HTS driven by fluorescence lifetime detection of FRET identifies activators and inhibitors of cardiac myosin.

Small molecules that bind to allosteric sites on target proteins to alter protein function are highly sought in drug discovery. High-throughput screening (HTS) assays are needed to facilitate the direct discovery of allosterically active compounds. We have developed technology for high-throughput time-resolved fluorescence lifetime detection of fluorescence resonance energy transfer (FRET), which enables the detection of allosteric modulators by monitoring changes in protein structure. We tested this approach at the industrial scale by adapting an allosteric FRET sensor of cardiac myosin to high-throughput screening (HTS), based on technology provided by Photonic Pharma and the University of Minnesota, and then used the sensor to screen 1.6 million compounds in the HTS facility at Bristol Myers Squibb. The results identified allosteric activators and inhibitors of cardiac myosin that do not compete with ATP binding, demonstrating high potential for FLT-based drug discovery.

Magnesium ions: activity in seawater.

The activity of magnesium ions in seawater was determined from solubility data and found to be between the values determined by Platford and by Garrels and Thompson. Our value may result from extensive formation of magnesium sulfate ion pairs.

Adenovirus-mediated p53 gene transfer in advanced non-small-cell lung cancer.

BACKGROUND: Preclinical studies in animal models have demonstrated tumor regression following intratumoral administration of an adenovirus vector containing wild-type p53 complementary DNA (Ad-p53). Therefore, in a phase I clinical trial, we administered Ad-p53 to 28 patients with non-small-cell lung cancer (NSCLC) whose cancers had progressed on conventional treatments. METHODS: Patients received up to six, monthly intratumoral injections of Ad-p53 by use of computed tomography-guided percutaneous fine-needle injection (23 patients) or bronchoscopy (five patients). The doses ranged from 10(6) plaque-forming units (PFU) to 10(11) PFU. RESULTS: Polymerase chain reaction (PCR) analysis showed the presence of adenovirus vector DNA in 18 (86%) of 21 patients with evaluable posttreatment biopsy specimens; vector-specific p53 messenger RNA was detected by means of reverse transcription-PCR analysis in 12 (46%) of 26 patients. Apoptosis (programmed cell death) was demonstrated by increased terminal deoxynucleotide transferase-mediated biotin uridine triphosphate nick-end labeling (TUNEL) staining in posttreatment biopsy specimens from 11 patients. Vector-related toxicity was minimal (National Cancer Institute's Common Toxicity Criteria: grade 3 = one patient; grade 4 = no patients) in 84 courses of treatment, despite repeated injections (up to six) in 23 patients. Therapeutic activity in 25 evaluable patients included partial responses in two patients (8%) and disease stabilization (range, 2-14 months) in 16 patients (64%); the remaining seven patients (28%) exhibited disease progression. CONCLUSIONS: Repeated intratumoral injections of Ad-p53 appear to be well tolerated, result in transgene expression of wild-type p53, and seem to mediate antitumor activity in a subset of patients with advanced NSCLC.

Adenovirus-mediated p53 gene transfer in sequence with cisplatin to tumors of patients with non-small-cell lung cancer.

PURPOSE: To determine the safety and tolerability of adenovirus-mediated p53 (Adp53) gene transfer in sequence with cisplatin when given by intratumor injection in patients with non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients with advanced NSCLC and abnormal p53 function were enrolled onto cohorts receiving escalating dose levels of Adp53 (1 x 10(6) to 1 x 10(11) plaque-forming units [PFU]). Patients were administered intravenous cisplatin 80 mg/m(2) on day 1 and study vector on day 4 for a total of up to six courses (28 days per course). Apoptosis was determined by the terminal deoxynucleotidyl- transferase-dUTP nick-end labeling assay. Evidence of vector-specific sequences were determined using reverse-transcriptase polymerase chain reaction. Vector dissemination and biodistribution was monitored using a series of assays (cytopathic effects assay, Ad5 hexon enzyme-linked immunosorbent assay, vector-specific polymerase chain reaction assay, and antibody response assay). RESULTS: Twenty-four patients (median age, 64 years) received a total of 83 intratumor injections with Adp53. The maximum dose administered was 1 x 10(11) PFU per dose. Transient fever related to Adp53 injection developed in eight of 24 patients. Seventeen patients achieved a best clinical response of stable disease, two patients achieved a partial response, four patients had progressive disease, and one patient was not assessable. A mean apoptotic index between baseline and follow-up measurements increased from 0.010 to 0.044 (P =.011). Intratumor transgene mRNA was identified in 43% of assessable patients. CONCLUSION: Intratumoral injection with Adp53 in combination with cisplatin is well tolerated, and there is evidence of clinical activity.

Charged beads enhance cutaneous wound healing in rhesus non-human primates.

Enhanced cutaneous wound healing by positively charged cross-linked diethylaminoethyl dextran beads (CLDD) was studied in a standardized incisional wound model in 20 adult and 20 geriatric Macaca mulatta (rhesus) partitioned equally over five time periods. Physiologic saline served as a control. Soft-tissue linear incisions were prepared between and 1 cm inferior to the scapulae. There were four incisions per rhesus; each incision was 1.5 cm long with 1 cm of undisturbed tissue between incisions, and both the experimental CLDD and physiologic saline treatments were administered to each rhesus. The incision treatments were either CLDD and soft-tissue closure with 4-0 BioSyn sutures or sterile physiologic saline and closure with 4-0 BioSyn sutures. The hypothesis was CLDD would enhance cutaneous wound repair. Verification of the hypothesis consisted of clinical examinations and histologic and tensiometric evaluations on biopsy specimens at 10 and 15 days, whereas 5-day and 2- and 4-month groups were assessed clinically and biopsy specimens were assessed histologically. The clinical course of healing for all groups was unremarkable. At 10 days, incisions in adult rhesus treated with CLDD had a 30-percent greater tensile strength compared with the physiologic saline-treated incisions (p = 0.01), whereas for geriatric rhesus, the CLDD treatment proved to be 15 percent greater in tensile strength compared with the physiologic saline cohort (p = 0.11). By day 15, incisions in adult rhesus were 26 percent stronger than the saline treatment group (p = 0.07), and the difference was 36 percent (p = 0.02) for the geriatric rhesus. From 5 through 15 days, histologic observations revealed a gradual decrease in quantity and integrity of CLDD, with no remnants of CLDD at either 2 or 4 months. Macrophages and multinucleated giant cells were localized in the dermis and were associated with the CLDD. These cells decreased commensurately with the decrease of CLDD beads. The data suggest that CLDD can enhance significantly the tensile properties of healing cutaneous wounds in both adult and geriatric rhesus. Moreover, if the wound healing is enhanced in geriatric patients, this finding may be clinically germane to conditions where wound healing is compromised, such as in diabetics and patients on steroids.

Pseudoaneurysm: diagnosis with color Doppler ultrasound.

Increasing numbers of interventional radiologic procedures, often with large arterial catheters, have led to increasing total numbers of complications. Pseudoaneurysm is not an infrequent complication. It is often difficult to differentiate pseudoaneurysm clinically from hematoma. In the past, we have used gray scale duplex scanning to aid in this differentiation. We have recently found color Doppler scanning to allow quicker, easier, and more accurate diagnosis. Color Doppler imaging also can often demonstrate the track between the artery and the aneurysm.

A continuum of researcher-participant relationships: an analysis and critique.

In this article, three nursing research studies are analyzed by focusing on the nature of the researcher-participant relationship. This analysis is rooted in the assumptions, goals, and language of "new paradigm" research. The importance of making the researcher-participant relationship a central focus of nursing inquiry is addressed.

Women's "sickness": a case of secondary gains or primary losses.

The functionalist view of the sick role is analyzed in terms of its applicability to women. Rather than focusing on the so-called secondary gains of the sick role, attention is given to the primary losses incurred when women's problems are subject to medical definitions and interventions. Women's "sickness" is placed in a historical and sociopolitical context. The "sickness" of the nursing profession and the "sickness" of women are seen as sharing similar symptoms, the same etiology, and hence a common cure.

Critique of "The use of animals in nursing research".

The authors present a critique of an article by Cunningham and Mitchell that advocates the use of animals in nursing research. Moral, political, social, and scientific arguments are presented to develop a case in opposition to the use of animals for experimentation. In conclusion, the authors assert that nurse scientists can make a choice at this critical juncture to take another path leading to the maturity of nursing as a research-based profession.

Continuous pH and Pco2 monitoring during respiratory failure in children with the Paratrend 7 inserted into the peripheral venous system.

CONTEXT: The Paratrend monitor provides continuous arterial blood gas monitoring after insertion through a >/=20-gauge arterial cannula. OBJECTIVE: To determine the correlation of arterial blood gas values and the Paratrend monitor placed through a peripheral intravenous catheter. DESIGN: Prospective, open-label evaluation. SETTING: University-based pediatric intensive care unit. PATIENTS: Infants and children with respiratory failure and arterial access. RESULTS: The cohort included 23 infants and children. A total of 100 sample sets (Paratrend/ABG Pco(2) and pH values) were collected. The absolute difference between the arterial and Paratrend Pco(2) was 2. 9 +/- 1.8 mm Hg (range 0 to 9 mm Hg). Linear regression analysis of Paratrend Pco(2) versus arterial Pco(2) resulted in r = 0.97 and r(2) = 0.9479 (P <.001). Bland-Altman analysis of Pco(2) values demonstrated a bias +/- precision of -2.1 +/- 2.7 mm Hg. The absolute difference between arterial and Paratrend pH was 0.04 +/- 0. 02 units (range 0 to 0.15 units). Linear regression analysis of Paratrend pH versus arterial pH resulted in r = 0.83 and r(2) = 0. 7016 (P <.0001). Bland-Altman analysis of pH values revealed a bias +/- precision of 0.03 +/- 0.03 units. CONCLUSIONS: Inserted through a peripheral intravenous cannula, the Paratrend monitor can be used to provide an accurate estimation of arterial blood gas values in children with respiratory failure.

Increase in wound breaking strength in rats in the presence of positively charged dextran beads correlates with an increase in endogenous transforming growth factor-beta1 and its receptor TGF-betaRI in close proximity to the wound.

We have previously shown that positively charged beads (DEAE A25) increase wound breaking strength in linear incisions in rats and nonhuman primates at days 10-14 post-wounding. The increased wound strength may result in part from a stimulation of cells adjacent to the DEAE A25 beads to produce growth factors important for wound healing. In this report, we investigate this hypothesis by comparing the relative expression levels of transforming growth factor-beta1 and its receptor transforming growth factor-beta receptor type I in DEAE A25-treated and contralateral untreated rat linear incisions. DEAE A25-treated incisions were stronger than untreated control wounds at 3 days post-wounding, and the difference in breaking strength reached statistical significance at days 5, 7 and 10. Immunohistochemical analysis revealed a significant increase in transforming growth factor-beta1 and transforming growth factor-beta receptor type I expression in DEAE A25-treated incisions, up to 7 days post-wounding, as compared to untreated control wounds. FACS analysis revealed that macrophage cell lines exposed to DEAE A25 in vitro upregulate transforming growth factor-beta1 and transforming growth factor-beta receptor type I expression by 2-3 fold. Therefore, the increase in expression of transforming growth factor-beta1 and transforming growth factor-beta receptor type I in DEAE A25-treated incisions may be due to an increase in the concentration of macrophages adjacent to DEAE A25 beads, as well as the stimulation of individual macrophages to produce greater amounts of transforming growth factor-beta1 and transforming growth factor-beta receptor type I. This study also supports the significance of transforming growth factor-beta1 in wound healing.

Differential effects of oxygen on human dermal fibroblasts: acute versus chronic hypoxia.

The observation that many chronic wounds are ischemic has spurred a series of studies evaluating the response of cells exposed to hypoxia. To date, these studies have shown largely beneficial effects from hypoxia, such as increased cellular replication and procollagen synthesis. These findings are counter-intuitive from a clinical standpoint because cellular growth and synthetic function are known to be retarded in chronic ischemic wounds. We have established an in vitro system in which human dermal fibroblasts grown chronically at 5 +/- 3 mm Hg will proliferate at a rate three times slower than those fibroblasts grown under standard culture conditions (namely an oxygen partial pressure of 150 mm Hg). No phenotypic changes are noted in chronically hypoxic cells, and the growth-retarding effects are reversible when the cells are returned to standard oxygen conditions. Competitive reverse transcription-polymerase chain reaction showed that acute exposure to hypoxia (up to 1 week) results in a 6.3-fold increase in the relative expression of transforming growth factor-beta1 messenger RNA, whereas chronic exposure to hypoxia leads to a 3.1-fold decrease in this message. Collagen production measured at both the mRNA and protein level is also decreased in the setting of chronic hypoxia. We propose that this system may be the most appropriate setting for studying the role of oxygen on dermal fibroblasts in ischemic, nonhealing wounds.

Stimulation of wound healing by positively charged dextran beads depends upon clustering of beads and cells in close proximity to the wound.

We have previously shown that positively charged dextran (DEAE A25) increases wound breaking strength in linear incisions in rats and nonhuman primates at days 10-14 postwounding. In this article, we examined the cellular responses to different types of charged dextran beads (DEAE A50 and Cytodex-1) in culture studies and in rat incisional wounds. We show that Cytodex 1 and DEAE A50 beads also increased wound breaking strength in a rat linear incisional model. However, the increase was approximately 30-40% less than that observed in wounds treated with DEAE A25 beads. The main distinction between the three types of beads was the presence of bead clusters observed in tissue sections. Wounds treated with DEAE A25 beads formed distinct clusters while both Cytodex 1 and DEAE A50 beads clustered to a lesser extent or failed to cluster at all. We propose that the different types of charged dextran beads improve healing by promoting cell adhesion and encouraging proliferation in close proximity to the wound. We also hypothesize that the 30-40% improvement in wound breaking strength seen with DEAE A25 beads compared to other types of charged dextran beads (DEAE A50 and Cytodex-1) originates from the unique characteristic of DEAE A25 beads in forming cell-bead aggregates adjacent to the wounded area. This clustering, in turn, affects the distribution of cells infiltrating the wounded area (such as macrophages) during the healing process and, as a consequence, alters the distribution of matrix molecules and growth factors secreted by these cells.

Verbal behavior and initial exposure to delayed reinforcement.

Ten subjects responded under a tandem fixed-ratio 1 not-responding-greater-than-t schedule of point delivery during one 75-min session in which the delay was either 10 or 20 s. Subjects were asked to describe the contingencies throughout the session. Although studies with non-humans have demonstrated response acquisition under similar delayed-consequence procedures, a minority of subjects in the current study demonstrated sensitivity to delayed consequences convincingly. All subjects exhibited inefficient patterns of responding and descriptions of nonexistent contingencies. Subjects who demonstrated learning were more likely to verbalize the actual contingencies, but this was not true in all cases. Furthermore, some subjects who demonstrated learning did not describe the delay contingency. Results suggest that learning may occur in the absence of a person's ability to describe environment-behavior relations.

A one-step monoclonal antibody typing procedure that simplifies HLA class I and class II typing.

We have developed monoclonal antibodies to most HLA specificities, making it possible for us to devise a simple, rapid, one-step microcytotoxicity test. The test is performed by adding 1 microliter of cells to 1 microliter of antibody-complement mixture predotted on the microtest tray. The reactions are read following a 1-hour incubation period (30 minutes in some instances). The analysis of reactions seen on testing 105 class I antibodies and 50 class II antibodies is shown. A comparison of typing by the standard NIH method and the new one-step procedure showed a > 96% concordance in the 500 T cells and 200 B cells we examined. Class I and class II typing could be performed using B cells, thus obviating the need to isolate both T and B cells for HLA typing.

Biodegradable positively charged ion exchange beads: a novel biomaterial for enhancing soft tissue repair.

Previous work in the area of vulnerary agents is extensive. One material of focus has been positively charged ion exchange beads, which have been shown to promote a variety of wound-healing responses in several models. The goal of this work was to improve upon the clinical utility of positively charged dextran beads by creating a biodegradable version that maintains the material's inherent efficacy. A chemical method consisting of a sodium periodate oxidation was used to create a degradable diethylaminoethanol crosslinked dextran bead. The ability of this process to create a degradable bead was verified in vitro and in vivo. Furthermore, efficacy was shown in a rat linear incision model for a variety of beads exhibiting different degradation rates. The results show that efficacy is maintained by a degradable bead, but there is a diminution of the magnitude of the response as the mass loss rate is increased. Efficacy also was investigated for a moderate but completely degrading bead material over time and dose. Wound breaking strength was evaluated at days 7, 10, 14, 21, and 28 for degradable beads at doses of 10 mg/mL and 50 mg/mL. Although little difference in efficacy was noted for the increased dose, statistically significant increases over control were seen at days 7, 10, and 14 for the 10 mg/mL dose and at days 10 and 14 for the 50 mg/mL dose. At days 21 and 28 there were no differences between treated and control wounds.