Structural changes of yellow Cameleon domains observed by quantitative FRET analysis and polarized fluorescence correlation spectroscopy.

Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand binding.

Cytosine methylation in the EcoRI site of active and inactive herpesvirus thymidine kinase promoters.

The herpesvirus thymidine kinase (tk) gene integrated in the human cell line, 2.1-a, can be inactivated by limited de novo methylation. All these TK- clones show partial EcoRI digestion of the recognition site (cGAATTCg) in the tk promoter in contrast to complete digestion of this site in the original cell line. Studies on well-defined substrates prepared in vitro showed that methylation of one cytosine in the EcoRI recognition sequence resulted in partial and methylation of both cytosines in severe inhibition of digestion by EcoRI. This characteristic was used to determine whether no, one or both cytosines in the EcoRI site of the tk promoter were methylated in various TK- clones derived from 2.1-a and in TK+ clones re-expressing the gene after 5-azacytidine treatment. A high correlation was found between inactivity of the tk gene and methylation of only one of the two cytosines in the EcoRI recognition site. The results also show that the tk promoter can be active despite the presence of a methylated cytosine.

Gene amplification in a human osteosarcoma cell line results in the persistence of the original chromosome and the formation of translocation chromosomes.

Although gene amplification, a process that is markedly enhanced in tumor cells, has been studied in many different cell systems, there is still controversy about the mechanism(s) involved in this process. It is still unclear what happens to the DNA sequences that become amplified, whether they remain present at their original location (conservative gene amplification) or whether gene amplification necessarily results in a deletion at the original location (non-conservative gene amplification). We have studied gene amplification in a human osteosarcoma cell line, starting from a cell clone which contains only one copy of a plasmid integrate. Independent amplificants, originating from this clone and containing elevated plasmid copy numbers, were isolated and analyzed. Based on previous observations, encompassing the persistence of single-copy DNA sequences besides amplified DNA sequences clustered at a different location in the independent amplificants, we proposed an amplification pathway including a local duplication step and transposition of the duplicated DNA to other chromosomal positions. Now we have extended our study to more independent amplificants. We prove that the single-copy plasmid-containing chromosomes in the different amplificants and the single-copy plasmid-containing chromosome in the original parental cell clone are indeed identical, namely a translocation chromosome composed of at least three parts of which two originate from chromosomes 14 and 17. We show that the unit of amplification and the unit of the proposed transposition event are at least 1.5 Mb. We also demonstrate that the amplified DNA sequences, present at genomic locations other than the original single-copy DNA sequences, are preferentially associated with chromosome 16. We find that the amplified DNA sequences are often located at or near a site of chromosome translocation involving chromosome 16. In one cell clone we detect the amplified DNA sequences in most of the cells to be located within a complete chromosome 16 while in a minority of cells the amplified sequences are located at or near a breakpoint on a translocation chromosome 16. This indicates that this amplification region is highly unstable and frequently gives rise to translocation events.

Association of early bilateral middle ear effusion with language at age 5 years.

OBJECTIVE: This study tested the hypothesis that children with early persistent middle ear effusion (MEE) are at risk for later language deficit. METHOD: We recruited 698 newborns and monitored them for MEE every 2 to 4 weeks at home until age 3 years. Language skills were assessed on 294 subjects at age 5, while controlling for 8 demographic and environmental factors. Language outcomes at age 5 years were studied as a function of duration of bilateral MEE from birth to age 3 years. RESULTS: A significant relation was found between duration of bilateral MEE and speech sound sensitivity (Carrow Elicited Language Inventory) and articulation (Goldman-Fristoe Articulation). Children's ability to discriminate speech sounds in a quiet environment (Carrow Auditory Visual Abilities Test) was less affected by early prolonged MEE in homes that provided more cognitive stimulation. CONCLUSIONS: These exploratory results indicate that prolonged early MEE may predispose children to language deficits at age 5 years. The language deficits are of small magnitude and may or may not be clinically significant. Language stimulation at home may protect against some of the effects of prolonged MEE.

Tumescent liposuction. A surgeon's perspective.

The tumescent technique consists of injection of large amounts of dilute lidocaine and epinephrine into the subcutaneous fat prior to performing liposuction. This preinjection provides local anesthesia and reduction in postoperative pain. Blood loss, bruising, and swelling are significantly reduced. The large volume of injectate is a complete fluid replacement, eliminating the need for intravenous fluid support. In the authors' experience, patients are most comfortable when the tumescent technique is combined with monitored intravenous sedation.

The effect of pentoxifylline on random-pattern skin-flap necrosis induced by nicotine treatment in the rat.

Cigarette smoke, and specifically nicotine, has been shown to reduce skin-flap survival. The purpose of this study was to determine if the preoperative administration of pentoxifylline can counteract the deleterious effects of nicotine on skin-flap survival in the rat. Sixty rats were distributed into four groups (n = 15). The survival of modified McFarlane skin flaps was assessed on postoperative day 7. The administration of nicotine (0.6 mg/kg) for 24 weeks preoperatively produced an average skin-flap survival of 59 percent; this was significantly decreased compared with controls (p < 0.05). When similarly treated animals were given pentoxifylline (20 mg/kg) for 30 days preoperatively, the mean skin-flap survival improved significantly to 80 percent (p < 0.05). Withholding nicotine for 2 weeks preoperatively also was found to significantly improve skin-flap survival to 73 percent (p < 0.05). Blood filterability was measured as an indicator of viscosity. The blood filterability in rats that received nicotine for 24 weeks was significantly decreased compared with controls (p < 0.05). Both the addition of pentoxifylline preoperatively and the withholding of nicotine for 2 weeks preoperatively were found to significantly improve blood filterability compared with rats that received nicotine alone for 24 weeks postoperatively (p < 0.05).

New composite resins: comparison of their resistance to toothbrush abrasion and characteristics of abraded surfaces.

Two of the newer composite resins, Miradapt and Visar-Fil, have resistance to toothbrush abrasion similar to that of Concise. After abrasion, the surface of these two resins appeared to be as rough as that of Concise. Prisma-Fil, Finesse, and Silar were abraded approximately six times as much as Concise. The abraded surface of Prisma-Fil was slightly granular, but the surface of Finesse and Silar was quite smooth. Surface characteristics after abrasion are attributable to the size of the largest particles used to make the composite resin. The blending of large and submicron particles may produce a composite that can initially be finished to an enamel-like smoothness, but eventually, toothbrush abrasion will produce a surface similar to that of a large particle resin, with the same potential for trapping stains and causing gingival irritation.

Effect of methods of tooth enamel preparation on the retentive strength of acid-etch composite resins.

The effect of three procedures of preparing enamel surface on the retentive strengths of Concise Enamel Bond. Adaptic acid etch, Restodent and Nurva-Seal/Nurva-Fil was investigated. Resins using an unfilled-filled resin combination(Concise Enamel Bond, Adaptic acid etch, and Nurva-Fil) had a significantly higher retentive strength when the enamel was prepared with a coarse diamond bur than when the surface was unprepared or prepared with a carbide bur. The different procedures of tooth preparation did not affect the retentive strength of the resin when only filled resin was used (Restodent).

A clinical taxonomy for the categorization of pragmatic language functions in normal preschool children.

The present study assessed the pragmatic language skills in normal preschool children. A specially designed taxonomy was used to classify the subjects' responses. Findings revealed (1) high reliability in the use of the taxonomy; (2) a decrease in the inappropriate responses used by children with an increase in age; (3) generally, inappropriate responses were confined to the same categories across age levels. Findings are discussed relative to patterns of pragmatic language development as a function of age.

The use of a pragmatic protocol with normal preschool children.

The purpose of the present study was to examine the use of language functions in a structured setting in normal preschool children. Subjects were 240 males and females between the ages of two and five years. Results indicated statistically significant differences in the appropriateness of responses among several different age groups. The results are discussed relative to the assessment of the preschool child with a possible language disorder.

AANA journal course: update for nurse anesthetists--current concepts in cardiopulmonary resuscitation.

Developmental strides in cardiopulmonary resuscitation were paralleled by acquired knowledge of pulmonary and cardiac physiology. The evolution of the current recommendations for cardiopulmonary resuscitation have evolved over the past 40 years as a result of human trial and error and have been authenticated through laboratory research and clinical experience. Current research efforts in cardiopulmonary resuscitation are examining the mechanisms of blood flow during external cardiac massage and techniques to optimize myocardial and cerebral perfusion during the resuscitative period. The restoration of circulation with preservation of myocardial and neurological function is the essential goal during the resuscitative effort. Prior to the 1950s, the occurrence of cardiac arrest was associated with the conduct of anesthesia and surgery. Although infrequent today by comparison, cardiac arrest continues to occur with the conduct of regional and general anesthesia. This course will review the etiology of cardiac arrest during anesthesia, detail the mechanisms of blood flow during resuscitation, and review the pharmacological importance of epinephrine for the preservation of myocardial and neurologic function.

How does the peer review process influence AANA journal article readability?

This study examined the readability of the AANA Journal, quantifying the effect of peer review on case and research reports published from 1992 to 1994. Gunning and Flesch index-based computer analysis, as well as human comparative analysis, was undertaken. Computer and human assessment of readability revealed improvement as papers evolved from submitted to published versions; however, at publication the manuscripts remained in the "difficult" readability range. Although this study provides evidence that peer review improves readability, it may be that, due to a professed need for scientific purity and an imposed sense of scholarship, nursing and other biomedical journals may overemphasize a style and approach that paradoxically make transfer of information unreasonably difficult.

AANA Journal Course: update for nurse anesthetists-brain death: terminology, clinical criteria, and diagnostic testing.

Developmental strides in biomedical technology and the growth and availability of intensive care units have paralleled the development of organ transplantation programs. The establishment of these programs required the development of a new definition, criteria, and test of death to facilitate the procurement of suitable organs. The historical definition of death, in instances of organ transplantation, is no longer compatible as cardiopulmonary support can be readily applied to individuals without spontaneous circulation and respiration. This AANA Journal course will examine the definitions of death and the evolution of the term "brain death." The essential clinical criteria and confirmatory diagnostic testing for the determination of adult "whole-brain death" will be reviewed.

AANA journal course: update for nurse anesthetists--current concepts in pediatric resuscitation.

The evolution of the current recommendations for pediatric cardiopulmonary resuscitation have slowly evolved following the development of cardiopulmonary resuscitation techniques for the adult patient. Anatomical and physiological differences along with the pathophysiology of pediatric cardiopulmonary arrest mandate special consideration when approaching pediatric cardiopulmonary resuscitation.