An intrinsic timer that controls cell-cycle withdrawal in cultured cardiac myocytes

Developing cardiac myocytes divide a limited number of times before they stop and terminally differentiate, but the mechanism that stops their division is unknown. To help study the stopping mechanism, we defined conditions under which embryonic rat cardiac myocytes cultured in serum-free medium proliferate and exit the cell cycle on a schedule that closely resembles that seen in vivo. The culture medium contains FGF-1 and FGF-2, which stimulate cell proliferation, and thyroid hormone, which seems to be necessary for stable cell-cycle exit. Time-lapse video recording shows that the cells within a clone tend to divide a similar number of times before they stop, whereas cells in different clones divide a variable number of times before they stop. Cells cultured at 33 degrees C divide more slowly but stop dividing at around the same time as cells cultured at 37 degrees C, having undergone fewer divisions. Together, these findings suggest that an intrinsic timer helps control when cardiac myocytes withdraw from the cell cycle and that the timer does not operate by simply counting cell divisions. We provide evidence that the cyclin-dependent kinase inhibitors p18 and p27 may be part of the timer and that thyroid hormone may help developing cardiac myocytes stably withdraw from the cell cycle.     

Both soluble and membrane-bound forms of Flt3 ligand enhance tumor immunity following “suicide” gene therapy in a murine colon carcinoma model

In prodrug-activated (suicide) gene therapy, tumor cells are transfected with the gene for an enzyme that converts an inactive prodrug, such as ganciclovir (GCV), to a toxic compound. Transfected cells are killed on administration of GCV, as also are untransfected bystander cells. The ability of the dendritic cell stimulatory cytokine Flt3 ligand (Flt3-L) to modulate prodrug-activated gene therapy has been investigated. Transfectants of the murine colon carcinoma MC26 were generated expressing soluble (FLS) and membrane-bound forms of Flt3-L. They were inoculated together with wild-type MC26 cells and cells expressing herpes simplex virus-1 (HSV1) thymidine kinase into BALB/c mice, which were then administered GCV. Expression of Flt3-L or FLS prevented regrowth of tumor in most mice, which was comparable to the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), while tumors recurred in all mice receiving suicide gene therapy alone. Recurring tumor cells were resistant to direct killing by GCV but sensitive to bystander killing in vitro. Mice without tumor recurrence were rechallenged with unmodified MC26 cells. Of those mice given transfectants expressing GM-CSF, Flt3-L, or FLS, approximately 50% were immune to rechallenge. These mice also showed cytotoxic and proliferative responses to MC26 cells. These experiments show that both soluble and membrane-bound forms of Flt3-L were able to induce a protective immune response to colon carcinoma cells in a fashion similar to GM-CSF.Abdul-Razzak Alsheikhly and Jehad Zweiri contributed equally to this work and are listed in alphabetical order.     

Changes in public order after the opening of a medically supervised safer injecting facility for illicit injection drug users

Background

North America''s first medically supervised safer injecting facility for illicit injection drug users was opened in Vancouver on Sept. 22, 2003. Although similar facilities exist in a number of European cities and in Sydney, Australia, no standardized evaluations of their impact have been presented in the scientific literature.

Methods

Using a standardized prospective data collection protocol, we measured injection-related public order problems during the 6 weeks before and the 12 weeks after the opening of the safer injecting facility in Vancouver. We measured changes in the number of drug users injecting in public, publicly discarded syringes and injection-related litter. We used Poisson log-linear regression models to evaluate changes in these public order indicators while considering potential confounding variables such as police presence and rainfall.

Results

In stratified linear regression models, the 12-week period after the facility''s opening was independently associated with reductions in the number of drug users injecting in public (p < 0.001), publicly discarded syringes (p < 0.001) and injection-related litter (p < 0.001). The predicted mean daily number of drug users injecting in public was 4.3 (95% confidence interval [CI] 3.5–5.4) during the period before the facility''s opening and 2.4 (95% CI 1.9–3.0) after the opening; the corresponding predicted mean daily numbers of publicly discarded syringes were 11.5 (95% CI 10.0–13.2) and 5.4 (95% CI 4.7–6.2). Externally compiled statistics from the city of Vancouver on the number of syringes discarded in outdoor safe disposal boxes were consistent with our findings.

Interpretation

The opening of the safer injecting facility was independently associated with improvements in several measures of public order, including reduced public injection drug use and public syringe disposal.Many cities are experiencing epidemics of bloodborne diseases as a result of illicit injection drug use,^1,2,3 and drug overdoses have become a leading cause of death in many urban areas.^4,5,6 Public drug use also plagues many inner city neighbourhoods, and the unsafe disposal of syringes in these settings is a major community concern.^{7,8,9,10,11,12,13}In over 2 dozen European cities and, more recently, in Sydney, Australia, medically supervised safer injecting facilities, where injection drug users (IDUs) can inject previously obtained illicit drugs under the supervision of medical staff, have been established in an effort to reduce the community and public health impacts of illicit drug use.¹⁴ Inside these facilities IDUs are typically provided with sterile injecting equipment, emergency care in the event of overdose, as well as primary care services and referral to addiction treatment.^13,15 Although anecdotal reports have suggested that such sites may improve public order,¹² reduce the number of deaths from overdose¹⁶ and improve access to care,¹⁷ no standardized evaluations of their impact are available in the scientific literature.¹⁸On Sept. 22, 2003, health officials in Vancouver opened a government-sanctioned safer injecting facility as pilot project. The facility, the first in North America, is centrally located in Vancouver''s Downtown Eastside, which is the most impoverished urban neighbourhood in Canada and home to well-documented overdose and HIV epidemics among the estimated 5000 IDUs who reside there.^19,20 Federal approval for the 3-year project was granted on the condition that the health and social impacts of the facility be rigorously evaluated. Although evaluation of the facility''s impact on certain outcomes (e.g., HIV incidence) is ongoing and will take several years, it is now possible to examine the impacts of the site on public order. Therefore, we conducted this study to test the hypothesis that changes in improperly discarded syringes and public drug use would be observed after the opening of the safer injecting facility.     

The investigation of cytosolic phospholipase A2 using ELISA

Two studies of the behaviour of cytosolic phospholipase A(2) (cPLA(2)) in the red blood cell (RBC), as measured by ELISA, are described. In the first study we show a significant increase in cPLA(2) in patients with schizophrenia compared to controls and suggest that this measure, if corroborated, could be used as a diagnostic marker. In a second study we found that washing the RBC introduced an unknown confounding variable which led us to reject this study. A subsequent investigation of washing red cells showed that the washing effect may be due to a plasma factor likely to be more than 5kDa MW which can be removed from red cells by washing with buffers. When the cells are washed, the concentration of cPLA(2) in the red cell, as measured by ELISA, significantly increases. We advise against washing the red cell in any study that involves measuring cPLA(2) by ELISA.     

High-level genotypic variation and antibiotic sensitivity among Escherichia coli O157 strains isolated from two Scottish beef cattle farms

Escherichia coli O157:H7 is a human pathogen that is carried and transmitted by cattle. Scotland is known to have one of the highest rates of E. coli O157 human infections in the world. Two hundred ninety-three isolates were obtained from naturally infected cattle and the environment on two farms in the Scottish Highlands. The isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI restriction endonuclease enzyme, and 19 different variations in patterns were found. There was considerable genomic diversity within the E. coli O157 population on the two farms. The PFGE pattern of one of the observed subtypes matched exactly with that of a strain obtained from a Scottish patient with hemolytic-uremic syndrome. To examine the stability of an individual E. coli O157 strain, continuous subculturing of a strain was performed 110 times. No variation from the original PFGE pattern was observed. We found three indistinguishable subtypes of E. coli O157 on both study farms, suggesting common sources of infection. We also examined the antibiotic resistance of the isolated strains. Phenotypic studies demonstrated resistance of the strains to sulfamethoxazole (100%), chloramphenicol (3.07%), and at a lower rate, other antibiotics, indicating the preservation of antibiotic sensitivity in a rapidly changing population of E. coli O157.     

Rapid detection of `rare' actinomycetes in environmental samples

Natural products continue to be a useful source of new leads for the pharmaceutical industry. Actinomycetes are prolific producers of natural products and one strategy to increase the possibility of discovering novel chemical entities is to screen actinomycetes considered 'rare' in the environment and previously under-represented in natural product screening collections. We describe a method using bacteriophage as a marker to detect these actinomycetes in environmental samples. This method allows samples to be pre-screened for the presence of target actinomycetes before lengthy isolation programmes are undertaken.     

Visibility of the impact of environmental noise: a response to Kaitala and Ranta

Based on simulation modelling, Kaitala and Ranta (2001 Proc. R. Soc. Lond. B 268, 1769-1774) have argued that detecting the statistical relationships between environmental variability and population fluctuations will be difficult. However, their study was limited in that only one pattern of density dependence and one detection method were used. Here, we show that their conclusion is in part a consequence of their choice of population model and in part a consequence of using relatively weak or inappropriate statistical methods. Other patterns of density dependence respond differently to environmental fluctuations, and the impact of the disturbance on these is clearly visible using their methods. For some patterns of population dynamics, environmental impacts are more readily detectable by correlating running-average environmental conditions with the population time-series or by correlating the first differences of the population time-series with environmental noise. When more appropriate statistical methods are used, environmental forcing is detectable in the majority of cases used by Kaitala and Ranta. The interplay between environmental stochasticity and density-dependent population growth means that there is no single best method to detect the influence of environmental forcing, even when population dynamics are approximately linear. But environmental forcing will often be detectable, contrary to Kaitala and Ranta's assertions.     

Structure-function analysis of the presumptive Arabidopsis auxin permease AUX1

We have investigated the subcellular localization, the domain topology, and the amino acid residues that are critical for the function of the presumptive Arabidopsis thaliana auxin influx carrier AUX1. Biochemical fractionation experiments and confocal studies using an N-terminal yellow fluorescent protein (YFP) fusion observed that AUX1 colocalized with plasma membrane (PM) markers. Because of its PM localization, we were able to take advantage of the steep pH gradient that exists across the plant cell PM to investigate AUX1 topology using YFP as a pH-sensitive probe. The YFP-coding sequence was inserted in selected AUX1 hydrophilic loops to orient surface domains on either apoplastic or cytoplasmic faces of the PM based on the absence or presence of YFP fluorescence, respectively. We were able to demonstrate in conjunction with helix prediction programs that AUX1 represents a polytopic membrane protein composed of 11 transmembrane spanning domains. In parallel, a large aux1 allelic series containing null, partial-loss-of-function, and conditional mutations was characterized to identify the functionally important domains and amino acid residues within the AUX1 polypeptide. Whereas almost all partial-loss-of-function and null alleles cluster in the core permease region, the sole conditional allele aux1-7 modifies the function of the external C-terminal domain.