Self-deleting suicide vectors (SDSV): selective killing of p53-deficient cancer cells.

A self-deleting retrovirus vector carrying a herpes simplex virus (HSV)-thymidine kinase suicide gene has been developed to selectively kill cancer cells expressing a dysfunctional p53 tumor suppressor protein. When cells containing functional p53 are infected with the virus, the integrated provirus and the HSV-thymidine kinase gene are deleted from the genome by site-specific recombination (Cre/loxP). In contrast, cells without p53 or cells expressing a DNA-binding mutant of p53 retain the provirus and become susceptible to killing by ganciclovir. This strategy provides a new concept for the selective killing of cancer cells that can be adapted to any other dysfunctional transcription factor expressed by different tumors.

Mutations in the novel protocadherin PCDH15 cause Usher syndrome type 1F.

We have determined the molecular basis for Usher syndrome type 1F (USH1F) in two families segregating for this type of syndromic deafness. By fluorescence in situ hybridization, we placed the human homolog of the mouse protocadherin Pcdh15 in the linkage interval defined by the USH1F locus. We determined the genomic structure of this novel protocadherin, and found a single-base deletion in exon 10 in one USH1F family and a nonsense mutation in exon 2 in the second. Consistent with the phenotypes observed in these families, we demonstrated expression of PCDH15 in the retina and cochlea by RT-PCR and immunohistochemistry. This report shows that protocadherins are essential for maintenance of normal retinal and cochlear function.

Interleukin 3 improves the ex vivo expansion of primitive human cord blood progenitor cells and maintains the engraftment potential of scid repopulating cells.

In umbilical cord blood (UCB) transplantation, the number of nucleated cells per kilogram is a major predictive and critical factor of hematopoietic recovery. Thus, ex vivo expansion of hematopoietic UCB progenitors could potentially accelerate engraftment. Whereas Flt-3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO) are considered indispensable, the role of interleukin 3 (IL-3) is still controversial: it has been reported either to support or abrogate the reconstituting ability of stem cells. By adding IL-3 we aimed to enhance the amplification of early and committed progenitor cells without impairing the long-term engraftment of stem cells. Demonstrating a positive impact of IL-3 on the proliferation of all progenitor subsets, the amplification of CD34+ UCB cells was increased 20.9-fold +/- 5.4 (mean +/- standard error) in serum-free culture with FL, SCF, TPO, and IL-3 as opposed to 9.3-fold +/- 3.2 without IL-3 after 7 days. If IL-3 was included, primitive long-term culture-initiating cells and committed colony-forming cells were expanded 16.3-fold +/- 5.5 and 18.1-fold +/- 2.4, respectively, compared to 12.6-fold +/- 5.6 and 9.1-fold +/- 2.0 without IL-3. Analysis of cultured CD34+ UCB cells in sublethally irradiated nonobese diabetic/severe combined immunodeficient mice confirmed that cultured cells had preserved their repopulating potential. After 6 weeks, all mice showed multilineage engraftment with their bone marrow containing an average of 45% human CD45+ cells of the unmanipulated sample, 43% of cells after culture in the presence of IL-3, and 27% of cells after culture without IL-3. In combination with early acting cytokines, IL-3 therefore improves the ex vivo expansion of UCB stem and progenitor cells without impairing their engraftment potential.

Yeast SUB1 is a suppressor of TFIIB mutations and has homology to the human co-activator PC4.

Activation of transcription in eukaryotes depends upon the interplay between transcriptional activators and general transcription factors. While direct contacts between activators and general factors have been demonstrated in vitro, an additional class of proteins, termed co-activators, is also required of transcriptional activation. Here we describe a yeast protein, SUB1, that was isolated as a suppressor of the cold-sensitive TFIIB R78H mutant. The N-terminal third of SUB1 is highly similar to the mammalian co-activator PC4. We show that increased expression of SUB1 suppresses two alleles of TFIIB (E62G, R78H) specifically and that the deletion of SUB1 is lethal in combination with these same two alleles. We show that SUB1 binds to TFIIB in vitro and that it specifically inhibits the formation of TBP-TFIIB-promoter complexes. Furthermore we show that increasing the copy number of SUB1 stimulates transcriptional activation in vivo. Based on our results and recent observations of others, we propose that SUB1 plays a role in the release of TFIIB from the transcription complex during transcription initiation.

Context-dependent effects of upstream A-tracts. Stimulation or inhibition of Escherichia coli promoter function.

Phased A-tract sequences were inserted in the upstream region of three synthetic promoters known to encompass different rate-limiting steps within the pathway of RNA polymerase-promoter interaction (Ellinger et al., accompanying paper). Promoter PS1, which is rate-limited in complex formation, was stimulated by A-tracts in vivo. Permanganate probing showed that the stimulation is due to an enhanced ability to compete for limiting RNA polymerase in vivo, leading to the increased formation of open complexes. By contrast, promoters PS2 and PS3, which are rate-limited in steps following open complex formation, were inhibited in vivo by A-tracts. Permanganate probing showed that the inhibition was accompanied by an A-tract-dependent accumulation of stalled initial transcribing complexes. A single A-tract was as effective as three. The phasing of the A-tracts with respect to the core promoter sequence was found to be important for promoter function. The position that caused maximal activation at one promoter caused maximal inhibition at another. These results suggest that the same molecular interaction gives rise to both inhibition and activation. This is likely to be due to facilitated RNA polymerase binding in the presence of A-tracts, which stimulates binding-limited promoters but inhibits promoter function in which polymerase escape and promoter clearance is rate limiting.

Laparoscopic nephrectomy: the learning-curve experience.

OBJECTIVE: To report the learning-curve experience with laparoscopic nephrectomy. DESIGN: Case series. SETTING: A tertiary-care referral centre serving southern Saskatchewan. PATIENTS: Sixteen patients (7 men, 9 women), selected to undergo laparoscopic nephrectomy. They ranged in age from 19 to 83 years. Indications for surgery were: recurrent atrophic pyelonephritis with pain (three patients), obstruction at the ureteropelvic junction with pain (three), small ectopic kidney with pain (one), renovascular hypertension (two), a solid renal mass confirmed by computed tomography (four), Staghorn calculus (one), transitional cell tumour of upper ureter (one) and pyonephrosis with a nonfunctioning kidney (one). INTERVENTIONS: Laparoscopic nephrectomy. MAIN OUTCOME MEASURES: Postoperative morbidity, complications of the procedure and duration of postoperative hospitalization. RESULTS: Kidneys were removed laparoscopically in 13 patients. Open nephrectomy was necessary in three patients, owing to a lack of experience in patient selection in two cases and to intraoperative hemorrhage in the third. All patients resumed oral intake on the 1st postoperative day, and most did not require analgesics for relief of pain beyond 36 hours postoperatively. Complications of laparoscopic nephrectomy included pneumonia (one patient), low-grade fever (two patients), need for blood transfusion (three patients) and transient ischemic attack (one patient). The mean postoperative hospital stay was 4.3 days. CONCLUSIONS: When patients are properly selected, laparoscopic nephrectomy provides decreased postoperative morbidity, a shorter convalescence, and thus cost savings, compared with open nephrectomy.

The Saskatchewan Clinical Stroke Prevention Project: design.

The Saskatchewan Clinical Stroke Prevention Project aims to examine the process and impact of incorporating enhanced stroke prevention into the routine clinical practice of family physicians. Twenty-four physicians in three practices in Saskatchewan are participating in a staged series of educational interventions over two years to enhance their management of smoking, transient ischemic attack/stroke, atrial fibrillation and hypertension. The components of each intervention include a seminar, printed materials, a one-to-one case discussion or "academic detailing," a self-documented chart audit and changes to the office system. Patients for whom this intervention is targeted are those 55 to 75 years of age presenting for a periodic health examination with one or more of the four main risk factors for stroke. Intervention will consist of counselling on relevant risk factors by the doctor and nurse teams, supported by appropriate patient education materials. Patient follow-up will be carried out according to clinical indications and will be done at least 3, 6, 12 and 24 months after entry into the study. Evaluation comprises measures of the process of implementing change in preventive practice as well as of the impact of such change. The former includes semi-structured interviews and focus groups with physicians, support staff and patients. The latter includes an assessment of knowledge, attitudes and charted practice before and after intervention.

A primer on graduate medical education financing.

Medical education is recognized as a legitimate cost of doing business in a teaching hospital. This article outlines the general principles and concepts currently used to determine the actual amount of financial reimbursement that a hospital receives for its graduate medical education (GME) programs. Directors of Medical Education should have a solid understanding of these principles if they are to work successfully with the hospital reimbursement experts to maximize the teaching hospital's revenue. To that end, the authors detail 10 rules to ensure that all essential elements are included in the reimbursement formulas. Also considered are the nonfinancial benefits of GME of which hospital leadership must be aware so that they may understand the total contribution that GME makes to the teaching hospital.

Insect blood meal studies using radiosodium 24Na and 22Na.

Rabbits were infused with 3.7 GBq (100 mCi) of [24Na]Na ion in a 100-ml sodium carbonate solution. Beta particles were detected using a Tennelec Counting System; background counts were 1.6 +/- 1 counts per minute (cpm). Counts for one nanoliter of blood ranged from 22 to 30 cpm. Blood volumes on the mouthparts of tabanids following a 15-sec interrupted feeding were estimated to be 12.5 nl for Tabanus fuscicostatus, 10.8 nl for T. nigrovittatus and 6.12 nl for Chrysops fuliginosus. Estimates of the quantity of blood adhering to 22-gauge needles and insect pins (size 2) following a percutaneous intramuscular needle-stick were 8.8 +/- 1.0 nl and 5.7 +/- 1.8 nl, respectively. Mosquitoes, Aedes aegypti females, were fed to repletion with a 22Na-artificial diet, and radioactivity was measured using a Packard Autogamma 5650. The estimated average blood meal size was 2.80 +/- 0.94 microliters.

PL of coliphage lambda: an alternative solution for an efficient promoter.

Promoter PL of coliphage lambda is highly active in vivo although it is recognized 15-30 times less efficiently by RNA polymerase when compared with promoters of similar strength. Moreover, it differs significantly from the consensus sequence for Escherichia coli promoters. Sequence variants of PL which are more homologous to consensus promoters bind RNA polymerase with increased efficiency. They are nevertheless significantly reduced in their in vivo strength. High activity can be restored by a downstream sequence of a typical consensus-like promoter. Evidently, such elements are required for the efficient release of a stably bound RNA polymerase into a transcriptional elongation complex. We propose that the functional programme encoded in a promoter sequence can be optimized in alternative ways.

Radiocesium-137 movement in a southern coastal plain ecosystem.

The movement of radiocesium (137Cs) in an open ecosystem under natural conditions was investigated at the Louisiana State University Radioecology Field Laboratory. Approximately 80 mCi of 137Cs were transferred to an experimental plot in September 1979. Observations over a six-month period beginning February 1981 indicated that the 137Cs content of plants growing naturally in the plot was low (0.98 Bq/g of plant tissue, dry wt). The vertical mobility of 137Cs to more than 10 cm was also found to be low (up to 1.32 Bq/g of soil, dry wt). The horizontal migration of 137Cs along water drainage directions from the plot was limited to the area of a small ditch that surrounds the experimental plot. No 137Cs above background was found in the fields beyond the 10 m X 10 m plot boundary. The overall results demonstrated that greater than 99.9% of the 137Cs remained within the top 10 cm of the soil profile of the experimental plot under natural field conditions.

Avian retrovirus pp32 DNA binding protein. Preferential binding to the promoter region of long terminal repeat DNA.

The avian retrovirus pp32 protein possesses DNA endonuclease activity and unique DNA binding properties. An improved purification procedure was developed for pp32, resulting in a severalfold increase in the yield of this virion protein. By use of the nitrocellulose filter binding assay, the protein retains approximately 2-fold more supercoiled (form I) DNA molecules than equivalent linear duplex DNA molecules. Single-stranded DNA is only slightly preferred over double-stranded DNA for pp32 binding. The pp32 DNA binding sites on form I pBR322 DNA which contained an insert of avian retrovirus long terminal repeat (LTR) DNA were determined. A preformed protein-DNA complex was digested with one of several different multicut restriction enzymes and filtered through nitrocellulose filters. Fragments containing viral LTR DNA sequences and plasmid DNA containing promoter sequences for the ampicillin and tetracycline genes, sequences for the "left-end" inverted repeat of transposon 3, and sequences encompassing the carboxyl terminus of the beta-lactamase gene were preferentially retained on the filter by pp32. Partial mapping of pp32 DNA binding sites on LTR DNA was accomplished by generation of deletions in LTR DNA sequences. The pp32 protein preferentially bound viral DNA fragments which contain the viral promoter (TATTTAA) and the adjacent "R" repeat sequences. Computer analysis revealed that three of the four plasmid DNA fragments retained by pp32 contained LTR DNA promoter-like sequences (one mismatch only) which were part of statistically significant and thermodynamically stable hairpin structures.

Antibodies against a synthetic peptide of the avian retrovirus pp32 protein and the beta DNA polymerase subunit.

Antibodies directed against a synthetic peptide (14 amino acids in length), whose amino acid sequence was predicted from the nucleotide sequence of the polymerase gene of Rous sarcoma virus (RSV), specifically immunoprecipitated the RSV beta polymerase subunit and the pp32 protein but not the alpha polymerase subunit. The first amino acid of the synthetic peptide is located approximately 30,800 Da from the predicted carboxyl terminus of the polymerase gene. These studies confirm the correct reading frame predicted for the polymerase gene and establish a minimal NH2 terminus for the pp32 DNA binding protein, which was previously shown to be derived from the carboxyl terminus of the polymerase gene.