CNS Hypomyelination Disrupts Axonal Conduction and Behavior in Larval Zebrafish.

Myelination is essential for central nervous system (CNS) formation, health and function. As a model organism, larval zebrafish have been extensively employed to investigate the molecular and cellular basis of CNS myelination, because of their genetic tractability and suitability for non-invasive live cell imaging. However, it has not been assessed to what extent CNS myelination affects neural circuit function in zebrafish larvae, prohibiting the integration of molecular and cellular analyses of myelination with concomitant network maturation. To test whether larval zebrafish might serve as a suitable platform with which to study the effects of CNS myelination and its dysregulation on circuit function, we generated zebrafish myelin regulatory factor (myrf) mutants with CNS-specific hypomyelination and investigated how this affected their axonal conduction properties and behavior. We found that myrf mutant larvae exhibited increased latency to perform startle responses following defined acoustic stimuli. Furthermore, we found that hypomyelinated animals often selected an impaired response to acoustic stimuli, exhibiting a bias toward reorientation behavior instead of the stimulus-appropriate startle response. To begin to study how myelination affected the underlying circuitry, we established electrophysiological protocols to assess various conduction properties along single axons. We found that the hypomyelinated myrf mutants exhibited reduced action potential conduction velocity and an impaired ability to sustain high-frequency action potential firing. This study indicates that larval zebrafish can be used to bridge molecular and cellular investigation of CNS myelination with multiscale assessment of neural circuit function.SIGNIFICANCE STATEMENT Myelination of CNS axons is essential for their health and function, and it is now clear that myelination is a dynamic life-long process subject to modulation by neuronal activity. However, it remains unclear precisely how changes to myelination affects animal behavior and underlying action potential conduction along axons in intact neural circuits. In recent years, zebrafish have been employed to study cellular and molecular mechanisms of myelination, because of their relatively simple, optically transparent, experimentally tractable vertebrate nervous system. Here we find that changes to myelination alter the behavior of young zebrafish and action potential conduction along individual axons, providing a platform to integrate molecular, cellular, and circuit level analyses of myelination using this model.

The decision-making processes of pharmacists in inland Australia--a pilot study.

INTRODUCTION: Little research has been conducted analysing the organisational risks that compound and trigger dispensing and medication errors. This pilot study appraises the attitudes to and behaviours related to the dispensing errors of pharmacists practising in diverse venues and roles in inland Australia. METHODS: Twelve pharmacists working in the Riverina (Wiradjuri country) participated in a structured interview consisting of a brief survey and open-ended questions. The interviews were audio-recorded for transcription, then analysed by the interviewer for emerging themes. In this pilot study, the attitudes and actions of pharmacists in response to dispensing errors were explored to determine the nature of organisational strategies implemented to detect and recover 'slips, lapses and mistakes'. The rationale behind investigating attitudes and actions stems from the theory of planned behaviour. RESULTS: While many common themes emerged, the attitudes of each pharmacist were unique. The strategies implemented to prevent errors were venue-specific and purpose-designed to the training level of the staff and physical environment. A diverse mix of attitudes was represented by the sample, with no correlation between worksite, sex, age or role identified. Trends may emerge because, in regard to dispensing errors, subjective norms and perceived behavioural control play a greater role in forming the intention to act, rather than personal attitudes. The majority of examples given by participants was discussion of recorded errors and near misses, which included changes to procedures implemented to prevent the same error occurring. This culture of continuous quality improvement was the overarching common theme. Other common themes were the role of technology in the supply of medicines, privacy implications when drawing staff from a rural or regional centre, workload concerns with regard to management responsibility and the impact of the way error management was demonstrated during the formative early years of practice. Distraction from dispensing, through management roles in pharmacies with moderate prescription volumes, was a common contributor to errors. CONCLUSION: A culture of continuous quality improvement exists amongst pharmacists in Inland Australia, which would benefit from improved dialogue about the impact of organisational risks on the rate of dispensing errors. The safety culture, and behaviour modelling experienced during the internship program has a profound impact on the perceived behavioural control of young pharmacists. This year instils mores, which may be the result of independent survival in remote and regional settings, rather than compliance with professional practice standards. While many of the pressures and demands of minimising errors are common across the profession; unique, venue specific strategies are commonly implemented in the cycle of continuous quality improvement in regional and remote settings.

Jarosite as an indicator of water-limited chemical weathering on Mars.

The Mars Exploration Rover Opportunity identified the ferric sulphate mineral jarosite and possible relicts of gypsum at the Meridiani Planum landing site. On Earth, jarosite has been found to form in acid mine drainage environments, during the oxidation of sulphide minerals, and during alteration of volcanic rocks by acidic, sulphur-rich fluids near volcanic vents. Jarosite formation is thus thought to require a wet, oxidizing and acidic environment. But jarosite on Earth only persists over geologically relevant time periods in arid environments because it rapidly decomposes to produce ferric oxyhydroxides in more humid climates. Here we present equilibrium thermodynamic reaction-path simulations that constrain the range of possible conditions under which such aqueous alteration phases are likely to have formed on Mars. These calculations simulate the chemical weathering of basalt at relevant martian conditions. We conclude that the presence of jarosite combined with residual basalt at Meridiani Planum indicates that the alteration process did not proceed to completion, and that following jarosite formation, arid conditions must have prevailed.

Distribution of Na+,K+-ATPase in rat exocrine pancreas as monitored by K+-NPPase cytochemistry and [3H]-ouabain binding: a plasma membrane protein found primarily to be ductal cell associated.

We investigated the distribution of Na+,K+-ATPase in rat exocrine pancreas. By use of enzymatic dissociation techniques, pancreatic acini (containing acinar cells and centroacinar ductal cells in a ratio of about 10:1) and all major classes of pancreatic ducts were isolated and analyzed for the presence of Na+,K+-ATPase using K+-NPPase cytochemistry and [3H]-ouabain binding assays. Ultrastructural analysis demonstrated a basolateral localization of ouabain-sensitive enzyme activity in all classes of pancreatic ducts, although the degree of activity varied among the various classes. Qualitative analysis (scale of 0 to + + +) indicated the following enzyme distribution: centroacinar ductal cells (+); intralobular ducts (+ +); interlobular ducts (+ + +); main duct (+ +). In contrast, no reaction product was associated with pancreatic acinar cells even when observed adjacent to enzyme-positive centroacinar ductal cells. Parallel experiments monitoring [3H]-ouabain binding supported the cytochemical studies. When expressed as femtomoles [3H]-ouabain/microgram DNA, the following values were obtained: whole pancreas, 100.3; ducts (pooled intralobular and interlobular), 337.0; acini, 48.2. The acinar value is complicated by the fact that acini contain both acinar and centroacinar cells, but in light of the cytochemical observations we suggest that most of the [3H]-ouabain binding is due to the few ductal cells present in acini. The results suggest that Na+,K+-ATPase is primarily associated with the ductal epithelium of the exocrine pancreas and is differentially distributed among the different classes of ducts.

The pancreatic ductal system of the rat: cell diversity, ultrastructure, and innervation.

The morphological features and innervation of the pancreatic ductal epithelium of the rat was investigated. For the purposes of this study, pancreatic ducts were classified as either intercalated, intralobular, interlobular, or main. Epithelial cells of these different classes were classified as either principal or specialized. Principal cells make up the majority of epithelial cells in all classes of ducts. Ultrastructural analysis indicated that cytoplasmic vesicles, some containing amorphous material, were often seen in the apical portion of principal cells. Similarly, a common feature of these cells was the appearance of apical membrane projections containing various cytoplasmic organelles. These vesicles and apical membrane projections became larger as one progressed through the ductal system to the main duct. Junctional complexes contained well-developed tight junctions that, when analyzed by freeze-fracture analysis, were found to consist of three to five sealing strands in a parallel arrangement. Specialized cells could be morphologically divided into five categories: light cells, basal cells, goblet cells, endocrine cells, and brush cells. Light cells, which only differed from principal cells by their clear, lightly staining cytoplasm, were found in all classes of ducts. Basal cells, which were attached to the basement membrane of the ductal epithelium but did not extend to the lumen, were found in all classes of ducts except the intercalated ducts. Goblet cells and endocrine cells were observed in the main and interlobular ducts, while cells very similar in morphologic appearance to the brush cells of the lung were found restricted to the interlobular ducts. Localization of biogenic amines by histofluorescence indicated that adrenergic nerve fibers were associated with the main ducts and interlobular ducts. Histochemical localization of acetylcholinesterase activity indicated that cholinergic fibers were also associated with the main and interlobular ducts. These results indicate that in the rat, the pancreatic ductal system is composed of a number of cell types that are differentially distributed in the various classes of ducts, and pancreatic ducts are associated with adrenergic and cholinergic nerves.

Morphological and biochemical characterization of a human pancreatic ductal cell line (PANC-1).

Study of the products secreted by pancreatic ductal cells and analysis of the mechanisms involved in the discharge of these products have been limited by a lack of in vitro models available to experimentally approach this problem. To this aim, this investigation has been designed to determine if a human pancreatic carcinoma cell line of ductal origin (PANC-1) has maintained some of the differentiated characteristics of normal mammalian pancreatic ductal epithelium. Morphological and immunocytochemical studies indicated that, similar to isolated rat pancreatic ducts, the PANC-1 cell line contained (a) intermediate filaments of the epithelial class, (b) a basolateral plasma membrane localization of Na+, K+-ATPase, (c) complete tight junctions based on freeze-fracture analysis, (d) a cuboidal morphology when grown on Type I collagen-coated nitrocellulose filters or isolated amnion basement membrane, and (e) normal ductal epithelial ultrastructural features. Biochemical analysis indicated that, also similar to isolated rat and human pancreatic ducts, the PANC-1 cell line contained (a) gamma-glutamyltranspeptidase, (b) carbonic anhydrase, and (c) Na+, K+-ATPase based on [3H]ouabain binding assays. Comparative studies with other transformed lines indicated that PANC-1 cells have similarities to ductal lines such as MDCK cells but are markedly different from mesenchymally derived lines such as L cells. In addition, as with isolated rat and human ducts, PANC-1 cells synthesize and secrete sulfated proteins with a MW range of approximately 180K to 1 million daltons, with the predominant species being 660K daltons as indicated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These results indicate that the PANC-1 cell line has maintained at least some of the differentiated characteristics of normal pancreatic ductal epithelial cells and may be a useful system for study of ductal secretory products as well as the mechanisms involved in the discharge of these products.

Comparative analysis of a human pancreatic undifferentiated cell line (MIA PaCa-2) to acinar and ductal cells.

Pancreatic ductal cell secretion has not been well characterized due to the difficulty in obtaining sufficient quantities of purified ductal cells. To determine if the MIA PaCa-2 cell line would provide a useful model for in vitro studies of pancreatic ductal cell secretion, the present study was designed to characterize these cells in greater detail. In this investigation, the human pancreatic undifferentiated cell line, MIA PaCa-2, was compared with PANC-1 cells (a human ductal cell line previously characterized), isolated rat and human ducts, acinar cells, and nonpancreatic cell lines. The results indicate that while the morphology of the MIA PaCa-2 cell line is nonpolarized and generally atypical of either ductal or acinar cells, the cell line has retained certain biochemical similarities to ductal cells. Additional morphological studies indicated (a) the presence of intermediate filaments characteristic of epithelial cells, (b) the absence of zymogen granules, and (c) an apparent basolateral plasma membrane localization of Na+, K+-ATPase. Similar to ductal cells, biochemical analyses indicated (a) the presence of Na+, K+-ATPase based on [3H]-ouabain binding assays, (b) high levels of carbonic anhydrase, (c) low levels of gamma-glutamyl transpeptidase, (d) nondetectable levels of amylase, and (e) protein composition and protein synthetic patterns comparable to PANC-1 cells. Finally, as with PANC-1 cells and isolated rat and human ducts, the major sulfated secretory product of MIA PaCa-2 cells was a protein with a molecular weight of approximately 660,000 to 1 million.(ABSTRACT TRUNCATED AT 250 WORDS)

Perceived vulnerability and control of martial arts and physical fitness students.

Anecdotal reports and limited research suggest that enrolling in self-defense courses can enhance feelings of control and reduce feelings of vulnerability; however, much self-defense is taught in the context of martial arts courses. To assess the effects of martial arts courses on perceptions of vulnerability and control, 83 students in physical fitness and 59 students in martial arts courses at 10 randomly chosen large universities responded to questionnaires. Martial arts students scored lower on control, higher on vulnerability, and higher on perceived likelihood of being injured than fitness students while enrolled in their courses. A year later, regardless of whether they had continued training, they scored higher on control and lower on vulnerability. Neither gender nor prior history of assault was related to responses. Enrolling in martial arts courses may not enhance people's feelings of control, at least in the initial stages of training.

Fiber optic sensing technology for detecting gas hydrate formation and decomposition.

A fiber optic-based distributed sensing system (DSS) has been integrated with a large volume (72 l) pressure vessel providing high spatial resolution, time-resolved, 3D measurement of hybrid temperature-strain (TS) values within experimental sediment-gas hydrate systems. Areas of gas hydrate formation (exothermic) and decomposition (endothermic) can be characterized through this proxy by time series analysis of discrete data points collected along the length of optical fibers placed within a sediment system. Data are visualized as an animation of TS values along the length of each fiber over time. Experiments conducted in the Seafloor Process Simulator at Oak Ridge National Laboratory clearly indicate hydrate formation and dissociation events at expected pressure-temperature conditions given the thermodynamics of the CH(4)-H(2)O system. The high spatial resolution achieved with fiber optic technology makes the DSS a useful tool for visualizing time-resolved formation and dissociation of gas hydrates in large-scale sediment experiments.

The variety of emotional reactions to miscarriage.

The theme that emotions following miscarriage are more varied and less uniformly negative than accounts imply is examined through a review of popular and professional literature and evidence from sixty-five women who had had spontaneous abortions who were interviewed about their experiences and emotions. Emotions remembered from immediately following the miscarriage were sadness, frustration, disappointment, and anger towards themselves. At the interview time, about four months after the miscarriage, the most common emotions were hopefulness, sadness, and happiness. Implications for health care and research are discussed.

Development of an apical plasma membrane domain and tight junctions during histogenesis of the mammalian pancreas.

The role of tight junctions (zonula occludens) in the formation of apical plasma membrane (PM) domains was investigated in the embryonic rat pancreas. In the present study, lectin-rhodamine (WGA-TRITC and RCAII-TRITC) and lectin-gold (WGA-Au and RCAII-Au) conjugates were used to monitor apical PM domain formation and freeze-fracture analysis was used to monitor tight junction formation in the pancreatic epithelium of embryonic, neonatal, and adult rats. Fluorescent and TEM analysis of WGA and RCAII binding indicated that an apical PM domain is formed as early as Day 13 of gestation in the pancreatic epithelium. While apical WGA binding remained into adult life, RCAII binding was lost by 1 day after birth. In contrast, tight junctions were not observed until Day 14 of gestation. At this time, tight junctions were found to be incomplete in formation and typically consisted of linear arrays of IMPs or discontinuous arrays of sealing strands (focal adherens). Continuous tight junctions were not completely formed until Day 15 of gestation. Continued development of tight junctions during gestation was characterized by (1) an increase in the number of sealing strands and (2) a more parallel arrangement of sealing strands within each junctional complex. By 8 weeks after birth, tight junctions were more loosely organized and contained fewer sealing strands as compared to that observed in the fetus. These results suggest that lateral diffusion of apical PM glycoconjugates may be restricted even in the absence of complete tight junctional complexes during development of the rat pancreas.

Extracellular matrix (mesoglea) of Hydra vulgaris. I. Isolation and characterization.

Hydrozoans such as Hydra vulgaris, as with all classes of Cnidaria, are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. The present study was undertaken to determine what extracellular matrix (ECM) components are associated with Hydra mesoglea. Using polyclonal antibodies generated from vertebrate ECM molecules, initial light and electron microscopic immunocytochemical studies indicated the presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin immunoreactive components in Hydra mesoglea. These immunocytochemical observations were in part supported by biochemical analyses of isolated Hydra mesoglea which indicated the presence of fibronectin and laminin based on Western blot analysis. Amino acid analysis of total mesoglea and some of its isolated components confirmed the presence of collagen molecules in mesoglea. Additional studies indicated the presence of (1) a gelatin binding protein in Hydra which was immunoreactive with antibodies raised to human plasma fibronectin and (2) a noncollagen fragment extracted from mesoglea which was immunoreactive to antibodies raised to the NC1 domain (alpha 1 subunit) of bovine glomerular basement membrane type IV collagen. These observations indicate that Hydra mesoglea is evolutionarily a primitive basement membrane that has retained some properties of interstitial ECM.