The stability and characteristics of a DNA Holliday junction switch.

A Holliday junction (HJ) consists of four DNA double helices, with a branch point discontinuity at the intersection of the component strands. At low ionic strength, the HJ adopts an open conformation, with four widely spaced arms, primarily due to strong electrostatic repulsion between the phosphate groups on the backbones. At high ionic strength, screening of this repulsion induces a switch to a more compact (closed) junction conformation. Fluorescent labelling with dyes placed on the HJ arms allows this conformational switch to be detected optically using fluorescence resonance energy transfer (FRET), producing a sensitive fluorescent output of the switch state. This paper presents a systematic and quantitative survey of the switch characteristics of such a labelled HJ. A short HJ (arm length 8 bp) is shown to be prone to dissociation at low switching ion concentration, whereas an HJ of arm length 12 bp is shown to be stable over all switching ion concentrations studied. The switching characteristics of this HJ have been systematically and quantitatively studied for a variety of switching ions, by measuring the required ion concentration, the sharpness of the switching transition and the fluorescent output intensity of the open and closed states. This stable HJ is shown to have favourable switch characteristics for a number of inorganic switching ions, making it a promising candidate for use in nanoscale biomolecular switch devices.

Role of leucocytes in damage to the vascular endothelium during ischaemia-reperfusion injury.

During this investigation, a model of tourniquet-induced forearm ischaemia-reperfusion injury is employed to investigate the role of leucocytes in damage to the vascular endothelium during ischaemia-reperfusion injury. Leucocyte entrapment is investigated by measuring the concentration of leucocytes in venous blood leaving the arm. Neutrophil and monocyte leucocyte subpopulations are isolated by density gradient centrifugation techniques. Cell surface expression of CD11b and the intracellular production of hydrogen peroxide are measured via flow cytometry. Plasma concentrations of elastase and von Willebrand factor (vWF) are measured using enzyme-linked immunosorbemt assay (ELISA) techniques. During ischaemia-reperfusion, there was an increase in CD11b cell surface expression on neutrophils (P=0.040) and monocytes (P=0.049), and a decrease in peripheral blood leucocytes (P=0.019). There was an increase in the intracellular production of hydrogen peroxide by leucocyte subpopulations (P=0.027 [neutrophils], P=0.091 [monocytes]) and in the plasma elastase concentration (P=0.05). There was also a trend to increasing plasma concentration of vWF (P=0.0562), which was measured as a marker of endothelial damage. Ischaemia-reperfusion results in increased adhesiveness, entrapment and activation of leucocytes. Even following a mild ischaemic insult, this leucocyte response was followed immediately by evidence of endothelial damage. These results may have important implications for understanding the development of chronic diseases that involve mild ischaemic episodes.

The value of decision tree analysis in planning anaesthetic care in obstetrics.

The use of decision tree analysis is discussed in the context of the anaesthetic and obstetric management of a young pregnant woman with joint hypermobility syndrome with a history of insensitivity to local anaesthesia and a previous difficult intubation due to a tongue tumour. The multidisciplinary clinical decision process resulted in the woman being delivered without complication by elective caesarean section under general anaesthesia after an awake fibreoptic intubation. The decision process used is reviewed and compared retrospectively to a decision tree analytical approach. The benefits and limitations of using decision tree analysis are reviewed and its application in obstetric anaesthesia is discussed.

cDNA sequence for bovine biglycan (PGI) protein core.

The nucleotide sequence of the protein core for bovine aortic smooth muscle cell biglycan was determined using recombinant DNA technology. Analysis of the deduced amino acid sequence for bovine biglycan revealed a striking homology, 94.6% and 95.7%, to human and rat biglycan, respectively. The bovine biglycan protein core has four potential O-linked and two potential N-linked glycosylation sites and is composed of 11 leucine-rich repeat units.

Nurse-physician collaboration: solving the nursing shortage crisis.

The current severe nursing shortage in the United States has many causes and its solution requires new strategies. Collaboration among the American Association of Critical-Care Nurses (AACN), the Society of Critical Care Medicine (SCCM) and the American College of Cardiology (ACC) has provided a model for the multidisciplinary approach needed. Nurse-physician collaboration is an important strategy to address the ongoing shortage.

Homeostatic responses to caloric restriction: influence of background metabolic rate.

The biological responses to caloric restriction (CR) are generally examined in rats with elevated metabolic rates due to being housed at ambient temperatures (T(a)) below the zone of thermoneutrality. We determined the physiological and behavioral responses to 2 wk of 30-40% CR in male FBNF1 rats housed in cool (T(a) = 12 degrees C) or thermoneutral (TMN; T(a) = 30 degrees C) conditions. Rats were instrumented with telemetry devices and housed continuously in home-cage calorimeters for the entire experiment. At baseline, rats housed in cool T(a) had reduced rate of weight gain; thus a mild CR (5%) group at thermoneutrality for weight maintenance was also studied. Rats housed in cool T(a) exhibited elevated caloric intake (cool = 77 +/- 1; TMN = 54 +/- 2 kcal), oxygen consumption (Vo(2); cool = 9.9 +/- 0.1; TMN = 5.5 +/- 0.1 ml/min), mean arterial pressure (cool = 103 +/- 1; TMN = 80 +/- 2 mmHg), and heart rate (cool = 374 +/- 3; TMN = 275 +/- 4 beats/min). Cool-CR rats exhibited greater CR-induced weight loss (cool = -62 +/- 3; TMN = -42 +/- 3 g) and reductions in Vo(2) (cool = -2.6 +/- 0.1; TMN = -1.5 +/- 0.1 ml/min) but similar CR-induced reductions in heart rate (cool = -59 +/- 1; TMN= -51 +/- 7 beats/min). CR had no effect on arterial blood pressure or locomotor activity in either group. Unexpectedly, weight maintenance produced significant reductions in Vo(2) and heart rate. At thermoneutrality, a single day of refeeding effectively abolished CR-induced reductions in Vo(2) and heart rate. The results reveal that rats with low or high baseline metabolic rate exhibit comparable compensatory reductions in Vo(2) and heart rate and suggest that T(a) can be used to modulate the metabolic background on which the more prolonged effects of CR can be studied.

The nucleotide sequence of the gene encoding the F protein of canine distemper virus: a comparison of the deduced amino acid sequence with other paramyxoviruses.

The nucleotide sequence of the gene encoding the fusion protein of canine distemper virus was determined from cDNA clones derived from virus genome RNA and poly(A)+ RNA extracted from infected cells. The mRNA encoding the F protein is about 2300 nucleotides in length including the 3' poly(A) tail. There is a large open reading frame from nucleotides 86 to 2071 which begins at the first AUG codon in the F mRNA. This reading frame encodes a protein of 662 amino acid residues with a calculated mol. wt. of 73001. The first major hydrophobic domain in the amino acid sequence of the deduced protein (residues 104 to 130) may represent all or part of a signal sequence for cleavage of the N terminal part of the F2 protein. There are four potential N glycosylation sites in the F protein located within the F2 part of the molecule or the putative signal sequence, and one in the F1 portion. A second hydrophobic region corresponds to the proteolytic cleavage site which generates the F2 and F1 subunits. This stretches from residue 225 to 262 and the N terminal part of the F1 protein shows sequence conservation with the other paramyxoviruses. A third major hydrophobic domain near the C terminus of the F protein probably represents the membrane anchor for the F protein (residues 602 to 630). The F1 proteins of six paramyxoviruses are compared and shown to have substantial conservation of those residues important in the maintenance of tertiary structure of this protein.

Analgesic efficacy and safety of single-dose oral and intramuscular ketorolac tromethamine for postoperative pain.

The efficacy and safety of the analgesic drug ketorolac tromethamine in the treatment of moderate to very severe postoperative pain was assessed in five dose-ranging studies with single-dose, double-blind, randomized, parallel-group designs. The drug was administered orally (2.5-200 mg, 352 patients in three trials) and intramuscularly (5-90 mg, 395 patients in two trials), and compared with placebo and reference drugs. Patients subjectively evaluated pain intensity and relief using verbal categoric and visual analog scales; efficacy values included pain intensity difference (PID), summed PID, and total pain relief. Oral ketorolac 10, 12.5, 100, and 200 mg were each statistically significantly superior to placebo in all efficacy measurements, and 10 mg was equivalent to intramuscular morphine 10 mg. Intramuscular ketorolac 90 mg was superior to and 10 and 30 mg were similar to intramuscular morphine 12 mg, and all of these ketorolac doses were superior to intramuscular morphine 6 mg. Intramuscular ketorolac 10 and 30 mg were superior to intramuscular meperidine 50 and 100 mg. Ketorolac was well tolerated, with rates of adverse events generally lower than those of the opiate comparators. Ketorolac doses of 2.5 and 5 mg were less effective than higher doses; 10 mg or more resulted in faster onset of action and greater peak efficacy; 90 mg or more gave more prolonged analgesic effects.

Audit of a complete home nebulizer service provided by a respiratory nurse specialist.

A retrospective audit was performed of a home nebulizer service managed entirely by a Respiratory Nurse Specialist (RNS). We examined (i) the role and workload of the RNS, (ii) the outcome of referrals to the service and (iii) the subsequent compliance with treatment of patients prescribed home nebulized bronchodilators. The case notes and RNS records of all patients referred for consideration of home nebulized bronchodilators over a 9-month period were reviewed. The nebulizer service encompassed patient assessment, trials of nebulized therapy, nebulizer tissue and patient registration and an exchange and servicing system. The non clerical aspects of the service took 9 h per week, 24% of total RNS time. A further 15 h weekly of clerical time was also required. Of 76 referrals, 57 (75%) patients had chronic airflow obstruction (CAO). In CAO patients, 41 had a trial of nebulized therapy and in 27 (66%) such therapy was considered beneficial. In the other 16 CAO patients the RNS deemed a trial inappropriate or impractical and 12 such patients were prescribed nebulized therapy. Overall, therefore 39/57 (68%) of CAO referrals were prescribed home nebulized therapy. At 3-6 months, seven (18%) patients were using their nebulizer infrequently or not at all, 25 (64%) one to four times daily, and seven (18%) four times daily. We conclude that a RNS can manage a complete nebulizer service, but at a considerable cost in professional time. Clerical assistance is essential if appropriate use is to be made of the skills of an RNS.(ABSTRACT TRUNCATED AT 250 WORDS)

Validity of skinfold equations for estimating body density in youth wrestlers.

The present investigation examined the validity of 16 skinfold equations for predicting body density (BD) in youth wrestlers (mean age +/- SD = 11.0 +/- 1.3 yr) by comparing the values to those obtained from underwater weighing. Forty-eight members of youth wrestling clubs (mean BD +/- SD = 1.0634 +/- 0.0125 g.cm-3) volunteered to serve as subjects. The statistical analyses included examination of the constant error (CE), standard error of estimate (SEE), correlation coefficient (r), and total error (TE). The results of this investigation indicated that all of the equations resulted in TE values that were > or = 0.0106 g.cm-3 (range = 0.0106-0.0229 g.cm-3) which corresponded to > or = 4.9% body fat. The TE values were too large to provide accurate estimates of body composition in the present sample of youth wrestlers. Future studies should use the CE values from the present investigation to adjust the intercepts of the skinfold equations in the present study and cross-validate the modified equations on young male athletes.

Breathlessness and exercise capacity in heart failure: the role of bronchial obstruction and responsiveness.

The cause of the breathlessness and reduced exercise capacity that occur in patients with chronic heart failure remains obscure. We examined the hypothesis that airway obstruction and bronchial hyper-responsiveness, which are recognised features of chronic heart failure, might contribute to the breathlessness and reduced exercise capacity in this condition. We studied 37 patients (7 female) with chronic heart failure, of mean age 61 years. Each patient underwent: (i) lung function testing with spirometry and expiratory flow volume loops. (ii) Measurement of bronchial responsiveness to methacholine. (iii) Symptom-limited treadmill exercise capacity using both incremental and fixed workload protocols, with measurement of Borg scores for breathlessness. Lung function was not significantly related to either exercise time, or Borg symptom scores in either exercise protocol. Bronchial hyper-responsiveness to methacholine was demonstrated in 12 patients. Exercise time did not correlate with the degree of bronchial hyper-responsiveness in these 12 patients. Group mean exercise time and Borg scores were not significantly different in these 12 patients when compared to the 25 patients in whom bronchial hyper-responsiveness was not found. We conclude that airway obstruction and bronchial hyper-responsiveness are not likely to be important determinants of reduced exercise capacity and breathlessness in chronic heart failure.

Effect of inflammation on the proliferation of human gingival epithelial cells in vitro.

The adaptive or pathologic responses of epithelial cells to inflammation are poorly characterized. The purpose of this study was to determine if epithelial cells cultured from clinically healthy and inflamed human gingival tissues express differences in proliferation rate and viability. Briefly, the inflammation status of individual donor sites from 101 patients was visually assessed at the time of periodontal surgery and categorized as either non-to-slightly inflamed, moderately inflamed, or severely inflamed. Discarded gingival tissues were then processed to obtain primary cell cultures, for which proliferation rates were determined by calculating the ratio of mean population doublings to the number of days required for cultures to become confluent. In general, the cells in the minimally inflamed group exhibited characteristics different than cells in the moderately and severely inflamed groups. Specifically, the cells obtained from clinical sites which exhibited no-to-slight inflammation had a significantly higher mean proliferation rate than cells in either the moderate inflammation group or the severe inflammation group. Based on trypan blue exclusion, the cells obtained from clinical sites which exhibited no-to-slight inflammation also were more viable than cells obtained from sites with moderate inflammation or severe inflammation. Microscopic evaluation showed morphological changes associated with increased inflammation. Cell cycle analysis by fluorescent-activated cell sorting (FACS) revealed a directly proportional relationship between the degree of inflammation and apoptosis, and a strong inversely proportional trend between the degree of inflammation and the numbers of cells undergoing mitosis. Taken together, these data suggest that epithelial cell proliferation and viability are inversely associated with the degree of gingival inflammation, once a putative "adaptive threshold" is exceeded. Elucidation of the underlying mechanisms will likely lead to improvements in clinical diagnosis and treatment.

Utilising a defective IBV RNA for heterologous gene expression with potential prophylactic application.

Based on the natural ability of coronaviruses to undergo homologous RNA recombination, we are working to produce infectious bronchitis virus (IBV) recombinants using RNA generated from recombinant fowlpox viruses (FPV). The aim is to replace the spike (S) gene of an existing IBV vaccine strain with the S gene of a heterologous strain. CD-61 is an IBV defective RNA (D-RNA) derived from a naturally occurring IBV D-RNA (CD-91). CD-61 D-RNA is being investigated as an RNA vector for the expression of heterologous genes. T7-derived RNA transcripts of CD-61 can be replicated and passaged in the presence of helper virus, following electroporation into IBV-infected cells. CD-61 cDNA was modified by the addition of the hepatitis delta virus ribozyme plus T7 terminator downstream of the 3'UTR. This allowed the synthesis of discreet RNA transcripts. The complete cassette was cloned into an FPV transfer vector (pEFL10) for generating recombinant fowlpox viruses. FPV/CD-61 recombinants will be assessed for D-RNA production in IBV-infected cells. The luciferase reporter gene sequence has been inserted into the modified CD-61, under the control of the IBV transcription associated sequence (TAS) from gene 5. Luciferase has been successfully expressed from CD-61 in helper virus-infected cells.

Nurse/physician collaboration: solving the nursing shortage crisis.

This summary article discusses the status of the nursing shortage in the United States, with emphasis on successful strategies to address it. Liaisons between the American Association of Critical-Care Nurses (AACN) and the Society for Critical Care Medicine, as well as with the American College of Cardiology, are highlighted, with primary emphasis on the strategy of nurse-physician collaboration.

Conflict resolution: a strategy for growth.

We have ample opportunities to practice negotiation skills every day. Our goal is to create an environment in critical care where confrontation regarding patient care issues, collaborative practice, shared governance, and standards of professional practice is the norm. Winning and losing are not the important factors. We should instead strive for establishing mutual interests, shared values, and a framework for decision making. Mutual gain is the goal of confrontation and conflict resolution. We must develop our skills to address conflicting interests at the bedside. It is essential that we have a better process to accept our difference and foster our mutual and shared interest, caring for the critically ill patient.

Who lives, who dies, who decides?

The financial costs of the ethical dilemmas in the health care industry are profound, and the emotional ramifications for the patient and family are even more costly. As critical care nurses and as health care consumers, we must create an impetus for change. We need to ensure that we contribute to the quality of life while dealing with the conflicting values inherent in the right-to-die question. Together, we can create an ethical practice environment for ourselves, our families, and the patients for whom we care.

Characterization of leukocytes by filtration of diluted blood.

From the known numbers and properties of normal blood cells, it is apparent that the analysis of the flow of diluted blood, through micropore filters, should allow calculation of the properties of leukocytes without the need for their prior purification. The number and transit time of a "slow" leukocyte population can be deduced by fitting the flow, of diluted blood through 5 microns membranes over about 150 s, to the appropriate mathematical model, which is chosen by the use of a suitable statistical test--the runs test. This population of leukocytes equates numerically with the monocyte population of normal blood; cells have a transit time, through 5 microns pores, of 27.7 +/- 10.9 s. The remaining "fast" leukocytes represent the sum of granulocytes and lymphocytes; their flow properties can be deduced from the measured initial flow rate of diluted blood and the estimated properties of the red blood cells. The properties of the red cells can be assessed from filtration of purified suspensions with any concentration of cells from 0.52 x 10(9)/ml to 3.0 x 10(9)/ml. The transit times for red cells and granulocytes/lymphocytes, in blood diluted with about an equal volume of buffer, are 1.36 +/- 0.17 ms and 1.48 +/- 0.33 s, respectively. The transit times of blood cells, through 5 microns pores, are therefore inversely related to their numbers in blood.

The effects of temperature on the filtration of diluted blood through 3 mu m and 5 mu m filters.

The effect of temperature on the flow of diluted blood [Hct = 0.21], through 5 mu m Nuclepore filters, is described by the Arrhenius equation with an energy of activation of 27.7 kJ/mol. Plasma, diluted with PBS, is almost three times less sensitive to temperature, with an energy of activation of 9.8 kJ/mol, while red cells are of intermediate sensitivity, with an energy of activation of 14.7 kJ/mol. The most sensitive elements to changes in temperature are leukocytes, with energies of activation of 31 kJ/mol and 35 kJ/mol for fast-flowing leukocytes (granulocytes and lymphocytes) and slow-flowing leukocytes (monocytes) respectively. Hence, the major determinants of the decline in filterability of blood through micropore filters are the leukocytes. This effect is compounded when blood is kept for 10 minutes or more at 10 degrees C due to activation of granulocytes, which leads to permanent pore blocking when the affected blood is filtered at room temperature. The combination of increased passage time of leukocytes through peripheral areas at abnormally low temperatures and subsequent activation might influence the flow of blood in non-affected tissues. The effect of temperature on the filterability of red blood cells through 3 mu m filters is not described by the Arrhenius equation and the deviations are seen as a gradual change of slope rather than a sharp break between two straight lines. The data are consistent with a gradual shift in rate limiting step away from the entry event into pores, which dominates at low temperature but becomes progressively less important at elevated temperatures. The changing parameter is probably the volume of the red cell, which is less important when flow is measured through 5 mu m pores.

The filterability of leukocytes in undiluted blood.

A filtrometer is described for measuring the flow of fluids through microfilters. The flow of Newtonian fluids through the filters can be predicted from the diameter, length and number of pores. There are no physical artefacts such as turbulent flow or a significant lag period before steady-state flow is achieved. The instrument has been used as a viscometer and has been used to record and analyse the flow of undiluted blood through 5 microns polycarbonate filters. The calculated viscosity of Newtonian fluids agrees well with those measured by a more conventional viscometer (Ostwald). Flow profiles of blood have been analysed to give both the numbers and the flow properties of a small population of slow leukocytes which equate numerically with the monocytes. They are subdivided into three distinct sub-populations, according to their rheological properties, and these are termed SL1, SL2 and PB. The concentration of these cells, in blood, are 0.12 +/- 0.02 x 10(6) ml-1, 0.11 +/- 0.02 x 10(6) ml-1, 0.09 +/- 0.02 x 10(6) ml-1 in young females aged about 25 years. The transit time of these cells, through 5 microns pores, is 34.8 +/- 1.4 s, 147.5 +/- 2.5 s and > 300 s, respectively. Analysis of blood from older men (53-79 years) gives essentially the same results although the concentration of SL1 is slightly higher at 0.19 +/- 0.09 x 10(6) ml-1.

Monocytes are a rheologically heterogeneous population of cells.

The effect of increased adhesiveness and decreased deformability of leukocytes following activation can have a profound effect on flow through the microcirculation. Measurement of leukocyte deformability is therefore an important tool in the study of the pathology of vascular diseases. Although much work has been done on the rheological properties of lymphocytes and granulocytes, there is little information available on the larger mononuclear cells, the monocytes. To investigate monocyte rheology, attempts were made to purify monocytes by a variety of methods. Purified monocytes were then filtered through 5 microm polycarbonate filters, with flow profiles (change in volume with time) recorded over 300 seconds. The profiles were analysed by least squares fitting to an appropriate mathematical model. Analysis of filtration data demonstrated 3 distinct sub-populations of monocytes with differing rheological properties. Other workers have characterised monocytes into defined subsets on the basis of their size, phagocytic ability or expression of cell surface markers. The definition of monocytes into defined rheological subsets is a new and useful addition to these studies.