The TeloDIAG: how telomeric parameters can help in glioma rapid diagnosis and liquid biopsy approaches.

BACKGROUND: In glioma, TERT promoter mutation and loss of ATRX (ATRX loss) are associated with reactivation of telomerase or alternative lengthening of telomeres (ALT), respectively, i.e. the two telomere maintenance mechanisms (TMM). Strangely, 25% of gliomas have been reported to display neither or both of these alterations. MATERIALS AND METHODS: The C-circle (CC) assay was adapted to tumor (formalin-fixed paraffin-embedded and frozen) and blood samples to investigate the TMM. RESULTS: We constructed a CC-based algorithm able to identify the TMM and reported a sensitivity of 100% and a specificity of 97.3% (n = 284 gliomas). By combining the TMM, the mutational status of the isocitrate dehydrogenase 1/2 (IDH) gene (IDHmt), and the histological grading, we propose a new classification tool: TeloDIAG. This classification defined five subtypes: tOD, tLGA, tGBM_IDHmt, tGBM, and tAIV, corresponding to oligodendroglioma, IDHmt low-grade astrocytoma, IDHmt glioblastoma, and IDHwt glioblastoma (GBM), respectively; the last class gathers ALT+ IDHwt gliomas that tend to be related to longer survival (21.2 months) than tGBM (16.5 months). The TeloDIAG was 99% concordant with the World Health Organization classification (n = 312), and further modified the classification of 55 of 144 (38%) gliomas with atypical molecular characteristics. As an example, 14 of 69 (20%) of TERTwt, ATRXwt, and IDHwt GBM were actually tAIV. Outstandingly, CC in blood sampled from IDHmt astrocytoma patients was detected with a sensitivity of 56% and a specificity of 97% (n = 206 gliomas and 30 healthy donors). CONCLUSION: The TeloDIAG is a new, simple, and effective tool helping in glioma diagnosis and a promising option for liquid biopsy.

Mass or total surface area with aerosol size distribution as exposure metrics for inflammatory, cytotoxic and oxidative lung responses in rats exposed to titanium dioxide nanoparticles.

There is currently no consensus on the best exposure metric(s) for expressing nanoparticle (NP) dose. Although surface area has been extensively studied for inflammatory responses, it has not been as thoroughly validated for cytotoxicity or oxidative stress effects. Since inhaled NPs deposit and interact with lung cells based on agglomerate size, we hypothesize that mass concentration combined with aerosol size distribution is suitable for NP risk assessment. The objective of this study was to evaluate different exposure metrics for inhaled 5 nm titanium dioxide aerosols composed of small (SA < 100 nm) or large (LA > 100 nm) agglomerates at 2, 7, and 20 mg/m3 on rat lung inflammatory, cytotoxicity, and oxidative stress responses. We found a significant positive correlation ( r = 0.98, p < 0.01) with the inflammatory reaction, measured by the number of neutrophils and the mass concentration when considering all six (SA + LA) aerosols. This correlation was similar ( r = 0.87) for total surface area. Regarding cytotoxicity and oxidative stress responses, measured by lactate dehydrogenase and 8-isoprostane, respectively, and mass or total surface area as an exposure metric, we observed significant positive correlations only with SA aerosols for both the mass concentration and size distribution ( r > 0.91, p < 0.01), as well as for the total surface area ( r > 0.97, p < 0.01). These data show that mass or total surface area concentrations alone are insufficient to adequately predict oxidant and cytotoxic pulmonary effects. Overall, our study indicates that considering NP size distribution along with mass or total surface area concentrations contributes to a more mechanistic discrimination of pulmonary responses to NP exposure.

Impact of emerging pollutants on pulmonary inflammation in asthmatic rats: ethanol vapors and agglomerated TiO2 nanoparticles.

CONTEXT: Titanium dioxide nanoparticles (nano-TiO(2)) and ethanol vapors are air contaminants with increasing importance. The presence of a pathological pulmonary condition, such as asthma, may increase lung susceptibility to such contaminants. OBJECTIVE: This study aimed to investigate if exposure to inhaled ethanol vapors or nano-TiO(2) can modulate the rat pulmonary inflammatory response resulting from an allergic asthmatic reaction. MATERIALS AND METHODS: Brown Norway rats were sensitized (sc) and challenged (15 min inhalation, 14 days later) with chicken egg ovalbumin (OVA). Leukocytes were counted in bronchoalveolar lavages (BAL) performed at 6, 24, 36, 48 and 72 h following the challenge and either after ethanol exposures (3000 ppm, 6 h/day, daily) or at 48 h (peak inflammation) for nano-TiO(2) exposures (9.35 mg/m(3) aerosol for 6 and 42 h after the OVA challenge). For the nano-TiO(2) exposures, plasma and BAL cytokines were measured and lung histological analyzes were performed. RESULTS: Exposure to ethanol did not significantly affect BAL leukocytes after OVA challenge. Exposure to nano-TiO(2) significantly decreased BAL leukocytes compared to OVA-challenged controls. Plasma and BAL IL-4, IL-6, and INF-γ levels were also decreased in the nano-TiO(2) group. DISCUSSION: While ethanol vapors do not modify the pulmonary inflammation in rats during an asthmatic response, a surprising protective effect for agglomerated nano-TiO(2) was observed. A putative mechanistic basis involving a decrease in the Th2 response caused by OVA is proposed. CONCLUSION: Allergic pulmonary inflammation is not up-regulated by inhalation of the pollutants ethanol and nano-TiO(2). On the contrary, nano-TiO(2) decreases lung inflammation in asthmatic rats.

Alteration of infant attention and activity by polychlorinated biphenyls: unravelling critical windows of susceptibility using physiologically based pharmacokinetic modeling.

Pre- and postnatal exposure to polychlorinated biphenyls (PCBs) can impair behavioural function in animal models at doses within the range at which humans are commonly exposed. Yet, epidemiologic studies conducted in the US and Europe are inconsistent with regard to the developmental effects of lactational exposure to these chemicals. This inconsistency may be due to limitations in the current methodological approaches for assessing postnatal exposure to PCBs. Our study used a physiologically based pharmacokinetic (PBPK) model to simulate blood PCB levels during specific pre- and postnatal periods and to evaluate the relation of those levels to infant behaviour. A previously validated PBPK model was used to simulate infant blood PCB-153 levels at delivery and on a month-by-month basis during the first year of life for Inuit infants enrolled in a longitudinal birth cohort. Infant behaviour was assessed using the Behaviour Rating Scales (BRS) of the Bayley Scales of Infant Development (BSID-II) at 11 months of age and video coding of inattention and activity measured during the administration of the mental development subscale of the BSID-II. The estimated pre- and postnatal PCB exposure measures predicted significant increases in inattention and activity at 11 months. Whereas inattention was related to prenatal exposure, activity level, measured by non-elicited activity, was best predicted by postnatal exposure, with the strongest association obtained for simulated PCB levels during the 4th month of life. These findings are consistent with previous reports indicating PCB-induced behavioural alteration in attention and activity level. Simulated infant toxicokinetic profiles for the first year of life revealed windows of susceptibility during which PCBs may impair infant attention and activity.

Toxicokinetics of p-tert-octylphenol in male and female Sprague-Dawley rats after intravenous, oral, or subcutaneous exposures.

This study was undertaken to characterize the toxicokinetics of p-tert-octylphenol (OP), a weak estrogenic compound, in male and female rats. Male and female Sprague-Dawley rats were given a single dose of OP either by oral gavage (50, 125 or 250 mg/kg), by intravenous (iv) injection (2, 4, or 8 mg/kg), or by subcutaneous (sc) injection (125 mg/kg). In a repeated dosing experiment, rats were given OP (oral) daily (25, 50, or 125 mg/kg) for 35 d (female) or 60 d (male). Blood and tissue samples were collected and analyzed for OP content using gas chromatography with detection by mass spectrometry. Blood OP concentrations were generally higher in female than male rats following a single oral or sc administration but were similar following a single iv injection. Tissue OP concentrations were also higher in female than male rats following oral exposure, consistent with the faster metabolism of OP observed in male rat liver microsomes. After subchronic administration, blood OP concentrations were higher at the end of exposure for female (33 d) (2.26-fold, not significant) and male (57 d) (3.47-fold) rats than single dosing but there was no change in the tissue OP concentrations. Gender differences in tissue OP concentrations may contribute, in part, to gender differences in the toxicity of OP in rats. The fact that OP was found in all reproductive tissues confirms its potential for direct endocrine-like effects.

Influence of ALDH2 polymorphism on ethanol kinetics and pulmonary effects in male and female rats exposed to ethanol vapors.

Ethanol is being added in various proportions to fuel in order to reduce greenhouse gas emissions. This is likely to result in involuntary exposure to ethanol vapors. Whether or not such exposure might cause health effects is still unknown. Acetaldehyde, an important metabolite of ethanol detoxified by aldehyde dehydrogenase (ALDH2) is more toxic that ethanol. This study assessed the impact of genetic ALDH2 polymorphism in male and female Sprague-Dawley rats on ethanol kinetics and pulmonary effects following sub-chronic exposure to ethanol vapors. Homozygote rats ALDH2(Q)/2(Q) (fast ALDH2 activity) and ALDH2(R)/2(R) (ALDH2 deficiency) were exposed to 1000 or 3000 ppm, 6 h/day, 5 days/week for 13 weeks. Blood ethanol concentrations (BEC) were measured at various post-exposure times. Cellularity in bronchoalveolar lavages (BAL) and lung histological evaluation were performed at week 13. Results showed that BEC in males were systematically lower than in females, e.g. BEC in ALDH2(Q)/2(Q) males (2 min, 1,000 ppm, day 1) was significantly (p < 0.05) lower (66.8 +/- 10.7 microM) compared to females (87.6 +/- 15.3 microM). BEC for ALDH2(Q)/2(Q) rats were different from ALDH2(R)/2(R) only for males exposed for more than 64 days. Repeated exposures resulted in a significant decrease of BEC, e.g. for ALDH2(Q)/2(Q) males (3,000 ppm) BEC on day 1 and day 85 were 324.6 +/- 102.6 microM and 187.5 +/- 32.1 microM, respectively. BAL and histological evaluation revealed no pulmonary toxicity for all groups. Overall, results showed that 3,000 ppm of ethanol vapors represents no observed adverse effect level (NOAEL) for pulmonary toxicity in the rat.

Determination of p-tert-octylphenol in blood and tissues by gas chromatography coupled with mass spectrometry.

A sensitive and reproducible procedure using gas chromatography coupled with mass spectrometry is described for the determination of p-tert-octylphenol (OP), a persistent degradation product of alkylphenol ethoxylates that binds to the estrogen receptor in blood and tissues. The first step involved the extraction of blood (200 microL) or tissue homogenate (400 microL) with methyl tert-butyl ether, including p-tert-butylphenol (BP) as internal standard. After extraction, the sample was evaporated to dryness with a gentle stream of nitrogen at 45 degrees C, and OP and BP were derivatized with an acetylation reaction involving acetic anhydride and catalyzed by pyridine. Samples were then analyzed by a gas chromatograph equipped with a mass spectrometer (single ion monitoring) with a Varian VF-5ms capillary column. The limit of detection and the limit of quantification of the method in blood were 4.6 and 15.5 ng/mL, respectively. The linearity and reproducibility of the method were acceptable, with coefficients of variation of approximately 10% for blood and ranging between 9% and 27% for tissues. This method was applied to the determination of unchanged OP in blood and tissues obtained from Sprague-Dawley rats after oral and IV OP administration.

Effects of postnatal exposure to mixtures of non-ortho-PCBs, PCDDs, and PCDFs in prepubertal female rats.

There are concerns that postnatal exposure to organochlorines present in breast milk could lead to adverse health effects. We reconstituted four mixtures of aryl-hydrocarbon receptor (AhR) agonists (3 non-ortho polychlorinated biphenyls [PCBs], 6 polychlorinated dibenzodioxins [PCDDs], 7 polychlorinated dibenzofurans [PCDFs], or all 16 chemicals together [referred to as AhRM]) based on their concentrations in breast milk, and examined their effects following exposure by gavage from day 1 until day 20 of age. Female neonates received dosages of AhRM equivalent to 1, 10, 100, or 1000 times the amount consumed by an infant over the first 24 days of life. Other groups received the PCBs, the PCDDs, or the PCDFs at the 1000x level. All rats were sacrificed at 21 days of age. Changes in ethoxyresorufin-o-deethylase hepatic activity, thymus and body weights, and serum thyroxin were linked to the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalents (TEQ) of the four mixtures (1000x-AhRM > PCDDs > PCBs > PCDFs). To test for AhRM antiestrogenicity, two additional groups received 1.5 microg/kg of 17alpha-ethynyl estradiol (EE) with or without the 1000x-AhRM. The AhRM had no effect on uterine weight or EE-stimulated uterine growth. The actions of the combined EE and AhRM treatments suggest additive effects in decreasing pentoxyresorufin-o-deethylase activity and spleen weight, but nonadditive/antagonistic effects on adrenal weight and serum thyroxin. In conclusion, (1) 10x-AhRM had no detectable effects, (2) TEQ values relate to observed toxicities, even when testing complex mixtures of AhR agonists, and (3) indications of tissue-specific additive and nonadditive/antagonistic effects, but no synergism, were observed when doses of AhRM were increased, or combined with EE.

Cdc13 prevents telomere uncapping and Rad50-dependent homologous recombination.

Cdc13 performs an essential function in telomere end protection in budding yeast. Here, we analyze the consequences on telomere dynamics of cdc13-induced telomeric DNA damage in proliferating cells. Checkpoint-deficient cdc13-1 cells accumulated DNA damage and eventually senesced. However, these telomerase-proficient cells could survive by using homologous recombination but, contrary to telomerase-deficient cells, did so without prior telomere shortening. Strikingly, homologous recombination in cdc13-1 mec3, as well as in telomerase-deficient cdc13-1 cells, which were Rad52- and Rad50-dependent but Rad51-independent, exclusively amplified the TG(1-3) repeats. This argues that not only short telomeres are substrates for type II recombination. The Cdc13-1 mutant protein harbored a defect in its association with Stn1 and Ten1 but also an additional, unknown, defect that could not be cured by expressing a Cdc13-1- Ten1-Stn1 fusion. We propose that Cdc13 prevents telomere uncapping and inhibits recombination between telomeric sequences through a pathway distinct from and complementary to that used by telomerase.

Hsp90 levels affect telomere length in yeast.

Cdc13 is a Saccharomyces cerevisiae protein that binds to telomeric single-stranded DNA and regulates telomerase activity. Stnl has been shown by two-hybrid analysis to form a physical complex with Cdc13. Temperature-sensitive mutations in CDC13 and STN1, which are both essential genes, activate a DNA damage-dependent checkpoint which is the cause of the arrest seen in the mutant strains. The stn1-13 mutation induces dramatic telomere elongation which is telomerase dependent, as shown here. Additional mutants for STN1, which show a tighter arrest phenotype than stn1-13, were generated in order to perform genetic screens aiming at uncovering new regulators of telomerase. HSC82, which encodes a conserved molecular chaperone of the Hsp90 family, was thus isolated as a high-dosage suppressor of a temperature-sensitive mutation in STN1. Overexpression of HSC82 also partially suppressed the growth defect of cdc13-1 cells. Overexpression of HSC82 was found to correct the telomeric defect associated with stn1 mutations. Shortening of telomeres was also observed in wild-type cells upon overexpression of HSC82, or of its temperature-inducible homologue, HSP82. These results identify Hsc82/Hsp82 as potential regulators of telomerase in yeast cells.

Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13.

In Saccharomyces cerevisiae, Cdc13 has been proposed to mediate telomerase recruitment at telomere ends. Stn1, which associates with Cdc13 by the two-hybrid interaction, has been implicated in telomere maintenance. Ten1, a previously uncharacterized protein, was found to associate physically with both Stn1 and Cdc13. A binding defect between Stn1-13 and Ten1 was responsible for the long telomere phenotype of stn1-13 mutant cells. Moreover, rescue of the cdc13-1 mutation by STN1 was much improved when TEN1 was simultaneously overexpressed. Several ten1 mutations were found to confer telomerase-dependent telomere lengthening. Other, temperature-sensitive, mutants of TEN1 arrested at G(2)/M via activation of the Rad9-dependent DNA damage checkpoint. These ten1 mutant cells were found to accumulate single-stranded DNA in telomeric regions of the chromosomes. We propose that Ten1 is required to regulate telomere length, as well as to prevent lethal damage to telomeric DNA.

Cdc13 cooperates with the yeast Ku proteins and Stn1 to regulate telomerase recruitment.

The Saccharomyces cerevisiae CDC13 protein binds single-strand telomeric DNA. Here we report the isolation of new mutant alleles of CDC13 that confer either abnormal telomere lengthening or telomere shortening. This deregulation not only depended on telomerase (Est2/TLC1) and Est1, a direct regulator of telomerase, but also on the yeast Ku proteins, yKu70/Hdf1 and yKu80/Hdf2, that have been previously implicated in DNA repair and telomere maintenance. Expression of a Cdc13-yKu70 fusion protein resulted in telomere elongation, similar to that produced by a Cdc13-Est1 fusion, thus suggesting that yKu70 might promote Cdc13-mediated telomerase recruitment. We also demonstrate that Stn1 is an inhibitor of telomerase recruitment by Cdc13, based both on STN1 overexpression and Cdc13-Stn1 fusion experiments. We propose that accurate regulation of telomerase recruitment by Cdc13 results from a coordinated balance between positive control by yKu70 and negative control by Stn1. Our results represent the first evidence of a direct control of the telomerase-loading function of Cdc13 by a double-strand telomeric DNA-binding complex.

Unsuspected intrathyroidal parathyroid adenoma: mimic of lymphocytic thyroiditis in fine-needle aspiration specimens-a case report.

Fine-needle aspirations (FNAs) of parathyroid adenomas (PA) are infrequently encountered, but the scant literature on this topic emphasizes the difficulties in distinguishing them from thyroid neoplasms. We report on a case of an unsuspected intrathyroidal PA whose two FNA specimens mimicked almost perfectly the features of lymphocytic thyroiditis (LT). The smears from two FNAs of a "thyroid nodule" in a 22-yr-old woman were received with a clinical diagnosis of "LT." The cytological features of both specimens were similar and consisted of groups of epithelial cells in a background of numerous "naked" nuclei, interpreted as Hurthle cells and lymphocytes respectively, and leading to a cytological diagnosis of LT. Subsequent surgical excision of the "nodule" revealed a large intrathyroidal PA. The oxyphil cells and chief cells (the latter devoid of cytoplasm) present in the PA resembled Hurthle cells and lymphocytes respectively, in the FNA specimens. In conclusion, PA can give a cytological picture almost identical to that of LT in FNA material. Important clues to the diagnosis of PA in FNA specimens include the presence of prominent capillaries and the knowledge of a clinical history of hyperparathyroidism. Diagn. Cytopathol. 1999;21:276-279.

Maximal exercise capacity and peripheral skeletal muscle function following lung transplantation.

BACKGROUND: There have been many suggestions that diminished exercise capacity in patients that have undergone lung transplantation is due, in part, to peripheral muscle dysfunction, brought on by either detraining or immunosuppressive therapy. There is limited data quantifying skeletal muscle function in this population, especially in those more than 18 months post-procedure. The present study sought to quantitate skeletal muscle function and cardiopulmonary responses to graded exercise in 19 lung transplant recipients, 15 of which were mostly more than 18 months post-procedure. METHODS: Ten single- (SLT) and 9 double-lung transplantation (DLT) underwent anthropometric measures and performed expiratory spirometry, whole body plethysmography to assess lung volumes, static maximal mouth pressures to assess respiratory muscle strength, progressive exercise testing on a cycle ergometer (with cardiac output measurements being performed every second workload) and isokinetic cycling to assess peripheral muscle power and work capacity. RESULTS: The DLT group was younger than the SLT group (23.0 [21.0-32.0] vs 47.5 [43.0-55.0] median [interquartile range], p < .05) with no differences in height, weight, or BMI. Despite the DLT group having significantly better spirometric values (FEV1: 86% vs 56.5% median) and less airtrapping (RV/TLC: 30% vs 53.5%), both groups were equally limited in exercise capacity (Wmax)(38.0 percent predicted [30.0-65.0] vs 37.5 percent predicted [30.0-44.0], SLT vs DLT), leg power (76.1 percent predicted [53.8-81.4] vs 69.0 percent predicted [58.3-76.0]) and leg work capacity (63.3 percent predicted [34.7-66.8] vs 38.4 percent predicted [27.5-57.3]). This lack of difference in performance persisted when the analysis was limited to those more than 18 months post-procedure. Respiratory muscle strength was also not different for the two groups, and was within normal limits. Wmax was best correlated with leg work capacity (r = .84), but also with leg power, RV/TLC, FEV1 (r = .49, -.52, .58). When normalized for age, height, and sex, percent predicted Wmax only correlated with percent predicted leg work capacity (r = .58). Cardiac output was appropriate for the work performed. CONCLUSIONS: We conclude that peripheral skeletal muscle work capacity is reduced following lung transplantation and mostly responsible for the limitation of exercise performance. While the causes of muscular dysfunction have yet to be clarified, the preservation of respiratory muscle strength with the concomitant reduction in leg power and work capacity suggests that most of the muscular dysfunction post-transplantation is attributable to detraining.

Dbf2 is implicated in a Cbt1-dependent pathway following a shift from glucose to galactose or non-fermentable carbon sources in Saccharomyces cerevisiae.

Dbf2, a cell cycle-regulated protein kinase, has been shown recently to be part of the CCR4 transcriptional regulatory complex in the yeast Saccharomyces cerevisiae. We report here that temperature-sensitive (ts) dhf2-2 mutant cells can be rescued by overexpression of CDC14, which encodes a dual-specificity protein phosphatase, when grown on glucose-containing medium, as reported previously, but not on galactose. Screening of two S. cerevisiae cDNA libraries led to the identification of CBT1 as a gene which, if overexpressed simultaneously with CDC14, results in the rescue of the dbJ2-2 mutation at restrictive temperature on galactose-based medium, as well as on media containing non-fermentable carbon sources such as glycerol, lactate and acetate. Cbt1 has been shown previously to be essential for formation of cytochrome b in the mitochondria. On the other hand, the ts defects of ccr4delta and caf1delta mutants on glycerol medium at 37 degrees C (Ccr4 and Caf1/Pop2 are two other members of the CCR4 complex) could not be complemented by simultaneous overexpression of CBT1 and CDC14. CCR4 and CAF1 have been shown to play an essential role in activating the expression of genes for non-fermentative growth. Our results strongly suggest that, within the CCR4 complex, Dbf2 is directly involved in some mitochondrial function that depends on cytochrome b or on one of its main regulators, Cbt1. Therefore, Dbf2 may be required not only during mitosis but also during growth on non-fermentable carbon sources.

The Saccharomyces cerevisiae Cdc14 phosphatase is implicated in the structural organization of the nucleolus.

Cdc14, a dual-specificity protein phosphatase, has been previously implicated in triggering exit from mitosis in the yeast Saccharomyces cerevisiae. Using immunofluorescence microscopy and immunogold labeling, we demonstrate that a functional HA-tagged version of the phosphatase Cdc14 localizes to the nucleolus. Moreover, Cdc14-HA co-localized with the nucleolar NOP2 and GAR1 proteins. By immunofluorescence, Cdc14-HA was found in the nucleolus during most of the mitotic cell cycle, except during anaphase-telophase when it redistributed along the mitotic spindle. While this work was in progress, the same pattern of Cdc14 localization was described by others (Visintin et al, Nature 398 (1999) 818). Constitutive overexpression of CDC14 was toxic and led to cell cycle arrest of cells, mainly in G1. This correlated with the appearance of abnormal nuclear structures. A genetic search for suppressors of the lethality associated with CDC14 overexpression identified YJL076W. Because overproduction of Yj1076w buffered the toxic effect of Cdc14 overproduction, this suggested that it might be a substrate of Cdc14. This has indeed been found to be the case by others who recently described Yj1076w/Netl as a nucleolar protein that physically associates with Cdc14 (Shou et al, Cell 97 (1999) 233). The present data confirm several recently uncovered aspects of the regulation of Cdc14 localization and activity and suggest that the level of expression of CDC14 influences the structural organization of the nucleolus.

The Cdc14 phosphatase is functionally associated with the Dbf2 protein kinase in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Cdc14 protein phosphatase and Dbf2 protein kinase have been implicated to act during late M phase, but their functions are not known. We report here that CDC14 is a low-copy suppressor of the dbf2-2 mutation at 37 degrees C. The kinase activity of Dbf2 accumulated at a high level, in vivo, during a cdc14 arrest and was also much higher in cdc14 mutant cells at the permissive temperature of growth, therefore in cycling mutant cells than in cycling wild-type cells. This correlated with the accumulation of the more slowly migrating form of Dbf2, previously shown to correspond to the hyperphosphorylated form of the protein. The finding that the dbf2-2 mutation could be rescued following overproduction of catalytically inactive forms of Cdc14 suggested that the control of Dbf2 activity by Cdc14 might be only indirect and independent of Cdc14 phosphatase activity. However, it was found that Cdc14 could form oligomers within the cell, thus leaving open the possibility that catalytically inactive Cdc14 might associate with wild-type Cdc14 and rescue dbf2-2 in a phosphatase-dependent manner. We confirmed that overexpression of CDC14 could rescue mutations in CDC15, which encodes another kinase also implicated to act in late M phase. Cells of a cdc15-2 dbf2-2 double mutant died at temperatures much lower than did either single mutant, whereas there was only a slight additive phenotype in the cdc14-1 dbf2-2 and cdc14-1 cdc15-2 double mutant cells. Finally, functional association between Cdc14 and Dbf2 (and also Cdc15) was confirmed by the finding that the cdc14, dbf2 and cdc15 mutations could be partially rescued by the addition of 1.2 M sorbitol to the culture medium. Our data are the first to demonstrate a functional link between Cdc14 and Dbf2 based on both biochemical and genetic information.

Inhaled nitric oxide in acute respiratory distress syndrome: a pilot randomized controlled study.

This pilot randomized controlled clinical trial of patients with ARDS was implemented to study the impact of inhaled nitric oxide (inhNO) on lung function, morbidity, and mortality. Thirty patients with ARDS were randomly allocated to usual care or usual care plus inhNO. The optimal dose of inhNO was determined to be between 0.5 and 40 parts-per-million daily. All therapeutic interventions were standardized. ARDS resulted mainly from sepsis (25 of the 30). During the first 24 h, the hypoxia score increased greatly in patients treated with inhNO +70.4 mm Hg (+59%) versus +14.2 mm Hg (+9.3%) for the control group (p = 0.02), venous admixture decreased from 25.7 to 15.2% in the inhNO group, and from only 19.4 to 14.9% in the control group (p = 0.05). After the first day of therapy no further beneficial effect of inhNO was detected. Forty percent of the patients treated with inhNO were alive and weaned from mechanical ventilation within 30 d after randomization compared with 33.3% in the control group (p = 0.83). The 30-d mortality rate was similar in the two groups; most deaths (11 of 17) were due to multiple organ dysfunction syndrome. This study shows that inhNO, in this population, may improve gas exchange but does not affect mortality.

Atypical squamous cells of undetermined significance. A cytohistologic study of 52 cases.

OBJECTIVE: To evaluate the significance of atypical squamous cells of undetermined significance (ASCUS) by correlating the histologic findings following a diagnosis of ASCUS on a cervical cytologic smear. STUDY DESIGN: Eighty-four smears that had been called ASCUS over a five-month period and that had corresponding histologic material were reviewed independently. Only 52 of the 84 cases on which a consensus was reached were retained for the current study. RESULTS: The breakdown of the follow-up histologic diagnoses was as follows: 28 cases (54%) were negative (without squamous intraepithelial lesions [SIL]); 22 cases (42%) showed SILs, of which 14 (27%) were low grade, 5 (10%) were high grade and 3 (5%) had SILs that could not be further classified because of fragmentation of the endocervical curettings. Finally, two cases (4%) proved to be invasive cervical carcinoma on histology despite smears that were satisfactory and not limited by the quantity or quality of material; in these the discrepancy was attributed to sampling error. CONCLUSION: Patients whose cervical cytologic smears fall into the category of ASCUS may, on follow-up, exhibit a wide spectrum of findings, ranging from no pathologic abnormality to frequent SIL and even to invasive carcinoma in rare instances. A diagnosis of ASCUS on smears warrants careful follow-up and investigation.