Influence of cell culture conditions on aromatase activity in human genital skin fibroblasts

Summary Because the measurement of aromatase activity in cultured human genital skin fibroblasts has been proposed as a means of studying estrogen production in men, we investigated the influence of culture conditions on aromatase activity. Genital skin fibroblasts were seeded onto culture plates at a density of 1×10⁶ cells/plate and aromatase activity was determined over a 1-mo. period. Enzyme activity rose slowly over the first 14 d but then rose rapidly to a 10-fold higher plateau by Day 28. The rise in aromatase activity was similar whether activity was normalized for protein or for DNA content. When cells were seeded at the usual density of 1×10⁶ or at 0.25×10⁶ cells/plate, aromatase activity was consistently lower during the first 2 wk in cells plated at lower density, but thereafter the levels of enzyme activity in the two groups converged. In cells plated at the lower density, the lower activity observed in the first 2 wk was associated with a lower V _max . Preincubation of cells plated at one density with conditoned medium from cells plated at the other density did not change the relatve levels of activity in the two groups. By contrast, dihydrotestosterone (DHT) receptor binding and 5α-reductase activity were similar at all time points, despite differences in plating density. In additional experiments, the culture medium was replaced daily rather than every 3rd d, and aromatase activity was assayed on Day 7. In cells fed daily, DNA and protein content were twice that of cells fed every 3rd d. By contrast, aromatase activity declined to 30% of the in the latter group. DHT and dexamethasone receptor binding and 5α-reductase activity were similar in the two groups. In summary, factors such as plating density, culture density, and frequency of media replacement dramatically influence aromatase activity in cultured human genital skin fibroblasts. Therefore, the interpretation of aromatase activity data obtained from cultured cells in relation to physiologic or pathologic states should be viewed with appropriate caution. The work was supported in part by grants R01 DK 35339 and R01 DK 00180 from the National Institutes of Health, Bethesda, MD, and by RR 00035 from CLINFO Systems at the Johns Hopkins University School of Medicine, Baltimore, MD.     

Postnatal Development of a Granule Cell-Enriched, Neurone-Specific Glycoprotein, gp50, in Normal and Thyroid-Deficient Rats

Glycoprotein gp50 is a neurone-specific, granule cell-enriched glycoprotein that is also a major component of isolated synaptic membranes. Here, we describe the use of a monoclonal antibody, mab SM gp50, to study the postnatal development of gp50 in the brain of normal and thyroid-deficient rats. Radioimmunoassay, enzyme-linked immunosorbent assay, and Western blotting show that gp50 is not detectable in brain until postnatal day 4 (P4) in both forebrain and cerebellum. In forebrain, the rate of increase of gp50 levels is maximal between P12 and P20. It is somewhat later in cerebellum, where peak levels are attained between P30 and P35. Immunocytochemical studies show little detectable gp50-like immunoreactivity before P16, and the staining is still weak, relative to adult tissue, at P25. The intense staining of the granule cell layer characteristic of adult cerebellum predominantly appears after P25. Development of gp50 is severely retarded in the cerebellum of thyroid-deficient rats, particularly during the second and third postnatal weeks. However, by the fourth postnatal week, gp50 levels in normal and hypothyroid animals are comparable. The results indicate that significant alterations in the pattern of gp50 expression continue to occur at a late stage of cerebellar development. In particular, the increase in immunocytochemical staining of the granule cells after P25 is striking in that by this time most major events associated with cerebellar development are essentially complete.     

The human cystatin C gene (CST3), mutated in hereditary cystatin C amyloid angiopathy,is located on chromosome 20

Summary Hereditary cystatin C amyloid angiopathy has recently been shown to be caused by a point mutation in the cystatin C gene. To determine the chromosomal localization of the gene, 20 human-rodent somatic cell hybrids and a fulllength cystatin C cDNA probe were used. Southern blot analysis of BamHI digested cell hybrid DNA revealed that the probe recognizes a 10.6 kb human specific fragment and that this fragment cosegregates with human chromosome 20. Therefore, the human cystatin C gene (CST3) was assigned to chromosome 20.     

(1–3)-β-Glucan synthesis ofNeurospora crassa

(1–3)--d-Glucan synthase activity ofNeurospora crassa was localized to the plasma membrane by autoradiography of colloidal gold-labeled plasma membranes. The active site of glucan synthase for substrate hydrolysis was determined to be cytoplasmic facing. However, glucan synthase activity present in intact protoplasts was partially sensitive to Novozym 234 and to glutaraldehyde treatments, suggestive that enzyme activity is transmembrane. Enzyme activity also directed the formation of microfibrils in vitro. Taken together, these and previous results support the following scheme for glucan synthesis: 1. The sequential addition of glucose residues from UDP-glucose to glucan chains occurs on the cytoplasmically facing portion of glucan synthase. 2. As each glucan chain is synthesized, it is extruded to the extracytoplasmic side of the plasma membrane. 3. As each chain is extruded, it forms interchain hydrogen bonds with adjacent chains, resulting in glucan microfibril assembly.     

A new bacterial alcohol dehydrogenase active on degraded lignin and several low molecular weight aromatic compounds

A new intracellular bacterial dehydrogenase has been purified. It was active in the reversible reduction by NADH of conjugated carbonyl groups in partially degraded lignin. It was also active on various aromatic aldehydes such as vanillin, syringaldehyde and cinnamaldehyde, but had no effect on acetovanillone and lignin models carrying a conjugated ketone. It is proposed that this enzyme functions as a broadly specific lignin dehydrogenase at the level of aldehydic groups that are present in the lignin preparations.     

The roles of calcium and manganese ions in the in vitro conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene by lentil root membranes

Ca²⁺ and Mn²⁺ activate the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) by root microsomes of Vicia lens as they do in other similar systems. The preparation of microsomes in the presence of Mn²⁺ greatly increases their ability to convert ACC into ethylene, without addition of Mn²⁺ in the reaction mixture. Ca²⁺ does not have this property. The effect could not be attributed to Mn²⁺ entrapping into membrane vesicles (sonication followed by repelleting had no effect) but, possibly, in part to Mn²⁺-mediated binding to microsomes of a soluble factor favouring the conversion of ACC to C₂H₄. Although no direct correlation could be established in vitro between ethylene-forming-enzyme (EFE) and peroxidase activities, some soluble peroxidases might be this soluble factor. Mn²⁺ favoured attachment to membranes of some peroxidase activity from the soluble fraction and from commercial HRP and lipoxygenase. This binding effect of Mn₂₊ cannot be readily distinguished from its role in the generation of a chain of free radicals and in redox mechanisms.     

Effects of rapid transfer from sea water to fresh water on respiratory variables,blood acid-base status and O2 affinity of haemoglobin in Atlantic salmon (Salmo salar L.)

Summary Oxygen consumption, gill ventilation, blood acid-base/ionic status and haemoglobin oxygen affinity were studied in seawater-adapted adult salmon (Salmo salar) during five weeks after transfer into fresh water. Freshwater exposure induced the following changes: Standard oxygen consumption ( ) and ventilatory flow ( ) decreased markedly during the first days after transfer, then decreased more gradually until a new steady-state was achieved at which and were about 80% and 56% of the control values, respectively. The marked increase in oxygen extraction coefficient (Ew _{O 2}) and the marked decrease in the oxygen convection requirement ( ) were associated with a reduction in the partial pressure of carbon dioxide in arterial blood (Pa _{CO 2}), in spite of a decrease of both ventilatory flow and water CO₂ capacitance. These results suggested that transfer into fresh water induced an increase in branchial diffusive conductance. A biphasic pattern was observed in the time-course of the changes in both plasma ion concentration and acid-base status. During the first 10 days, plasma Na⁺, K⁺, and Cl^– concentrations fell abruptly, then more gradually. [Cl^–] decreased more than [Na⁺] resulting in a progressive increase in the [Na⁺]/[Cl^–] ratio. During the second phase of acclimation to fresh water plasma Na⁺, K⁺, and Cl^– concentrations progressively increased. [Cl^–] increased more than [Na⁺], so that [Na⁺]/[Cl^–] ratio decreased. Transfer into fresh water did not significantly change plasma lactate concentration. Upon exposure to fresh water, blood pH increased from 7.94±0.04 to 8.43±0.06 at day 10 and then decreased to 8.08±0.03 at day 34. The increase in blood pH induced by transfer to fresh water initially represented a mixed metabolic/respiratory alkalosis. However, after 15 days Pa _{CO 2} had returned to pretransfer values and the alkalosis was purely metabolic. The metabolic component of the alkalosis was associated with appropriate changes in the plasma strong ion difference (S.I.D.). Blood alkalosis moved the oxygen dissociation curve to the left, so that P₅₀ was decreased by 30% below the value in seawater for the maximal increase in blood pH. This rise in haemoglobin affinity for O₂, associated with a marked increase in blood buffer capacity, are regarded as adaptative processes allowing the salmon to cope with the markedly increased energy expenditure required for upstream migration.