Delivery of DNA into mammalian cells by receptor-mediated endocytosis and gene therapy

The correction of genetically based disorders by the introduction of a therapeutic genetic construct into the appropriate cell type (“gene therapy”), has become a distinct possibility in recent years. In order for gene therapy to be a practical alternative to more conventional pharmaceutical approaches to treatment, it must be administrable in vivo. This demands that a system be developed that can specifically target the DNA to the desired cell type once introduced into the patient. Among the procedures that are currently being pursued, the delivery of DNA to cells by receptor mediated endocytosis (RME), comes closest to fulfilling this crucial requirement. The natural physiological process of RME can be exploited to deliver genetic material to cells. An antibody or ligand to a cell surface receptor that is known to undergo endocytosis, is complexed with DNA through a covalently linked polycationic adjunct (e.g., polylysine, protamines). Such complexes retain their binding specificity to the cell surface and are taken up into the cell where they enter the endosomal compartment via normal endocytotic processes. In addition, steps must be taken to avoid degradation of the DNA within the endosome-lysosome. Cells can be treated with the lysosomatropic agent chloroquine during the transfection procedure. Alternatively, the components of viruses that enter cells by endocysis and possess an endosomal “break out” capacity can be used. Replication defective adenovirus coupled to the ligand-DNA complex gives transfection efficiencies of virtually 100% on tissue culture cells in vitro. Synthetic peptides that mimic the membrane fusing region of influenza virus hemagglutinin, have also been successfully used as part of the ligand-DNA complex to bring about endosomal escape. Preliminary studies have demonstrated the potential of this method to specifically target DNA to the cell type of choice in vivo. Delivery of genes by receptor-mediated endocytosis offers the greatest hope that gene therapy can be an inexpensive, easily applicable, widespread technology.     

Further development of a transcervical technique for artificial insemination in sheep using previously frozen semen

A transcervical technique (the Guelph System for transcervical AI) was used to inseminate 2060 ewes on 65 farms (average 31 ewes, range 5 to 107) in Ontario, Canada, from October 1990 to September 1992, using previously frozen semen. Estrus was synchronized using progestagen pessaries and PMSG with median inseminations done at 54 h from pessary removal. Maiden ewes were not included. Only ewes in which the cervix could be penetrated were inseminated with 150 million spermatozoa per insemination. A total of 1809 were penetrated and inseminated (penetration rate 87.8%). Success of penetration increased from 76.3% in the first 500 ewes to 97.9% in the last 500 (P=0.01). Cervical penetration was more successful in ewes in the accelerated lambing program (92.3%, average 3.1 mo since the previous lambing) than those in the annual lambing program (82.4%, average 7.0 mo since the previous lambing; P=0.06). The lambing rate for ewes bred during the combined traditional breeding seasons (Fall of 1990, 1991, 1992) was 50.7% compared to 24.4% for ewes bred at other periods (P=0.00001). The average time required for handling and insemination decreased from 8.62 min in the first 500 ewes to 3.62 min in the last 500 ewes. The Guelph System for Transcervical AI was found to be successful for cervical penetration in most ewes. Penetration success was affected by period since the last lambing and by inseminator experience. The lambing rate was higher for ewes bred during the traditional Fall breeding seasons than during other times of the year.     

The role of Mg2+ in the hydrolytic activity of the isolated chloroplast ATPase: Study by high-performance liquid chromatography

The influences of total magnesium ion concentration at different total ATP concentrations, and of total ATP concentration, for different total magnesium ion concentrations, on the enzymatic rate of the isolated chloroplast F₁ ATPase, have been followed by a chromatographic method consisting in the separation and determination of ADP. From the various series of curves, it is concluded that the experimental results (position of the maxima,K _m values) are better fitted by a mechanism involving the activation of the enzyme by magnesium ion and hydrolysis of free ATP, rather than by the classical mechanism, for which the enzyme hydrolyzes the MgATP complex and is inhibited by Mg²⁺. Although the equations giving the reaction rate are similar in the two cases, the calculated values ofK _m are widely different. The value obtained from the classical mechanism does not agree withK _D , the dissociation constant of the enzyme-substrate complex, measured by the Hummel and Dreyer method. Moreover, when the total ATP concentration tends toward the total magnesium ion concentration, the nucleotide binding to the enzyme tends toward zero, although it should be maximum if MgATP were the true substrate. Finally, the inhibitory effect of Na⁺ is more easily explained as a competition between this ion and the activating Mg²⁺, than by the classical mechanism.     

On the inducibility of nitrate transport by tobacco cells

The question as to whether the nitrate transport system is induced by nitrate was addressed using a cell suspension of the XD line of Nicotiana tabacum L. cv. Xanthi as an experimental system. The cells were grown on area as the sole nitrogen source, and tungstate was used to render nitrate reductase non-functional. To avoid shock due to vacuum filtration, the cells, were harvested by gravity filtration. Nitrate uptake by cells, which were harvested, transferred to fresh medium, and immediately exposed to nitrate (freshly harvested cells), displayed a lag period of about 3 h.
In cells which were given incubation periods in fresh medium before exposure to nitrate (preincubated cells), the lag period was considerably shortened. After 3 h of preincubation in the absence of nitrate (recovered cells), the lag period was almost completely eliminated. Cycloheximide inhibited nitrate uptake by recovered cells within minutes, and prevented the development of nitrate uptake in freshly harvested cells. Cycloheximide did not affect uptake of α-aminoisobutyric acid (AIB) within the first 2 h after its addition. Recovery of the membrane potential from a low value just after the harvest of the cells to a maximal value 3 h later, was observed using the lipophilic cation methyltriphenylphosphonium (MTPP⁺), supplied at low concentrations, as a probe. Depolarization of the membrane potential by MTPP⁺, at the millimolar range, caused a rapid inhibition of nitrate uptake by recovered cells. The results indicate that nitrate transport by the XD cells depends on the membrane potential and on protein components with short half life. In addition, it requires a continuous protein synthesis. The effects of physical manipulation on nitrate uptake are discussed.     

The effect of a depot injection of recombinant bovine somatotropin on follicular development and embryo yield in superovulated Holstein heifers

Superovulated Holstein heifers (n = 32) were given a depot injection of 500 mg recombinant bovine somatotropin (rBST) or vehicle at Day 4 of the estrous cycle (7 days before the first FSH injection); at Day 11, coincidentally with the first FSH injection; or at Day 15, the time of artificial insemination. Embryos were collected nonsurgically, and the number of corpora lutea was counted by ultrasonography at Day 7 after insemination. Blood samples were taken every second day, from Day 2 of the superovulatory cycle until the day of embryo collection, and were analyzed for progesterone, somatotropin and insulin-like growth factor-1 (IGF-1). Somatotropin-treated heifers at Day 11 had a significantly higher mean number of corpora lutea than the controls (18.1 vs 13.4; P 0.63), but it was negatively correlated with progesterone (P 相似文献   

Evaluation of the role of State transitions in determining the efficiency of light utilisation for CO2 assimilation in leaves

Wheat leaves were exposed to light treatments that excite preferentially Photosystem I (PS I) or Photosystem II (PS II) and induce State 1 or State 2, respectively. Simultaneous measurements of CO₂ assimilation, chlorophyll fluorescence and absorbance at 820 nm were used to estimate the quantum efficiencies of CO₂ assimilation and PS II and PS I photochemistry during State transitions. State transitions were found to be associated with changes in the efficiency with which an absorbed photon is transferred to an open PS II reaction centre, but did not correlate with changes in the quantum efficiencies of PS II photochemistry or CO₂ assimilation. Studies of the phosphorylation status of the light harvesting chlorophyll protein complex associated with PS II (LHC II) in wheat leaves and using chlorina mutants of barley which are deficient in this complex demonstrate that the changes in the effective antennae size of Photosystem II occurring during State transitions require LHC II and correlate with the phosphorylation status of LHC II. However, such correlations were not found in maize leaves. It is concluded that State transitions in C₃ leaves are associated with phosphorylation-induced modifications of the PS II antennae, but these changes do not serve to optimise the use of light absorbed by the leaf for CO₂ assimilation.Abbreviations Fm, Fo, Fv maximal, minimal and variable fluorescence yields - Fm, Fv maximal and variable fluorescence yields in a light adapted state - LHC II light harvesting chlorophyll a/b protein complex associated with PS II - q_P photochemical quenching - A₈₂₀ light-induced absorbance change at 820 nm - _{PS I}, _{PS II} relative quantum efficiencies of PS I and PS II photochemistry - _{CO 2} quantum yield of CO₂ assimilation     

MAP kinase kinase: A node connecting multiple pathways

Summry— Numerous studies have been published these last few years on the involvement of MAP kinases in signal transduction reflecting their importance in cell cycle and cell growth controls. The identification and the characterization of their direct upstream activator has considerably enlarged our understanding of the phosphorylation network. The MAP kinase kinases (MAPKKs) are dual-specificity protein kinases which phosphorylate and activate MAP kinases. To date, MAPKK homologues have been found in yeast, invertebrates, amphibians, and mammals. Moreover, the MAPKK/MAPK phosphorylation switch constitutes a basic module activated in distinct pathways in yeast and in vertebrates. MAPKK regulation studies have led to the discovery of at least four MAPKK convergent pathways in higher organisms. One of these is similar to the yeast pheromone response pathway which includes the ste11 protein kinase. Two other pathways require the activation of either one or both of the serine/threonine kinase-encoded oncogenes c-Raf-I and c-Mos. Additionally, recent studies suggest a possible effect of the cell cycle control regulatory cyclin-dependent kinase 1 (cdc2) on MAPKK activity. Finally, MAPKKs seem to be essential transducers through which signals must pass before reaching the nucleus.