Bicistronic expression of ecdysone-inducible receptors in mammalian cells.

The recent emergence of inducible expression systems for mammalian cells has greatly facilitated the in vivo analysis of gene function. The ecdysone-inducible expression system is particularly attractive because of (i) extremely low basal expression and high-level induced expression, (ii) the lack of pleiotropic effects caused by the inducer or activator, and (iii) the rapid penetrance and clearance of the inducer. Here, we describe an improved receptor expression vector. The required ecdysone receptor proteins (VgEcR and RXR) are co-expressed from a bicistronic cytomegalovirus (CMV) expression cassette in the vector pERV3. The CMV promoter in this vector can be readily replaced with a cell type-specific promoter of interest. Using the ecdysone analogs, muristerone A or ponasterone A, induction ratios of up to three orders of magnitude were attained in the transient transfection assays and in a cell line stably transformed with both pERV3 and an ecdysone-inducible reporter vector. Fine control of luciferase expression was achieved bv varying both the induction time and inducer concentration. Here, we describe a set of cell lines stably transformed with the vector pERV3, in which the ecdysone receptors are expressed at optimal levels for the high-level induction of gene expression.

The digital transformation of oral health care. Teledentistry and electronic commerce.

BACKGROUND: Health care is being changed dramatically by the marriage of computers and telecommunications. Implications for hospitals and physicians already have received extensive media attention, but comparatively little has been said about the impact of information technology on dentistry. This article illustrates how the digital transformation will likely affect dentists and their patients. CONCLUSIONS: Based on recent experiences of hospitals and medical practices, dentists can expect to encounter revolutionary changes as a result of the digital transformation. The Internet, the World Wide Web and other developments of the information revolution will redefine patient care, referral relationships, practice management, quality, professional organizations and competition. PRACTICE IMPLICATIONS: To respond proactively to the digital transformation of oral health care, dentists must become familiar with its technologies and concepts. They must learn what new information technology can do for them and their patients and then develop creative applications that promote the profession and their approaches to care.

Epidermal lesions and mortality caused by vibriosis in deep-sea Bahamian echinoids: a laboratory study.

When significant mortality of the bathyal spatangoid echinoid Paleopneustes cristatus occurred under laboratory conditions, we investigated the cause and course of the disease by culturing and identifying internal pathogens, then experimentally infecting healthy urchins with isolates of the suspected disease organism. The pathogen was determined to be the gram-negative halophilic bacterium Vibrio alginolyticus. This species was also recovered from frozen post-challenge specimens of P. cristatus and from moribund individuals of Archaeopneustes hystrix, another spatangoid reared under similar in vitro conditions. This is the first experimental study of bacterial disease in any deep-sea invertebrate.

An Escherichia coli expression vector that allows recovery of proteins with native N-termini from purified calmodulin-binding peptide fusions.

We describe a T7-based Escherichia coli expression vector in which protein coding sequence is seamlessly fused to the N-terminal calmodulin-binding peptide (CBP) purification tag. We combined the use of the site-specific protease enterokinase (EK) and the type IIs restriction enzyme Eam1104 I, which cleave outside their respective (amino acid and nucleotide) target sequences, such that any amino acid sequence may be fused directly C-terminal to the EK cleavage site without codon constraints conferred by the cloning method. PCR products are cloned using ligation-dependent or ligation-independent methods with high cloning efficiencies (>10(6) cfu/microg vector), allowing production of insert quantities sufficient for several cloning experiments with a limited number of PCR cycles, resulting in a significant time-savings and reduced likelihood of accumulating PCR-derived mutations. CBP fusion proteins are expressed to high levels when the CBP peptide is positioned at the N-terminus. CBP binds to calmodulin with nanomolar affinity, and fusion proteins are purified to near homogeneity from crude extracts with one pass through calmodulin affinity resin using gentle binding and elution conditions. We show high efficiency seamless cloning of three inserts into the pCAL-n-EK vector, including one encoding the protein c-Jun N-terminal kinase (JNK). CBP-EK-JNK fusion protein was synthesized to 10-20 mg/liter culture and purified to near homogeneity in one step with calmodulin affinity resin. The fusion tag was efficiently removed with EK to yield active JNK with native N-terminal amino acid sequence.

Using Schizosaccharomyces pombe as a host for expression and purification of eukaryotic proteins.

We have established a eukaryotic protein expression and purification system by using the yeast Schizosaccharomyces pombe as the host and the glutathione S-transferase (GST) as a protein purification tag. This system provides opportunities for rapid, inexpensive, and high yield production of proteins in a eukaryotic organism. Unlike E. coli, S. pombe provides for post-translational modifications of the proteins, which are often critical for the structure and function of eukaryotic proteins. Two vectors have been constructed for protein expression in S. pombe, pESP-1 and pESP-2. Both vectors use the nmt1 promoter for constitutive or induced expression of the gene of interest. Expressed GST-tagged proteins are easily and rapidly purified using glutathione agarose beads. The GST tag can be removed from the fusion proteins by treatment with either the thrombin or enterokinase protease. Proteins expressed from the pESP-2 vector will yield native amino acid sequence when the GST tag is removed by treatment with enterokinase. Nine proteins have been purified by using the system with yields ranging from 1.0 mg/l to 12.5 mg/l of induced culture.

Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction.

We have developed a facile procedure for rapid PCR-based site-directed mutagenesis of double-stranded DNA. Increasing the initial template concentration and decreasing the PCR cycles to 5-10 allows us to reduce the rate of undesired second-site mutations and dramatically increase the time savings. Following PCR, DpnI treatment is used to select against parental DNA molecules. The DpnI (target sequence 5'-Gm6ATC) is specific for methylated and hemimethylated DNA and is used to digest parental DNA and select for mutation-containing amplified DNA. DNA isolated from almost all common Escherichia coli strains is Dam methylated and therefore susceptible to DpnI digestion. Pfu DNA polymerase is used, prior to intramolecular ligation of the linear template, to remove any bases extended onto the 3' ends of the PCR product by Taq DNA polymerase. The recircularized vector DNA incorporating the desired mutations is transformed into E. coli. This method can be used independently of any host strain and vector.

Clinical program renovation management.

The day-to-day management of medical practice is so heavily influenced by tactical imperatives that few physicians have the time to think strategically. Traditionally, the difference between tactics and strategy is not a major concern of most physician executives. The realm of tactics is the short run, when managers must make do with the fixed resources at hand. Strategy addresses the long run, when all resources and markets are variable. A tactical focus is understandable, maybe even acceptable, in industries where nothing challenges traditional approaches to production of an established product or service. However, medical care in the 1990s is changing so fast that a physician executive must devote an incredible amount of time and effort just to stay confused about what is going on.

The future of the rural hospital: assessing the alternatives.

Rural hospitals have new opportunities to create health systems that mesh with their own limitations and potential. Trustees who see the provision of high quality health care as their ultimate responsibility (and not the maintenance of a building with beds) will help their hospitals successfully meet the health needs of rural America.