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.

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.

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.

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.

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.

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.

Assessment of the role of glutathione conjugation in the protection afforded by anethol dithiolthione against hexachloro-1,3-butadiene-induced nephrotoxicity.

Administration of anethol dithiolthione (ADT) to rodents can afford protection against some chemically induced toxicities. The aim of the present study was to assess the effects of ADT on hexachloro-1,3-butadiene (HCBD)-induced nephrotoxicity in the rat and to determine the mechanism of its action. Renal integrity was evaluated by measuring urinary excretion of glucose, protein, and gamma-glutamyl transpeptidase and by histological evaluation. A 3-day pretreatment with ADT (300 mg/kg/day) protected against the toxicity of various doses of HCBD (ranging from 15.6 to 62.5 mg/kg). The pretreatment increased (1.4-fold) the nonprotein sulfhydryl content (NPSH) of the liver. However, it did not modify the biliary excretion of radiolabeled materials in [14C]HCBD- treated (20 mg/kg) rats, nor that of the bioactivated HCBD metabolite, S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-glutathione (PCBG). Moreover, ADT pretreatment protected rats against the nephrotoxicity induced by PCBG (20 mg/kg) itself. The extent of covalent binding to kidney proteins of [14C]HCBD-derived metabolites was not modified by pretreatment with ADT. Incubation of rat kidney cortical slices in a medium containing 0.1 mM of the nephrotoxic glutathione (PCBG) or cysteine (PCBC, S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-L-cysteine) conjugates of HCBD for 30 min resulted in a 75% reduction in the slice/medium ratio of p-aminohipurate (PAH) compared to that seen in controls. When the cortical slices were incubated with ADT (30 min, 0.2 mM) prior to incubation with the nephrotoxic conjugates, the reduction was only 33%. Neither the in vitro nor the in vivo treatments did modify the activity of renal cytosolic beta-lyase; however, the latter treatment caused an increase in NPSH content. A 15-min incubation of kidney cortical slices with glutathione (10 mM) resulted in a 5-fold increase of NPSH, but failed to prevent the reduction in PAH uptake caused by PCBG and PCBC. Altogether, the in vivo and renal slice data suggest that ADT protects rats against HCBD-induced nephrotoxicity by a mechanism that does not involve the modulation of HCBD conjugation with liver GSH, nor the modulation of the kidney NPSH level and beta-lyase activity. The mechanism of protection conferred to rats by an ADT pretreatment against HCBD-induced nephrotoxicity appears to take place in the kidney at a step beyond the generation of ultimate toxic metabolites derived from PCBC.

Sex-related difference in hepatic glutathione conjugation of hexachlorobenzene in the rat.

Hexachlorobenzene (HCB) induces hepatic porphyria and liver cancer in female rats, whereas toxicity is minimal in male rats. HCB is biotransformed to sulfur-containing metabolites originating from conjugation to glutathione (GSH). This study aimed to assess differences in GSH conjugation of HCB between male and female rats. Sprague-Dawley rats of both sexes were given (po, 10 ml/kg in corn oil) five consecutive doses of 100 mg/kg HCB [2 bid (7:30, 15:30) + 1 sid (7:30)]. This cumulative dose produced porphyria in female but not male rats after a delay period of 6 weeks. Animals were killed 0, 6, 12, 18, or 24 hr after the last dose. Hepatic GSH level showed a diurnal cycle in rats of both sexes, but it was more pronounced in males; the minimum level was observed at 12 hr after dosing. The GSH level in HCB-treated male rats was significantly lower than control at 6, 18, and 24 hr, whereas no significant differences were observed for HCB-treated female rats. Biliary excretion of pentachlorothiophenol, a metabolite originating from GSH conjugation of HCB, was higher in male than female rats. Liver cytosolic GSH transferase activity toward 3,4-dichloronitrobenzene was significantly higher than control level in male but not female rats given HCB. GSH transferase activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane in male and female rats was not increased by HCB treatment. The liver HCB concentration at 24 hr after dosing was higher in male rats than in female rats but decreased faster thereafter. These results suggest that hepatic GSH conjugation of HCB is more important in male than in female rats. This may be related to the reduced liver porphyria observed in HCB-treated male rats compared to female rats.

Male rat specific nephrotoxicity resulting from subchronic administration of hexachlorobenzene.

Male rats are more sensitive to the nephrocarcinogenic effect of hexachlorobenzene (HCB) than are female rats. The purpose of this study was to shed light on this phenomenon by investigating mechanisms of subchronic nephrotoxicity of HCB. Groups of rats were administered HCB in corn oil (po) at 100 mg/kg, 5 days per week for 15 days or at 50 mg/kg, 5 days per week for 50 days. Urine was collected on Days 1, 8, and 15 for the 15-day treatment and on Day 50 for the 50-day treatment. Glucosuria, proteinuria, and enzymuria (gamma-glutamyl transpeptidase) were measured to assess renal function. Twenty-four hours after the last HCB treatment, the animals were killed and kidneys were removed for histopathological evaluation. Urine analyses showed no indication of renal dysfunction in treated animals compared to controls during the 15-day treatment. However, histology of male rat kidneys revealed degenerative and regenerative cellular foci accompanied by an increased accumulation of protein droplets in epithelial cells of the proximal tubules. The same histological observations were also made in male rats after a 50-day HCB treatment but this time they were accompanied by renal function alterations. In female rats, no such renal functional or histological alterations were observed. The histopathological observations in male rats correspond well with the protein droplet nephropathy; the latter is characteristic of the accumulation in kidney cells of alpha 2u-globulin probably caused by the reversible binding of a chemical to alpha 2u that renders the protein indigestible to kidney proteases. alpha 2u-Globulin was measured in the cytosol of male rats and was found to be increased 11-fold compared to controls. Also, HCB was found to be bound reversibly to alpha 2u. These results suggest that HCB induces a male rat specific nephropathy that could explain the higher incidence of kidney tumors in male rats compared to female rats.

Thoracoscopy for the diagnosis of pleural disease.

OBJECTIVE: To assess the accuracy and safety of thoracoscopy for the evaluation of pleural disease. DESIGN: Prospective evaluation of patients referred for thoracoscopy. SETTING: University hospital specializing in chest diseases. PATIENTS: We studied 102 patients with pleural disease, the cause of which had not been determined after initial investigation, including thoracentesis and needle biopsy. Eighty-six patients had pleural effusion, 11 had pleural mass, and 5 had pleural effusion in association with a known primary lung carcinoma. INTERVENTION: All patients had thoracoscopy under local anesthesia with mild sedation. Visually directed biopsies were done of parietal pleura. MEASUREMENTS: We recorded clinical characteristics, laboratory data, findings and duration of thoracoscopy, and any complications associated with the procedure. Hospital and clinic follow-up records were reviewed, and patients were contacted by telephone 12 and 24 months after thoracoscopy to assess their health status. MAIN RESULTS: One hundred and four thoracoscopies were done in 102 patients. A definitive diagnosis was established in 95 patients: 42 had malignant pleural disease and 53 had benign pleural disease. A diagnosis of benign pleural disease using thoracoscopy could not be confirmed in the remaining 7 patients because of insufficient follow-up information. Overall, thoracoscopy was 96% accurate with a sensitivity of 91%, a specificity of 100% and a negative predictive value of 93% for the diagnosis of pleural malignancy. Thoracoscopy was well tolerated under local anesthesia and entailed hospitalization for less than 24 hours in most cases. No deaths occurred, although 1.9% of patients had major complications, and 5.5% had minor complications. CONCLUSIONS: Among patients with pleural disease remaining undiagnosed after usual initial investigation, thoracoscopy done under local anesthesia is a rapid, safe, and well-tolerated procedure with an excellent diagnostic yield that is equivalent to that of thoracotomy.

Patterns of protein synthesis during Xenopus oocyte maturation differ according to the type of stimulation.

We examined the qualitative patterns of protein synthesis in fully grown prophase-blocked oocytes of Xenopus laevis and after meiosis reinitiation accompanying maturation of the oocytes. Newly synthesized proteins labelled with [35S]methionine were run on isoelectric focusing gels and further separated in the second dimension on SDS-polyacrylamide slab gels. Three types of maturation inducer were compared: progesterone, considered as the natural inducer of Xenopus oocyte maturation, hCG (human chorionic gonadotropin) and insulin. Three polypeptides with apparent molecular masses of 37 kDa (pI 4.7-4.8), 78 kDa (pI 4.7) and 138 kDa (pI 4.6-4.7) were found to be always synthesized in all three types of stimulation, while the synthesis of a fourth one (molecular mass 116 kDa, pI 4.7) was arrested during oocyte maturation. Moreover, when the follicular cells surrounding the oocytes were part of the stimulating pathway, which is the case during hCG-induced maturation, an additional polypeptide was synthesized by the oocytes (molecular mass 106 kDa, pI 6.0-6.2). This polypeptide was not synthesized during progesterone- or insulin-induced oocyte maturation, two types of stimulation which do not require the presence of the follicular cells. The biological significance of the hCG-induced polypeptide, not necessary for oocyte maturation, is discussed. On the other hand, the four other modifications in protein synthesis taking place during all three types of maturation-inducing stimulation appear to be necessary for oocyte maturation, since oocytes which failed to mature in response to stimulation always missed one or several of these four polypeptides.

Involvement of reversible binding to alpha 2u-globulin in 1,4-dichlorobenzene-induced nephrotoxicity.

Similarly to unleaded gasoline, 1,4-dichlorobenzene (1,4-DCB) administered for 2 years caused a dose-related increase in the incidence of renal tumors in male but not in female rats or in either sex of mice. Unleaded gasoline and 2,2,4-trimethylpentane (TMP), a component of unleaded gasoline, increased protein droplet formation and cell proliferation in male but not in female rat kidneys. These protein droplets contained, alpha 2u-globulin, a male rat-specific low-molecular-weight protein and 2,4,4-trimethyl-2-pentanol, a metabolite of TMP that was reversibly bound to this protein. Studies were undertaken to determine if 1,4-DCB produced similar effects; 1,2-DCB was used for comparison since it did not produce renal carcinogenesis in male rats. Gel filtration chromatography of a 116,000g supernatant prepared from kidneys of 1,4-[14C]DCB-treated rats showed that radiolabel coeluted with alpha 2u-globulin as one sharp peak as opposed to a multipeak pattern observed for 1,2-[14C]DCB; the maximal quantity of radiolabel for 1,4-DCB was twice that for 1,2-DCB. Equilibrium dialysis of kidney cytosol in the presence or absence of sodium dodecyl sulfate demonstrated that the radiolabel was reversibly bound to alpha 2u-globulin; the amount for 1,4-[14C]DCB-treated rats was almost twice as much as that for 1,2-[14C]DCB-treated rats. 1,2-DCB was also shown to be covalently bound to renal alpha 2u-globulin, and covalently bound to liver and plasma high-molecular-weight proteins. 1,4-DCB and, to a minor extent, 2,5-dichlorophenol, the major metabolite of 1,4-DCB, were reversibly bound to renal alpha 2u-globulin from 1,4-DCB-treated rats. 1,4-DCB increased protein droplet formation in male but not in female rat kidneys, whereas equimolar doses of 1,2-DCB showed no effect in either sex. Renal cell proliferation, measured by [3H]thymidine incorporation into renal DNA, was increased after 1,4-DCB but not after 1,2-DCB treatment. Nephrotoxicity and biochemical alterations induced by 1,4-DCB resemble those of unleaded gasoline and suggest that a similar mechanism is involved in the induction of alpha 2u-globulin nephropathy in male rats.

The comparative pathobiology of alpha 2u-globulin nephropathy.

alpha 2u-Globulin nephropathy is an important toxicologic syndrome that occurs in male rats following exposure to a number of important industrial and environmental chemicals. A low, but significant incidence of renal neoplasia develops in male rats as a chronic sequela to the disease. Studies on the pathogenesis of alpha 2u-globulin nephropathy have demonstrated that this protein is produced in large amounts in the male rat, that reversible binding occurs between chemicals and/or their metabolites and alpha 2u-globulin, and that this complex is resistant to proteolytic hydrolysis, leading to accumulation in renal lysosomes and subsequent cytotoxicity and cell death. This results in marked exposure-related increases in cell proliferation that persist for at least one year, providing exposure continues. This sustained increase in renal cell proliferation can promote initiated cells to form preneoplastic foci and renal neoplasia in male rats. Since this syndrome is highly species and sex specific, it is important to determine the relevance of these data for human risk assessment. The scientific considerations involved in high to low dose and species to species extrapolation are discussed.

A study on the inheritance of thyroxine-binding globulin (TBG) deficiency from data obtained in 13 families detected by a neonatal screening program.

Thirteen families with thyroxine-binding globulin deficiency, detected through probands screened by the Quebec Network of Genetic Medicine, were investigated. These families were divided into two groups, depending on whether hemizygous males had low or undetectable serum thyroxine-binding globulin levels. Five families belonged to the low (hypo-TBGnemic) type while the remaining 8 families belonged to the absent (a-TBGnemic) type. On pedigree analysis, 10 families satisfied the requirements for an X-linked co-dominant mode of transmission. Three families, 2 a-TBGnemic and 1 hypo-TBGnemic, did not satisfy these requirements and three hypotheses had to be put forward to explain these situations: an autosomal recessive mode of transmission, a spontaneous mutation, or an extreme lyonisation of the defective X. Since the thyroxine-binding globulin gene has been localized to the X-chromosome, the two latter explanations appear to be the most plausible.

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.

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.

The onset of activation responsiveness during maturation coincides with the formation of the cortical endoplasmic reticulum in oocytes of Xenopus laevis.

Immature, Stage VI oocytes of Xenopus laevis fail to activate (i.e., to propagate a cortical reaction and elevate a fertilization envelope) when pricked or exposed to A23187. We determined the times during maturation when immature oocytes treated with progesterone in vitro developed the capacity to respond to pricking and to ionophore. Responsiveness to ionophore first appears at about 3.5-4.5 hr after progesterone treatment; all oocytes are activated by 8-9 hr after progesterone. The capacity to respond to pricking appears about 1.0-1.5 hr after first signs of ionophore responsiveness. We examined the cortical endoplasmic reticulum (CER) by TEM to determine whether the morphology of this component could be correlated with the development of responsiveness during maturation. Fully mature oocytes exhibit an extensive CER that (1) forms a "shell" around most cortical granules, (2) appears to interconnect cortical granules, and (3) forms junctions with the plasma membrane. The CER-plasma membrane junctions are especially obvious in preparations of isolated cortex. The elaborate CER is not present in immature oocytes. It first appears during maturation of progesterone-treated oocytes at 4.5-5.0 hr, coincident with the time when maturing oocytes develop their responsiveness to ionophore and to pricking. This temporal correlation is consistent with the hypothesis that the CER is one of the components required for regulation of intracellular free calcium in oocytes.