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.     

Rabbit Ig kappa 1b6 gene structure

Previous studies employing Southern blot analyses have detected multiple kappa-homologous sequences within EcoRI-digested DNA isolated from kappa 1b6 homozygous rabbits and kappa 1b6 L chain secreting RMH H158 cell line. These results are very unexpected because the published partial protein sequence for the kappa 1b6 C region is incompatible with an EcoRI restriction endonuclease recognition sequence at the nucleotide level for this allotype. To determine their identity, the kappa-homologous sequences were isolated from DNA extracted from a kappa 1b6 L chain secreting RMH H158 cell line by molecular cloning. Structural analyses demonstrated these sequences to contain genetic information encoding the majority of the kappa 1b6 L chain gene locus. The protein sequence deduced from the kappa 1b6 C region gene was shown to differ from the published partial kappa 1b6 C region protein sequence at five amino acid positions. One of these differences results in a glycine to serine interchange that introduces an EcoRI restriction endonuclease recognition site within the kappa 1b6 C region gene. Subsequent genomic Southern blot analyses confirmed this structural assignment. Based on these data, the EcoRI-sensitive kappa-homologous fragments present within the genomes of the RMH H158 cell line and kappa 1b6 homozygous rabbits represent the nominal kappa 1 gene and not an alternative kappa isotype or kappa pseudogene. Rabbit Ig kappa 1 allelic nucleotide sequence homology comparisons have shown the isolated kappa 1b6 J-C gene locus to display common structural features previously identified in other kappa 1 alleles.     

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.     

Intraluminal proteolytic activation plays an important role in replication of type 1 reovirus in the intestines of neonatal mice.

Oral inoculation of suckling mice with reovirus serotype 1 (strain Lang) results in the conversion of intact virions to intermediate subviral particles (ISVPs) in the intestinal lumen. Digestion of virus in vitro with chymotrypsin or trypsin reveals two distinct forms of ISVPs, while the predominant species of ISVPs found in the small intestinal lumen appears to be identical to the chymotrypsin product. The in vivo conversion of virions to ISVPs was blocked by pretreatment of mice with protease inhibitors, resulting in inefficient replication of reovirus in intestinal tissue. The early inhibition of viral replication in suckling mice pretreated with protease inhibitors was not observed when suckling mice were inoculated with ISVPs generated by in vitro digestion with either chymotrypsin or trypsin. However, replication was decreased during secondary rounds of replication in mice receiving repeated doses of protease inhibitors, suggesting that luminal proteolytic digestion is important in rendering progeny virions infectious in the gut.     

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.     

Duplication of an Xp segment that includes the ZFX locus causes sex inversion in man

Summary Two 46,XY females with tandem duplications of an X short arm segment were studied by cytogenetic and Southern blot analysis. The results show that the duplicated segment in each case included the Xp21.2–Xp22.2 interval, resulting in a double dose of ZFX on the single active X chromosome. The results from our two cases, in conjunction with those reported by other workers, lead us to conclude that the duplication is the reason for the sex inversion. If ZFY and ZFX are indeed sex-determining gene loci, these findings favour a model of sex determination characterized by antagonistic interaction between these genes.     

(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.     

Synthesis and properties of 5-azido-UDP-glucose. Development of photoaffinity probes for nucleotide diphosphate sugar binding sites

A new active site directed photoaffinity probe, which is a model compound for studying nucleotide diphosphate sugar binding proteins, has been synthesized by coupling 5-azido-UTP and [32P]Glc-1-P using yeast UDP-glucose pyrophosphorylase to produce [beta-32P]5-azidouridine 5'-diphosphoglucose (5N3UDP-Glc). This probe has photochemical properties similar to that of 5-azidoUTP (Evans, R. K., and Haley, B. E. (1987) Biochemistry 26, 269-276). The efficacy of 5N3UDP-Glc as an active site directed probe was demonstrated using yeast UDP-Glc pyrophosphorylase. Saturation effects of photoinsertion were observed with an apparent Kd of 51 microM and the natural substrate, UDP-Glc, prevented photoinsertion of [beta-32P]5N3UDP-Glc with an apparent Kd of 87 microM. Prevention of photoinsertion was also seen with UTP and pyrophosphate with apparent Kd values less than 200 microM. UMP, UDP, ATP, and GTP were much less effective competitors. Selective photoinsertion was observed with several partially purified enzymes including UDP-Glc dehydrogenase, UDP-Gal-4-epimerase, Gal-1-P uridyltransferase, and phosphorylase a. The absence of nonselective photoinsertion into bulk proteins was demonstrated with crude homogenates of rabbit liver as well as with several UDP-Glc binding proteins. Of the six purified enzymes tested, only phosphoglucomutase has been shown to incorporate radiolabel from the photoprobe in the absence of UV irradiation. These results and a discussion of the utility of 5N3UDP-Glc for detecting UDP-Glc binding proteins and isolating active site peptides are presented.