Congenital adrenal hyperplasia caused by mutant P450 oxidoreductase and human androgen synthesis: analytical study.

Background: Congenital adrenal hyperplasia with apparent combined P450C17 and P450C21 deficiency is associated with accumulation of steroid metabolites, indicating impaired activity of 17alpha-hydroxylase and 21-hydroxylase. However, no mutations have been reported in the CYP17 and CYP21 genes, which encode these P450 enzymes. Affected girls are born with ambiguous genitalia, but their circulating androgens are low, and virilisation does not progress.

Mutations in the genes encoding 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to cause cortisone reductase deficiency.

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.

Reduced placental 11beta-hydroxysteroid dehydrogenase type 2 mRNA levels in human pregnancies complicated by intrauterine growth restriction: an analysis of possible mechanisms.

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.

Measurement of hybridoma cell number, viability, and morphology using fully automated image analysis.

A novel automated image analysis method is described for characterizing the viability and morphology of animal cells from suspension cultures. With the aid of the exclusion dye Trypan Blue, the total and viable cell counts and the percentage of dead cells present are found. The area, perimeter, equivalent diameter, and circularity of the projected image of each cell are also measured, allowing the estimation of cell volume.

A flow cytometric study of hydrodynamic damage to mammalian cells.

Flow cytometry has been used to study the mechanisms of damage to mammalian cells by hydrodynamic forces. Cell damage resulted from the stresses created both by bubble entrainment and by bubble bursting caused by vortex formation in highly agitated culture. Damage to the antigen molecules on the cell surface together with increasing leakage and passive transport were observed.

The effect of Pluronic F-68 on hybridoma cells in continuous culture.

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.

Specific monoclonal antibody productivity and the cell cycle-comparisons of batch, continuous and perfusion cultures.

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.

Cell cycle, cell size and mitochondrial activity of hybridoma cells during batch cultivation.

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.

Flow cytometric study of cultured mammalian cells.

Flow cytometry provides a rapid, sensitive and accurate analytical means to monitor hybridoma cell cultures. The use of flow cytometry has enabled us to study the changes in DNA, RNA, protein, IgG, mitochondrial activity and cell size that take place during the growth cycle of batch culture. The temporal changes in the levels of these analytes and their heterogeneity have been related to the growth/death kinetics.

Rheological action of drugs that prevent erythrocyte dehydration.

The water content of the human erythrocyte is a major determinant of its cytoplasmic viscosity and thus deformability. Loss of cell water may be either primary or secondary to loss of erythrocyte cations (K+). Several existing drugs (cetiedil citrate, pentoxifylline and piracetam) have recently been shown to inhibit K+ loss from erythrocytes and thus have the potential to prevent erythrocyte dehydration.

KCl cotransport in HbAA and HbSS red cells: activation by intracellular acidity and disappearance during maturation.

Low intracellular pH was shown to be a potent activator of the KCl cotransport system in HbSS red cells, and in reticulocyte-rich fractions of HbAA red cells. Rheological experiments indicated that cell dehydration via the KCl cotransporter in response to low pH decreased the filterability of HbSS red cells. In vitro maturation experiments showed that the KCl cotransport system was rendered cryptic rapidly, in contrast to choline transport, and serine transport via system ASC, which disappeared much more slowly.

Erythrocyte heterogeneity in sickle cell disease: effect of deoxygenation on intracellular polymer formation and rheology of sub-populations.

Erythrocytes from 12 patients with homozygous sickle cell disease in the steady state were fractionated on a Percoll-Stractan density gradient. Erythrocyte deformability was measured by initial-flow-rate filtration through pores of 5 microns diameter and erythrocyte polymer content was calculated as a function of oxygen saturation. Density fractionated sub-populations of sickle cells showed distinct rheological characteristics, the filterability of dense cells being impaired by minimal oxygen desaturation with the apparent formation of little or no intracellular polymer.