Development of a bioavailable Hg(II) sensing system based on MerR-regulated visual pigment biosynthesis.

Engineered microorganisms have proven to be a highly effective and robust tool to specifically detect heavy metals in the environment. In this study, a highly specific pigment-based whole-cell biosensor has been investigated for the detection of bioavailable Hg(II) based on an artificial heavy metal resistance operon. The basic working principle of biosensors is based on the violacein biosynthesis under the control of mercury resistance (mer) promoter and mercury resistance regulator (MerR). Engineered biosensor cells have been demonstrated to selectively respond to Hg(II), and the specific response was not influenced by interfering metal ions. The response of violacein could be recognized by the naked eye, and the time required for the maximum response of violacein (5 h) was less than that of enhanced green fluorescence protein (eGFP) (8 h) in the single-signal output constructs. The response of violacein was almost unaffected by the eGFP in a double-promoter controlled dual-signals output construct. However, the response strength of eGFP was significantly decreased in this genetic construct. Exponentially growing violacein-based biosensor detected concentrations as low as 0.39 µM Hg(II) in a colorimetric method, and the linear relationship was observed in the concentration range of 0.78-12.5 µM. Non-growing biosensor cells responded to concentrations as low as 0.006 µM Hg(II) in a colorimetric method and in a Hg(II) containing plate sensitive assay, and the linear relationship was demonstrated in a very narrow concentration range. The developed biosensor was finally validated for the detection of spiked bioavailable Hg(II) in environmental water samples.

Comparative physiological and transcriptomic analyses of photosynthesis in Sphagneticola calendulacea (L.) Pruski and Sphagneticola trilobata (L.) Pruski.

Sphagneticola trilobata (L.) Pruski is one of the fast-growing malignant weeds in South China. It has severely influenced local biodiversity and native plant habitat. Photosynthesis is the material basis of plant growth and development. However, there are few reports on the photosynthetic transcriptome of S. trilobata. In this study, S. trilobata had a relatively large leaf area and biomass. The gas exchange parameters per unit area of leaves, including net photosynthetic capacity (Pn), intercellular CO2 (Ci), stomatal conductance (Gs), transpiration rate (Tr), water use efficiency (WUE), photosynthetic pigment and Rubisco protein content were higher than those of the native plant Sphagneticola calendulacea (L.) Pruski. On this basis, the differences in photosynthesis pathways between the two Sphagneticola species were analyzed by using the Illumina HiSeq platform. The sequencing results for S. trilobata and S. calendulacea revealed 159,366 and 177,069 unigenes, respectively. Functional annotation revealed 119,350 and 150,846 non-redundant protein database annotations (Nr), 96,637 and 115,711 Swiss-Prot annotations, 49,159 and 60,116 Kyoto Encyclopedia of Genes and Genomes annotations (KEGG), and 83,712 and 97,957 Gene Ontology annotations (GO) in S. trilobata and S. calendulacea, respectively. Additionally, our analysis showed that the expression of key protease genes involved in the photosynthesis pathway, particularly CP43, CP47, PsbA and PetC, had high expression levels in leaves of S. trilobata in comparison to native species. Physiological and transcriptomic analyses suggest the high expression of photosynthetic genes ensures the high photosynthetic capacity of leaves, which is one of the inherent advantages underlying the successful invasion by S. trilobata.

[Characteristics and Sources of Water-soluble Organic Carbon/Nitrogen in PM2.5 During Spring in Changzhou].

To understand the characteristics and sources of water-soluble organic carbon (WSOC) and organic nitrogen (WSON) in atmospheric aerosols during spring in Changzhou, 84 fine particle (PM2.5) samples were collected from March 1 to May 30, 2017, in Changzhou. The water-soluble components, including water-soluble organic carbon, water-soluble total nitrogen (WSTN), water-soluble ions, and carbonaceous components (OC and EC), were analyzed. The levels of WSOC and WSON and their source characteristics were discussed. The results show that the average concentrations of PM2.5, WSOC, and WSON are 101.97, 7.63, and 1.50 µg·m-3, respectively, during the sampling period. The WSON accounts for 12.9% of the WSTN and the water-soluble inorganic nitrogen mainly exists in two forms, that is, NH4+ and NO3-, accounting for 86.15% of the WSTN. The WSOC is weakly correlated with WSON (r=0.58), indicating that WSOC and WSON do not have the same sources. The WSOC is related to SOC, K+, and secondary ions (SO42-, NH4+, and NO3-), indicating that it is mainly derived from biomass burning and secondary conversion; WSON is strongly correlated to secondary ions, indicating that it is mainly derived from secondary conversion. The wind speed is the main factor affecting the WSOC and WSON concentration levels. Furthermore, the WSON is positively correlated with the air pressure and negatively correlated with the temperature. The results of the principal component analysis show that PM2.5 mainly originates from four sources:secondary formation, dust, coal combustion, biomass burning, and the ocean. The backward trajectory analysis indicates that the total concentrations of PM2.5, WSOC, and WSON in air masses from long-distance transformation are higher than that from short-distance transmission, whereas there is no significant difference in the WSON/WSTN ratio from different transmission paths.

Synthesis and in vivo fungicidal activity of some new quinoline derivatives against rice blast.

BACKGROUND: Quinoline derivatives possess excellent fungicidal activity against rice blast, but quinoline derivatives have not been thoroughly explored as fungicides. In the process of designing new fungicides, the 1,1,1,2,3,3,3-heptafluoropropan-2-yl group was introduced in order to find new structure quinoline derivatives. RESULTS: Seventeen new quinoline derivatives containing 1,1,1,2,3,3,3-heptafluoropropan-2-yl moiety were designed and synthesised. In vivo fungicidal activities of these compounds were tested against rice blast. Some of the compounds provided effective control at 100 mg L-1 , and a few compounds were effective at 10 mg L-1 . Furthermore, a density functional theory study established the structure-activity relationships of the synthesised compounds. CONCLUSION: Quinoline derivatives, especially benzyl (2,3,8-trimethyl-6-(perfluoropropan-2-yl)quinolin-4-yl) carbonate, which possess good control effective against rice blast and cucumber powdery mildew, may become new lead compounds for the development of fungicides with further structure modification. © 2017 Society of Chemical Industry.

[Degradation Characteristics of Spectral Analysis of Straw Nursery Containers in Situ Soil Culture].

This paper aimed to reveal the degradation behavior of a new type of biodegradable containers. The biodegradable containers, which was made of modified soybean adhesive and straw, was processed in situ biodegradation under natural condition. The physicochemical property and microstructure of straw nursery containers treated and untreated were characterized with Cellulose Tester, Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscope - Energy Dispersive X-ray Spectroscopy (SEM-EDS), and Thermo-gravimetry Analysis (TGA), respectively. The results indicated that the content of cellulose, hemicellulose and lignin of treated specimen decreased to 21.43%, 21.41% and 9.54% from 29.03%, 30.44% and 12.52%, respectively, comparing with those of untreated straw nursery container. FTIR analysis revealed that the ester and fat bond have been ruptured, and the aromatic characteristic peak became weakened. SEM-EDS spectrum showed the microfibril chain in the container has been fragmentation, and the soybean adhesive was also degradation. The surface of container appeared oxidization degradation. TGA analysis showed that a large number of small molecules have been produced in the process of degradation and the thermo-stability of treated samples improved.

[Inhibitory Effect of the Excretory/Scretory Proteins of Trichinella spiralis on Proliferation of Human Hepatocellular Carcinoma HepG2 Cell line].

Human hepatocellular carcinoma HepG2 Cell line were cultured with different concentrations of excretory/secretory proteins from Trichinella spiralis, and MTT assay was used to evaluate the cell inhibition rate. After co-cultured with 300 µg/ml excretory/secretory proteins for 24 h, the HepG2 cells were observed under a fluorescence microscope with AO and EB staining. When co-cultured with 75 µg/ml excretory/secretory proteins for 24 h, the HepG2 cells were quantified by flow cytometry using Annexin V-FITC/PI stain, and the expression of cleaved-caspase 9 was detected by immunofluorescence assay. The proliferation of HepG2 cells was inhibited significantly by excretory/secretory proteins in a dosage dependant manner. Under fluorescence microscope, some HepG2 cells presented typical apoptotic morphologic changes and the cleaved-caspase 9 protein expression was higher than that of the control. The early and late apoptotic cells and necrotic ones occupied 17.9%, 7.3%, and 6.6%, respectively.

Decomposition of dimethylamine gas with dielectric barrier discharge.

The decomposition of dimethylamine (DMA) with gas under high flow rate was investigated with dielectric barrier discharge (DBD) technology. Different parameters including removal efficiency, energy yield, carbon balance and CO2 selectivity, secondary products, as well as pathways and mechanisms of DMA degradation were studied. The experimental results showed that removal efficiency of DMA depended on applied voltage and gas flow rate, but had no obvious correlation with initial concentration. Excellent energy performance was obtained using present DBD technology for DMA abatement. When experiment conditions were controlled at: gas flow rate of 14.9 m(3)/h, initial concentration of 2104 mg/m(3), applied voltage of 4.8 kV, removal efficiency of DMA and energy yield can reach 85.2% and 953.9 g/kWh, respectively. However, carbon balance (around 40%) was not ideal due to shorter residence time (about 0.1s), implying that some additional conditions should be considered to improve the total oxidation of DMA. Moreover, secondary products in outlet gas stream were detected via gas chromatogram-mass spectrum and the amounts of NO3(-) and NO2(-) were analyzed by ion chromatogram. The obtained data demonstrated that NOx might be suppressed due to reductive NH radical form DMA dissociation. The likely reaction pathways and mechanisms for the removal of DMA were suggested based on products analysis. Experimental results demonstrated the application potential of DBD as a clean technology for organic nitrogen-containing gas elimination from gas streams.

Functionalization of dendritic polyethylene with cationic poly(p-phenylene ethynylene) enables efficient siRNA delivery for gene silencing.

A novel system based on a dendritic polyethylene-cationic poly(p-phenylene ethynylene) polyvalent nanocarrier was developed for siRNA delivery. By using the combination of a molecular wire and a "dendritic effects" strategy, this design provides the nanocarrier system with low cytotoxicity, cellular imaging and high siRNA delivery efficiency, allowing it to exhibit remarkable gene knockdown abilities as well as real-time monitoring of the siRNA delivery process.

Urothelial tumor initiation requires deregulation of multiple signaling pathways: implications in target-based therapies.

Although formation of urothelial carcinoma of the bladder (UCB) requires multiple steps and proceeds along divergent pathways, the underlying genetic and molecular determinants for each step and pathway remain undefined. By developing transgenic mice expressing single or combinatorial genetic alterations in urothelium, we demonstrated here that overcoming oncogene-induced compensatory tumor barriers was critical for urothelial tumor initiation. Constitutively active Ha-ras (Ras*) elicited urothelial hyperplasia that was persistent and did not progress to tumors over a 10 months period. This resistance to tumorigenesis coincided with increased expression of p53 and all pRb family proteins. Expression of a Simian virus 40 T antigen (SV40T), which disables p53 and pRb family proteins, in urothelial cells expressing Ras* triggered early-onset, rapidly-growing and high-grade papillary UCB that strongly resembled the human counterpart (pTaG3). Urothelial cells expressing both Ras* and SV40T had defective G(1)/S checkpoint, elevated Ras-GTPase and hyperactivated AKT-mTOR signaling. Inhibition of the AKT-mTOR pathway with rapamycin significantly reduced the size of high-grade papillary UCB but hyperactivated mitogen-activated protein kinase (MAPK). Inhibition of AKT-mTOR, MAPK and STAT3 altogether resulted in much greater tumor reduction and longer survival than did inhibition of AKT-mTOR pathway alone. Our studies provide the first experimental evidence delineating the combinatorial genetic events required for initiating high-grade papillary UCB, a poorly defined and highly challenging clinical entity. Furthermore, they suggest that targeted therapy using a single agent such as rapamycin may not be highly effective in controlling high-grade UCB and that combination therapy employing inhibitors against multiple targets are more likely to achieve desirable therapeutic outcomes.

The genetic profiling of preferentially expressed genes in murine splenic CD8α+ dendritic cells.

In the murine splenocytes, CD8α+ dendritic cells (abbreviated as 8+DC) and CD8α- dendritic cells (abbreviated as 8-DC) are identified with some vague features for each of them. 8+DCs but not 8-DCs cross-prime cytotoxic T cells in vivo. We aim to distinguish the two subtypes of DC based on gene expression profiling. Suppressive subtractive hybridization was undertaken to get differentially expressed genes from such subtracted cDNA library specific to 8+DC. A total of 114 sequences from the subtracted cDNA library specific to 8+DC library were analyzed. Most of them are known proteins, but some of them were novel, either totally novel genes or homologs to known genes, but with novel exon. About 55 probably novel exons were discovered, and 11 exons had longer length than those in gene bank. The clones 12, 44, 79, and 110 have no match with known sequences in gene bank. Then, semi-quantitative PCR was done to compare the expression of the enriched sequences between 8+DC and 8-DC. About 14 genes are differentially expressed in 8+DC. Therefore, SSH is an effective method to clone differentially expressed genes for 8+DC compared to 8-DC.

A novel agonistic anti-human death receptor 5 monoclonal antibody with tumoricidal activity induces caspase- and mitochondrial-dependent apoptosis in human leukemia Jurkat cells.

An agonistic antibody against TNF-related apoptosis-inducing ligand death receptor 5 (DR5) is a practicable candidate drug for antitumor therapy. In this study, a novel murine anti-human DR5 monoclonal antibody, mDRA-6(IgG1-κ), has been generated. This study aimed to explore the caspase-dependent and mitochondrial mechanisms of mDRA-6 in inducing apoptosis in human leukemia Jurkat cells. The apoptotic effects of mDRA-6 on Jurkat cells, which express DR5 on the cell surface, were detected by flow cytometry and western blot after exposure to different doses of mDRA-6 and at fixed doses of mDRA-6 at different times. It was demonstrated that mDRA-6 can induce Jurkat cell apoptosis via caspase- and mitochondrial-dependent pathways. These results indicate that the novel antibody mDRA-6 against DR5 has an antitumor function and may provide a new reagent for tumor therapy.

[Responses of Jatropha curcas L. seedlings to drought stress].

By adding different concentrations (5%-25%) of polyethylene glycol (PEG) into Hoagland solution to simulate a gradient of drought stress, the photosynthetic characteristics and drought resistance of Jatropha curcas L. seedlings were investigated. The results showed that when treated with lower concentrations (< or = 15%) of PEG, the photosynthetic rate (Pn), stomatal conductance (Gs), and intercellular carbon dioxide concentration (Ci) of the seedlings decreased with increasing PEG concentration, while the chlorophyll fluorescence parameters optimal/maximal quantum yield of PS II (Fv/Fm) decreased slightly and minimal fluorescence (Fo) was almost unaltered. The certain reduction of actual photochemical efficiency of PS II in the light (Phi(PS II)), coefficient of photochemical quenching (qP), and apparent electron transport rate (ETR) was associated with a significant increase in non-photochemical quenching (NPQ), whereas as the PEG concentration was increased to higher than 15%, Ci increased remarkably, Pn, Gs, and WUE had a sustained decrease, while Fv/Fm, Phi(PS II), qP and ETR decreased more significantly, Fo increased rapidly, and NPQ decreased. The reduction of Pn under low concentration PEG was mainly due to stomatal limitation, while that under high PEG concentration was referred to the combination of non-stomatal and stomatal limitation. When the PEG concentration was lower than 20%, Pn decreased but photosynthetic structure was not damaged. The recovery of PS II activity and the survival of plant were found after the severe drought stress was removed 15 days later. It was concluded that Jatropha curcas had stronger tolerance to drought stress.

[Clinical observation on treatment of cerebral infarction-induced broca aphasia by Tiaoshen Fuyin acupuncture therapy combined with language training].

OBJECTIVE: To observe therapeutic effect of Tiaoshen Fuyin acupuncture therapy on cerebral infarction-induced Broca aphasia. METHODS: Sixty cases were randomly divided into an observation group and a control group, 30 cases in each group. The observation group were treated with Tiaoshen Fuyin acupuncture therapy and language training, and the control group with simple language training. For the acupuncture treatment, Sishencong (EX-HN 1), Benshen (GB 13), Shenting (GV 24), Lianquan (CV 23), Xinshu (BL 15), Shentang (BL 44), Shendao (GV 11), Lingtao (HT 4) were selected and language training included training of phonatory organs in mouth, the mouth shape, sound, spoken language expression and practical exchange ability. They were treated for 5 therapeutic courses. Language examination was conducted once each before and after treatment with "Aphasia Battery of Chinese" (ABC method). RESULTS: The therapeutic effect of Tiaoshen Fuyin acupuncture therapy combined with language training was better than that of simple language training. CONCLUSION: Tiaoshen Fuyin acupuncture therapy combined with language training can significantly improve language function and increase life quality in the patient of cerebral infarction-induced Broca aphasia.

[Comparative study of the resistance of bacteriophageT4, PhiX174D, MS2 and f2 to gamma radiation].

OBJECTIVE: To screen the suitable bacteriophage as virus indicator in irradiation sterilization. METHODS: Suspensions of bacteriophage T4, phiX174D, MS2, and f2, Escherichia coli 8099, and Bacillus subtilis var.niger.sp. ATCC9372 were irradiated with (60)Co-gamma ray. The mean log(10) inactivation value (LIV) and killing log value (KL) were calculated. RESULTS: (1) Under 100 Gy of gamma-radiation, the LIV levels of the bacteriophage T4, PhiX174, f2, and MS2 were 6.31, 6.92, 5.74, and 4.46 log(10) respectively, all reaching the disinfection level (LIV >/= 4.00 log(10)), (2) Under the same absorbed dose, the KL of Escherichia coli 8099 was > 7.97 log(10); (3) Under the same absorbed dose, the KL of the Bacillus subtilis var.niger.sp. ATCC9372 was 1.61 log(10). CONCLUSION: The order of resistance of the above six microorganisms to gamma-radiation from the biggest to the smallest is as follows: Bacillus subtilis var. niger. sp. > bacteriophage MS2 > bacteriophage f2 > bacteriophage T4 > bacteriophage phiX 174D > E. coli.

Transcriptional regulation of the androgen receptor cofactor androgen receptor trapped clone-27.

Cofactors modulate nuclear receptor activity and impact human health and disease, yet surprisingly little is known about their transcriptional regulation. Androgen receptor trapped clone-27 (ART-27) is a cofactor that binds to androgen receptor (AR) amino terminus and modulates AR-dependent transcription. Interestingly, ART-27 displays both a cell type- and developmental stage-specific expression pattern. However, the cis-acting elements and trans-acting factors affecting ART-27 gene expression have not been elucidated. We found that ART-27 gene expression is repressed and its promoter is histone H3-K27 tri-methylated in human embryonic kidney cells, but not prostate cells, and the histone deacetylase inhibitor, trichostatin A, relieves this inhibition. The DNA response elements that control the induction of ART-27 gene expression were also characterized. The major cis-acting element corresponds to a consensus cAMP-responsive element (CRE) and binds the CRE-binding protein (CREB) as shown by EMSA and chromatin immunoprecipitation assays. Furthermore, ART-27 promoter activity is induced upon CREB overexpression. Epidermal growth factor, which activates CREB via phosphorylation, also induces ART-27 expression, whereas a reduction in CREB phosphorylation or expression blocks this induction in prostate cells. In human prostate development, both epithelial and stromal cells express CREB; however, active phosphorylated CREB is restricted to epithelial cells where ART-27 is expressed. Based on these findings, we propose a transcriptional regulatory circuit for the developmental expression of ART-27 that includes repression by chromatin modification through a trichostatin A-sensitive factor and activation upon growth factor stimulation via CREB.

Persistent uroplakin expression in advanced urothelial carcinomas: implications in urothelial tumor progression and clinical outcome.

As the terminal differentiation products of human urothelium, uroplakins (UPs) would be expected to diminish during urothelial tumorigenesis. Surprisingly, recent studies found UPs to be retained even by well-advanced urothelial carcinomas, suggesting that the loss of UPs does not strictly parallel urothelial transformation. Little is known, however, about whether the status of UPs is associated with a particular pathologic parameter, the tumor's biological behavior, or patient outcome. Here we assessed UP expression by immunohistochemistry on tissue arrays from 285 patients with bladder urothelial carcinomas or nontumor conditions. UPs were expressed in all 9 normal urothelial specimens, 63 of 74 (85%) patients with non-muscle-invasive urothelial carcinomas on transurethral resection, 104 of 202 (51.5%) patients who underwent radical cystectomy for advanced urothelial carcinomas, and 33 of 50 (66%) lymph node metastases. Normally associated with urothelial apical surface, UPs were localized aberrantly in tumors, including microluminal, basal-laminal, cytoplasmic, or uniform patterns. In non-muscle-invasive diseases, there was no association between UP expression and disease recurrence, progression, or mortality. In contrast, in invasive diseases, absent UP expression was significantly associated with advanced pathologic stage, lymph node metastases, disease recurrence, and bladder cancer-specific mortality (P = .042, P = .035, P = .023, and P = .022, respectively) in univariate analyses. Furthermore, UP status was independent of key cell-cycle regulators, including p53, pRb, p27, and cyclin D1, thus excluding a functional link between these 2 groups of proteins. Our data demonstrate for the first time that persistent UP expression is associated with a favorable clinical outcome and that UPs may be used as adjunct markers for predicting the prognoses of patients with invasive and metastatic bladder carcinomas. Our results also suggest that UP-positive and -negative carcinomas have different clonal origins or may be derived from different cancer stem cells.

Decreased DOC-2/DAB2 expression in urothelial carcinoma of the bladder.

PURPOSE: DOC-2/DAB2 (differentially expressed in ovarian carcinoma-2/disabled-2), a potential tumor suppressor gene, is underexpressed in several cancers. Little is known about the expression of this gene in urothelial carcinoma of the bladder (UCB). We profiled DOC-2/DAB2 expression in mouse and human normal and neoplastic urothelia. EXPERIMENTAL DESIGN: Immunohistochemical staining for DOC-2/DAB2 was carried out on tissue specimens from two transgenic mouse models with urothelium-specific molecular alterations and on a tissue microarray containing cores from 9 normal controls, 44 patients who underwent transurethral resection of the bladder tumor (TURBT), 195 patients who underwent radical cystectomy for UCB, and 39 lymph nodes with metastatic UCB. RESULTS: Normal mouse urothelium stained uniformly with DOC-2/DAB2. Weaker staining was observed in low-grade, superficial papillary bladder tumors from transgenic mice harboring constitutively active Ha-Ras, whereas carcinoma in situ-like lesions and high-grade bladder tumors from transgenic mice expressing a SV40 T antigen completely lacked DOC-2/DAB2 expression. In human tissues, DOC-2/DAB2 expression was decreased in 11% of normal bladder specimens, 59% of TURBT specimens, 65% of radical cystectomy specimens, and 77% of the metastatic lymph node specimens. Decreased DOC-2/DAB2 expression was associated with advanced pathologic stage (P = 0.023), lymph node metastases (P = 0.050), and lymphovascular invasion (P < 0.001). In univariable, but not in multivariable analysis, decreased DOC-2/DAB2 was associated with an increased probability of bladder cancer recurrence (log-rank test, P = 0.020) and bladder cancer-specific mortality (log-rank test, P = 0.023). CONCLUSIONS: Decreased DOC-2/DAB2 expression seems to occur early in bladder tumorigenesis and becomes more prominent in advanced stages of UCB.

Hyperactivation of Ha-ras oncogene, but not Ink4a/Arf deficiency, triggers bladder tumorigenesis.

Although ras is a potent mitogenic oncogene, its tumorigenicity depends on cellular context and cooperative events. Here we show that low-level expression of a constitutively active Ha-ras in mouse urothelium induces simple urothelial hyperplasia that is resistant to progression to full-fledged bladder tumors even in the absence of Ink4a/Arf. In stark contrast, doubling of the gene dosage of the activated Ha-ras triggered early-onset, rapidly growing, and 100% penetrant tumors throughout the urinary tract. Tumor initiation required superseding a rate-limiting step between simple and nodular hyperplasia, the latter of which is marked by the emergence of mesenchymal components and the coactivation of AKT and STAT pathways as well as PTEN inactivation. These results indicate that overactivation of Ha-ras is both necessary and sufficient to induce bladder tumors along a low-grade, noninvasive papillary pathway, and they shed light on the recent findings that ras activation, via point mutation, overexpression, or intensified signaling from FGF receptor 3, occurs in 70%-90% of these tumors in humans. Our results highlight the critical importance of the dosage/strength of Ha-ras activation in dictating its tumorigenicity--a mechanism of oncogene activation not fully appreciated to date. Finally, our results have clinical implications, as inhibiting ras and/or its downstream effectors, such as AKT and STAT3/5, could provide alternative means to treat low-grade, superficial papillary bladder tumors, the most common tumor in the urinary system.

Cell-specific regulation of androgen receptor phosphorylation in vivo.

The biological ramifications of phosphorylation of the androgen receptor (AR) are largely unknown. To examine the phosphorylation of AR at serine 213, a putative substrate for Akt, a phosphorylation site-specific antibody was generated. The use of this antibody indicated that AR Ser-213 is phosphorylated in vivo and that phosphorylation is tightly regulated in a cell type-specific manner. Furthermore, Ser-213 phosphorylation took place with rapid kinetics and was inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. Phosphorylation occurred in response to R1881 and dihydrotestosterone but weakly if at all in response to testosterone. It did not occur in response to AR antagonists or growth factor stimulation in the absence of an AR agonist. Transcription assays using an AR-responsive reporter gene construct showed that activated phosphatidylinositol 3-kinase inhibited transcription mediated by wild type AR but not that of a mutant AR variant (S213A), which could not be phosphorylated at Ser-213. By immunohistochemistry, the AR Ser(P)-213 antigen was detected in prostate epithelial but not stromal cells despite the fact that an antibody recognizing both phosphorylated and non-phosphorylated forms of AR demonstrates that AR is present in both cell types as expected. In fetal tissue the AR-Ser(P)-213 antigen was present in epithelial cells of the urogenital sinus when endogenous androgen levels were high and activated Akt was prevalent, but absent at a later stage of development when endogenous androgen levels were low and Akt activation was minimal. Immunoreactivity was evident in differentiated cells lining the lumen of the urogenital sinus but not in rapidly dividing, Ki67 positive cells within the developing prostate or stromal tissue, suggesting that site-specific phosphorylation of AR Ser-213 by cellular kinases occurs in a non-proliferating cellular milieu.

Tamm-Horsfall protein is a critical renal defense factor protecting against calcium oxalate crystal formation.

BACKGROUND: The tubular fluid of the mammalian kidney is often supersaturated with mineral salts, but crystallization rarely occurs under normal conditions. The unique ability of the kidney to avoid harmful crystal formation has long been attributed to the inhibitory activity of the urinary macromolecules, although few in vivo studies have been carried out to examine this hypothesis. Here we examined the role of Tamm-Horsfall protein (THP), the principal urinary protein, in urinary defense against renal calcium crystal formation, using a THP knockout model that we recently developed. METHODS: Wild-type and THP knockout mice were examined for the spontaneous formation of renal calcium crystals using von Kossa staining. The susceptibility of these mice to experimentally induced renal crystal formation was evaluated by administering mice with ethylene glycol, a precursor of oxalate, and vitamin D(3), which increases calcium absorption. Renal calcium crystals were visualized by von Kossa stain, dark field microscopy with polarized light and scanning electron microscopy. RESULTS: Inactivating the THP gene in mouse embryonic stem cells results in spontaneous formation of calcium crystals in adult kidneys. Excessive intake of calcium and oxalate, precursors of the most common type of human renal stones, dramatically increases both the frequency and the severity of renal calcium crystal formation in THP-deficient, but not in wild-type mice. Under high calcium/oxalate conditions, the absence of THP triggers a marked, adaptive induction in renal epithelial cells of osteopontin (OPN), a potent inhibitor of bone mineralization and vascular calcification. Thus, OPN may serve as an inducible inhibitor of calcium crystallization, whereas THP can serve as a constitutive and apparently more effective inhibitor. CONCLUSION: These results provide the first in vivo evidence that THP is a critical urinary defense factor and suggest that its deficiency could be an important contributing factor in human nephrolithiasis, a condition afflicting tens of millions of people in the world annually.