The Octavius1500 2D ion chamber array and its associated phantoms: dosimetric characterization of a new prototype.

PURPOSE: The purpose of the study is to characterize the prototype of the new Octavius1500 (PTW, Freiburg, Germany) 2D ion chamber array, covering its use in different phantom setups, from the most basic solid water sandwich setup to the more complex cylindrical Octavius® 4D (Oct4D) (PTW) phantom/detector combination. The new detector houses nearly twice the amount of ion chambers as its predecessors (Seven29 and Octavius729), thereby tackling one of the most important limitations of ion chamber (or diode) arrays, namely the limited detector density. The 0.06 cm3 cubic ion chambers are now arranged in a checkerboard pattern, leaving no lines (neither longitudinally nor laterally) without detectors. METHODS: All measurements were performed on a dual energy (6 MV and 18 MV) iX Clinac (Varian Medical Systems, Palo Alto, CA) and all calculations were done in the Eclipse treatment planning system (Varian) with the Anisotropic Analytical Algorithm. First, the basic characteristics of the 2D array, such as measurement stability, dose rate dependence and dose linearity were investigated in the solid water sandwich setup. Second, the directional dependence was assessed to allow the evaluation of the new Octavius2D phantom (Oct2D(1500)) for planar verification measurements of composite plans. Third, measurements were performed in the Oct4D phantom to evaluate the impact of the increased detector density on the accuracy of the volumetric dose reconstruction. RESULTS: While showing equally good dose linearity and dose rate independence, the Octavius1500 outperforms the previous models because of its instantaneous measurement stability and its twofold active area coverage. Orthogonal field-by-field measurements immediately benefit from the increased detector density. The 3.9 cm wide compensation cavity in the new Oct2D(1500) phantom prototype adequately corrects for directional dependence from the rear, resulting in good agreement within the target dose. Discrepancies may arise towards the sides of the array because of uncompensated lateral beam incidence. The beneficial impact of the detector density is most prominent in the Oct4D system, for which the average pass rate (PR) is now nearly 100% (99.31±0.37) when using gamma criteria of 2%G,2 mm (10% dose threshold). In search of gamma analysis criteria that are not too lenient to detect possibly relevant deviations, the authors conclude that for our radiotherapy environment, the authors choose to adopt 3%L,3 mm PR97% (threshold 10%) criteria for the Oct2D(1500)/Octavius1500 system and 2%L,3 mm PR97% (threshold 10%) for the Oct4D/Octavius1500 system. These are first line pass/check criteria and plans that fail are not necessarily rejected, but submitted to a more detailed investigation. CONCLUSIONS: When irradiated from the front, the Octavius1500 array has two main advantages over its 729 predecessors: its instantaneous measurement stability and--most importantly-its twofold detector density. In the Oct2D1500 phantom, these advantages are counterbalanced by the more pronounced directional dependence. The measurement-based 3D dose reconstruction in the Oct4D system, however, benefits considerably from the higher detector density in the checkerboard panel design.

Expression of the glucose transporter HXT1 involves the Ser-Thr protein phosphatase Sit4 in Saccharomyces cerevisiae.

We studied the effect of the loss of the Ser-Thr protein phosphatase Sit4, an important post-translational regulator, on the steady-state levels of the low-affinity glucose transporter Hxt1p and observed a delay in its appearance after high glucose induction, slow growth, and diminished glucose consumption. By analyzing the known essential pathway necessary to induce Hxt1p, we observed a partial inhibition of casein kinase I activity. In both WT and sit4Δ strains, the transcript was induced with no significant difference at 15 min of glucose induction; however, after 45 min, a clear difference in the level of expression was observed being 45% higher in WT than in sit4Δ strain. As at early time of induction, the HXT1 transcript was present but not the protein in the sit4Δ strain we analyzed association of HXT1 with ribosomes, which revealed a significant difference in the association profile; in the mutant strain, the HXT1 transcript associated with a larger set of ribosomal fractions than it did in the WT strain, suggesting also a partial defect in protein synthesis. Overexpression of the translation initiation factor TIF2/eIF4A led to an increase in Hxt1p abundance in the WT strain only. It was concluded that Sit4p ensures that HXT1 transcript is efficiently transcribed and translated thus increasing protein levels of Hxt1p when high glucose levels are present.

The regulatory inputs controlling pleiotropic drug resistance and hypoxic response in yeast converge at the promoter of the aminocholesterol resistance gene RTA1.

Aminosterols possessing potent fungicidal activity are attractive alternatives to currently available antifungals. Although their precise mechanism of action is not fully understood, the effect of 7-aminocholesterol (7-ACH) involves a partial block of Δ8-Δ7 isomerase and C-14 reductase. The function of RTA1 encoding the 7-transmembrane helix protein, cloned as the multicopy suppressor of 7-ACH toxicity in yeast, remains unclear. In this report, we show that Rta1p is localized in the plasma membrane and has a high rate of metabolic turnover, as revealed by fluorescence microscopy, cell fractionation and pulse-chase experiments. Analysis of the RTA1-lacZ reporter activity and deletion mapping of the promoter allowed the identification of the regions responsible for negative regulation by Tup1 and the two synergistically acting repressors of hypoxic genes, Rox1p and Mot3p. This was in line with increased RTA1-mediated resistance to 7-ACH under hypoxic conditions, associated with increased Rta1p level. Overexpression of RTA1 also affected the response to the signalling sphingolipid precursor phytosphingosine. Positive inputs of two transcriptional activators Pdr1p and Upc2p were also detected, indicating a regulatory link common to sterol biosynthetic genes as well as those involved in pleiotropic drug resistance and sphingolipid metabolism.

The Arabidopsis multistress regulator TSPO is a heme binding membrane protein and a potential scavenger of porphyrins via an autophagy-dependent degradation mechanism.

TSPO, a stress-induced, posttranslationally regulated, early secretory pathway-localized plant cell membrane protein, belongs to the TspO/MBR family of regulatory proteins, which can bind porphyrins. This work finds that boosting tetrapyrrole biosynthesis enhanced TSPO degradation in Arabidopsis thaliana and that TSPO could bind heme in vitro and in vivo. This binding required the His residue at position 91 (H91), but not that at position 115 (H115). The H91A and double H91A/H115A substitutions stabilized TSPO and rendered the protein insensitive to heme-regulated degradation, suggesting that heme binding regulates At-TSPO degradation. TSPO degradation was inhibited in the autophagy-defective atg5 mutant and was sensitive to inhibitors of type III phosphoinositide 3-kinases, which regulate autophagy in eukaryotic cells. Mutation of the two Tyr residues in a putative ubiquitin-like ATG8 interacting motif of At-TSPO did not affect heme binding in vitro but stabilized the protein in vivo, suggesting that downregulation of At-TSPO requires an active autophagy pathway, in addition to heme. Abscisic acid-dependent TSPO induction was accompanied by an increase in unbound heme levels, and downregulation of TSPO coincided with the return to steady state levels of unbound heme, suggesting that a physiological consequence of active TSPO downregulation may be heme scavenging. In addition, overexpression of TSPO attenuated aminolevulinic acid-induced porphyria in plant cells. Taken together, these data support a role for TSPO in porphyrin binding and scavenging during stress in plants.

The serine/threonine protein phosphatase Sit4p activates multidrug resistance in Saccharomyces cerevisiae.

Multidrug resistance in Saccharomyces cerevisiae is frequently associated with gain-of-function mutations in zinc finger-containing transcription factors Pdr1p and Pdr3p. These regulatory proteins activate the expression of several ATP-binding cassette transporter genes, leading to elevated drug resistance. Here, we report that loss of the type 2A-related serine/threonine protein phosphatase Sit4p renders yeast cells sensitive to cycloheximide, azoles, daunorubicin and rhodamine 6G. This effect is a consequence of the decreased transcriptional levels of mainly PDR3 and its target genes, PDR5, SNQ2 and YOR1, which encode multidrug efflux pumps. The multidrug sensitivity of sit4 mutant cells is suppressed by the PDR1-3 mutant allele, which encodes a hyperactive form of Pdr1p. Sit4p is known to associate with regulatory proteins Sap155p, Sap4p, Sap185p and Sap190p. We found that the sap155 mutant strain is sensitive to azoles, but not to cycloheximide, while the sap155sap4 and sap185sap190 mutant strains are sensitive to both drugs. This finding indicates that the Sit4p-Sap protein complex subtly modulates the expression of drug efflux pumps. Drug resistance conferred by the expression of the Candida albicans CDR1 gene, an ortholog of PDR5 in S. cerevisiae, is also positively modulated by Sit4p. These data uncover a new regulatory pathway that connects multidrug resistance to Sit4p function.

The lipid-translocating exporter family and membrane phospholipid homeostasis in yeast.

The fungal lipid-translocating exporter family consists of conserved membrane proteins, with six or seven transmembrane spans. Phylogenetic trees and conserved gene order relationships show that the common ancestor of five closely related hemiascomycetous yeast species contained the RSB1 and PUG1 paralogous genes. In Saccharomyces cerevisiae, Rsb1 functions as a transporter or translocase of sphingoid bases, whereas Pug1 facilitates the inducible transport of protoporphyrin IX and hemin. The budding yeast contains two other paralogs, Ylr046p, of unknown function, and Rta1p, overexpression of which confers resistance to an ergosterol biosynthesis inhibitor. Large-scale mRNA expression profiling has shown that transcription of PUG1, RTA1 and YLR046 is induced under hypoxic conditions. Ergosterol biosynthesis is impaired under low-oxygen conditions as a consequence of the decreased synthesis of heme and heme-containing proteins. These genes may encode transporters or sensors that facilitate the excretion of excessive or aberrant biosynthetic intermediates, either directly or indirectly. The expression of RSB1 and RTA1 is under the control of pleiotropic drug resistance transcription factors, suggesting that the encoded proteins may have additional roles in cell resistance to xenobiotics. This review summarizes current knowledge concerning the lipid-translocating exporter family and its potential functions, focusing on multidrug resistance and membrane phospholipid homeostasis.

Diagnosis of vulvar lesions by non-invasive optical analysis: a pilot study.

A procedure that could allow an early in vivo and non-invasive detection of vulvar lesions would be extremely useful. We tested an innovative optical method (Optiprobe), which uses a harmless, visible light source for the in vivo, on-line detection of minimal alterations in the structure of vulvar epithelium. A group of 3 female volunteers without gynecological symptoms were first screened to evaluate optical properties of normal vulvar tissue. Next, a group of 16 patients undergoing gynecological examination for vulvar lesions was evaluated by the Optiprobe at suspected sites before these sites were biopsied for histological analysis. Adjacent, non-involved sites were also measured to provide internal controls. Histological analysis of the biopsies identified one case that did not show obvious alterations, 4 cases of high-grade vulvar intraepithelial neoplasia (VIN), 5 cases of vulvitis, and 6 cases of lichen sclerosis (LS).The optical properties of the VIN cases were significantly different from those of controls, due to a decrease in the absorption spectra and an increase in the scattering spectra. In contrast, a significant increase in the absorption spectra and a decrease in the scattering spectra were observed in the cases of vulvitis. In the LS cases, the absorption spectra were as in controls, whereas the scattering spectra were significantly decreased. We conclude that the Optiprobe provides a useful tool for a rapid and non-invasive detection of vulvar alterations. The method should contribute to reduce the number of biopsies and to facilitate the long-term follow-up of vulvar lesions.

Substrate-binding sites of UBR1, the ubiquitin ligase of the N-end rule pathway.

Substrates of a ubiquitin-dependent proteolytic system called the N-end rule pathway include proteins with destabilizing N-terminal residues. N-recognins, the pathway's ubiquitin ligases, contain three substrate-binding sites. The type-1 site is specific for basic N-terminal residues (Arg, Lys, and His). The type-2 site is specific for bulky hydrophobic N-terminal residues (Trp, Phe, Tyr, Leu, and Ile). We show here that the type-1/2 sites of UBR1, the sole N-recognin of the yeast Saccharomyces cerevisiae, are located in the first approximately 700 residues of the 1,950-residue UBR1. These sites are distinct in that they can be selectively inactivated by mutations, identified through a genetic screen. Mutations inactivating the type-1 site are in the previously delineated approximately 70-residue UBR motif characteristic of N-recognins. Fluorescence polarization and surface plasmon resonance were used to determine that UBR1 binds, with a K(d) of approximately 1 microm, to either type-1 or type-2 destabilizing N-terminal residues of reporter peptides but does not bind to a stabilizing N-terminal residue such as Gly. A third substrate-binding site of UBR1 targets an internal degron of CUP9, a transcriptional repressor of peptide import. We show that the previously demonstrated in vivo dependence of CUP9 ubiquitylation on the binding of cognate dipeptides to the type-1/2 sites of UBR1 can be reconstituted in a completely defined in vitro system. We also found that purified UBR1 and CUP9 interact nonspecifically and that specific binding (which involves, in particular, the binding by cognate dipeptides to the UBR1 type-1/2 sites) can be restored either by a chaperone such as EF1A or through macromolecular crowding.

Pdr5-mediated multidrug resistance requires the CPY-vacuolar sorting protein Vps3: are xenobiotic compounds routed from the vacuole to plasma membrane transporters for efflux?

In Saccharomyces cerevisiae several members of the ATP-binding cassette transporter superfamily efflux a broad range of xenobiotic substrates from cells. The vacuole also plays a critical role in multidrug resistance. Mutations in genes such as VPS3 that are essential for vacuolar acidification and carboxypeptidase Y vacuolar protein-sorting are multidrug sensitive. A similar phenotype is also observed with deletions of VPS15, VPS34, and VPS38, which encode essential members of the carboxypeptidase Y vacuolar protein-sorting pathway. Prior to the work described herein, detoxification by transporters and the vacuole were presumed to function independently. We demonstrate that this is not the case. Significantly, Vps3 has an epistatic relationship with Pdr5, a major yeast multidrug transporter. Thus, a double pdr5, vps3 deletion mutant is no more multidrug sensitive than its isogenic single-mutant counterparts. Subcellular fractionation experiments and analysis of purified plasma membrane vesicles indicate, however, that a vps3 mutation does not affect the membrane-localization or ATPase activity of Pdr5 even though rhodamine 6G efflux is reduced significantly. This suggests that Vps3 and probably other members of the carboxypeptidase Y vacuolar protein-sorting pathway are required for relaying xenobiotic compounds to transporters in the membrane.

Subcellular trafficking of the yeast plasma membrane ABC transporter, Pdr5, is impaired by a mutation in the N-terminal nucleotide-binding fold.

The plasma membrane ATP-binding cassette (ABC) transporter, Pdr5p, mediates resistance to many different xenobiotic compounds in yeast. We have isolated several mutated forms that fail to confer resistance to cycloheximide and itraconazole. Here, we examined two variants, the expression of which was abnormally low when cells reach the stationary phase of growth. The Pdr5(1157) variant lacked the C-terminal transmembrane domain due to the presence of a nonsense mutation at codon 1158. The second variant, Pdr5(L183P), contained a Leu183Pro substitution close to the Walker A motif in the N-terminal nucleotide-binding domain. This substitution impaired UTPase activity as well as protein stability. The Pdr5(L183P) variant induced the unfolded protein response and was targeted to the proteasome for degradation. Fluorescence microscopy showed that the highly unstable Pdr5(L183P) was mislocalized to endoplasmic reticulum (ER)-associated compartments, whereas the truncated Pdr5(1157) protein was retained in the ER. When threonine 363 (located in the first nucleotide-binding domain, close to the Walker B motif) in Pdr5(L183P) was replaced with isoleucine, this double mutant conferred partial drug resistance. These results suggest that Pdr5p requires a properly folded nucleotide-binding domain for trafficking to the plasma membrane.

ER-associated degradation of membrane proteins in yeast.

Proteins destined for the secretory pathway are translocated into the endoplasmic reticulum (ER), where they are subjected to a variety of post-translational modifications before they reach their final destination. Newly synthesized proteins that have defect in polypeptide folding or subunit assembly are recognized by quality control systems and eliminated by the 26S proteasome, a cytosolic ATP-dependent proteolytic machinery. Delivery of non-native ER proteins to the proteasome requires retrograde transport across the ER membrane and depends on a protein-unfolding machine consisting of Cdc48p, Ufd1p, and Npl4p. Recent studies in yeast have highlighted the possible function of the Sar1p/COPII machinery in ER-associated degradation of some lumenal and membrane proteins.

Diagnosis and grading of gastritis by non-invasive optical analysis.

OBJECTIVES: The precise identification of many diseases of the gastrointestinal tract requires the histological analysis of multiple biopsies of the lining mucosae, thus preventing an immediate diagnosis and the safe screening of the entire organ. To address these limitations, we developed a novel spectroscopic procedure for a real-time, non-invasive optical analysis of mucosae. METHODS: We have used a fibre-optic probe that monitors light propagation through small tissue volumes to evaluate the antral and fundic mucosa of 51 patients that underwent gastroscopy for symptoms of dyspepsia. Several optical coefficients were computed from the recorded light reflectance, and confronted to the diagnosis made by an expert gastroscopist at the time of the clinical examination. Both evaluations were then validated by comparison with the histological diagnosis of a pathologist who screened biopsies taken at the sites of the optical measurements. RESULTS: We report that the optical procedure discriminated normal and pathological gastric mucosae with a higher sensitivity and specificity than endoscopic diagnosis. We also show that the changes in light-scattering coefficient, which permitted the optical diagnosis of gastritis alterations, were indirectly correlated with the extent of inflammatory infiltration of the mucosa and detected mucosal alterations mild enough to escape endoscopic detection. CONCLUSIONS: The results show that, in a normal clinical setting, the optical in vivo analysis provided by our system detects alterations typical of gastritis, and allow for their graded scoring with a specificity and sensitivity that compare well with those of standard histology, while avoiding the invasiveness of the latter procedure. The method is adaptable to the screening of other types of lesions and mucosae and, hence, should prove useful in improving available diagnostic approaches.

Binding of Cdc48p to a ubiquitin-related UBX domain from novel yeast proteins involved in intracellular proteolysis and sporulation.

The Cdc48/p97 AAA-ATPase functions in membrane fusion and ubiquitin-dependent protein degradation. Here, we show that, in yeast, Cdc48p interacts with three novel proteins, Cuil-3p, which contain a conserved ubiquitin-related (UBX) domain. Cui2p and Cui3p are closely related, interact with each other, and are localized at the perinuclear membrane. Cdc48p binds directly the UBX domain of Cui3p in vitro. Multiple deletions of the CUI1, CUI2 and CUI3 genes confer deficiency in sporulation and degradation of model ubiquitin-protein fusions. The Cuil-3 proteins were also found to interact with Ufd3p, a WD repeat protein known to associate with Cdc48p. Together, these results indicate that the Cuil-3 proteins form complexes that are components of the ubiquitin-proteasome system.

A new optical method for the non-invasive detection of minimal tissue alterations.

Histological analysis, which is used to detect and diagnose most tissue alterations, requires an invasive biopsy procedure and a time-consuming tissue treatment, which limit its efficiency in providing rapid, cost-effective diagnosis and hinder the longitudinal study of tissue alteration. To address these limitations, we have developed a novel procedure, using the features of elastic-scattering spectroscopy, for a real-time, non-invasive analysis of tissues. We have tested whether this approach can detect in vivo changes in mouse skin induced by a single exposure to either complete Freund's adjuvant or 12-O-tetradecanoylphorbol-13-acetate, two drugs known to induce discrete alterations of epidermis and dermis, without obvious changes on the skin surface. Here we report that the evaluation of localized absorption and reduced scattering coefficients permitted the detection of changes in skin regions that showed histological alterations, but not in regions which failed to be modified by the drugs. Results show that the optical in vivo analysis of small regions has sufficient specificity and sensitivity to detect minimal alterations of superficial tissues. In view of the prominent involvement of mucosal alterations in most human diseases, including carcinomas, the method provides a useful complement to standard biopsy, notably for the in vivo screening of early in situ epithelial alterations.

Control of 26S proteasome expression by transcription factors regulating multidrug resistance in Saccharomyces cerevisiae.

In eukaryotic cells, intracellular proteolysis occurs mainly via the ubiquitin-proteasome system. Expression of the yeast proteasome is under the control of the transcription factor, Rpn4p (also known as Son1p/Ufd5p). We show here that the RPN4 gene promoter contains regulatory sequences that bind Pdr1p and Pdr3p, two homologous zinc finger-containing transcription factors, which mediate multiple drug resistance through the expression of membrane transporter proteins. Mutations in the RPN4 Pdr1p/Pdr3p binding sites lead to decreased expression of the proteasome RPT6 gene and to defective ubiquitin-mediated proteolysis. Pdr3p, but not Pdr1p, is required for normal levels of intracellular proteolysis, indicating that the two transcription factors have distinct functions in the control of RPN4 expression. The RPN4 promoter contains an additional sequence that binds Yap1p, a bZIP-type transcription factor that plays an important role in the oxidative stress response and multidrug resistance. We also show that the Yap1p response element is important in the transactivation of RPN4 by Yap1p. In yeast cells lacking Pdr1p, ubiquitin-Pro-beta-galactosidase, a short-lived protein used to assay proteasome activity, is stabilized by the loss of Yap1p. These data demonstrate that the ubiquitin-proteasome system is controlled by transcriptional regulators of multidrug resistance via RPN4 expression.

Identification and functional analysis of the Saccharomyces cerevisiae nicotinamidase gene, PNC1.

Nicotinamidase (NAMase) from the budding yeast, Saccharomyces cerevisiae, was purified by Ni(2+) affinity chromatography and gel filtration. N-terminal microsequencing revealed sequence identity with a hypothetical polypeptide encoded by the yeast YGL037C open reading frame sharing 30% sequence identity with Escherichia coli pyrazinamidase/nicotinamidase. A yeast strain in which the NAMase gene, hereafter named PNC1, was deleted shows a decreased intracellular NAD(+) concentration, consistent with the loss of NAMase activity in the null mutant. In wild-type strains, NAMase activity is stimulated during the stationary phase of growth, by various hyperosmotic shocks or by ethanol treatment. Using a P(PNC1)::lacZ gene fusion, we have shown that this stimulation of NAMase activity results from increased levels of the protein and requires stress response elements in the 5'non-coding region of PNC1. These results suggest that NAMase helps yeast cells to adapt to various stress conditions and nutrient depletion, most likely via the activation of NAD-dependent biological processes.