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.

Intracellular free calcium oscillates during cell division of Xenopus embryos.

In Xenopus embryos, previous results failed to detect changes in the activity of free calcium ions (Ca2+i) during cell division using Ca2(+)-selective microelectrodes, while experiments with aequorin yielded uncertain results complicated by the variation during cell division of the aequorin concentration to cell volume ratio. We now report, using Ca2(+)-selective microelectrodes, that cell division in Xenopus embryos is accompanied by periodic oscillations of the Ca2+i level, which occur with a periodicity of 30 min, equal to that of the cell cycle. These Ca2+i oscillations were detected in 24 out of 35 experiments, and had a mean amplitude of 70 nM, around a basal Ca2+i level of 0.40 microM. Ca2+i oscillations did not take place in the absence of cell division, either in artificially activated eggs or in cleavage-blocked embryos. Therefore, Ca2+i oscillations do not represent, unlike intracellular pH oscillations (Grandin, N., and M. Charbonneau. J. Cell Biol. 111:523-532. 1990), a component of the basic cell cycle ("cytoplasmic clock" or "master oscillator"), but appear to be more likely related to some events of mitosis.

Cycling of intracellular pH during cell division of Xenopus embryos is a cytoplasmic activity depending on protein synthesis and phosphorylation.

In Xenopus embryos, the successive and rapid cell divisions that follow fertilization are accompanied by periodic oscillations of intracellular pH (pHi). Cycling of pHi occurs in phase with several other oscillatory activities, namely nuclear divisions, M phase-promoting factor (MPF) activity, and surface contraction waves (SCWs). We report that treatments that abolish cycling of MPF activity and the SCWs also suppress the pHi oscillations, whereas those that block cell division without affecting neither MPF activity nor the SCWs do not suppress the pHi oscillations. Experiments on enucleated oocytes, matured in vitro and activated, demonstrated that the activity governing the rhythmicity of the pHi oscillations resided in the cytoplasm of the oocyte. In this respect, the activity responsible for the pHi oscillations was different from that which drives the SCWs, which necessitated the presence of the oocyte germinal vesicle (Ohsumi et al., 1986), but more closely resembled MPF activity that did not require the presence of the oocyte germinal vesicle (Dabauvalle et al., 1988). In mature eggs enucleated at the time of egg activation, the pHi oscillations were similar to those in control nucleated eggs, whereas the period between two peaks of SCWs was 35-60 min vs. 20-35 min in nucleated control eggs. Previous studies had shown that the periodicity of SCWs was larger in anucleate egg fragments than in their nucleate counterparts (Sakai and Kubota, 1981), the difference being on the order of 6-15 min (Shinagawa, 1983). However, in these previous studies, enucleation was performed 30-50 min after fertilization. Our results clearly demonstrate that the periodicity of the SCWs is lengthened when the interval between egg activation and enucleation is shortened, thereby providing an easier way to assess the nuclear dependency of the SCWs. Finally, the various possibilities concerning the role of pHi cycling during cell division are discussed.

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.

Two new inherited defects of the thyroxine-binding globulin (TBG) molecule presenting as partial TBG deficiency.

Serum-denatured TBG (dnTBG) measured in 32 families deficient in native TBG (nTBG) was undetectable in all subjects with complete nTBG deficiency and was high in 2 of 16 families with partial nTBG deficiency. nTBG (in mean micrograms per decaliter +/- SD) in members of the Quebec and Montreal families, respectively were: 258 +/- 54 and 230 in affected men, 747 +/- 190 and 927 +/- 90 in affected women, and 1568 +/- 151 and 1300 +/- 195 in unaffected relatives. Corresponding mean dnTBG levels were: 14.3 +/- 2.9 and 21.3 in affected men, 8.6 +/- 1.0 and 11.6 +/- 3.1 in affected women, and less than 2.1 and less than 2.6 in unaffected relatives. All were euthyroid with normal free thyroxine and thyrotropin levels. In comparison to common type TBG, TBG-Quebec was more heat labile by 10 degrees C and TBG-Montreal by 12 degrees C. The degree of dnTBG elevation and nTBG lability at 37 degrees C were correlated (r = 0.99). Isoelectric focusing showed cathodal shift of all TBG bands: TBG-Quebec by 0.06 isoelectric points (pI) and TBG-Montreal by 0.02 pI. These two TBG variants represent different mutations most likely affecting the polypeptide chain of the molecule. Their inheritance is X-chromosome linked. The instability of these TBGs at 37 degrees C may lead to more rapid degradation in vivo resulting in low nTBG and high dnTBG concentrations in serum.

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.

Changes in intracellular free calcium activity in Xenopus eggs following imposed intracellular pH changes using weak acids and weak bases.

The aim of this work was to determine the potential relationships between rises in intracellular pH (pHi) and intracellular free calcium activity (Cai2+) during cell activation in Xenopus eggs. To do this, we used two weak bases, NH4Cl and procaine, and a weak acid, CO2, and measured Cai2+ variations in response to these imposed pHi variations. NH4Cl and procaine increased Cai2+ in both unactivated and activated eggs. Procaine was found to alkalinize the egg cytoplasm, whereas the other weak base, NH4Cl, acidified the egg cytoplasm. On the other hand, CO2 was found to acidify the cytoplasm and to substantially decrease Cai2+, also in unactivated and activated eggs. In addition, CO2 triggered an increase in the conductance of the plasma membrane to Cl- ions, similarly to what had been found previously with weak bases (Charbonneau, M. (1989) Cell Differ. Develop. 26, 39-52). These Cl- channels, similarly to the sperm-triggered Cl- channels during the fertilization potential, are supposed to be Ca2(+)-sensitive. Therefore, the changes in Ca2+ observed in response to CO2 do not seem to be responsible for the opening of these Cl- channels, which would rather be triggered by an increase in Cai2+ localized near the plasma membrane. We conclude therefore that weak acids and bases represent appropriate tools for studying cytosolic Ca2+ homeostasis, but not for dissecting the complex pathways involved in signal transduction.

Replacement of Leu227 by Pro in thyroxine-binding globulin (TBG) is associated with complete TBG deficiency in three of eight families with this inherited defect.

The T4-binding globulin (TBG) gene is a single copy located on the X-chromosome. Previous studies have failed to elucidate the molecular defect in individuals with complete TBG deficiency (TBG-CD). Indeed, no major deletions, insertions, or other rearrangements were observed in the TBG gene of six unrelated males with this defect. To clarify the molecular basis of TBG-CD, we have cloned and sequenced the TBG gene of an affected male (CD5) of French Canadian origin. The sequence of the exons encoding the mature protein, adjacent introns, and the promoter region revealed two nucleotide substitutions: CTA(Leu)----CCA(Pro) at codon 227 and TTG(Leu)----TTT(Phe) at codon 283. The Leu----Phe substitution, a relatively conservative replacement, is a TBG polymorphism present in 16% (3 of 19) of French Canadian males. It has no effect on the serum concentration or properties of the common type TBG (TBG-C). The new Leu----Pro substitution, which is predicted to alter the higher order of TBG structure, is probably responsible for the TBG-CD phenotype of the individual studied and two other families with TBG-CD. It possibly impairs TBG biosynthesis or secretion or perhaps alters TBG structure to such a degree that the molecule is not recognized by antibodies against native or denatured TBG and does not bind T4.

Detection of the thyroxine-binding globulin (TBG) gene in six unrelated families with complete TBG deficiency.

T4-binding globulin (TBG) is a glycoprotein of hepatic origin which transports thyroid hormone in serum. Inherited TBG defects in man are X-chromosome linked and are expressed in hemizygotes as complete deficiency, partial deficiency, or excess. Since TBG is not necessary for thyroid hormone action, affected subjects are healthy. Using DNA probes for human TBG, we searched for restriction fragment length polymorphisms in six affected males belonging to 6 unrelated families with inherited complete TBG deficiency and an equal number of normal males. TBG could not be detected in the serum of any of the TBG-deficient males by a specific and sensitive RIA capable of detecting as little as 5 micrograms TBG/L or 0.031% of the average normal serum TBG concentration. DNA isolated from white blood cells was digested with 11 restriction endonucleases, and the digests were submitted to DNA blot analysis using two cloned TBG-DNA probes which together covered the entire protein coding and the 5'-flanking sequences of the TBG gene. A total of 26 different bands were detected on DNA blots, identifying 18 restriction sites located within the 4.2-kilobase TBG gene, which includes intronic, exonic, and 5'-flanking sequences. This analysis, which sampled 2.3% of the total TBG genome, failed to reveal differences in fragment size among the 6 TBG-deficient and 6 normal males examined. One restriction endonuclease (NcoI) identified normal sequences at the putative promoter region of the gene, and four other endonucleases (TaqI, SstII, MspI, and HpaII) recognized the cytosine-guanine dinucleotide phosphate sequences representing potential mutation hot spots. Although C was methylated at these sites, no C to T (thymidine) transitions were found. These data suggest that large deletions, insertions, or rearrangements of the TBG gene, or mutations at sites of methylated cytosine-guanine dinucleotide phosphate dimers are not common mechanisms for inherited complete TBG deficiency in man.

Effect of estradiol on the induction of porphyria by hexachlorobenzene in the rat.

Hexachlorobenzene (HCB) is porphyrinogenic in adult female but not in male rats. This study aimed to assess the role of 17beta-estradiol in the induction of porphyria by HCB in both sexes by adding or removing the hormone. Groups of intact females, ovariectomized females (Ova), castrated males (Cas), and Cas receiving 17beta-estradiol (4 mg/kg, i.m., once a week beginning 2 weeks prior to HCB) were given five consecutive daily doses of HCB (100 mg/kg in corn oil, p.o.). Porphyria was assessed by urinary uroporphyrin excretion measured at days 16, 31, 38, 45, 52, 59, and 87. The percentage of porphyric rats in intact females increased from day 31 (58%) to day 87 (75%), whereas none of the Ova or Cas rats responded. However, administration of estradiol (days 120-169) and another sequence of HCB doses (days 134-138) to the same Ova rats caused porphyria (50% at day 186). Cas rats given estradiol also developed porphyria (43 and 86% on days 31 and 87, respectively). HCB-treated Ova rats given two doses of estradiol at either days 1 and 8 or days 22 and 29 developed a porphyria of similar magnitude (day 52). The role of estradiol cannot be explained by a reduction of pentachlorothiophenol formation, a putative detoxication pathway. Overall, results show that both sexes have the ability to respond to HCB when 17beta-estradiol is present and suggest that the sexual dimorphism in HCB-induced porphyria in the rat is related to the hormonal status.

High- to low-dose extrapolation: critical determinants involved in the dose response of carcinogenic substances.

Recent investigations on mechanism of carcinogenesis have demonstrated important quantitative relationships between the induction of neoplasia, the molecular dose of promutagenic DNA adducts and their efficiency for causing base-pair mismatch, and the extent of cell proliferation in target organ. These factors are involved in the multistage process of carcinogenesis, including initiation, promotion, and progression. The molecular dose of DNA adducts can exhibit supralinear, linear, or sublinear relationships to external dose due to differences in absorption, biotransformation, and DNA repair at high versus low doses. In contrast, increased cell proliferation is a common phenomena that is associated with exposures to relatively high doses of toxic chemicals. As such, it enhances the carcinogenic response at high doses, but has little effect at low doses. Since data on cell proliferation can be obtained for any exposure scenario and molecular dosimetry studies are beginning to emerge on selected chemical carcinogens, methods are needed so that these critical factors can be utilized in extrapolation from high to low doses and across species. The use of such information may provide a scientific basis for quantitative risk assessment.

Changes in obstructive sleep apnea characteristics through the night.

It was our impression that the respiratory parameters in obstructive sleep apnea (OSA) worsened as the night progressed. To confirm this, we review polysomnographic studies from 66 patients with apnea-hypopnea indices (AHI) of 40 to 125 events per hour, dividing bed time into equal quartiles. As the night progressed, the mean apnea duration (MAD) increased from 27.2 s to 34.6 s (p < 0.0001), mainly from increases during NREM sleep. The proportion of time spent in apnea increased from 54 to 71% (p < 0.0001) due to increases in both MAD and the proportion of REM sleep (from 2.8 to 14.7% of the total sleep time). The AHI did not change significantly between quartiles. Even though preapneic oxygen saturation did not change and apnea duration increased as the night progressed, the end-apneic saturation did not decrease, hence the rate of oxygen desaturation decreased. Also, it was found that patients with an AHI greater than 65 events per hour increased their proportion of time spent in apnea significantly more than those with an AHI smaller than 65, as the night progressed. In the patients with an AHI greater than 85, this was due to both an increase in MAD and AHI. In conclusion, in patients with an AHI greater than 40 events per hour, the severity of apnea increased as the night progressed due to lengthening of MAD, increased proportion of REM sleep, and in the most severe patients, also an increase in AHI. Even though the exact pathophysiologic mechanisms for the observed changes are unknown, a decrease in respiratory muscle effort with consequent decrease in oxygen consumption may explain both the lengthening of the apneas and the decrease in the rate of oxygen desaturation.

Early Events in Anuran Amphibian Fertilization: An Ultrastructural Study of Changes Occurring in the Course of Monospermic Fertilization and Artificial Activation: (anuran/egg cortex/exocytosis/fertilization/microvilli).

In unfertilized frog eggs, the plasma membrane displays an animal vegetal polarity characterized by the presence of short microvilli in the vegetal hemisphere and long microvilli or ridge-like protrusions in the animal hemisphere. The densities of microvilli are similar in the two hemispheres. The fertilizing sperm always fuses with the animal hemisphere of the egg and induces a wave of exocytosis of cortical granules from its site of penetration. Similar spreading of the cortical reaction is seen on activation by pricking the egg cortex. The integration of the cortical granule membrane with the plasma membrane is rapidly followed by elongation of microvilli, which is progressively realized all over the egg surface from the site of sperm entry or the site of pricking. At this time, the length and shape of the microvilli in the animal and vegetal hemispheres are similar and their densities are the same as in unfertilized eggs. A "smoothing" wave can be seen on the living egg, 40-60 seconds after pricking, starting around the site of pricking. This wave of microvillar elongation is accompanied by changes in intensity of diffracted light spots observed at the surface of the egg. This pattern might result from rapid and progressive thickening of the cortex that would drive pigment granules into the cytoplasm. The Brownian movement of these granules is thought to be responsible for the observed diffracted light spots. Electrical stimulus or the ionophore A23187 induced activation reactions similar to those triggered by the sperm or by pricking, except that the cortical reaction began simultaneously in several distinct sites of the cortex.

Voltage Noise Changes during Monospermic and Polyspermic Fertilization of Mature Eggs of the Anuran, Rana temporaria: (anuran egg/electrical membrane properties/fertilization/polyspermy/voltage noise).

The electrical response of mature anuran eggs to the fertilizing sperm consists of a rapid depolarization and a decrease in resistance of the plasma membrane (fertilization potential) and serves as a fast block to polyspermy. We report here that the fertilization potential, previously thought to be the earliest electrical response of the egg, is preceded in Rana temporaria by changes in voltage noise. Voltage noise was recorded after insemination and compared in monospermic and NaI-induced polyspermic eggs. Fertilization potential in monospermic eggs arised at 1 min 45 sec to 2 min 15 sec after insemination, and that in NaI-induced polyspermic eggs did at 3 min to 3 min 30 sec after insemination. However, the increase in voltage noise was detected at the similar time (1-2 min 30 sec) after insemination in both the eggs. The duration of voltage noise increase before the fertilization potential was larger in polyspermic eggs (50-105 sec) than in monospermic eggs (10-40 sec). Polyspermic fertilization in Rana temporaria induced by NaI was checked by visualizing multiple sperm entry sites with the scanning microscope. The process of sperm entry and the development of the fertilization body are similar to those occurring with monospermic fertilization; furthermore all supernumerary sperm fuse only with the animal hemisphere of the egg. Although the physiological basis of the changes in voltage noise is unclear, these alterations appear to be the earliest electrical response to sperm yet reported.

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.

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.