Involved field radiotherapy for locally advanced non-small cell lung cancer: isolated mediastinal nodal relapse.

The current standard of care for locally advanced inoperable non-small cell lung cancer is high dose radiotherapy with concurrent chemotherapy. We report on a patient with stage IIIA NSCLC treated with concurrent chemoradiotherapy on the primary tumor and the 18-fluorodeoxyglucose positron emission tomography ((18)FDG-PET) positive hilar and mediastinal lymph nodes. Six months after treatment this patient developed a single isolated contralateral mediastinal nodal relapse outside but in the proximity of the irradiated target volume. This patient was successfully re-irradiated to this isolated nodal relapse after reconstruction of the dose given to the localisation of this regional recurrence. This case describes the clinical problem of a regional recurrence after involved field radiotherapy that occasionally occurs. A possible explanation for those regional recurrences is an under staging of extension of the disease because the time-interval between the staging (18)FDG-PET-CT scan and the start of the irradiation was too long. If the time-interval is 4 weeks or more, we strongly recommend a new (18)FDG-PET-CT because of the possibility of upstaging of the disease.

Interactions of oxidosqualene cyclase (Erg7p) with 3-keto reductase (Erg27p) and other enzymes of sterol biosynthesis in yeast.

In Saccharomyces cerevisiae and Candida albicans, two enzymes of the ergosterol biosynthetic pathway, oxidosqualene cyclase (Erg7p) and 3-keto reductase (Erg27p) interact such that loss of the 3-keto reductase also results in a concomitant loss of activity of the upstream oxidosqualene cyclase. This interaction wherein Erg27p has a stabilizing effect on Erg7p was examined to determine whether Erg7p reciprocally has a protective effect on Erg27p. To this aim, three yeast strains each lacking the ERG7 gene were tested for 3-ketoreductase activity by incubating either cells or cell homogenates with unlabeled and radiolabeled 3-ketosteroids. In these experiments, the ketone substrates were effectively reduced to the corresponding alcohols, providing definitive evidence that oxidosqualene cyclase is not required for the 3-ketoreductase activity. This suggests that, in S. cerevisiae, the protective relationship between the 3-keto reductase (Erg27p) and oxidosqualene cyclase (Erg7p) is not reciprocal. However, the absence of the Erg7p, appears to affect other enzymes of sterol biosynthesis downstream of lanosterol formation. Following incubation with radiolabeled and non-radiolabeled 3-ketosteroids we detected differences in hydroxysteroid accumulation and ergosterol production between wild-type and ERG7 mutant strains. We suggest that oxidosqualene cyclase affects Erg25p (C-4 sterol oxidase) and/or Erg26p (C-3 sterol dehydrogenase/C-4 decarboxylase), two enzymes that, in conjunction with Erg27p, are involved in C-4 sterol demethylation.

Genetic analyses involving interactions between the ergosterol biosynthetic enzymes, lanosterol synthase (Erg7p) and 3-ketoreductase (Erg27p), in the yeast Saccharomyces cerevisiae.

Protein-protein interaction studies in the Saccharomyces cerevisiae ergosterol biosynthetic pathway suggest that enzymes in this pathway may act as an integrated multienzyme complex. The yeast sterol 3-ketoreductase (Erg27p) required for C-4 demethylation of sterols has previously been shown to also be required for the function of the upstream oxidosqualene cyclase/lanosterol synthase (Erg7p); thus, erg27 mutants accumulate oxidosqualenes as precursors rather than 3-ketosterones. In the present study, we have created various mutations in the ERG27 gene. These mutations include 5 C-terminal truncations, 6 internal deletions, and 32 point mutants of which 14 were obtained by site-directed mutagenesis and 18 by random mutagenesis. We have characterized these ERG27 mutations by determining the following: Erg27 and Erg7 enzyme activities, presence of Erg27p as determined by western immunoblots, ability to grow on various sterol substrates and GC sterol profiles. Mutations of the predicted catalytic residues, Y202F and K206A, resulted in the endogenous accumulation of 3-ketosterones rather than oxidosqualenes suggesting retention of Erg7 enzyme activity. This novel phenotype demonstrated that the catalytic function of Erg27p can be separated from its Erg7p chaperone ability. Other erg27 mutations resulted in proteins that were present, as determined by western immunoblotting, but unable to interact with the Erg7 protein. We also classify Erg27p as belonging to the SDR (short-chain dehydrogenase/reductase) family of enzymes and demonstrate the possibility of homo- or heterodimerization of the protein. This study provides new insights into the role of Erg27p in sterol biosynthesis.

Influence of the structure of cornstarch dispersions on kinetics of aroma release.

This study deals with the impact of food structure and texture on aroma release. This was done with cornstarch dispersions with constant concentrations in starch but differing in their structures. The structure parameters of the cornstarch dispersions were varied by changing the shearing conditions during the pasting process. Linalool and isoamyl acetate were chosen as reference aroma compounds. Linalool is known to form complexes with amylose while isoamyl acetate does not. The release of aroma compounds from starch dispersions under stirring was studied at 2 temperatures by discrete sampling of the headspace. Aroma release curves were modeled and the kinetic and thermodynamic parameters were extracted. The release of linalool seemed to be governed mainly by the interactions (complexation) with starch. In contrast, the interactions between starch and isoamyl acetate were feebler, and the release of this aroma compound was governed by the structure of the starch dispersion. The observed interactions were better established at 20 degrees C than at 32 degrees C.

Cancer in the Norwegian printing industry.

OBJECTIVES: The aim of this study was to investigate cancer risk among Norwegian workers in the printing industry, particularly lung and bladder cancer. METHODS: Cancer incidence was investigated from 1953 through 1998 in a cohort of 10 549 male members of a trade union in the printing industry in Oslo and nearby areas. Rates from the region, were used to calculate standardized incidence ratios (SIR) separately for the skilled and unskilled workers. Smoking data from a sample of the cohort were utilized for evaluating the risk estimates of smoking-related cancers. Specific exposure data were not available. RESULTS: Among the skilled workers, significantly elevated risks of cancer of the urinary bladder [standardized incidence ratio (SIR) 1.47, 95% confidence interval (95% CI) 1.19-1.79], liver (SIR 1.92, 95% CI 1.15-2.99), pancreas (SIR 1.46, 95% CI 1.07-1.94) and colon (SIR 1.27, 95% CI 1.05-1.55) were observed, whereas an increased risk of lung cancer in this group was confined to those born before 1910. Among the unskilled workers, there were significantly increased risks of cancer of the mouth, esophagus, stomach, larynx, lung, and all sites. CONCLUSIONS: The study showed that workers in the printing industry were at increased risk of several types of cancer. In particular the increased risk of bladder cancer among the skilled workers is suggestive of an occupational cause. However, no specific agent could be identified as an occupational carcinogen. The results did not support the hypothesis of a generally increased risk of lung cancer. The risk pattern for unskilled workers may reflect confounding by nonoccupational factors.

Isolation, characterization, and regulation of the Candida albicans ERG27 gene encoding the sterol 3-keto reductase.

The Candida albicans ERG27 gene which encodes the 3-keto reductase enzyme required for sterol C-4 demethylation was isolated and found to encode a 349 amino acid protein that is 60% identical at the amino acid level to the Saccharomyces cerevisiae Erg27p. A C. albicans erg27 null was created in a strain containing an integrated ERG27 rescue cassette under the control of the pMAL2 inducible promoter. The C. albicans erg27 strain was able to grow only in the presence of maltose indicating that the ERG27 gene is essential. The C. albicans erg27 null showed complete loss of both 3-keto reductase and oxidosqualene cyclase (Erg7p) activities compromising all sterol synthesis. These results suggest that Erg27p inhibitors might be effective antifungals. To explore ERG27 regulation, an erg11 null strain was generated. C. albicans erg6 and erg24 mutants were also employed along with the inhibitors, itraconazole and zaragozic acid A, to characterize ERG27 expression using Northern analysis. Expression was increased two- to fourfold in erg11, erg6 and erg24 backgrounds. However, itraconazole which targets Erg11p (lanosterol demethylase) increased ERG27 expression 10-fold and zaragozic acid A which targets the Erg9p (squalene synthase) increased ERG27 expression fivefold. The azole and erg11 results support other observations that azoles may affect non-sterol targets.

Ergosterol gene expression in wild-type and ergosterol-deficient mutants of Candida albicans.

The ergosterol pathway is the major target of the azole antifungals. We have developed a panel of five viable ergosterol biosynthetic mutants (erg2, erg3, erg6, erg11 and erg24) and have performed Northern analyses to study transcriptional regulation using probes to four ergosterol biosynthetic genes (ERG2, ERG7, ERG11 and ERG25), as well as probes to two additional genes encoding ergosterol cytochrome coenzymes (CYB5 and NCP1). ERG11, which encodes the sterol 14-demethylase, the direct target of the azole antifungals, was the most up-regulated gene followed by ERG7 and ERG25. Transcription of the four ergosterol genes was most up-regulated in erg24 and erg6 mutant backgrounds, deficient in C-14 reductase and the C-24 sterol transmethylase, respectively. Unexpectedly, we also found that the two cytochrome genes, CYB5 encoding cytochrome b5 and NCP1 encoding the cytochrome P450 reductase, were not regulated markedly different from wild-type in the erg2, erg3, erg6, erg11 and erg24 strains of Candida albicans.

Disruption of the Candida albicans CYB5 gene results in increased azole sensitivity.

Sterol synthesis in fungi is an aerobic process requiring molecular oxygen and, for several cytochrome-mediated reactions, aerobically synthesized heme. Cytochrome b(5) is required for sterol C5-6 desaturation and the encoding gene, CYB5, is nonessential in Saccharomyces cerevisiae. Cyb5p and Ncp1p (cytochrome P-450 reductase) appear to have overlapping functions in this organism, with disruptions of each alone being viable. The cytochrome P-450 reductase phenotype has also been shown to demonstrate increased sensitivity to azole antifungals. Based on this phenotype, the CYB5 gene in the human pathogen Candida albicans was investigated to determine whether the cyb5 genotype was viable and would also demonstrate azole sensitivity. Sequential disruption of the CYB5 alleles by direct transformation resulted in viability, presumably conferred by the presence of a third copy of the CYB5 gene. Subsequent disruption procedures with a pMAL2-CYB5 rescue cassette and a CYB5-URA3 blaster cassette resulted in viable cyb5 strains with no third copy. The C. albicans CYB5 gene is concluded to be nonessential. Thus, the essentiality of this gene and whether we observed two or three alleles was dependent upon the gene disruption protocol. The C. albicans cyb5 strains produced a sterol profile containing low ergosterol levels and sterol intermediates similar to that reported for the S. cerevisiae cyb5. The C. albicans cyb5 shows increased sensitivity to azoles and terbinafine, an inhibitor of squalene epoxidase, and, unexpectedly, increased resistance to morpholines, which inhibit the ERG2 and ERG24 gene products. These results indicate that an inhibitor of Cyb5p would not be lethal but would make the cell significantly more sensitive to azole treatment.

In yeast sterol biosynthesis the 3-keto reductase protein (Erg27p) is required for oxidosqualene cyclase (Erg7p) activity.

In Saccharomyces cerevisiae, the 3-keto reductase (Erg27p) encoded by ERG27 gene is one of the key enzymes involved in the C-4 demethylation of the sterol intermediate, 4,4-dimethylzymosterol. The oxidosqualene cyclase (Erg7p) encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study, we found that erg27 strains grown on cholesterol- or ergosterol-supplemented media did not accumulate lanosterol or 3-ketosterols but rather squalene, oxidosqualene, and dioxidosqualene intermediates normally observed in ERG7 (oxidosqualene cyclase) mutants. These results suggested a possible interaction between these two enzymes. In this study, we present evidence that Erg27p interacts with Erg7p, facilitating the association of Erg7p with lipid particles (LPs) and preventing digestion of Erg7p both in the endoplasmic reticulum (ER) and LPs. We demonstrate that Erg27p is required for oxidosqualene cyclase (Erg7p) activity in LPs, and that Erg27p co-immunoprecipitates with Erg7p in LPs but not in microsomal fractions. While Erg27p is essentially a component of the ER, it can also be detected in LPs. In erg27 strains, a truncated Erg7p mislocalizes to microsomes. Restoration of Erg7p enzyme activity and LPs localization was achieved in an erg27 strain transformed with a plasmid containing a wild-type ERG27 allele. We suggest that the physical interaction of Erg27p with Erg7p is an essential regulatory tool in yeast sterol biosynthesis.

Protein-protein interactions among C-4 demethylation enzymes involved in yeast sterol biosynthesis.

A Saccharomyces cerevisae microarray expression study indicated that an ORF, YER044C, now designated ERG28, was strongly coregulated with ergosterol biosynthesis. Disruption of the ERG28 gene results in slow growth and accumulation of sterol intermediates similar to those observed in erg26 and erg27 null strains, suggesting that the Erg28p may interact with Erg26p and/or Erg27p. In this study, a peptide from human hemagglutinin protein (HA) epitope tag was added to ERG26 and ERG27 genes, and a Myc tag was added to the ERG28 gene to detect interactions between Erg28p and Erg26p/Erg27p. Differential centrifugation showed that Erg26p, Erg27p, and Erg28p are all membrane-associated proteins. Green fluorescent protein-fusion protein localization studies showed that Erg26p, Erg27p, and Erg28p are all located in the endoplasmic reticulum. Solubilized membrane protein coimmunoprecipitation studies using rabbit anti-Erg25p indicated that Erg25p coimmunoprecipitates with both Erg27p and Erg28p. Erg28p was also shown to reciprocally coimmunoprecipitate with Erg27p. However, no coimmunoprecipitation was observed with Erg26p, most likely because of the poor solubilization of this protein. Sucrose gradient ultracentrifugation studies suggested that Erg25p/Erg26p/Erg27p/Erg28p, along with other proteins in sterol biosynthesis, might form a complex between 66 and 200 kDa. Using an anti-HA column with Erg27p-HA and Erg26p-HA as target proteins, a complex containing Erg25p/Erg26p/Erg27p/Erg28p was identified. Thus, we suggest that Erg28p works as a transmembrane scaffold to tether Erg27p and possibly other C-4 demethylation proteins (Erg25p, Erg26p), forming a demethylation complex in the endoplasmic reticulum.

[Malignant pleural mesothelioma. From diagnosis to prognosis]. / Le mésothéliome pleural malin. Du diagnostic au pronostic.

EPIDEMIOLOGY: The incidence of malignant pleural mesothelioma has constantly increased over the past forty years. The recent measures of ban on the use of asbestos and the long latency of this tumor after exposure means that its peak incidence can be foreseen for the years 2010-2020. DIAGNOSIS: Various health professionals are involved in the care of this tumor, which benefits equally from progresses in clinical and fundamental research. Some progress has been made in understanding its oncogenesis as well as its histopathologic analysis. PROGNOSIS: Malignant pleural mesothelioma symptoms are rapidly invalidating and the patient's prognosis is bad at short-term. However, hope may come from the detection of early stages of the disease and from the individualization of good prognosis factors, permitting the selection of patients for whom some curative therapies are in course of evaluation.

[Malignant pleural mesothelioma. Present data and perspectives for treatment]. / Le mésothéliome pleural malin. Données actuelles et perspectives pour le traitement.

CHEMOTHERAPY: With regard to the efficacy of mono-chemotherapy and according to the literature, no cytotoxic substance, apart from methrotrexate at high doses, leads to a response rate of more than 20%. With regard to cyrotoxic associations, the published results show slightly betterresponse rates. IMMUNOTHERAPY: Interleukine 2 as well as various interferons have been tested alone or in association with chemotherapy. Fairly encouraging response rates have been reported. However, the possibility of severe adverse events must be taken into account. RADIOTHERAPY: The interest of prophylactic parietal radiation following invasive thoracic treatment has been demonstrated. Palliative use of radiotherapy is possible for pain, and more rarely for decompression. With curative aim, the results of isolated radiotherapy are disappointing. SURGERY: Palliative surgery is aimed at reducing the tumour and pleural symphysis. Curative surgery consists in wide extrapleural pneumonectomy, permitting total resection of the visceral pleura, or decortication pleurectomy leaving the lung in place. Mean survival of patients having undergone surgery is of 10 to 17 months with rates between 10 and 30% at 2 years ASSOCIATED THERAPY: For the first time, despite high morbidity rates, associated therapy has led to prolonged survival, whereas isolated therapy has not. This is the case with an association of radical surgery, radiotherapy and adjuvant chemotherapy. IN THE FUTURE: In the treatment of malignant pleural mesothelioma, genetic, anti-proliferative and immune therapy, that attempt to use the immune system of the patient to obtain an anti-tumour cytotoxic reaction, appear promising.

Candida albicans sterol C-14 reductase, encoded by the ERG24 gene, as a potential antifungal target site.

The incidence of fungal infections has increased dramatically, which has necessitated additional and prolonged use of the available antifungal agents. Increased resistance to the commonly used antifungal agents, primarily the azoles, has been reported, thus necessitating the discovery and development of compounds that would be effective against the major human fungal pathogens. The sterol biosynthetic pathway has proved to be a fertile area for antifungal development, and steps which might provide good targets for novel antifungal development remain. The sterol C-14 reductase, encoded by the ERG24 gene, could be an effective target for drug development since the morpholine antifungals, inhibitors of Erg24p, have been successful in agricultural applications. The ERG24 gene of Candida albicans has been isolated by complementation of a Saccharomyces cerevisiae erg24 mutant. Both copies of the C. albicans ERG24 gene have been disrupted by using short homologous regions of the ERG24 gene flanking a selectable marker. Unlike S. cerevisiae, the C. albicans ERG24 gene was not required for growth, but erg24 mutants showed several altered phenotypes. They were demonstrated to be slowly growing, with doubling times at least twice that of the wild type. They were also shown to be significantly more sensitive to an allylamine antifungal and to selected cellular inhibitors including cycloheximide, cerulenin, fluphenazine, and brefeldin A. The erg24 mutants were also slightly resistant to the azoles. Most importantly, erg24 mutants were shown to be significantly less pathogenic in a mouse model system and failed to produce germ tubes upon incubation in human serum. On the basis of these characteristics, inhibitors of Erg24p would be effective against C. albicans.

Phase II trial of ZD0473 as second-line therapy in mesothelioma.

A phase II, open-label, non-comparative, multicentre trial of the platinum analogue ZD0473 as second-line therapy for pleural mesothelioma has been completed. The objectives were to evaluate the activity and tolerability of ZD0473 in patients with relapsed or progressive disease who had received one prior chemotherapy regimen. Forty-seven patients were recruited onto the trial, all aged > 18 years with a life-expectancy > 12 weeks, and World Health Organization (WHO) performance status < or = 2. A starting dose of 120 mg/m2 was administered to 14 patients, six of whom subsequently had their dose escalated to 150 mg/m2. Thirty-three patients received a starting dose of 150 mg/m2. In total, 147 treatment cycles were administered (median number of cycles 3 [range 1-6]). The main toxicity of ZD0473 was haematological (thrombocytopenia) and the most common non-haematological adverse event was nausea. There was no clinically significant nephro-, neuro-, or oto-toxicity. Of the 43 patients evaluable for response, 12% had a minor response (defined by a reduction in lesion size > or = 10% but < 50%), 44% had stable disease, 40% had disease progression, and two patients died before an objective response could be assigned. Median time to progression and death in evaluable patients was 77 days (95% confidence interval [CI]: 44, 105 days) and 203 days (95% CI: 165, 277 days), respectively. In conclusion, although ZD0473 demonstrated a manageable tolerability profile, no complete or partial responses were seen in second-line treatment of mesothelioma. This trial also demonstrates that clinical trials in second-line mesothelioma patients are feasible.

Functional identification of sterol-4alpha-methyl oxidase cDNAs from Arabidopsis thaliana by complementation of a yeast erg25 mutant lacking sterol-4alpha-methyl oxidation.

Specific primers derived from both genomic sequence data and EST cDNA sequences were used to polymerase chain reaction amplify two full-length cDNA sequences (AtSMO1 and AtSMO2), 801 and 783 bp, respectively, from an Arabidopsis thaliana cDNA library. The predicted proteins show 32 and 29% identity to the ERG25 gene from Saccharomyces cerevisiae which encodes the sterol-4alpha-methyl oxidase (SMO), a membrane-bound non-heme di-iron oxygenase involved in lipid metabolism. Heterologous expression of AtSMO1 and AtSMO2 in a yeast erg25 ergosterol auxotroph, lacking SMO activity, restored growth and endogenous ergosterol synthesis. These results represent the first functional identification of SMO genes from plants.

Transcriptional regulation of the two sterol esterification genes in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae transcribes two genes, ARE1 and ARE2, that contribute disproportionately to the esterification of sterols. Are2p is the major enzyme isoform in a wild-type cell growing aerobically. This likely results from a combination of differential transcription initiation and transcript stability. By using ARE1 and ARE2 promoter fusions to lacZ reporters, we demonstrated that transcriptional initiation from the ARE1 promoter is significantly reduced compared to that from the ARE2 promoter. Furthermore, the half-life of the ARE2 mRNA is approximately 12 times as long as that of the ARE1 transcript. We present evidence that the primary role of the minor sterol esterification isoform encoded by ARE1 is to esterify sterol intermediates, whereas the role of the ARE2 enzyme is to esterify ergosterol, the end product of the pathway. Accordingly, the ARE1 promoter is upregulated in strains that accumulate ergosterol precursors. Furthermore, ARE1 and ARE2 are oppositely regulated by heme. Under heme-deficient growth conditions, ARE1 was upregulated fivefold while ARE2 was down-regulated. ARE2 requires the HAP1 transcription factor for optimal expression, and both ARE genes are derepressed in a rox1 (repressor of oxygen) mutant genetic background. We further report that the ARE genes are not subject to end product inhibition; neither ARE1 nor ARE2 transcription is altered in an are mutant background, nor does overexpression of either ARE gene alter the response of the ARE-lacZ reporter constructs. Our observations are consistent with an important physiological role for Are1p during anaerobic growth when heme is limiting and sterol precursors may accumulate. Conversely, Are2p is optimally required during aerobiosis when ergosterol is plentiful.

Physician executives report high job satisfaction. Summary of findings from a survey of senior physician executives.

The role of the senior physician executive is well established in American hospitals and health systems. There is little research, however, on overall physician executive job satisfaction, their perceptions of their organizational role and job performance, or their views of the medical staffs with which they work. A recent survey of physician executives examined these and other areas. It found physician executives to be quite satisfied with their jobs. What follows is a summary of the findings. An article based on the survey will be featured in a future issue of The Physician Executive.

Positive and negative regulation of squalene synthase (ERG9), an ergosterol biosynthetic gene, in Saccharomyces cerevisiae.

To identify regulatory cis-elements in the proximal promoter of the yeast ERG9 squalene synthase gene, promoter deletion analysis was performed. This approach identified two regulatory elements, one an upstream repressing cis-element (URS), and the other an upstream activating cis-element (UAS). Electromobility shift assays (EMSAs) demonstrated that distinct proteins bind each element. Genetic screens were performed to identify yeast mutants that altered expression of ERG9 promoter-reporter gene fusions. Three non-ergosterol biosynthetic pathway genes were identified. A mutation in TPO1(YLL028W) led to a 5.5-fold increase in ERG9 expression while mutations in YER064C and SLK19 (YOR195W) led to a 3.1- and 5.6-fold decrease, respectively. Deletion analysis of these genes demonstrated that TPO1 and SLK19 specifically regulated ERG9 expression when tested with several different promoter-reporter gene fusions. Additionally, EMSAs demonstrated that extracts derived from the TPO1 deletion strain was unable to shift the repressing cis-element while protein extracts from the SLK19 deletion strain had a reduced shift of the activating cis-element. Furthermore, these two mutants showed quantitative differences in sterols and antifungal drug susceptibilities consistent with their role in regulating ERG9 expression.