Human arterial smooth muscle cells in culture: Inverse relationship between proliferation and expression of contractile proteins

Summary Human arterial smooth muscle cells (hASMC) from explants of the inner media of uterine arteries were studied in secondary culture. We had previously found that these cells depend on exogenous platelet-derived growth factor (PDGF) for proliferation in vitro. Deprivation of the serum mitogen(s) by culture in plasma-derived serum or bovine serum albumin (BSA) caused a true growth arrest that was reversible upon reexposure to the mitogen(s). When added to serum-containing medium, heparin caused a reversible growth arrest which could be competed for by increasing concentrations of serum. In the current study we used a set of smooth muscle-specific actin and myosin, antibodies to study the expression of contractile proteins in stress fibers under indirect immunofluorescence on hASMC in culture. Even in sparse culture, grwoth-arrested hASMC expressed stress fibers containing these actin and myosin epitopes. This was true irrespective of whether growth arrest was achieved by culture in media containing only BSA or a combination of heparin and whole blood serum. hASMC proliferating in whole blood serum in sparse culture did not express such strees fibers, as judged by immunofluorescent staining. This was true also for cells that were restimulated to proliferate in serum after a growth arrest. Utilizing a monoclonal antibody against a nuclear antigen expressed in proliferating human cells, we were able to demonstrate an inverse relationship between the expression of this antigen and the SMC-specific contractile proteins, respectively. Under these culture conditions, the reversible transition between defifferentiated and differentiated hASMC was almost complete and terminated about 1 wk after the change in culture condition. We conclude that hASMC in vitro respond, to exogenous PDGF by proliferation and dedifferetiation as a single population of cells. We also conclude that this modulation is reversible, because the cells become uniformly quiescent and differentiated when the mitogenic stimulus is blocked or removed. This study was supported by grants from the Swedish Medical Research Council (Project no. 4531 and 6816), the Swedish Association against Heart and Chest Diseases, the King Gustaf V and Queen Victoria Foundation, the National Institutes of Health, Bethesda, MD (grant HL 29873) and the Swedish National Board for Laboratory Animals.     

Helix formation in reduced,S-carboxymethylated human choriogonadotropin β subunit and tryptic peptides

The β subunit of human choriogonadotropin (hCGβ) and its asialoderivative were digested with trypsin and then reduced and S-carboxymethylated. A series of peptides were purified which corresponded to residues 1–43, 44–95, 96–114, and 123–145 of the 145 amino acid residue glycoprotein. The two N-linked oligosaccharides were present on the amino terminal peptide, and three of the four O-linked oligosaccharides were present on the carboxy terminal peptide. Circular dichroic spectra between 190–240 nm were obtained on reduced, S-carboxymethylated (RCM) hCGβ and the above peptides, both in aqueous solution and in the helicogenic solvent 80% (vol/vol) trifluoroethanol (TFE). In aqueous solution there was evidence of only limited helicity in the peptides and RCM-hCGβ however, in the presence of TFE, peptides 1–43 and 44–95 exhibited significant helicity, as did the full-length linear chain. The helicity developed in TFE by RCM-hCGβ appears much greater than that which occurs in the native, disulfide-intact form, thus suggesting that the disulfides prevent expression of helicity in regions with α-helix potential. Application of the Chou-Fasman secondary structure predictive algorithm to hCGβ suggested that several regions of helix potential, in particular regions 14–21, 59–69, and perhaps 80–88, may account for much of the helicity observed in peptides 1–43 and 44–95, respectively, in TFE. The region from 96–145 has no significant potential for helicity, consistent with the measured circular dichroic spectra of peptides 96–114 and 123–145. These results demonstrate that helicity can occur in the linear form of hCGβ, and this secondary structure can best be attributed to the amino terminal and the middle portion of the molecule. Several potential regions of β-structure and β-turns were also suggested.     

Early estrogen treatment of female zebra finches masculinizes the brain pathway for learned vocalizations

Telencephalic nucleus HVC and its two efferent targets, RA and X, play essential roles in the production of complex, learned vocalizations in the male zebra finch. Normal females do not produce these learned vocalizations; HVC, RA, and X are small in volume, and HVC and RA are not synaptically connected. We have shown that estrogen treatment during development causes females to learn and produce male-like vocalization. This article describes the neural masculinization of these E2 females, replicating and extending the work of others. Female zebra finches were treated with 17β-estradiol (E2) at hatching, at 14–22 days of age, or as adults. In adulthood, the volumes of nucleus RA and area X were measured and the efferent projections of nucleus HVC examined using the anterograde tracer PHA-L. Early, sustained E2 treatment caused the greatest increase in the volume of RA and X, the innervation of RA and X by HVC axons, and the masculinization of auditory responses of cells in RA. Treatments that lasted for a shorter period or started later in development resulted in different patterns of partial brain masculinization. E2 treatment in adulthood had no effect on the volume of RA or X or their innervation by HVC. Bilateral lesions of the tracheosyringeal nerves or of HVC had the same effects on the male-typical vocalizations produced by E2 females as they do on the vocalizations produced by males. These results demonstrate that the neural masculinization of telencephalic nuclei induced by E2 treatment sets up a functional circuit in females similar to one in males that enables the learning and production of complex vocalizations.     

Genetic transformation of apple (Malus pumila Mill.) using a disarmed Ti-binary vector

The disamed Ti-binary vector pBIN 6 in Agrobacterium tumefaciens has been used in leaf disc transfomations to produce transgenic apple (Malus pumila Mill.) plants with a nomal phenotype except for a somewhat reduced capacity to root. The presence of the genes for nopaline synthase and neomycin phosphotrans ferase (conferring kanamycin resistance), inserted into the host genome by the vector, was confirmed by Southern blot analysis, the detection of nopaline synthase activity and rooting in the presence of the antibiotic.The nopaline synthase gene continued to be expressed in glasshouse-grown plants several months after removal from in vitro growth conditions.     

Triacylglycerol fatty acid and sterol composition of sediment microorganisms from McMurdo Sound,Antarctica

Summary The triacylglycerol fatty acid and sterol profiles of microorganisms from three McMurdo Sound sediment sites, collected during the austral summer of 1984–1985, were determined using gas chromatography and gas chromatography-mass spectrometry. Comparison of the three sites indicated that Cape Evans contained the greatest concentration of triacylglycerol (TG) (220 nmoles/gram dry weight (gdw) of sediment), approximately six to seven times that determined for sediment microorganisms from the Cape Armitage and New Harbor sites. The relative proportion of triacylglycerolderived polyunsaturated fatty acids (PUFA) revealed a somewhat different trend. New Harbor sediment contained the greatest relative proportion of PUFA (22% of triacylglycerol fatty acids), followed by Cape Evans (16%) and Cape Armitage (11%). The proportion of unsaturated fatty acids (poly-and monounsaturated) was relatively constant and ranged from 63% to 71% of the triacylglycerol fatty acids for the three sites. Sterol concentrations varied from 610 pmoles/gdw at Cape Evans, to 370 and 240 pmoles/gdw for Cape Armitage and New Harbor respectively, and was approximately 1% of the total determined lipid. Cholesterol was the major sterol component detected, occurring at similar relative levels (29%) for all three sites. Other sterols present in decreasing order of abundance were 22-dehydrocholesterol, brassicasterol, 24-ethylcholesterol and 24-methylcholesterol. 5-stanols were only minor components of the three sediments, indicating that in situ biohydrogenation of stenols was not a major sterol transformation process in these recent surface oxic sediments.Part 3 in the series: Microbial Ecology in Antarctic Sea-Ice and Benthic Communities