Serial propagation of human ovarian surface epithelium in tissue culture

Most human ovarian cancers are thought to arise in the ovarian surface epithelium (OSE). The precise role of OSE in carcinogenesis has not been defined because no appropriate animal models for the study of this tissue exist and culture of human OSE has been limited to primary outgrowths. In this report, we describe conditions for serial cultivation of normal human OSE. Premenopausal ovarian tissue was obtained at surgery. OSE growth was compared in media MCDB 202, 199 and Waymouth's 752/1 (WM) supplemented with 5, 15, or 25% fetal bovine serum (FBS), with/without 20 ng/ml epidermal growth factor (EGF) and 0.4 micrograms/ml hydrocortisone (HC). The rate and extent of OSE outgrowths from explants in primary culture were greatest in either WM or 199/202 (1:1), supplemented with 15% FBS/EGF/HC. In early passage cultures, cell proliferation was most rapid and extensive in 199/202 with 15% FBS, EGF, and HC. In this medium, OSE cells were subcultured up to 10 times and underwent 20-25 population doublings over 5 weeks. The population doubling time during rapid growth was approximately 48 h. Seeding efficiencies of up to 53% and cloning efficiencies of up to 13% were obtained. Early passage OSE cells reversibly modulated from a slow growing, epithelial, intensely keratin-positive form in 199/202 medium lacking EGF/HC, to a rapidly proliferating, elongate, less keratin-positive form in medium with EGF/HC. OSE cells grown in WM/5-15% FBS were epithelial and near-stationary. Thus, culture conditions have been defined for ovarian carcinogen assays requiring either proliferating or stationary cell populations, and for further studies of the role of OSE in ovarian biology.     

Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

Plant regeneration from mesophyll protoplasts of Matthiola incana (L.) R. Br.

Summary A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of three lines of Matthiola incana is described. Protoplasts were isolated from leaves of 21–28 days old Matthiola plants grown in controlled environment. Sustained divisions were achieved when protoplasts were embedded in sodium alginate. Up to 2.0 % of the protoplasts developed into colonies which could be transferred to shoot regeneration media. More than 25 % of the obtained calluses regenerated shoots. About 4 % of these shoots could be rooted and after transfer to soil phenotypically normal plants have been obtained.Abbreviations 2,4-D 2,4-dichlorphenoxyacetic acid - NAA naphthalene acetic acid - IAA indole-3-acetic acid - BAP 6-banzylaminopurine - IPA isopentenyladenine - IPAR isopentenyladenosine - MES (2-[N-morpholino]) ethanesulfonic acid     

Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain

Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30-40% using the synthetic detergent 3[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate. Kinetic analysis employing the high-affinity antagonist 125I-Sar1,Ile8-Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r = 0.99) between the respective pIC50 values of a series of agonist and antagonists competing for 125I-Sar1,Ile8-Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I-Ang II to solubilized receptors with the homo-bifunctional cross-linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross-linking of 125I-Ang II to solubilized brain membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I-Sar1,Ile8-Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isoelectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.     

Differential effects of pentobarbital and ethanol in mice

The duration of the loss of righting reflex (RR) after ethanol, 4 g/kg, intraperitoneally (i.p.), was significantly longer in “long-sleep” (LS) than in “short-sleep” (SS) mice. This effect was shown to be correlated with differences in brain sensitivities to ethanol. In contrast, pentobarbital sodium (PB), 50 mg/kg, i.p., produced a significantly longer loss of RR in SS than in LS mice. The PB concentrations in the brain were the same in both mouse strains at the time of RR recovery suggesting equal sensitivities of the central nervous systems to PB. The rates of disappearance of PB from the blood were the same in both strains, but the apparent volume of distribution of PB in the LS strain was greater than in SS mice.In addition, C57BL/6J mice were found to be more sensitive than DBA/2J mice to PB, 50 mg/kg. In contrast, C57BL mice are known to be less sensitive than the DBA strain to ethanol. The PB concentration in the brain of DBA mice at the recovery of the RR was significantly greater than in C57BL mice. The apparent volumes of distribution of PB were not different in the two strains, but the rate of disappearance of PB from the blood of C57BL mice was significantly greater than for the DBA strain. In conclusion, factors which govern the brain sensitivities of selected mouse strains to ethanol and pentobarbital may not be equivalent.     

Activation of phosphatidylinositol-3 kinase,AMP-activated kinase and Akt substrate-160 kDa by trans-10, cis-12 conjugated linoleic acid mediates skeletal muscle glucose uptake

Conjugated linoleic acid (CLA), a dietary lipid, has been proposed as an antidiabetic agent. However, studies specifically addressing the molecular dynamics of CLA on skeletal muscle glucose transport and differences between the key isomers are limited. We demonstrate that acute exposure of L6 myotubes to cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12) CLA isomers mimics insulin action by stimulating glucose uptake and glucose transporter-4 (GLUT4) trafficking. Both c9,t10-CLA and t10,c12-CLA stimulate the phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit and Akt substrate-160 kDa (AS160), while showing isomer-specific effects on AMP-activated protein kinase (AMPK). CLA isomers showed synergistic effects with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Blocking PI3-kinase and AMPK prevented the stimulatory effects of t10,c12-CLA on AS160 phosphorylation and glucose uptake, indicating that this isomer acts via a PI3-kinase and AMPK-dependent mechanism, whereas the mechanism of c9,t11-CLA remains unclear. Intriguingly, CLA isomers sensitized insulin-Akt-responsive glucose uptake and prevented high insulin-induced Akt desensitisation. Together, these results establish that CLA exhibits isomer-specific effects on GLUT4 trafficking and the increase in glucose uptake induced by CLA treatment of L6 myotubes occurs via pathways that are distinctive from those utilised by insulin.     

Evolutionary constraint on low elevation range expansion: Defense‐abiotic stress‐tolerance trade‐off in crosses of the ecological model Boechera stricta

Most transplant experiments across species geographic range boundaries indicate that adaptation to stressful environments outside the range is often constrained. However, the mechanisms of these constraints remain poorly understood. We used extended generation crosses from diverged high and low elevation populations. In experiments across low elevation range boundaries, there was selection on the parental lines for abiotic stress‐tolerance and resistance to herbivores. However, in support of a defense‐tolerance trade‐off, extended generation crosses showed nonindependent segregation of these traits in the laboratory across a drought‐stress gradient and in the field across the low elevation range boundary. Genotypic variation in a marker from a region of the genome containing a candidate gene (MYC2) was associated with change in the genetic trade‐off. Thus, using crosses and forward genetics, we found experimental genetic and molecular evidence for a pleiotropic trade‐off that could constrain the evolution of range expansion.     

Cutting edge: restoration of the ability to generate CTL in mice immune to adenovirus by delivery of virus in a collagen-based matrix

Viruses are commonly used for the delivery of genes coding for tumor-associated Ags to elicit tumor-specific immune responses. The success of viral vectors has been limited in preclinical and clinical trials in part because of antiviral immunity. We investigated the ability of a collagen-based matrix (Gelfoam; Pharmacia and Upjohn, Kalamazoo, MI) to improve CTL activation by recombinant adenovirus. The data show that coinjection of Gelfoam with type 5 adenovirus recombinant for prostate-specific Ag (Ad5-PSA) enhanced CTL activation. Ad5-PSA priming in Gelfoam also abrogated the inhibitory effects of adenoviral immunity on CTL activation in mice naive to PSA but immune to adenovirus. Finally, Gelfoam enhanced immunization in a self-Ag model using type 5 adenovirus recombinant for membrane-bound OVA (Ad5-mOVA) in rat insulin promoter (RIP)-mOVA-transgenic mice. Thus, Gelfoam enhances CTL activation by recombinant viral vectors in a setting where preformed Ab to the virus is present and also in a tolerant self-Ag model.