Efficient lipid-mediated transfection of DNA into primary rat hepatocytes

Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids, however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic (EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio. Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions for transfecting 5 μg of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration of 15 μg DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical, easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes.     

Genetic transfer of clumping phenotype inAnacystis nidulans

Occasionally a mutation occurs in liquid cultures ofAnacystis nidulans, spreading quickly through the population and causing cells to adhere together in clumps. This phenotype is stable indefinitely and is an inherited characteristic of all cells within a clumping culture. Inoculation with a few living cells from a clumping culture quickly produces the clumping genotype in a majority of cells within a previously non-clumping culture. Killed cells, broken cell extracts, or media from clumping cultures do not produce aggregation in non-clumping cultures. Actively growing cells in clumping cultures do not affect non-clumping cultures when separated by 0.4 μm Millipore filter. Apparently transfer of the clumping trait requires direct contact between living cells. Pili-like projections connect individual cells within clumps, but no slime layer or capsule is seen. Clumps can be dispersed without cell damage; reaggregation requires photosynthesis.     

Effects of acute and chronic restraint and estrus cycle on pituitary-adrenal function in the rat

Female Long-Evans hooded rats with 5-day estrus cycles were subjected to 4 hr of continuous restraint for either 1 or 20 days. On the last day of the stress regimen, plasma and adrenal corticosterone concentrations were determined and classified according to the stage of the estrous cycle. The results indicated that acute stress produced greater plasma corticosterone concentrations than controls only during estrus, whereas in response to chronic stress significant stress-induced increments were observed during estrus and proestrus. The results suggest that the estrous cycle influences the magnitude of the stress-induced increments for both acute and chronic stress. In addition, the pituitary-adrenal system did not show habituation to repeated administration of this stress, but sensitization was observed during proestrus.     

Membrane and protein properties of freeze-dried mouse platelets

Membrane properties and the overall protein secondary structure of freeze-dried trehalose-loaded mouse platelets were studied using steady state fluorescence anisotropy and Fourier transform infrared spectroscopy (FTIR). FTIR results showed that fresh control mouse platelets have a main phase transition at ~14°C, whereas, freeze-dried platelets exhibited a main phase transition ~12°C. However, the cooperativity of the transition of the rehydrated platelets was greatly enhanced compared to that of control platelets. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed that the lipid order in the core of the membrane was affected by freeze-drying procedures. Similar experiments with trimethyl ammonium 1,6 diphenyl-1,3,5 hexatriene (TMA-DPH), a membrane surface probe, indicated that membrane properties at the membrane/water interface were less affected by freeze-drying procedures than the core of the membrane. Lyophilization did not result in massive protein denaturation, but the overall protein secondary structure was altered, based on in situ assessment of the amide-I and amide-II band profiles. Lyophilization-induced changes to endogenous platelet proteins were further investigated by studying the protein's heat stability. In fresh control platelets, proteins denatured at 42°C, whereas proteins in the rehydrated platelets denatured at 48°C.     

Melanoma central nervous system metastases: An update to approaches,challenges, and opportunities

In February 2018, the Melanoma Research Foundation and the Moffitt Cancer Center hosted the Second Summit on Melanoma Central Nervous System (CNS) Metastases in Tampa, Florida. In this white paper, we outline the current status of basic science, translational, and clinical research into melanoma brain metastasis development and therapeutic management. We further outline the important challenges that remain for the field and the critical barriers that need to be overcome for continued progress to be made in this clinically difficult area.     

Dynamics of cytokine production during coccidial infections in chickens: colony-stimulating factors and interferon

Abstract We assayed two classes of immunoregulatory cytokines, colony-stimulating factors (CSF) and interferon (IFN), during and immediately after a primary coccidial infection in chickens. Coccidial infection induces significant alterations in serum colony-stimulating activity (CSA) and these alterations immediately precede the characteristic biphasic leukocytosis. CSA rose sharply during the first 24 h post-inoculation (PI), but returned to control levels by 48 h PL. At this time, we detected an increase in peripheral blood leukocytes which peaked at 96 h PI. A second phase of CSA increase began 96 h PI and peaked at 120–144 h PI which again preceded the second phase of leukocytosis. We also examined the production of IFN during the first 20 days PI. Splenic T cells from Eimeria maxima -infected chickens produced significantly less IFN on day 5 PI compared to T cells from the coccidia-free controls. By days 10 and 15 PI, there was no significant difference in IFN production between the T cells of infected and non-infected chickens. However, by day 20 PI, IFN production by the T cells of the infected birds produced significantly more IFN than the control T cells. The results of our studies indicated the differential production of two different cytokines by chickens during and following a primary coccidial infection. Based on these experiments, CSF may be some of the first cytokines produced during an E. maxima -infection, while IFN may be one of the later cytokines produced.     

Language revitalization in Native North America--issues of intellectual property rights and intellectual sovereignty

Language revitalization, oral tradition and epistemology are expressions of Native peoples intellectual sovereignty, and thus the foundation for indigenous intellectual property rights. As the people of California move towards language and cultural revitalization the question arises: What constitutes or constructs the definitions of intellectual property and how can appropriation of indigenous knowledge be protected? Looking at the issues faced by the California's indigenous populace and by implication, other indigenous peoples in the United States, this essay examines how protection may be afforded under the United Nations definition of 'heritage'. Given that the holding safe of a 'culture' or 'heritage' is inclusive of language, and thus has been determined to be a human right.     

Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of 340 and 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The K_m of TID for indene was 380 M. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The K_m of TID for naphthalene was 28 M. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.