Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids

The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors. Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination.     

Role of gap junctions in lung neoplasia

Reduced gap junctional intercellular communication (GJIC) has been noted in many types of neoplastic cells and may contribute to the neoplastic phenotype. This study assessed GJIC (by fluorescent dye-coupling) and gap junction protein (connexin) expression in mouse and human lung carcinoma cell lines and investigated whether reduced GJIC was involved in their neoplastic phenotype. Dye-coupling and connexin43 (Cx43) expression were much lower in most of the carcinoma lines (16 of 22) compared to nontransformed lung epithelial cells. Other connexins were not detected. A poorly communicating mouse lung carcinoma cell line (E9) was transfected with Cx43 or transduced with Cx32 and several stable clones were isolated that had 2- to 4-fold increased dye coupling. When evaluated for growth in vitro, the population doubling times were increased and the saturation densities were decreased in the clones. When assessed for tumorigenicity, the parental E9 cells formed tumors with a 100% incidence (6/6 mice), whereas the clones varied in tumorigenic response (0-88% incidence). The best communicating clone (E9-2) was not tumorigenic. The highly communicating Cx32 clone, E9/32-9, gave a tumor incidence of 88%. These results suggest that restoration of GJIC by forced connexin expression can reduce the growth and tumorigenicity of lung carcinoma cells in a connexin-specific manner.     

Leprosy (Hansen''s Disease): Summary notes

This article takes three different approaches to the question of whether the failing heart is in an energy-starved state. A brief historical overview introduces the issue and points out problems in both models and methods. Second, current information regarding the energetic state of the failing heart is examined. Finally, the mechanistic and therapeutic implications of a defect in energy production are described.     

Effect of the selective phosphodiesterase type 5 inhibitor sildenafil on erectile dysfunction in the anesthetized dog

The role of the sensitivity of bone marrow cells to, and the pharmacokinetics of granulocyte colony-stimulating factor (G-CSF) on the rhythm of leukocyte-increasing effect was investigated in ICR male mice housed under a standardized light-dark cycle (lights on at 0700, off at 1900). A significant circadian rhythm was demonstrated for leukocyte counts at 24 hr after G-CSF (250 microg/kg, s.c.) injection at six different circadian times (P < .01). The leukocyte counts of mice given G-CSF at 0500, 0900, 1300 or 1700 were significantly higher than those of mice given G-CSF at 2100 (P < .01, respectively). The rhythmic pattern resembled overall the rhythm occurring after saline injection. In the comparison between leukocyte counts after G-CSF injection at 0700 and 1900, the time when leukocyte counts are equal in nondrugged state, the leukocyte counts at 24 hr after G-CSF (250 microg/kg, i.v.) injection were approximately 50% higher in mice injected with the drug at 0700 than at 1900 (P < .01). Bone marrow cultures obtained at two times of day resulted in different numbers of myeloid colonies even when treated with the same concentrations of G-CSF in vitro. The colony-forming activity of G-CSF was significantly more potent at 0700 than at 1900 (P < .01). The plasma G-CSF concentrations after G-CSF (250 or 5 microg/kg, i.v.) injection were significantly higher in mice receiving injections with the drug at 0700 than at 1900 (P < .05, respectively). The area under the curve and mean residence time were significantly larger in mice injected with the drug at 0700 than at 1900 (P < .01, P < .05, respectively). Our suggests that the rhythm of G-CSF activity is caused by that of the sensitivity of bone marrow cells to, and the pharmacokinetics of the drug.     

The effect of Magic Angle Spinning on proton spin-lattice relaxation times in some organic solids

Proton spectra of solids are usually broadened by strong proton homonuclear dipolar interactions. However, substantial line narrowing may be achieved by Magic Angle Spinning (MAS) in systems of low proton density or in systems in which rapid molecular motions occur. In such conditions, T1(H) measurements are often used to characterise the dynamics of each resolved proton site. We show that T1(H) values measured for solid organic compounds with high proton abundance, such as adamantane and glycine, may be strongly dependent on the spinning rate employed, so that care is required when values are compared. The effects of molecular motion and proton density on T1(H) and its dependence on spinning rate were investigated. We found that an increase in molecular motion leads to an increase of T1(H) at higher spinning rates. The opposite is found for systems with low proton densities which show relatively lower T1(H), at higher spinning rates. A possible interpretation is suggested in terms of the reduced spin diffusion efficiency at higher spinning rates.     

Osteolysis associated with cemented total knee arthroplasty

Osteolysis has not been mentioned as a complication or cause of failure of cemented total knee arthroplasties in long-term follow-up studies. We are aware of a single case report of osteolysis after cemented total knee arthroplasty. We report the case of an 87-year-old woman with massive osteolysis beneath a cemented tibial component.