Simultaneous Pretreatment Research Articles

Current perspectives in enzymatic saccharification of lignocellulosic biomass

With the depletion of fossil fuel reserves, there is an urgent need to search for renewable and cost effective strategies for biofuel production. Lignocellulosic biomass has been perceived as a potential feedstock, wherein effective pretreatment and saccharification is necessary prerequisite for developing viable biofuel processes. Recent approaches in this context are, (i) studying enzymes from extremophilic organisms, particularly thermophiles which are gaining importance in this aspect as they are found to be stable and catalytically more effective under harsh conditions; (ii) usage of ionic liquids for pretreatment is emerging as a greener technology due to their non toxic nature. Developing/screening for ionic liquid tolerant lignocellulosic enzymes in order to attain simultaneous pretreatment and saccharification, offer an interesting option; and (iii) engineering/manipulating the existing lignocellulosic enzymes for desirable traits and viable saccharification and biofuel generation processes. The review encompasses these approaches and the focus on the recent development in the area.

Simultaneous pretreatment and saccharification: Green technology for enhanced sugar yields from biomass using a fungal consortium

Two different biomasses were subjected to simultaneous pretreatment and saccharification (SPS) using a cocktail of hydrolytic and oxidizing enzymes. Application of a novel laccase as a detoxifying agent caused the removal of 49.8% and 32.6% of phenolic contents from the soaked rice straw and willow, respectively. Hydrolysis of soaked substrates using a newly developed fungal consortium resulted in saccharification yield of up to 74.2% and 63.6% for rice straw and willow, respectively. A high saccharification yield was obtained with soaked rice straw and willow without using any hazardous chemicals. The efficiency of each step related to SPS was confirmed by atomic force microscopy. The suitability of the developed SPS process was further confirmed by converting the hydrolysate from the process into bioethanol with 72.4% sugar conversion efficiency. To the best of our knowledge, this is the first report on the development of a less tedious, single-pot, and eco-friendly SPS methodology.

최종 하수처리장 슬러지의 추가감량을 위한 슬러지 전처리 연구

본 연구는 부산시 남부하수처리장의 탈수슬러지를 이용하여 전처리에 따른 분해 및 혐기성 상태에서의 최종 메탄 및 이산화탄소 발생량을 평가하기 위해 Biochemical Methane Potential(BMP) 실험을 실시하였다. 초기 1 % TS 농도의 탈수슬러지를 각각 30분 동안 5M NaOH를 이용하여 알칼리 전처리를 실시하였고 초음파 전처리는 30분 동안 조사밀도를 1.5 W/mL로 조절하여 실시하였으며 알칼리와 초음파 전처리를 병합한 동시 전처리를 시행하였다. 그에 따라 SCOD 값이 초기 33.1 mg/L에서 최대 494 mg/L로 증가하였다. 또한 BMP 실험을 실시한 결과 31.2~84.2 mL <TEX>$CH_4/g$</TEX> VS, 9.2~13.5 mL <TEX>$CO_2/g$</TEX> VS로 나타났다. 영양배지와 상등액 슬러지를 주입여부에 따른 BMP 실험을 실시한 결과 각각 73.1, 73.8 mL <TEX>$CH_4/g$</TEX> VS, 11.2, 13.6 mL <TEX>$CO_2/g$</TEX> VS으로 나타났으며 모델값과는 큰 차이를 보이지 않았다. BMP 실험을 종료 후 VS를 분석한 결과 초기 62 %에서 33.8~45.4 %로 감소하였다. 알칼리, 초음파 전처리보다는 동시 전처리가 메탄발생량과 SCOD 값을 증가시켰으며 VS 감소율이 증가하여 혐기성 소화율이 향상되었다. In this study, biochemical methane potential test was conducted to estimate ultimate methane and carbon dioxide yield for anaerobic digestion and pretreatment with sewage sludge cake. Two of 0.2 % TS of sewage sludge cakes were treated with 5M NaOH or sonication of 0.51 W/mL during 30 min respectively. Another sample was treated simultaneously with NaOH and sonication in same condition. Then, initial soluble COD increased from 33.1 mg/L to 494 mg/L. After BMP test, methane production ranged from 3.12 and 84.2 mL <TEX>$CH_4$</TEX> per g of Volatile Solid (VS) and 9.2 and 13.5 mL <TEX>$CO_2$</TEX> per g of Volatile Solid (VS) for carbon dioxide. In other tests, injection of nutrient media or sludge supernatant produced 73.1 and 73.8 mL <TEX>$CH_4$</TEX> per g of Volatile Solid (VS) and 11.2 and 13.6 mL <TEX>$CO_2$</TEX> per g of Volatile Solid (VS) respectively. When BMP test finished, 62 % of initial volatile solids decreased to 33.8~45.4 %. Simultaneous pretreatment increased soluble COD, reduction rate of volatile solids and digestion efficiency than those for alkaline and ultrasonic pretreatment.

Collagen gel contraction assay using human bronchial smooth muscle cells and its application for evaluation of inhibitory effect of formoterol.

Collagen gel contraction assay is a method for evaluating contraction of cells embedded in collagen gel matrices through measuring the gel size. In the present study, we established a protocol for collagen gel contraction assay using human bronchial smooth muscle cells obtained commercially, and applied it for evaluation of inhibitory effect of formoterol on histamine-induced contraction. Human bronchial smooth muscle cells were embedded in collagen gel in wells of 24-well plates, and gel contraction against histamine or acetylcholine was observed. Gel size was measured at an interval of 10 min for 60 min from the addition of a stimulant. Both acetylcholine and histamine caused gel contraction in a concentration-dependent manner and the contraction by histamine was apparently potent than that by acetylcholine. Formoterol at concentrations of 10(-10)-10(-7) M inhibited collagen gel contraction caused by histamine concentration-dependently. Pre-treatment with fluticasone at a concentration of 10(-8) M apparently potentiated the inhibitory effect of formoterol at 10(-10) and 10(-8) M on collagen gel contraction by histamine. Prolonged pre-treatment with 10(-8) M formoterol abolished the inhibitory effect of 10(-8) M formoterol. Furthermore, 4 h simultaneous pre-treatment with 10(-8) M formoterol and fluticasone partially but significantly recovered the inhibitory effect of 10(-8) M formoterol. Present results indicate that the collagen gel contraction assay using human bronchial smooth muscle cells is useful for evaluating the effects of bronchodilating drugs, and that fluticasone potentiates the inhibitory effect of formoterol on histamine-induced collagen gel contraction.

Optimization of sono-assisted dilute sulfuric acid process for simultaneous pretreatment and saccharification of rice straw

In this study, sono-assisted dilute sulfuric acid process was evaluated for its viability of simultaneous pretreatment and saccharification of rice straw. Three critical factors for simultaneous pretreatment and saccharification process, such as sonication time (30–50 min), temperature (70–90 °C), and acid concentration (5–10 %), were optimized to maximize reducing sugar yield using Box-Behnken design and response surface methodology. The response surface methodology model was found to be adequately fitted to the obtained data. Simultaneous pretreatment and saccharification factors were optimized at sonication of 50 min, 80 °C and an acid concentration of 10 % yielding the maximum sugar content (31.78 g/100 g of biomass). Scanning electron microscopy revealed that the smooth surface of raw biomass was altered into a rough and porous surface as a result of sugar release, which showed the prospective feasibility of simultaneous pretreatment and saccharification process. This process integration may lead to develop economical bioethanol production facility. However, further research is required to make this process industrially viable.

Simultaneous pretreatment and acidogenesis of solid food wastes by a rotational drum fermentation system with methanogenic leachate recirculation and andesite porphyry addition

A simultaneous pretreatment and acidogenesis process was developed by recirculating methanogenic leachate and adding andesite porphyry (WRS) powder to a rotational drum fermentation system to enhance the anaerobic digestion of solid food wastes. In the continuous operations, methanogenic leachate recirculation significantly increased hydrolysis rates and volatile solids (VS) degradation. The VS degradation ratio and the hydrolysis rate constant at a higher leachate recirculation ratio (2:1 weight ratio of methanogenic leachate to substrate) were increased by 2.1- and 1.4-fold, respectively, compared to those of the lower ratio (1:1 leachate recirculation ratio). A 10% (weight ratio of WRS to substrate solid content) WRS addition assisted the biochemical reactions in the process at the higher leachate recirculation ratio was employed. The hydrolysis rate constant and VS degradation were elevated by 54.7% and 63.9%, respectively, with the WRS addition. Besides, the WRS addition enhanced the VA formation and its conversion to biogas.

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Simultaneous pretreatment and sacchariffication of rice husk by Phanerochete chrysosporium for improved production of reducing sugars

Phanerochete chrysosporium, the white-rot fungus, (a best source for lignolytic enzymes system) was used in the biological pretreatment of rice husk for reducing sugars production. Usually reducing sugar production through biochemical process involves two steps: solid state fermentation (SSF) of fungal pretreatment for delignification, subsequently pretreated biomass subjected to enzymatic hydrolysis. During the fungal pretreatment of rice husk for reducing sugar production along with cellulase and xylanse, the activities of lignin degradation-related enzymes such as lignin peroxidases (LiP), GLOX (glyoxidase), and aryl alcohol oxidases (AAO), were observed. The fungal pretreated rice husk produced highest (895.9mg/ml/2g of rise husk) reducing sugars on 18th day of fungal treatment. This method may be good alternative to avoid operational costs associated with washing and the removal of inhibitors during the conventional pretreatment methods.

Vitamin D binding protein gene polymorphisms and baseline vitamin D levels as predictors of antiviral response in chronic hepatitis C

Vitamin D deficiency seems to predict the unsuccessful achievement of sustained viral response (SVR) after antiviral treatment in hepatitis C virus (HCV) difficult-to-treat genotypes. Vitamin D binding protein (GC) gene polymorphisms are known to influence vitamin D levels. This study was performed to assess whether the interaction between basal circulating vitamin D and the GC polymorphism plays a role in influencing the rate of antiviral responses in patients affected by chronic hepatitis C. In all, 206 HCV patients treated with a combination therapy of pegylated (PEG)-interferon plus ribavirin were retrospectively evaluated. GC rs7041 G>T, GC rs4588 C>A, and IL-28B rs12979860 C>T polymorphisms were genotyped. Frequencies of GC rs7041 G>T and rs4588 C>A polymorphisms were: G/G = 64 (31.1%), G/T = 100 (48.5%), T/T = 42 (20.4%) and C/C = 108 (52.4%), C/A = 84 (40.8%), A/A = 14 (6.8%). Patients were divided into those carrying ≥3 major alleles (wildtype [WT]+: G-C/G-C, G-C/T-C, G-C/G-A, N = 100) and the remaining (WT-: G-C/T-A, T-A/T-C, T-A/T-A, T-C/T-C, N = 106). Four groups were identified: vitamin D ≤20 ng/mL and WT-, vitamin D ≤20 and WT+, vitamin D >20 and WT-, vitamin D >20 and WT+. In difficult-to-treat HCV genotypes the proportion of patients achieving SVR significantly increased with a linear trend from the first to the last group: 6/25 (24.0%), 9/24 (37.5%), 12/29 (41.4%), 19/29 (65.5%) (P = 0.003). At multivariate analysis, having basal vitamin D >20 ng/mL plus the carriage of GC WT+ was found to be an independent predictor of SVR (odds ratio 4.52, P = 0.015). In difficult-to-treat HCV genotypes, simultaneous pretreatment normal serum vitamin D levels and the carriage of GC-globulin WT isoform strongly predicts the achievement of SVR after PEG-interferon plus ribavirin antiviral therapy.

Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity

Identification of regulatable mechanisms by which transcription factor NF-E2 p45-related factor 2 (Nrf2) is repressed will allow strategies to be designed that counter drug resistance associated with its up-regulation in tumours that harbour somatic mutations in Kelch-like ECH-associated protein-1 (Keap1), a gene that encodes a joint adaptor and substrate receptor for the Cul3-Rbx1/Roc1 ubiquitin ligase. We now show that mouse Nrf2 contains two binding sites for β-transducin repeat-containing protein (β-TrCP), which acts as a substrate receptor for the Skp1-Cul1-Rbx1/Roc1 ubiquitin ligase complex. Deletion of either binding site in Nrf2 decreased β-TrCP-mediated ubiquitylation of the transcription factor. The ability of one of the two β-TrCP-binding sites to serve as a degron could be both increased and decreased by manipulation of glycogen synthase kinase-3 (GSK-3) activity. Biotinylated-peptide pull-down assays identified DSGIS338 and DSAPGS378 as the two β-TrCP-binding motifs in Nrf2. Significantly, our pull-down assays indicated that β-TrCP binds a phosphorylated version of DSGIS more tightly than its non-phosphorylated counterpart, whereas this was not the case for DSAPGS. These data suggest that DSGIS, but not DSAPGS, contains a functional GSK-3 phosphorylation site. Activation of GSK-3 in Keap1-null mouse embryonic fibroblasts (MEFs), or in human lung A549 cells that contain mutant Keap1, by inhibition of the phosphoinositide 3-kinase (PI3K) – protein kinase B (PKB)/Akt pathway markedly reduced endogenous Nrf2 protein and decreased to 10-50% of normal the levels of mRNA for prototypic Nrf2-regulated enzymes, including the glutamate-cysteine ligase catalytic and modifier subunits, glutathione S-transferases Alpha-1 and Mu-1, heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1. Pre-treatment of Keap1−/− MEFs or A549 cells with the LY294002 PI3K inhibitor or the MK-2206 PKB/Akt inhibitor increased their sensitivity to acrolein, chlorambucil and cisplatin between 1.9-fold and 3.1-fold, and this was substantially attenuated by simultaneous pre-treatment with the GSK-3 inhibitor CT99021.

Comparative study of hollow-fiber liquid-phase micro-extraction and an aqueous two-phase system for determination of phytohormones in soil

Two methods, hollow-fiber liquid-phase micro-extraction (HF-LPME) and an aqueous two-phase system (ATPS), have been systematically optimized and compared for extraction and determination of phytohormones in soil by high-performance liquid chromatography (HPLC). The effects on extraction of conditions including solvent type and volume, extraction time, temperature, and amount of salt were evaluated. It was shown that ATPS was superior to HF-LPME for determination of paclobutrazol and uniconazole under the optimum conditions. The limits of detection (LODs) of ATPS were 0.002 μg g(-1) for uniconazole and 0.01 μg g(-1) for paclobutrazol, whereas LODs of HF-LPME were 0.005 μg g(-1) and 0.03 μg g(-1), respectively. Relative standard deviations (RSDs, n=5) and recovery were in the range 1.7-5.3 % and 86-102 %, respectively, for ATPS and 6.7-7.9 % and 40-60 % for HF-LPME. In addition, the advantages of ATPS were shorter extraction time, suitable for simultaneous pretreatment of batches of samples, and higher extraction capacity. ATPS was therefore applied to the determination of paclobutrazol and uniconazole in real soil samples. Uniconazole was detected in all the samples analyzed whereas paclobutrazol was not found.

Polysulfide-borohydride modification of southern pine alkaline pulping integrated with hydrothermal pre-extraction of hemicelluloses

We studied three process modifications to investigate their effects on the property and yield recovery capabilities of kraft pulping integrated with hemicellulose pre-extraction of southern pine. Loblolly pine chips were pre-extracted with hot water until the sugar extraction yield reached the targeted value of 10% and then subjected to conventional and modified kraft pulping. Modification included polysulfide pretreatment; polysulfide-sodium borohydride dual pretreatment, and polysulfide followed by polysulfide-sodium borohydride dual pretreatment two-stage pretreatments prior to kraft pulping. In the first modification, about 5% of the lost pulp yield (total 7%) caused by hemicellulose pre-extraction could be recovered with 15%-20% polysulfide pretreatment. Complete recovery (7%) was achieved with simultaneous pretreatment using 15% polysulfide and 0.5% sodium borohydride with 0.1% anthraquinone in polysulfide-sodium borohydride dual pretreatment. Two-stage pretreatment using recycled 15% polysulfide followed by simultaneous treatment of 6% polysulfide and 0.4%–0.5% sodium borohydride with 0.1% anthraquinone also achieved 100% yield recovery. Continuous recycling of 15% polysulfide employed in the two-stage process modification maintained its yield protection efficiency in a repeated recycling cycle. No significant changes in paper strength were found in handsheets prepared from the three process modifications, except for a minor reduction in tear strength.

Ethanol production from grass silage by simultaneous pretreatment, saccharification and fermentation: First steps in the process development

AbstractGrass silage provides a great potential as renewable feedstock. Two fractions of the grass silage, a press juice and the fiber fraction, were evaluated for their possible use for bioethanol production. Direct production of ethanol from press juice is not possible due to high concentrations of organic acids. For the fiber fraction, alkaline peroxide or enzymatic pretreatment was used, which removes the phenolic acids in the cell wall. In this study, we demonstrate the possibility to integrate the enzymatic pretreatment with a simultaneous saccharification and fermentation to achieve ethanol production from grass silage in a one‐process step. Achieved yields were about 53 g ethanol per kg silage with the alkaline peroxide pretreatment and 91 g/kg with the enzymatic pretreatment at concentrations of 8.5 and 14.6 g/L, respectively. Furthermore, it was shown that additional supplementation of the fermentation medium with vitamins, trace elements and nutrient salts is not necessary when the press juice is directly used in the fermentation step.

Investigating the role of endogenous opioids and KATP channels in glycerol‐induced acute renal failure

The present study was designed to investigate the possible role of endogenous opioids and K(ATP) channels in glycerol-induced acute renal failure (ARF) in rats. The rats were subjected to rhabdomyolytic ARF by single intramuscular injection of hypertonic glycerol (50% v/v; 8 mL/kg), and the animals were sacrificed after 24 h of glycerol injection. The plasma creatinine, blood urea nitrogen, creatinine clearance, and histopathological studies were performed to assess the degree of renal injury. Naltrexone (2.5, 5.0 and 10.0 mg/kg s.c.), glibenclamide (5.0 and 10.0 mg/kg i.p.), and minoxidil (25 and 50 mg/kg) were employed to explore the role of endogenous opioids and K(ATP) channels in rhabdomyolysis-induced ARF. Pretreatment with naltrexone and glibenclamide attenuated hypertonic glycerol-induced renal dysfunction in a dose-dependent manner, suggesting the role of endogenous opioids and K(ATP) channels in the pathogenesis of myoglobuniric renal failure. However, the simultaneous pretreatment with naltrexone (10 mg/kg s.c.) and glibenclamide (10 mg/kg i.p.) did not enhance the reno-protective effects of individual drugs, suggesting that release of endogenous opioids and opening of K(ATP) channels constitute a single pathway in acute renal injury triggered by hypertonic glycerol-induced rhabdomyolysis. Furthermore, administration of minoxidil abolished the attenuating effects of naltrexone in glycerol-induced renal failure, suggesting that opening of K(ATP) channels is downstream to opioid receptor activation. It is concluded that hypertonic glycerol-induced rhabdomyolysis may involve release of endogenous opioids that in turn modulate K(ATP) channels to contribute in the pathogenesis of ARF.

The cap’n’collar transcription factor Nrf2 mediates both intrinsic resistance to environmental stressors and an adaptive response elicited by chemopreventive agents that determines susceptibility to electrophilic xenobiotics

Transcription factor Nrf2 regulates genes encoding drug-metabolising enzymes and drug transporters, as well as enzymes involved in the glutathione, thioredoxin and peroxiredoxin antioxidant pathways. Using mouse embryonic fibroblast (MEF) cells from Nrf2 +/+ and Nrf2 −/− mice, in conjunction with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay, we have shown that loss of Nrf2 diminishes the intrinsic resistance of mutant fibroblasts towards isothiocyanates (i.e. sulforaphane), epoxides (i.e. (2S,3S)-(−)-3-phenylglycidol, ethyl 3-phenylglycidate and styrene-7,8-epoxide), peroxides, hydroquinones and quinones (i.e. tert-butylhydroperoxide, tert-butylhydroquinone and 2,3-dimethoxynaphthoquinone), NaAsO 2, and various mutagens, including β-propiolactone, cisplatin, mechlorethamine and methyl methanesulfonate to ∼50% of that observed in equivalent wild-type cells. Exposure of Nrf2 +/+ fibroblasts, but not Nrf2 −/− fibroblasts, to a non-toxic dose (3 μmol/l) of the chemopreventive agent sulforaphane (Sul) stimulated an adaptive response that, 18 h after first being subjected to the isothiocyanate, caused an induction of between 2- and 10-fold in the levels of mRNA for glutamate–cysteine ligase catalytic (Gclc) and modifier (Gclm) subunits, glutathione S-transferases and NAD(P)H:quinone oxidoreductase-1 (Nqo1); this was accompanied by an increase in total glutathione of between 1.5- and 1.9-fold. Pre-treatment of Nrf2 +/+ MEF cells with 3 μM Sul for 18 h prior to challenge with xenobiotics, conferred between 2.0- and 4.0-fold protection against isothiocyanates, reactive carbonyls, peroxides, quinones, NaAsO 2, and the anticancer nitrogen mustard chlorambucil, but pre-treatment with 3 μM Sul produced no such increased tolerance in Nrf2 −/− MEF cells. The inducible resistance towards acrolein, cumene hydroperoxide and chlorambucil, produced by pre-treating wild-type fibroblasts with 3 μM Sul, was dependent on glutathione because simultaneous pre-treatment with 5 μmol/l buthionine sulfoximine abolished the increased tolerance of these xenobiotics. However, inducible resistance towards menadione that occurred upon pre-treatment with 3 μM Sul was independent of glutathione and may be due to upregulation of Nqo1. Thus Nrf2 controls cellular resistance against electrophiles.

Simultaneous pretreatment of lignocellulose and hydrolysis of starch in mixtures to sugars

Mixtures of starch and lignocelluloses are available in many industrial, agricultural, and municipal wastes and residuals. In this work, dilute sulfuric acid was used for simultaneous pretreatment of lignocellulose and hydrolysis of starch, to obtain a maximum amount of fermentable sugar after enzymatic hydrolysis with cellulase and β-glucosidase. The acid treatment was carried out at 70-150°C with 0-1% (v/v) acid concentration and 5-15% (w/v) solids concentration for 0-40 minutes. Under the optimum conditions, obtained at 130°C, 1% acid, and 7.5% solids loading for 30 min, the starch was almost completely converted to glucose. However, the acid treatment was not successful for efficient hydrolysis of pure cellulose. A mixture of pine softwood and potato as representatives of lignocellulosic and starch components, respectively, were treated at the optimum conditions for acid hydrolysis of starch. The dilute-acid treatment resulted in 1.2, 60.5, and 23.6% hydrolysis of glucan, xylan, and mannan of pine wood and 67% of potato starch to fermentable sugars. After the acid treatment, the solid residue of the mixture was subjected to enzymatic hydrolysis. The enzymatic hydrolysis under the optimum conditions resulted in conversion of 76% of the glucan in the treated softwood. Therefore, using acid treatment of the mixture is a promising process for pretreatment of wood in addition to the hydrolysis of starch.

Investigative Clinical Study on Prostate Cancer Part II: On the Role of the Pretreatment Total PSA to Free Testosterone Ratio as a Marker Assessing Prostate Cancer Prognostic Groups after Radical Retropubic Prostatectomy

Objectives: To explore the significance of the pretreatment total prostate-specific antigen (PSA) to free testosterone (FT) ratio (PSA/FT) as a marker for assessing the pathologic Gleason sum (pGS) and levels of tumor extension (pT) in prostatectomy specimens. Patients and Methods: 128 of 135 consecutive patients diagnosed with prostate cancer underwent radical prostatectomy. Simultaneous pretreatment serum samples were obtained to measure serum total testosterone, FT and total PSA levels. The statistical design of the study included 2 sections: the first part trying to explore the role of the PSA/FT ratio in clustering patients with different pathologic prognostic factors, and the second to investigate the PSA/FT ratio distribution in different groups of patients according to the pathologic stage and pGS of the specimen after radical prostatectomy. Results: The average age was 65.80 (range 51.21–77.26) years, mean PSA was 8.88 (range 1.22–44.27) µg/l, mean FT was 35.32 (range 13.70–69.30) pmol/l, and the mean PSA/FT ratio was 0.27 (range 0.04–1.48). The PSA/FT ratio significantly clustered both the pT and pGS groups. Analysis of variance for the distribution of the PSA/FT ratio was significant for the pT model groups. The mean PSA/FT ratio increased as the tumor extended and grew through the prostate gland (high-stage disease). Analysis of variance for the different distributions of the PSA/FT ratio was significant for all model pGS groups. In our investigation we also found (data not shown) that a PSA/FT ratio of ≧0.40 was strongly correlated with large extensive (pT3b+pT4) and high-grade cancers (pGS8+pGS9). Conclusions: Prostate cancer patients may be classified into 3 different pathologic prognostic groups according to the PSA/FT ratio: low risk (PSA/FT ≤0.20), intermediate risk (PSA/FT >0.20 and ≤0.40), and high risk (PSA/FT >0.40 and ≤1.5). The PSA/FT ratio may be considered as the marker expressing different biology groups of prostate cancer patients, and it is strongly associated with pT and pGS.

Postnatal development of kidney function in rats receiving thyroid hormones.

In immature rats, renal excretion of p-aminohippurate (PAH) can be increased by daily pretreatment with triiodothyronine (T3) or tetraiodothyronine (T4) beginning on the 2nd day after birth (10 micrograms/100 g b.wt. i.p.). The increase of PAH excretion is nearly of the same extent, if 5-, 10-, 20-, 30-, 50-, and 105-day-old rats were pretreated with thyroid hormones (10 micrograms/100 g b.wt. i.p., 3 days, once daily). There is no strongly dose dependent renal effect of T3 and T4, respectively. The time course of stimulation of renal PAH excretion was also characterized in rats of different ages. Simultaneous pretreatment of young and adult rats with cyclopenthiazide and T3 is not distinctly more effective as both components given alone. Particularly the low degree of stimulation in young rats receiving cyclopenthiazide cannot be pronounced by additional T3 administration. Inhibitors of protein biosynthesis (azauracil, neomycin) can antagonize the stimulation of renal p-amino-hippurate excretion in rats receiving T3. The presented data indicate that T3 is effective in young rats. Furthermore, T4 can be converted to T3 in young rats, too. The importance of an intact protein synthesis seems to be a prerequisite to stimulate the kidney function by T3 pretreatment.

BOTH 5‐HYDROXYTRYPTAMINE 5‐HT2A AND 5‐HT1B RECEPTORS ARE INVOLVED IN THE VASOCONSTRICTOR RESPONSE TO 5‐HT IN THE HUMAN ISOLATED INTERNAL THORACIC ARTERY

1. The 5-hydroxytryptamine (5-HT, serotonin) receptor subtypes that mediate vasoconstriction in the human internal thoracic artery (ITA), which is frequently used as an arterial graft, remain unclear. The aim of the present study was to elucidate the 5-HT receptor subtypes responsible for 5-HT-induced contraction of the human ITA. 2. The contractile responses to 5-HT of endothelium-denuded human ITA obtained from patients undergoing coronary bypass surgery were examined. In addition, we investigated the effects of sarpogrelate and SB224289, antagonists of 5-HT(2A) and 5-HT(1B) receptors, respectively, on the 5-HT-induced vasoconstriction. Finally, 5-HT(2A) and 5-HT(1B) receptors in the human ITA were immunolabelled. 3. 5-Hydroxytryptamine (1 nmol/L-10 micromol/L) caused vasoconstriction in a concentration-dependent manner. Both sarpogrelate (1 micromol/L) and SB224289 (1 micromol/L) significantly, but not completely, inhibited 5-HT-induced vasoconstriction. 4. Conversely, simultaneous pretreatment with supramaximum concentrations (1 micromol/L for both) of sarpogrelate and SB224289 almost completely inhibited the 5-HT-induced vasoconstriction. 5. Immunopositive staining for 5-HT(2A) and 5-HT(1B) receptors was detected in smooth muscle cells of the human ITA. 6. These results demonstrate that, in human ITA, 5-HT-induced vasoconstriction is mediated by activation of both 5-HT(2A) and 5-HT(1B) receptors. Thus, when the human ITA is used as an arterial graft, a combination of 5-HT(2A) and 5-HT(1B) receptor antagonists would appear to be most useful to prevent 5-HT-induced vasospasm.

The essentiality of PKCα and PKCβI translocation for CD14+monocyte differentiation towards macrophages and dendritic cells, respectively

Human peripheral CD14(+)monocytes have been known to differentiate into monocyte-derived macrophages (MDMs) or dendritic cells (MoDCs) upon suitable stimulation. However, the key intracellular molecule(s) associated with their differentiation towards specific cell types was(were) not fully understood. This study was designated to determine the association of PKC isoenzymes with the differentiation of CD14(+)monocytes into MDMs or MoDCs. Purified human peripheral CD14(+)monocytes were cultured with GM-CSF, or GM-CSF plus IL-4 for 7 days to induce cell differentiation. The phenotypic changes were analyzed by Flow-Cytometry using various specific antibodies to cell type-specific surface markers. The immunological functions of these differentiated cells were determined by measuring the amounts of TNF-alpha secretion for MDMs, and the capacities of antigen-capturing and bacterial phagocytosis for MoDCs. The translocations of PKC isoenzymes in these cells from cytosol to plasma membrane were examined by Western Blot analysis and Confocal Microscopic observation. The treatment of CD14(+)monocytes with either GM-CSF or PMA elicited PKCalpha translocation and consequently induced their differentiation into MDMs. The inclusion of PKCalpha/beta(I) specific inhibitor, Go6976, greatly inhibited the GM-CSF-induced PKCalpha translocation and dose-dependently reduced the GM-CSF- induced MDM differentiation. On the other hand, the simultaneous pretreatment of CD14(+)monocytes with Go6976 and PKCbeta-specific inhibitor predominantly suppressed the GM-CSF/IL-4-induced generation of MoDCs. Further study demonstrated that GM-CSF/IL-4 selectively induced the translocation of PKCbeta(I), not PKCalpha or PKCbeta(II), in CD14(+)monocytes. In conclusion, the cell fate commitment of CD14(+)monocytes towards MDMs or MoDCs appears to be steered by the selective activation of PKCalpha or PKCbeta(I), respectively.