Nuclear markers confirm taxonomic status and relationships among highly endangered and closely related right whale species.

Right whales (genus: Eubalaena) are among the most endangered mammals, yet their taxonomy and phylogeny have been questioned. A phylogenetic hypothesis based on mitochondrial DNA (mtDNA) variation recently prompted a taxonomic revision, increasing the number of right whale species to three. We critically evaluated this hypothesis using sequence data from 13 nuclear DNA (nuDNA) loci as well as the mtDNA control region. Fixed diagnostic characters among the nuclear markers strongly support the hypothesis of three genetically distinct species, despite lack of any diagnostic morphological characters. A phylogenetics analysis of all data produced a strict consensus cladogram with strong support at nodes that define each right whale species as well as relationships among species. Results showed very little conflict among the individual partitions as well as congruence between the mtDNA and nuDNA datasets. These data clearly demonstrate the strength of using numerous independent genetic markers during a phylogenetics analysis of closely related species. In evaluating phylogenetic support contributed by individual loci, 11 of the 14 loci provided support for at least one of the nodes of interest to this study. Only a single marker (mtDNA control region) provided support at all four nodes. A study using any single nuclear marker would have failed to support the proposed phylogeny, and a strong phylogenetic hypothesis was only revealed by the simultaneous analysis of many nuclear loci. In addition, nu DNA and mtDNA data provided complementary levels of support at nodes of different evolutionary depth indicating that the combined use of mtDNA and nuDNA data is both practical and desirable.

Cooperative learning and feminist pedagogy--a model for classroom instruction in nursing education.

This article reports on a research study conducted as part of a doctoral dissertation on the development of a cooperative learning teaching model in nursing education. The subjects for the study were a convenience sample of registered nurses who were pursuing a baccalaureate degree in nursing at an urban university. Principles of feminist pedagogy were incorporated as part of the cooperative learning model. The teacher/researcher taught two sections of the same course and, through the use of action research, developed a model for using cooperative learning strategies as the primary teaching modality. End of class and end of semester evaluations provided feedback that suggested that this was an exciting and effective alternative to traditional classroom teaching.

Effect of low pH on single skeletal muscle myosin mechanics and kinetics.

Acidosis (low pH) is the oldest putative agent of muscular fatigue, but the molecular mechanism underlying its depressive effect on muscular performance remains unresolved. Therefore, the effect of low pH on the molecular mechanics and kinetics of chicken skeletal muscle myosin was studied using in vitro motility (IVM) and single molecule laser trap assays. Decreasing pH from 7.4 to 6.4 at saturating ATP slowed actin filament velocity (V(actin)) in the IVM by 36%. Single molecule experiments, at 1 microM ATP, decreased the average unitary step size of myosin (d) from 10 +/- 2 nm (pH 7.4) to 2 +/- 1 nm (pH 6.4). Individual binding events at low pH were consistent with the presence of a population of both productive (average d = 10 nm) and nonproductive (average d = 0 nm) actomyosin interactions. Raising the ATP concentration from 1 microM to 1 mM at pH 6.4 restored d (9 +/- 3 nm), suggesting that the lifetime of the nonproductive interactions is solely dependent on the [ATP]. V(actin), however, was not restored by raising the [ATP] (1-10 mM) in the IVM assay, suggesting that low pH also prolongs actin strong binding (t(on)). Measurement of t(on) as a function of the [ATP] in the single molecule assay suggested that acidosis prolongs t(on) by slowing the rate of ADP release. Thus, in a detachment limited model of motility (i.e., V(actin) approximately d/t(on)), a slowed rate of ADP release and the presence of nonproductive actomyosin interactions could account for the acidosis-induced decrease in V(actin), suggesting a molecular explanation for this component of muscular fatigue.

Hypertrophic and dilated cardiomyopathy mutations differentially affect the molecular force generation of mouse alpha-cardiac myosin in the laser trap assay.

Point mutations in cardiac myosin, the heart's molecular motor, produce distinct clinical phenotypes: hypertrophic (HCM) and dilated (DCM) cardiomyopathy. Do mutations alter myosin's molecular mechanics in a manner that is predictive of the clinical outcome? We have directly characterized the maximal force-generating capacity (F(max)) of two HCM (R403Q, R453C) and two DCM (S532P, F764L) mutant myosins isolated from homozygous mouse models using a novel load-clamped laser trap assay. F(max) was 50% (R403Q) and 80% (R453C) greater for the HCM mutants compared with the wild type, whereas F(max) was severely depressed for one of the DCM mutants (65% S532P). Although F(max) was normal for the F764L DCM mutant, its actin-activated ATPase activity and actin filament velocity (V(actin)) in a motility assay were significantly reduced (Schmitt JP, Debold EP, Ahmad F, Armstrong A, Frederico A, Conner DA, Mende U, Lohse MJ, Warshaw D, Seidman CE, Seidman JG. Proc Natl Acad Sci USA 103: 14525-14530, 2006.). These F(max) data combined with previous V(actin) measurements suggest that HCM and DCM result from alterations to one or more of myosin's fundamental mechanical properties, with HCM-causing mutations leading to enhanced but DCM-causing mutations leading to depressed function. These mutation-specific changes in mechanical properties must initiate distinct signaling cascades that ultimately lead to the disparate phenotypic responses observed in HCM and DCM.

Jewish beliefs, values, and practices: implications for culturally sensitive nursing care.

Providing culturally sensitive nursing care for the Jewish patient is a challenge for the non-Jewish nurse. Understanding the major values, ethics, and practices of Judaism that have relevance to nursing and medical care will give the advanced practice nurse the ability to provide care that is individualized to the patient and family.

Human complement C7 and C9 in fetal and newborn sera.

Using specific immune sera, C7, C9, and C3 activator were detected in sera from human fetuses more than 16 weeks old and in newborn samples. Levels of C9 in cord sera ranged between 10 and 30% of those present in sera from adult subjects. The mean value of Ce activator was about half that in maternal blood. The mean level of C7 in newborns was nearly 70% of the amount in normal adults.

Alpha-fetoprotein levels in different strains of mice during development.

The levels of alpha-fetoprotein (AFP) were measured at birth and at 7 days of age in plasma from Q, C3H/He-mg/Crc, BALB/c/Crc, A/Crc, CBA/Ca/Crc and C57BL/Mcl mice, and in adult blood samples and amniotic fluid at 14 days' gestation from Q, C3H and BALB/c mice. The AFP levels in amniotic fluid and neonatal plasma were significantly higher in BALB/c mice than in the other strains tested, but by postnatal day 7, C57BL and C3H mice had the highest plasma levels. It seems therefore that the genetic mechanisms controlling the synthesis of AFP prenatally may be distinct from those concerned with its reduction after birth. At 6 weeks of age the level in C3H mice was somewhat higher than in BALB/c, and more than double that in Q mice. An attempt to increase AFP levels in response to galactosamine-induced liver damage was successful only in Q adult mice. When amniotic fluids and day 0 plasma from several strains of mice were tested by polyacrylamide gel electrophoresis, no differences in AFP mobility were detected. Preliminary studies were also carried out to see if the administration of sodium butyrate, claimed to delay the timing of the fetal to adult haemoglobin switch in some mammalian species, could also affect the rate of synthesis of AFP during fetal life.

Levels of beta-trace protein and lysozyme in human amniotic fluids.

The levels of beta-trace protein and lysozyme were estimated in amniotic fluids from normal fetuses and from fetuses with neuraltube defects. The values of these proteins in normal amniotic fluids were found to be similar to those detected in fetuses with anencephaly and spina bifida. The levels of lysozyme were shown to be correlated with gestational age.

A fluorescence video-endoscopy technique for detection of gene transfer and expression.

The green fluorescent protein (GFP) has previously been adapted as a reported for gene transfer and expression in mammalian cells in culture and in tissue sections. Herein is described a new method for detecting GFP in situ within epithelia accessible to fiberoptic endoscopy by incorporating fluorescent filters for detection of GFP into an existing fiberoptic endoscopy system. This device was used to detect expression of GFP from adeno-associated virus (AAV; does of 3 x 10(7) IU) and adenovirus (Ad; does of l x 10(9) to 1 x 10(10) p.f.u.) vectors within the bronchial epithelium of New Zealand white rabbits. GFP expression was confirmed by fluorescence-activated cell sorting (FACS), direct fluorescence microscopy of cytospin preparations of brushed cells, and by fluorescence microscopy of fixed tissue sections. This reporter gene/detection system was then used to track the time course of expression of the AAV vector in the bronchial epithelium over the first 30 days after administration. The transduction frequency in the treated region of the epithelium peaked at around 50% at 21 days after transduction. Vector expression was still present at around 20% efficiency at 30 days after administration. This example indicates how this method could be used to reliably track gene transfer in living animals or patients.

Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung.

Gene therapy for cystic fibrosis (CF) would ideally be accomplished with a vector capable of long-term expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in the absence of a host inflammatory response. Recombinant adeno-associated virus (AAV)-CFTR vectors possess these characteristics in rabbits. Because the utility of AAV vectors as gene transfer agents has only been recognized recently, AAV vector-mediated transduction has never been modeled in a primate host, which is an important step before its use in humans. In order to test the safety and biological activity of AAV-CFTR, single doses of AAV-CFTR vector were administered by fiberoptic bronchoscopy to the posterior basal segment of the right lower lobe (RLL) of the lungs of 10 rhesus macaques with four matched vehicle-treated controls. Animals were followed for 10, 21, 90 or 180 days following vector instillation. Vector DNA transfer occurred in bronchial epithelial cells in the RLL of each animal that received vector as assessed by in situ DNA PCR. Vector mRNA was detectable for 180 days after administration as detected by RT-PCR and by RNase protection assay. Safety of vector administration was determined by measurements of pulmonary mechanics, arterial blood gas analysis, chest radiographs, and bronchoalveolar lavage (BAL) fluid analysis including cell count and quantification of inflammatory cytokines. Gross and microscopic pathologic examination were also performed. There was no evidence of inflammation or other toxicity, although vector DNA was found in extrapulmonary organs of some animals. These results indicate that transduction of the primate airway epithelium with the AAV-CFTR mediates long-term CFTR cDNA transfer and is relatively safe.

Repeated delivery of adeno-associated virus vectors to the rabbit airway.

Efficient local expression from recombinant adeno-associated virus (rAAV)-cystic fibrosis (CF) transmembrane conductance regulator (CFTR) vectors has been observed in the airways of rabbits and monkeys for up to 6 months following a single bronchoscopic delivery. However, it is likely that repeated administrations of rAAV vectors will be necessary for sustained correction of the CF defect in the airways. The current study was designed to test the feasibility of repeated airway delivery of rAAV vectors in the rabbit lung. After two doses of rAAV-CFTR to the airways, rabbits generated high titers of serum anti-AAV neutralizing antibodies. Rabbits then received a third dose of a rAAV vector containing the green fluorescent protein (GFP) reporter gene packaged in either AAV serotype 2 (AAV2) or serotype 3 (AAV3) capsids. Each dose consisted of 1 ml containing 5 x 10(9) DNase-resistant particles of rAAV vector, having no detectable replication-competent AAV or adenovirus. Three weeks later, GFP expression was observed in airway epithelial cells despite high anti-AAV neutralizing titers at the time of delivery. There was no significant difference in the efficiency of DNA transfer or expression between the rAAV3 and rAAV2 groups. No significant inflammatory responses to either repeated airway exposure to rAAV2-CFTR vectors or to GFP expression were observed. These experiments demonstrate that serum anti-AAV neutralizing antibody titers do not predict airway neutralization in vivo and that repeated airway delivery rAAV allows for safe and effective gene transfer.