Correlation between the clonogenic initial slope and the response of polykaryon-forming units: the behavior of strains defective in XRCC5 and ATM and the heritability of small variations in radioresponse.

The polykaryon-forming unit (PFU) assay measures the survival of multiple cycles of DNA synthesis after exposure to ionizing radiation, and it is known that there is a strong correlation between the slope of the PFU dose-response curve and the clonogenic initial slope. This suggests that DNA lesions expressed in clonogens are also important in PFU. Cells having a mutation in XRCC5 (also known as Ku80; strain xrs-6) and ATM (strain AT5BIVA) were hypersensitive in the PFU assay and in clonogens, while a strain of xrs-6 cells transfected with hamster wild-type XRCC5 cDNA displayed wild-type resistance in both assays. These data suggest that the DNA double-strand break (DSB) is an important lesion in PFU, although the relative radioresistance of PFU compared to clonogens indicates differential DSB toxicity. We propose that this results from the absence of cytokinesis-related loss of DNA fragments. Small variations in the radioresponse of PFU were observed between CHO K1 cell substrains, such that the xrs parental substrain RR-CHOK1 (carrying wild-type XRCC5) was more sensitive than an independent K1 substrain (E-CHOK1). Somatic hybridization showed that this variation is heritable and that the resistant E phenotype is dominant. In RR-CHOK1 cells there was a biphasic PFU radioresponse, which suggests that there may be transient expression at a locus selectively affecting PFU sensitivity.

Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms.

A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205-2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable.

Prediction of the initial slope of the acute clonogenic survival curve by the post-irradiation behaviour of cytochalasin-induced polykaryons.

We have investigated the behaviour of 11 lines of cultured cells in a survival assay whose endpoint is the ability of cells to become polyploid when incubated in the presence of cytochalasin B (CB). Single cells were induced by CB to form polykaryons after irradiation, and, by analogy with the colony-forming assay, the survival of polykaryon-forming units (PFUs) was defined as the fraction of cells able to achieve a given DNA content (at least 16C in most experiments). There was a radiation dose-dependent reduction in PFU survival, which, following the appearance of cells containing at least 16C DNA, was not markedly dependent upon the sampling time. In all cases, PFUs appeared to be more radioresistant than clonogens, especially at high dose. In 9/11 lines the PFU dose-response curves were exponential, while in two there was a pronounced curvature (quadratic parameter). There was a highly significant positive correlation between PFU response and the corresponding clonogenic initial slope. We suggest that in polykaryons the opportunity for the mechanical loss of DNA fragments may be reduced because the cells do not divide, and therefore that DNA damage resulting from chromosome aberrations may be less important in PFUs than in clonogens. Consequently, PFUs may express lesions not directly associated with mechanical gene loss. This assay may yield an alternative estimate for the clonogenic initial slope, and could be of use where colony-forming assays fail due to incomplete cell monodispersion.

Variation in radiosensitivity due to cell age and split-dose recovery in polykaryons induced by cytochalasin.

We have investigated the properties of an in vitro cell survival assay that uses as its endpoint the ability to form polyploid cells (polykaryons) in the presence of cytochalasin B (CB). The criterion for survival is that a polykaryon-forming unit (PFU) must reach the arbitrary DNA content of at least 16C. The age-dependence of PFU sensitivity to 137Cs irradiation was determined using V79-379A cells synchronized at mitosis. Cells assayed as PFUs demonstrated much less variation in radiosensitivity with age than did clonogens, but the changes in curve shape were qualitatively similar. In both assays mitotic cells yielded an exponential survival curve while that obtained at 5 h (mid-late S) had a marked quadratic component. Owing to the small overall variation in PFU survival with age, at doses greater than about 25 Gy the surviving fraction at 5 h was lower than in mitosis. In both V79-379A and HeLa S3 cells, PFUs demonstrated a capacity for split-dose recovery and yielded recovery ratios at 2.6 at 50 Gy in V79 and 1.5 at 20 Gy in HeLa. Since these ratios were much lower than in clonogens at the same dose, we suggest that this is consistent with an association that we have previously demonstrated between PFU response and the clonogenic initial slope. In an attempt to clarify the DNA lesions to which PFUs may be sensitive, we determined PFU response following exposure to 254-nm UV irradiation. In contrast with ionizing radiation, PFU response to UV was very similar to that of clonogens. This suggests that following UV exposure the absence of cytokinesis in polykaryons may confer less protection than in the case of ionizing radiation, possibly due to fundamental differences in the spectrum of DNA lesions produced.

Cycle delay in irradiated cytochalasin-induced polykaryons. Does cytoskeleton status affect cell cycle checkpoints?

Following exposure of CHO-K1 cells to 137Cs irradiation at doses up to 20Gy, a delay in G2 was observed to occur in cells permitted to divide normally, while cells induced to become giants by means of cytochalasin B demonstrated a minimal delay in the transition 2C-8C suggesting that the inhibition of cytokinesis results in modification of one or more cell cycle checkpoints. We postulate that this may occur as a consequence of damage tolerance, or by a feedback loop resulting from the reorganisation of the cytoskeleton that precludes cytokinesis.

Inputs to group II-activated midlumbar interneurones from descending motor pathways in the cat.

1. Connections from descending motor pathways to group II-activated interneurones in the midlumbar segments of the spinal cord have been examined by intracellular recording. Interneurones, many of which had axonal projections to the hindlimb motor nuclei, were tested for inputs from rubro-, reticulo-, vestibulo- or corticospinal fibres. 2. Of 138 cells, 113 were monosynaptically excited by electrical stimulation of at least one of the descending motor pathways. Monosynaptic excitation from reticulo-, vestibulo- and rubrospinal pathways was common. Monosynaptic corticospinal EPSPs were identified in fewer neurones. 3. Convergent monosynaptic inputs from pathways which descend in the ventrolateral and ventral funiculi were common. Although few neurones with monosynaptic input from the corticospinal tract were identified, most also had monosynaptic rubrospinal input. In contrast, few neurones (4.3%) had convergent monosynaptic input both from pathways in the dorsolateral funiculus and from fibres in the ventral/ventrolateral funiculi. 4. The patterns of convergence from the different descending motor pathways differ from the patterns expected if the descending connections were distributed independently. Thus there is a significant segregation between rubrospinal and reticulo- or vestibulospinal inputs, and a significant association of reticulo- and vestibulospinal inputs. 5. Since descending motor pathways make monosynaptic connections with most group II-activated midlumbar neurones, many of which project to the hindlimb motor nuclei, some of these neurones provide a disynaptic pathway for the supraspinal control of hindlimb movements. The distribution of descending connections is consistent with the hypothesis that pathways descending in the dorsal part of the lateral funiculus and those descending in the ventrolateral or ventral funiculi contact different sets of interneurones.

Serum 5-androstene-3 beta,17 beta-diol sulphate and 5 alpha-androstane-3 alpha,17 beta-diol sulphate in hirsute females with polycystic ovarian disease.

Serum sulphates of 5-androstene-3 beta,17 beta-diol (5-ADIOL-S), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-DIOL-S) and dehydroepiandrosterone (DHEA-S), unconjugated androstene-dione (AD) and testosterone (T), sex hormone binding globulin (SHBG), free androgen index (FAI), 17 alpha-hydroxyprogesterone (17OHP), luteinising hormone (LH) and follicle stimulating hormone (FSH) were measured by specific radioimmunoassay in 28 hirsute women with polycystic ovarian disease (PCO) and in normal women (n = 73). Mean levels of steroids measured were significantly elevated, and SHBG significantly depressed, in the women with PCO with values (mean +/- SE) for 5-ADIOL-S (516 +/- 51 vs 267 +/- 10 nmol/l), 3 alpha-DIOL-S (130 +/- 9 vs 52 +/- 2 nmol/l), DHEA-S (7.3 +/- 0.5 vs 4.4 +/- 0.2 mumol/l), AD (11.3 +/- 1.1 vs 3.4 +/- 0.2 nmol/l), T (3.3 +/- 0.2 vs 1.5 +/- 0.1 nmol/l) and 17OHP (5.1 +/- 0.8 vs 2.8 +/- 0.2 nmol/l). SHBG levels were 31 +/- 2.9 vs 65 +/- 2.5 nmol/l, and the free androgen index [100 x T (nmol/l) divided by (SHBG nmol/l)] was 12.5 +/- 1.4 vs 2.4 +/- 0.1. The mean LH to FSH ratio was also elevated at 2.8 +/- 0.3. These studies suggest that the measurement of 5-ADIOL-S and DHEA-S may indicate adrenal gland involvement in PCO while 3 alpha-DIOL-S appears to be a reflection of peripheral androgen metabolism. A comprehensive biochemical profile of PCO should thus include the analysis of these sulphoconjugates as well as unconjugated steroids.

Plasma C19 steroid sulphate levels and indices of androgen bioavailability in female pattern androgenic alopecia.

Female pattern androgenic alopecia (AA) is a relatively common endocrine abnormality in premenopausal women. However, unlike hirsutism, little is known about the androgen metabolism and plasma C19 steroid sulphate profiles in this disorder. We have therefore measured the plasma levels of dehydroepiandrosterone sulphate (DHEA-S), 5-androstene-3 beta,17 beta-diol sulphate (5-ADIOL-S), 5 alpha-androstane-3 alpha, 17 beta-diol sulphate (3 alpha-DIOL-S), androstenedione (AD), total testosterone (T), free testosterone (FT), sex hormone binding globulin (SHBG), non-SHBG bound T, luteinizing hormone (LH) and follicle stimulating hormone (FSH), and have calculated the free androgen index (FAI): 100 x T (nmol/l) divided by SHBG (nmol/l), in premenopausal women with AA (n = 25-45) and in normal premenopausal women (n = 17-73). While mean plasma concentrations of DHEA-S and T were not significantly different from controls, mean SHBG concentrations were significantly lower (47 +/- 3 vs 64 +/- 3 nmol/l) and the mean free androgen index (4.4 +/- 0.4 vs 2.4 +/- 0.2), and mean concentrations of free testosterone (45 +/- 5 vs 26 +/- 1.4 pmol/l), non-SHBG bound T (0.9 +/- 0.2 vs 0.6 +/- 0.1 nmol/l) and androstenedione (4.3 +/- 0.3 vs 3.4 +/- 0.2 nmol/l) were significantly elevated in women with AA. Furthermore, mean plasma concentrations of 5-ADIOL-S (512 +/- 42 nmol/l) and 3 alpha-DIOL-S (76 +/- 7 nmol/l) were significantly higher than levels found in normal women (272 +/- 12 nmol/l and 52 +/- 2 nmol/l respectively). The nature of the hyperandrogenism associated with AA may thus only be revealed by a comprehensive plasma androgen and androgen sulphate profile, which may explain apparently aberrant data for a given patient. In addition, 5-ADIOL-S and 3 alpha-DIOL-S may serve as excellent plasma markers of both the existence of the disorder and the efficacy of its treatment.

Serum C-19 steroid sulphates in females with clinical hyperandrogenism.

Serum sulphates of 5-androstene-3 beta, 17 beta-diol (5-ADIOL-S), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-DIOL-S) and dehydroepiandrosterone (DHEA-S), as well as unconjugated androstenedione (AD), testosterone (T) and 17 alpha-hydroxyprogesterone (17OHP), sex hormone binding globulin (SHBG) and the free androgen index (FAI) were measured by specific radioimmunoassay in girls with premature adrenarche (n = 9-16), and in hirsute women with (1) late onset 21 hydroxylase deficiency (n = 14), (2) polycystic ovarian disease (n = 28) and (3) idiopathic hirsutism (n = 74). Levels were also determined in females with androgenic alopecia (n = 35-45), in normal prepubertal girls (n = 9-14) and in normal adult women (n = 50-73). Mean serum concentrations of 5-ADIOL-S, 3 alpha-DIOL-S, DHEA-S, AD, T, and FAI were elevated and SHBG depressed, in all patient groups compared with controls, except for DHEA-S and T in patients with alopecia. We conclude that in addition to DHEA-S, 5-ADIOL-S may have a role as a pro-hormone in the synthesis of more potent androgens (T, DHT) in peripheral tissues such as skin; in addition, 3 alpha-DIOL-S may be a marker of peripheral androgen metabolism.

Diagnosis of 21-hydroxylase deficiency in newborn infants by GC-MS of urinary steroids.

In a study using gas chromatography-mass spectrometry (GC-MS) on urine specimens from 16 normal infants and 16 infants with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (aged 1 day to 4 weeks), the major steroids recognized in all infants were: 16 alpha-hydroxy-dehydroepiandrosterone, 16 beta-hydroxy-dehydroepiandrosterone, 16-oxo-androstenediol, androstenetriol, 15 beta,17 alpha-dihydroxy-pregnenolone and 16 alpha-hydroxy-pregnenolone. Pregnanetriol was detectable in three normal infants (aged 3, 6 and 15 days) but the levels seen in 15 CAH patients were in a higher range. Pregnanetriolone, 5 beta-17-hydroxy-pregnanolone and 15 beta,17 alpha-dihydroxy-pregnanolone were present in the urine of 15 CAH patients, but were not detectable in any of the normal infants. The older the patient, the higher the level was of each of these four steroids. The results indicate that, even on day 1, patients with CAH due to 21-hydroxylase deficiency may be positively identified using GC-MS of urine specimens. This does not preclude the possibility that a minority of patients with CAH, most likely those with mild 21-hydroxylase deficiency, may not exhibit the characteristic GC-MS findings on day 1, as seen in one of the 16 CAH patients.

Studies on the absorption of L-xyloascorbic acid (vitamin C) in young and elderly subjects.

Absorption of ascorbic acid was assessed by measuring its urinary excretion following the administration of a standard dose under pre-determined conditions. The absorption mechanism appeared to be a saturable one and with intakes of up to 1 g over 90 per cent of the subsequent urinary excretion occurred during the first 8 h. Significantly less ascorbic acid was absorbed by elderly persons than by young ones; after a 500 mg dose the mean urinary excretion by nine elderly females (mean age 82.6) was 25 mg and by six young females (mean age 21.8) 245 mg. It is suggested that impaired gastrointestinal absorption is an important factor in the aetiology of low blood concentrations of ascorbic acid in the elderly.

The effects of discontinuing long-term diuretic therapy in the elderly.

The effects of discontinuing long-term diuretic therapy were investigated by means of a double-blind randomized controlled trial. A total of 141 elderly patients in the long-stay wards of six hospitals were found to be taking maintenance diuretics, and for 33 of these the drugs were judged to be mandatory. Of the remaining patients, 52 continued to receive diuretics while 54 were given matching placebo tablets. Eight in the latter group required diuretic therapy to be resumed during the following 12 weeks. The main change observed in patients who completed the trial was a slight increase of ankle oedema in the placebo group. Plasma potassium levels below 3.5 mEq/1 were found in some patients taking diuretics but not when the drugs had been withdrawn for 12 weeks. Blood pressure rose slightly in the placebo group and plasma urea rose slightly in the diuretic group. It was concluded that old people receiving long-term diuretic therapy without obvious current indication should have the drugs withdrawn under careful supervision so that those needing them could be identified.