

A 63-year-old man with a retroperitoneal tumor found incidentally was referred to our hospital. Computed tomography showed a tumor ventrally adjacent to urinary bladder and prostate. Pathological examination of retroperitoneal tumor specimens obtained by percutaneous needle biopsy revealed hypercellularity of spindle cells positive for CD 34. Under the suspicion of solitary fibrous tumor (SFT) or stromal tumors of uncertain malignant potential (STUMP), we performed en bloc resection of tumor, urinary bladder and prostate because tumor was firmly fixed to urinary bladder and prostate. The final diagnosis of retroperitoneal tumor was SFT because pathological findings of the surgical specimen were the same as those of the biopsy specimens.

Glucose transporter 1-mediated vascular translocation of nanomedicines enhances accumulation and efficacy in solid tumors.

Nanomedicine modification with ligands directed to receptors on tumor blood vessels has the potential for selectively enhancing nanomedicine accumulation in malignant tissues by overcoming the vascular barrier of tumors. Nevertheless, the development of broadly applicable ligand approaches capable of promoting the transvascular transport of nanomedicines in a wide spectrum of tumors has been elusive so far. By considering the indispensable and persistent glycolytic fueling of tumors, we developed glucose-installed polymeric micelles loading cisplatin (Gluc-CDDP/m) targeting the glucose transporter 1 (GLUT1), which is overexpressed in most tumors and present on vascular endothelial cells, toward improving the delivery efficiency and therapeutic efficacy. The design of the glucose ligands on Gluc-CDDP/m was engineered to control the conjugation via the carbon 6 of the glucose moieties, as well as the ligand density on the poly (ethylene glycol) (PEG) shell of the micelles. The series of micelles was then studied in vitro and in vivo against GLUT1-high human squamous cell carcinoma of the head and neck OSC-19 cells and GLUT1-low human glioblastoma-astrocytoma U87MG cells. Our results showed that precisely tuning the micelles to have glucose ligands on 25% of their PEG chains increased the efficacy against the tumors by significantly enhancing the tumor accumulation, even in GLUT1-low U87MG tumors. The enhancement of the intratumoral levels of these micelles was hindered by concomitant administration of glucose, or the GLUT1 inhibitor STF-31, confirming a GLUT1/glucose-mediated increment of the accumulation. Intravital confocal laser scanning microscopy imaging of tumor tissues further demonstrated the rapid extravasation and penetration of Gluc-CDDP/m in OSC-19 tumors compared to non-targeted CDDP/m. These findings indicate GLUT1-targeting as a promising approach for overcoming the vascular barrier and boosting the delivery of nanomedicine in tumors.

[Preoperative magnetic resonance imaging in the management of the nonpalpable testis].

We evaluated the efficacy of magnetic resonance imaging (MRI) as the preoperative study in the management of the nonpalpable testis. From 1997 to 2005, a total of 41 MRI was performed for the patients with nonpalpable testis and 42 testes were evaluated. The MRI showed 19 testes from 18 patients in the inguinal region. Eighteen testes were found in the inguinal region and one was intraabdominal during groin exploration. Six testes were pointed out to be intra-abdominal by MRI. Five testes were identified in the abdomen and one was disclosed to be vanishing testis. Seventeen testes could not be detected by MRI and they underwent laparoscopic or groin exploration. Four abdominal testes were identified. Thirteen patients had a diagnosis of vanishing testis. The sensitivity and the specificity of our MRI studies in predicting the presence of nonpalpable testis were 85.7% and 92.9%, respectively. Enlarged lymph node could be misdiagnosed as abdominal testis. Inguinal undescended testes can be diagnosed accurately with MRI. MRI is informative regarding the testis location and useful for limiting the area of surgical exploration.

[Does carbonate originate from carbonate-calcium crystal component of the human urinary calculus?].

PURPOSE: It gives important information in selecting the appropriate treatment for urolithiasis to confirm the component of urinary calculus. Presently component analysis of the urinary calculus is generally performed by infrared spectroscopy which is employed by companies providing laboratory testing services in Japan. The infrared spectroscopy determines the molecular components from the absorption spectra in consequence of atomic vibrations. It has the drawback that an accurate crystal structure cannot be analyzed compared with the X-ray diffraction method which analyzes the crystal constituent based on the diffraction of X-rays on crystal lattice. The components of the urinary calculus including carbonate are carbonate apatite and calcium carbonate such as calcite. Although the latter is reported to be very rare component in human urinary calculus, the results by infrared spectroscopy often show that calcium carbonate is included in calculus. The infrared spectroscopy can confirm the existence of carbonate but cannot determine whether carbonate is originated from carbonate apatite or calcium carbonate. Thus, it is not clear whether calcium carbonate is included in human urinary calculus component in Japan. In this study, we examined human urinary calculus including carbonate by use of X-ray structural analysis in order to elucidate the origin of carbonate in human urinary calculus. MATERIALS AND METHODS: We examined 17 human calculi which were reported to contain calcium carbonate by infrared spectroscopy performed in the clinical laboratory. Fifteen calculi were obtained from urinary tract, and two were from gall bladder. The stones were analyzed by X-ray powder method after crushed finely. RESULTS: The reports from the clinical laboratory showed that all urinary culculi consisted of calcium carbonate and calcium phosphate, while the gallstones consisted of calcium carbonate. But the components of all urinary calculi were revealed to be carbonate apatite by X-ray diffraction. The components of gallstones were shown to be calcium carbonate (one calcite and the other aragonite) not only by infrared spectroscopy but by X-ray diffraction. CONCLUSIONS: It was shown that component analysis of the calculus could be more accurately performed by adding X-ray diffraction method to infrared spectroscopy. It was shown that calcium carbonate existed in a gallstone. As for the carbonate in human urinary calculi, present study showed that it was not calcium carbonate origin but carbonate apatite origin.