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In cortisone reductase deficiency (CRD), activation of cortisone to cortisol does not occur, resulting in adrenocorticotropin-mediated androgen excess and a phenotype resembling polycystic ovary syndrome (PCOS; refs. 1,2). This suggests a defect in the gene HSD11B1 encoding 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), a primary regulator of tissue-specific glucocorticoid bioavailability. We identified intronic mutations in HSD11B1 that resulted in reduced gene transcription in three individuals with CRD. In vivo, 11beta-HSD1 catalyzes the reduction of cortisone to cortisol whereas purified enzyme acts as a dehydrogenase converting cortisol to cortisone. Oxo-reductase activity can be regained using a NADPH-regeneration system and the cytosolic enzyme glucose-6-phosphate dehydrogenase. But the catalytic domain of 11beta-HSD1 faces into the lumen of the endoplasmic reticulum (ER; ref. 6). We hypothesized that endolumenal hexose-6-phosphate dehydrogenase (H6PDH) regenerates NADPH in the ER, thereby influencing directionality of 11beta-HSD1 activity. Mutations in exon 5 of H6PD in individuals with CRD attenuated or abolished H6PDH activity. These individuals have mutations in both HSD11B1 and H6PD in a triallelic digenic model of inheritance, resulting in low 11beta-HSD1 expression and ER NADPH generation with loss of 11beta-HSD1 oxo-reductase activity. CRD defines a new ER-specific redox potential and establishes H6PDH as a potential factor in the pathogenesis of PCOS.

A selection of mouse hybridoma cell lines showed a variation of approximately two orders of magnitude in intracellular monoclonal antibody contents. The different levels directly influenced apparent specific monoclonal antibody productivity during the death phase but not during the growth phase of a batch culture. The pattern of changes in specific productivity during culture remained basically similar even though at different levels for all cell lines tested. Arresting the cells in the G1 phase using thymidine increased the specific productivity, cell volume and intracellular antibody content but at the same time led to decreased viability. In continuous culture DNA synthesis decreased with decreasing dilution rate though without an accompanying change in cell cycle and cell size distributions. The data shows both the decrease in viability and intracellular antibody content to be important factors which influence the negative association between specific antibody productivity and growth rate. In high cell density perfusion culture, when the cell cycle was prolonged by slow growth, viability was low and dead, but not lysed, cells were retained in the system, the specific antibody productivity was nearly two fold higher than that obtained in either batch or continuous cultures. The results imply that the prolongation of G1 phase and the increase in death rate of cells storing a large amount of antibody together cause an apparent increase in specific antibody productivity.

11beta-Hydroxysteroid dehydrogenase type 2 (11beta-HSD2) inactivates cortisol to cortisone. In the placenta 11beta-HSD2 activity is thought to protect the fetus from the deleterious effects of maternal glucocorticoids. Patients with apparent mineralocorticoid excess owing to mutations in the 11beta-HSD2 gene invariably have reduced birth weight, and we have recently shown reduced placental 11beta-HSD2 activity in pregnancies complicated by intrauterine growth restriction. This is reflected in the literature by evidence of hypercortisolemia in the fetal circulation of small babies. In this study we have determined the levels of placental 11beta-HSD2 mRNA expression across normal gestation (n = 86 placentae) and in pregnancies complicated by intrauterine growth restriction (n = 19) and evaluated the underlying mechanism for any aberrant 11beta-HSD2 mRNA expression in intrauterine growth restriction. 11beta-HSD2 mRNA expression increased more than 50-fold across gestation, peaking at term. Placental 11beta-HSD2 mRNA levels were significantly decreased in intrauterine growth restriction pregnancies when compared with gestationally matched, appropriately grown placentae [e.g. at term DeltaCt (11beta-hydroxysteroid dehydrogenase type 2/18S) 12.8 +/- 0.8 (mean +/- SE) vs. 10.2 +/- 0.2, respectively, P < 0.001]. These differences were not attributable to changes in trophoblast mass in intrauterine growth restriction placentae, as assessed by parallel analyses of cytokeratin-8 mRNA expression. No mutations were found in the 11beta-HSD2 gene in the intrauterine growth restriction cohort, and imprinting analysis revealed that the 11beta-HSD2 gene was not imprinted. Although the underlying cause is unknown, 11beta-HSD2 gene expression is reduced in intrauterine growth restriction pregnancies. These data highlight the important role of 11beta-HSD2 in regulating fetal growth, a known factor in determining fetal morbidity but also the subsequent development of cardiovascular disease in adulthood.

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Cell cycle, cell size and rhodamine 123 fluorescence in cell populations of two batch cultures were analysed and quantified with a fluorescence-activated cell sorter (FACS). Two cultures derived from either exponential or stationary phase innocula were investigated in order to demonstrate the dependency of the subsequent cell growth on innoculum condition. The results demonstrated that the level of activity of cells in the innoculum culture could have a significant effect on cellular activity during the initial phase of the inoculated culture, as it advances through its growth cycle. Positive correlation was found between the cell size and mitochondrial activity (as measured by rhodamine 123 uptake) with S and G2 fractions as the cell progressed through the cell cycle. The enumeration of the fractions of cell cycle phases has helped in prediction of the changes in cell numbers following perturbation of the culture condition.

The comparative effects of Pluronic on cell growth of hybridomas were studied. The addition of 0.05% Pluronic decreased the steady-state cell number in continuous culture by about 12% compared to a non-supplemented culture. In short-term experiments, results demonstrated a gradual decrease in cell number with increasing concentration of Pluronic. Such growth inhibition was found to be a result of lowering the rate of DNA synthesis.