Usher syndrome is a frequent cause of the combination of deafness and blindness due to retinitis pigmentosa (RP). Five genes are known to underlie different forms of Usher syndrome type I (USH1). In the Ashkenazi Jewish population, the R245X mutation of the PCDH15 gene may be the most common cause of USH1 (Ben-Yosef T, Ness SL, Madeo AC, Bar-Lev A, Wolfman JH, Ahmed ZM, Desnick RK, Willner JP, Avraham KB, Ostrer H, Oddoux C, Griffith AJ, Friedman TB N Engl J Med 348: 1664-1670, 2003). To estimate what percentage of Ashkenazi Jewish children born with profound hearing loss will develop RP due to R245X, we examined the prevalence of the R245X PCDH15 mutation and its carrier rate among Ashkenazi Jews in Israel. Among probands diagnosed with nonsyndromic hearing loss not due to mutations of connexin 26 (GJB2) and/or connexin 30 (GJB6), and below the age of 10, 2 of 20 (10%) were homozygous for the R245X mutation. Among older nonsyndromic deaf individuals, no homozygotes were detected, although one individual was heterozygous for R245X. The carrier rate of the R245X mutation among the normal hearing Ashkenazi population in Israel was estimated at 1%. Ashkenazi Jewish children with profound prelingual hearing loss should be evaluated for the R245X PCDH15 mutation and undergo ophthalmologic evaluation to determine whether they will develop RP. Rehabilitation can then begin before loss of vision. Early use of cochlear implants in such cases may rescue these individuals from a dual neurosensory deficit.

The levels of the cGMP in smooth muscle of the gut reflect continued synthesis by soluble guanylate cyclase (GC) and breakdown by phosphodiesterase 5 (PDE5). Soluble GC is a haem-containing, heterodimeric protein consisting alpha- and beta-subunits: each subunit has N-terminal regulatory domain and a C-terminal catalytic domain. The haem moiety acts as an intracellular receptor for nitric oxide (NO) and determines the ability of NO to activate the enzyme and generate cGMP. In the present study the mechanism by which protein kinases regulate soluble GC in gastric smooth muscle was examined. Sodium nitroprusside (SNP) acting as a NO donor stimulated soluble GC activity and increased cGMP levels. SNP induced soluble GC phosphorylation in a concentration-dependent fashion. SNP-induced soluble GC phosphorylation was abolished by the selective cGMP-dependent protein kinase (PKG) inhibitors, Rp-cGMPS and KT-5823. In contrast, SNP-stimulated soluble GC activity and cGMP levels were significantly enhanced by Rp-cGMPS and KT-5823. Phosphorylation and inhibition of soluble GC were PKG specific, as selective activator of cAMP-dependent protein kinase, Sp-5, 6-DCl-cBiMPS had no effect on SNP-induced soluble GC phosphorylation and activity. The ability of PKG to stimulate soluble GC phosphorylation was demonstrated in vitro by back phosphorylation technique. Addition of purified phosphatase 1 inhibited soluble GC phosphorylation in vitro, and inhibition was reversed by a high concentration (10 microM) of okadaic acid. In gastric smooth muscle cells, inhibition of phosphatase activity by okadaic acid increased soluble GC phosphorylation in a concentration-dependent fashion. The increase in soluble GC phosphorylation inhibited SNP-stimulated soluble GC activity and cGMP formation. The results implied the feedback inhibition of soluble GC activity by PKG-dependent phosphorylation impeded further formation of cGMP.

We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae. Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation. Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min. The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+). Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity. It showed a good stability in organic solvents and especially in long chain-fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate. About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.

Previous findings were confirmed that C-reactive protein (C-RP) occurs in some vasculitis lesions, particularly those infiltrated mainly by neutrophils (necrotizing vasculitis). The C-RP was usually in lesions also containing complement CCC-RP/ml serum, C-RP was found in sera of many normal persons, and the amount was influenced by the occupation of the donor. Sera of thirty-one persons with vasculitis with mainly mononuclear cell-infiltrated lesions had about four-fold more C-RP (mean 28, 200 ng/ml serum) than found in normal persons, and sera of thirty-nine persons with mainly neutrophil-infiltrated lesions had eight times the normal amount (mean 56,400 ng C-RP/ml). The amount of C-RP was influenced by the severity, extent and duration of the disorder in most patients. Experimental data suggests that C-RP may contribute to the perpetuation of inflammation in chronic vasculitis.

Mutations in SNRPcause autosomal-dominant retinal disorder retinitis pigmentosa (RP). The protein product of SNRNP200 is BRR2, a DExD/H box RNA helicase crucial for pre-mRNA splicing. In this study, we prepared p.S1087L and p.R1090L mutations of human BRR2 using bacterial artificial chromosome recombineering and stably expressed them in human cell culture. Mutations in BRR2 did not compromise snRNP assembly and both mutants were incorporated into the spliceosome just as the wild-type (wt) protein. Surprisingly, cells expressing RP mutants exhibited increased splicing efficiency of the LDHA gene. Next, we found that depletion of endogenous BRR2 enhanced usage of a β-globin cryptic splice site while splicing at the correct splice site was inhibited. Proper splicing of optimal and cryptic splice sites was restored in cells expressing BRR2-wt but not in cells expressing RP mutants. Taken together, our data suggest that BRR2 is an important factor in 5'-splice-site recognition and that the RP-linked mutations c.3260C>T (p.S1087L) and c.3269G>T (p.R1090L) affect this BRR2 function.

Statistical differences between amperometric traces recorded from chromaffin cells using K(+) and Ba(2+) secretagogues support the assertion that readily releasable pool (RRP) and reserve pool (RP) vesicles can be probed with pool-specific secretagogues. Release from the RRP was evoked by K(+) while release from the RP was evoked by Ba(2+). Similar temperature-dependent changes in spike area and half-width for both pools suggest that the content of RRP and RP vesicles is similar and packaged in the same way. Differences between the vesicle pools were revealed in the temperature dependence of spike frequency. While the burst spike frequency of the RRP, which is comprised of pre-docked and primed vesicles, increased 2.8% per degrees C, the RP spike frequency increased 12% per degrees C. This difference is attributed to a temperature-dependent mobilization of the RP. Furthermore, the RP exhibited more foot events at room temperature than the RRP but this difference was not apparent at 37 degrees C. This trend suggests that RP vesicle membranes have a compromised surface tension compared to RRP vesicles. Collectively, the changes of release characteristics with temperature reveal distinctions between the RRP and the RP.

Sequence comparisons predicted a potential papain-like proteinase domain in the N-terminal cleavage product (NRP) of the large nonstructural replicase polyprotein (RP) of turnip yellow mosaic virus (TYMV). Replacement of the predicted catalytic amino acids, Cys-783 by Ser, or of His-869 by Glu, abolished cleavage of the 206K RP into a approximately 150 K NRP and a approximately 78 K C-terminal product in reticulocyte lysates, while other substitutions exerted no apparent influence on proteolysis. The proteinase-deficient mutant RPs could not be cleaved in trans by as much as an eight-fold molar excess of wild-type proteinase. Deletion experiments have excluded the possible influence on autoproteolysis of amino acid sequences 1-708 and 982-1204 flanking the proteinase domain. Thus, the proteinase of TYMV with a papain-like dyad of essential amino acids has been mapped just upstream from the putative NTPase domain. Statistically significant sequence similarities with the TYMV proteinase were found for the similarly located domains of the replicase polyproteins of carlaviruses, capilloviruses, apple stem pitting virus and apple chlorotic leaf spot virus as well as for those of other tymoviruses and for the domain located downstream from the putative NTPase domain of the large polyprotein of beet necrotic yellow vein furovirus. All these domains are not significantly similar to other known proteinases, although they conserve papain-like Cys- and His-containing motifs. Thus these domains constitute a compact group of related enzymes, the tymo-like proteinases, within the proposed papain-like proteinase supergroup. The resulting alignment of 10 tymo-like proteinase sequences has revealed a third highly conserved residue--Gly (Gly821 in TYMV RP) followed by a hydrophobic residue. We speculate that all the tymo-like proteinase domains of the viral replicative proteins may share common biochemical and biological features.

A retrospective analysis of computed tomographic (CT) and magnetic resonance (MR) images and clinical records of 39 patients with retropharyngeal space (RPS) lesions was completed. The review was undertaken to answer the following questions: (a) what is the spectrum of lesions of the RPS? (b) what imaging features mark a lesion as originating in the RPS? and (c) is there a difference between the radiologic pattern of the suprahyoid and infrahyoid portions of the neck? Of the 39 patients in the study, nine had RPS infections, 17 had RPS malignancies, two had benign tumors, seven had RPS pseudotumors, and four had hematoma or air in the RPS after trauma. RPS lesions demonstrated two distinct radiologic patterns: a nodal pattern and a nonnodal pattern. The nodal pattern, found only in the suprahyoid neck, occurs when infection or tumor begins in the lymph nodes of the RPS. The lesions may be unilateral or bilateral, but the middle part of the RPS is spared. The nonnodal pattern, found primarily in the infrahyoid RPS, results when the infection or tumor directly invades the RPS or goes beyond the nodes of the RPS. The nonnodal lesion appears rectangular and spans the RPS from side to side.

Enterobacter asburiae PSI3 is a rhizospheric isolate that solubilizes mineral phosphates by the action of a phosphate starvation-inducible GDH (EC 1.1.5.2). We report here that GDH activity of this isolate shows broad substrate range, being able to act on mono and disaccharides. Enterobacter asburiae PSI3 was proficient at bringing about a drop in pH and solubilization of RP with the use of 75 mmol/L of each of the GDH substrate sugars tested as the sole C source. It liberated amounts of P ranging from 450 micromol/L (on arabinose) to 890 micromol/L (on glucose). When grown on a mixture of 7 GDH substrates at concentrations of 15 mmol/L each, the bacterium solubilized RP equivalent to 46% of the value when 75 mmol glucose/L was the C source. HPLC analysis of the culture supernatant under these conditions showed that the acidification of the media is primarily due to the production of organic acids. The significance of these results on the efficacy of E. asburiae PSI3 at solubilizing phosphates under rhizospheric conditions is discussed.

Gender differences were studied in ventricular myocytes from insulin-deficient (Type 1) diabetic rats. Cells were obtained by enzymatic dispersion of hearts from control male and female rats and from rats made diabetic with streptozotocin (100 mg/kg) 7-14 days before experiments. ANG II content, measured by ELISA, was augmented in diabetic males but unaltered in diabetic females. In diabetic ovariectomized females, ANG II levels were augmented as in males. ANG II affects multiple cellular pathways including activation of protein kinase C (PKC) and several tyrosine kinases as well as inhibition of protein kinase A (PKA). The involvement of these pathways in modulating outward K(+) currents was studied. Transient and sustained outward K(+) currents were measured using the whole cell voltage-clamp method. In males, these currents are attenuated under diabetic conditions but are augmented by the ANG II-converting enzyme inhibitor quinapril. Activation of PKA by 8-bromo-cAMP enhanced both K(+) currents in cells from diabetic males. The augmentation of these currents by quinapril was blocked when PKA inhibition was maintained with the Rp isomer of 3',5'-cyclic monophosphorothioate. Inhibition of tyrosine kinases by genistein also augmented K(+) currents in cells from diabetic males. Action potentials were abbreviated by 8-bromo-cAMP and genistein. However, both genistein and 8-bromo-cAMP had no effect on K(+) currents in cells from diabetic females. In cells from ovariectomized diabetic females, 8-bromo-cAMP and genistein enhanced these K(+) currents as in males. Inhibition of PKC augmented the transient and sustained K(+) currents in cells from diabetic males and females. A contribution of non-ANG II-dependent activation of PKC is suggested. These results describe some of the mechanisms that may underlie gender-specific differences in the development of cardiac disease and arrhythmias.

In opossum kidney (OK) cells, L-dihydroxyphenylalanine (10 microM) raised dopamine to 10 nM and inhibited Na-inorganic phosphate (Pi) uptake 20% (P = 0.001). Inhibition was completely blocked by carbidopa or SCH23390. Dopamine (1 microM) inhibited uptake 55% (half-maximal inhibition, 0.03 microM). Fenoldopam (0.1 microM, DA1 agonist) inhibited uptake 45 +/- 2%. DA1 antagonists (SKF83566 and SCH23390), but not DA2-antagonist (sulpiride), blocked dopamine inhibition. Quinpirole (DA2 agonist) did not modify Pi uptake. Bisindolylmaleimide (10 microM), a protein kinase C inhibitor, blocked inhibition of Pi uptake by phorbol ester but had no effect on the response to dopamine. Dopamine inhibited Pi uptake in cells that had been exposed to phorbol ester for 18 to 24 h. Dopamine inhibition was not reduced by 1 microM U73,122 but was reduced 20% by 10 microM, which is 10 times the concentration reported to completely inhibit phospholipase C in OK cells. Adenylate cyclase inhibitors SQ 22536 (100 microM) and 2,5-dideoxyadenosine (100 microM) reduced dopamine-stimulated cAMP production, but not dopamine inhibition of Pi uptake. Rp-cAMPS counteracted the inhibition of Pi uptake by Sp-cAMPS but had no effect on the dopamine response. H-89 inhibited dopamine-stimulated protein kinase A activity, but neither H-89 nor H-9 alone or with bisindolylmaleimide altered dopamine inhibition of Pi uptake. Genistein and herbimycin A (tyrosine kinase inhibitors) reduced Pi uptake. However, dopamine, a benzoquinone like several tyrosine kinase inhibitors, did not inhibit tyrosine kinase activity. Thus, dopamine inhibited Pi uptake in this OK cell clone by activating a G protein-linked pathway that operates independently from adenylyl cyclase, protein kinase A, protein kinase C, and protein tyrosine kinase.

Single sodium-channel currents were measured in neuroblastoma cells after inhibition of inactivation by chloramine-T (CHL-T), sea anemone toxin II (ATX-II) and scorpion toxin (SCT). The decaying phase of the averaged single-channel currents recorded with 90-msec pulses in cell-attached patches was clearly slower than that of the ummodified channels, suggesting inhibition of macroscopic inactivation. Each substance caused repetitive openings and a moderate increase in the channel open time. At Vm = RP + 20 mV and T = 12 degrees C, the mean channel open times were 1.4, 1.6 and 1.8 msec for CHL-T, ATX-II and SCT, respectively, as opposed to 1.07 msec for native channels. Open-time histograms could be best fitted by the sum of two exponentials. The time constants of the fits were similar for histograms constructed from single openings and from openings during bursts. This suggests that the population of channels is homogeneous and that in bursts the same open conformations of channels occur as in single openings. Mean burst durations for bursts consisting of more than one opening at Vm = RP + 20 mV were 4.9, 5.8 and 6.1 msec for CHL-T, ATX-II and SCT, respectively. Burst open-time histograms constructed from two or three openings were fitted by the gamma function. The different time constants of the fits obtained for ATX-II and SCT suggested multiple open conformations of channels for openings of bursts. However, significantly different open-time histograms constructed from the first, second and third openings of bursts could not be obtained systematically. A positive correlation was found for the dwell time of the first and the second, as well as for the second and the third opening of bursts with each substance, but a negative one for the dwell time of an opening and the neighboring closing of bursts with ATX-II. The results suggest a model with multiple open and inactivated states. In this model the inactivated states are weakly absorbing.

Paris polyphylla Smith var. yunnanensis extracts, Rhizoma Paridis saponins (RPS) have been found to show strong antitumor activity. However, few studies have yet investigated pulmonary metastasis treatment with this herb. To detail the effective components in RPS and discuss the preliminary mechanism of antitumor effects in vivo and in vitro, a mixture isolated from RPS was investigated. The main constituents were identified as polyphyllin D, formosanin C, dioscin, Paris H, Paris VII and pennogennin 3-O-α-L-rhamnopyranosyl (1→4)-[β-L-rhamnopyranosyl (1→2)]-β-D-glucopyranoside. In our experiments, LA795 cells were exposed to the mixed compounds. Migration inhibition was evaluated by wound healing assay and migration assay in non-cytotoxic dose which was determined by MTT assay. The results demonstrated that the constituent in varying degrees inhibited the migration of the tumor cells in vitro. The mixture also showed antitumor effects on carcinoma in vivo. In conclusion, the mixture is a potent anticancer agent that elicits programmed cell death and inhibits the migration in murine lung adenocarcinoma, both in vitro and in vivo.

The let-7 microRNA (miRNA) regulates developmental timing at the larval-to-adult transition in Caenorhabditis elegans. Dysregulation of let-7 results in irregular hypodermal and vulval development. Disrupted let-7 function is also a feature of human lung cancer. However, little is known about the mechanism and co-factors of let-7. Here we demonstrate that ribosomal protein RPS-14 is able to modulate let-7 function in C. elegans. The RPS-14 protein co-immunoprecipitated with the nematode Argonaute homolog, ALG-1. Reduction of rps-14 gene expression by RNAi suppressed the aberrant vulva and hypodermis development phenotypes of let-7(n2853) mutant animals and the mis-regulation of a reporter bearing the lin-41 3'UTR, a well established let-7 target. Our results indicate an interactive relationship between let-7 miRNA function and ribosomal protein RPS-14 in regulation of terminal differentiation that may help in understanding the mechanism of translational control by miRNAs.

The diagnosis of digital artery vasospasm in the hand-arm vibration syndrome (HAVS) is clinically based, and the need for an accurate objective test to support the diagnosis has been highlighted. This study aims to analyse the potential of cold provocation thermography (CPT) to fulfill this role. CPT was performed on two groups of subjects: 10 controls and 21 patients with Raynaud's phenomenon (RP) secondary to HAVS. After taking a pre-cooling image, patients donned latex gloves and immersed their hands in water at a temperature of 5 degrees C for 1 min. The patients removed their hands from the water and discarded the gloves, and further images were taken every 30 s for 10 min. On each image, the temperatures of the tip and base were analysed for each digit. The sensitivity, specificity, positive and negative predictive values for fingertip temperatures only, fingertip and fingerbase temperatures combined, and fingertip temperature, fingerbase temperature and temperature gradient combined were determined. Patients with RP secondary to HAVS demonstrated significantly lower finger tip and base temperatures and lower digital temperature gradients at all time intervals when compared with controls (P < 0.01, Student's t-test). CPT has good sensitivity, specificity, positive predictive value and negative predictive value; it strongly supports the clinical diagnosis of digital vasospasm.

BACKGROUND

Ascorbate (vitamin C) has been evaluated as a potential treatment for cancer as an independent agent and in combination with standard chemotherapies. This review assesses the evidence for safety and clinical effectiveness of intravenous (IV) ascorbate in treating various types of cancer.

METHODS

Single arm and randomized Phase I/II trials were included in this review. The PubMed, MEDLINE, and Cochrane databases were searched. Results were screened by three of the authors (GN, RP, and CJP) to determine if they met inclusion criteria, and then summarized using a narrative approach.

RESULTS

A total of 23 trials involving 385 patients met the inclusion criteria. Only one trial, in ovarian cancer, randomized patients to receive vitamin C or standard of care (chemotherapy). That trial reported an 8.75 month increase in progression-free survival (PFS) and an improved trend in overall survival (OS) in the vitamin C treated arm.

CONCLUSIONS

Overall, vitamin C has been shown to be safe in nearly all patient populations, alone and in combination with chemotherapies. The promising results support the need for randomized placebo-controlled trials such as the ongoing placebo-controlled trials of vitamin C and chemotherapy in prostate cancer.

The major allergen (Tur c 1) in the muscle of the gastropod, Turbo cornutus, was isolated by Sephacryl S-300, Mono Q HR 5/5 and TSKgel Phenyl-5PW RP column chromatography. ELISA showed Tur c 1 to react strongly with sera from three individuals sensitive to both mollusks and crustaceans. SDS-PAGE showed Tur c 1 to produce a major band corresponding to a molecular mass of 35 kDa under the reduced condition. Its amino acid composition was characterized by the abundance of Glx, followed by Leu, Ala and Lys in decreasing abundance, and the absence of Trp. In addition to these properties, the determined partial amino acid sequence identified Tur c 1 to be a tropomyosin, as in the case of the known mollusk and crustacean allergens. However, the results of competitive ELISA inhibition experiments suggest that Tur c 1 has an IgE-binding epitope in the C-terminal region which is dissimilar to those proposed for Cra g 1 (the oyster Crassostrea gigas allergen) and Pen i 1 (the shrimp Penaeus indicus allergen).

In the present study, we investigate the coherence of signaling pathways leading to lipolysis in 3T3-L1 adipocytes. We observe two linear signaling pathways: one well known, acting via cAMP and protein kinase A (PKA) activation, and a second one induced by phorbol 12-myristate 13-acetate treatment involving protein kinase C (PKC) and MAPK. We demonstrate that both the PKA regulatory subunits RIalpha and RIIbeta are expressed in 3T3-L1 adipocytes and are responsible for the lipolytic effect mediated via the cAMP/PKA pathway. Inhibition of the PKA pathway by the selective PKA inhibitor Rp-8-CPT-cAMPS does not impair lipolysis induced by PKC activation, and neither PD98059 nor U0126, as known MAPK kinase inhibitors, changes the level of glycerol release caused by PKA activation, indicating no cross-talk between these two pathways when only one is activated. However, when both are activated, they act synergistically on glycerol release. Additional experiments focusing on this synergy show no involvement of MAPK phosphorylation and cAMP formation. Phosphorylation of hormone-sensitive lipase is similar upon stimulation of either pathway, but we demonstrate a difference in the ability of both PKA and the PKC pathway activation to phosphorylate perilipin, which in turn may be an explanation for the different maximal lipolytic effect of both pathways.

Fertile women have lower blood pressure and cardiovascular risk than age-matched men, which suggests that estrogens exert cardiovascular protective effects. However, whether 17 β-estradiol (E2) blunts aldosterone secretion, and thereby affects the gender dimorphism of blood pressure, is unknown. We therefore sought for the estrogen receptor (ER) subtypes in human adrenocortical tissues ex vivo by performing gene and protein expression studies. We also investigated the effect of E2 on aldosterone synthesis and the involved receptors through in vitro functional experiments in the adrenocortical cells HAC15. We found that in the human adrenal cortex and aldosterone-producing adenoma cells, the most expressed ERs were the ERβ and the G protein-coupled receptor-1 (GPER-1), respectively. After selective ERβ blockade, E2 (10 nmol/L) markedly increased both the expression of aldosterone synthase and the production of aldosterone (+5- to 7-fold vs baseline, P < .001). Under the same condition, the GPER-1 receptor agonist 1-[4-(6-bromo-benzo (1, 3)dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quinolin-8-yl]-ethanone (G-1) (10 nmol/L) mimicked this effect, which was abrogated by cotreatment with either the GPER-1 receptor antagonist (3aS*,4R*,9bR*)-4-(6-Bro-mo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline (G-15), or a selective protein kinase A inhibitor 8-Bromo-2-monobutyryladenosine-3,5-cyclic mono-phosphorothioate, Rp-isomer. Silencing of the ERβ significantly raised aldosterone synthase expression and aldosterone production. Conversely, silencing of the GPER-1 lowered aldosterone synthase gene and protein expression. Moreover, it blunted the stimulatory effect of E2 on aldosterone synthase that was seen during ERβ blockade. These results support the conclusion that in humans, E2 inhibits aldosterone synthesis by acting via ERβ. Pharmacologic disinhibition of ERβ unmasks a potent secretagogue effect of E2 that involves GPER-1 and protein kinase A signaling.

OBJECTIVE

To investigate the cellular expression of cis-acting splicing mutations in the rhodopsin gene (RHO) that lead to autosomal dominant or recessive retinitis pigmentosa (adRP/arRP) and the role of nonsense-mediated mRNA decay (NMD) in its pathogenic mechanism. To design a potential therapeutic RNAi-based suppression strategy for cis-acting adRP splicing mutants.

METHODS

Cells were transfected with genomic constructs encoding the human wild-type (WT) and c.531-2A>G, c.936+1G>T, c.937-1G>T and c.745G>T RHO mutants. Total RNA was quantified by RT-PCR and protein was analyzed by immunocytochemistry. Three small interfering (si)RNAs directed against adRP mutant transcripts were designed and assayed in COS7 cells.

RESULTS

The RHO cis-acting splicing mutations causing adRP, c.531-2A>G and c.937-1G>T, induce cryptic splicing. In contrast, the c.936+1G>T mutation, which causes arRP, results in exon skipping. Although the c.531-2A>G and c.745G>T RHO sequence predicted a premature termination codon (PTC) that should be a target for NMD, these mutant proteins were detected in transfected cells. The siRNAs designed to interfere with adRP mutants silenced the corresponding mRNA with varying efficiency.

CONCLUSIONS

Although two RHO mutations that cause different RP phenotypes were the target for the NMD mechanism, a fraction of mutant RNA transcript may circumvent the NMD mechanism and be translated into protein. Thus, different levels of mutant protein may be necessary to trigger the RP phenotype. The findings demonstrate the potential use of siRNA to interfere with cis-acting splicing RHO transcripts. However, limitations in the mutation sequence and incomplete mutant transcript elimination should be considered in a therapeutic approach for adRP.

Cinnamaldehyde is a natural antimicrobial that has been found to be effective against many food-borne pathogens, including Escherichia coli O157:H7. Although its antimicrobial effects have been well investigated, limited information is available on its effects at the molecular level. Sublethal treatment at 200 mg/liter cinnamaldehyde inhibited growth of E. coli O157:H7 at 37°C and for ≤2 h caused cell elongation, but from 2 to 4 h growth resumed and cells reverted to normal length. To understand this transient behavior, genome-wide transcriptional analysis of E. coli O157:H7 was performed at 2 and 4 h of exposure to cinnamaldehyde in conjunction with reverse-phase high-performance liquid chromatography (RP-HPLC) analysis for cinnamaldehyde and other cinnamic compounds. Drastically different gene expression profiles were obtained at 2 and 4 h. RP-HPLC analysis showed that cinnamaldehyde was structurally stable for at least 2 h. At 2 h of exposure, cinnamaldehyde induced expression of many oxidative stress-related genes and repressed expression of DNA, protein, O-antigen, and fimbrial synthetic genes. At 4 h, many cinnamaldehyde-induced repressive effects on E. coli O157:H7 gene expression were reversed, and cells became more motile and grew at a slightly higher rate. Data indicated that by 4 h, E. coli O157:H7 was able to convert cinnamaldehyde into the less toxic cinnamic alcohol using dehydrogenase/reductase enzymes (YqhD and DkgA). This is the first study to characterize the ability of E. coli O157:H7 to convert cinnamaldehyde into cinnamic alcohol which, in turn, showed that the antimicrobial activity of cinnamaldehyde is mainly attributable to its carbonyl aldehyde group.

OBJECTIVE

To evaluate (11)C-choline PET/CT as a diagnostic tool for restaging prostate cancer (PCa), in a large, homogeneous and clinically relevant population of patients with biochemical recurrence (BCR) of PCa after primary therapy. The secondary aim was to assess the best timing for performing (11)C-choline PET/CT during BCR.

METHODS

We retrospectively analysed 9,632 (11)C-choline PET/CT scans performed in our institution for restaging PCa from January 2007 to June 2015. The inclusion criteria were: (1) proven PCa radically treated with radical prostatectomy (RP) or with primary external beam radiotherapy (EBRT); (2) PSA serum values available; (3) proven BCR (PSA >0.2 ng/mL after RP or PSA >2 ng/mL above the nadir after primary EBRT with rising PSA levels). Finally, 3,203 patients with recurrent PCa matching all the inclusion criteria were retrospectively enrolled and 4,426 scans were analysed.

RESULTS

Overall, 52.8 % of the (11)C-choline PET/CT scans (2,337/4,426) and 54.8 % of the patients (1,755/3,203) were positive. In 29.4 % of the scans, at least one distant finding was observed. The mean and median PSA values were, respectively, 4.9 and 2.1 ng/mL at the time of the scan (range 0.2 - 50 ng/mL). In our series, 995 scans were performed in patients with PSA levels between 1 and 2 ng/mL. In this subpopulation the positivity rate in the 995 scans was 44.7 %, with an incidence of distant findings of 19.2 % and an incidence of oligometastatic disease (one to three lesions) of 37.7 %. The absolute PSA value at the time of the scan and ongoing androgen deprivation therapy were associated with an increased probability of a positive (11)C-choline PET/CT scan (p < 0.0001). In the ROC analysis, a PSA value of 1.16 ng/mL was the optimal cut-off value. In patients with a PSA value <1.16 ng/mL, 26.8 % of 1,426 (11)C-choline PET/CT scans were positive, with oligometastatic disease in 84.7 % of positive scans.

CONCLUSIONS

In a large cohort of patients, the feasibility of (11)C-choline PET/CT for detecting the sites of metastatic disease in PCa patients with BCR was confirmed. The PSA level was the main predictor of a positive scan with 1.16 ng/mL as the optimal cut-off value. In the majority of positive scans oligometastatic disease, potentially treatable with salvage therapies, was observed.

A stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed which can separate and accurately quantitate paracetamol, dantrolene, cetirizine and pseudoephedrine. The method was successfully validated for the purpose of conducting stability studies of the four analytes in quality control (QC) laboratories. The stability-indicating capability of the method was demonstrated by adequate separation of these four analytes from all the degradant peaks. A gradient mobile phase system consisting of (A) 50 mmol L(-1) sodium dihydrogen phosphate, 5 mmol L(-1) heptane sulfonic acid sodium salt, pH 4.2 and (B) acetonitrile was used with Discovery reversed-phase HS C(18) analytical column (250 mm x 4.6 mm i.d., 5 microm particle size). Quantitation was achieved with UV detection at 214 nm, based on peak area. The proposed method was validated and successfully applied for the analysis of pharmaceutical formulations and laboratory-prepared mixtures containing the two multicomponent combinations.

The betalain pattern of differently colored Swiss chard (Beta vulgaris L. ssp. cicla [L.] Alef. cv. Bright Lights) was investigated for the first time. Nineteen betaxanthins and nine betacyanins were identified by RP-HPLC and positive ion electrospray mass spectrometry, co-injection experiments with semisynthetic reference compounds, and standards derived from authentic plant material, respectively. Histamine-betaxanthin and alanine-betaxanthin were found to be novel betaxanthins, which to the best of our knowledge have not been reported as natural compounds until now. Furthermore, tyramine-betaxanthin (miraxanthin III) and 3-methoxytyramine-betaxanthin, which to date were known only from families other than the Chenopodiaceae, were detected for the first time in colored Swiss chard. The betacyanin pattern of purple petioles was composed of betanin, isobetanin, betanidin, and isobetanidin. Although phyllocactin was present in only trace amounts, further acylated structures such as betanidin-monoferuloyl-5-O-beta-diglucoside and lampranthin II, accompanied by their corresponding C(15)-epimers, were identified. In addition, quantification of betalains and CIE LCh degrees measurements were performed with the colored extracts to correlate the visual appearance with the respective pigment patterns. Besides the novel phytochemical findings, the present study is useful for the evaluation of betalainic Swiss chard as a potential coloring foodstuff.