Quantitative measurement of cell migration using time-lapse videomicroscopy and non-linear system analysis

Epithelial cells of the mammary gland possess the inherent capacity to form epithelial monolayers in vitro. This requires coordination of cell migration, cell-cell contact formation, and cell proliferation. Using time-lapse phase contrast videomicroscopy we have observed mammary gland epithelial cells over different time scales. We show the generation of a complete polarized epithelial monolayer in real-time, starting from a few cells. We subsequently concentrated on the early stages of this process by tracking epithelial cells during phases of polarized migration. We performed migration analysis using fractal measures. With this technology the structure of seemingly random processes not accessible to the usual methods of linear analysis can be measured. As a control and proof of principle approach we applied infection of cells with an adenoviral vector, which is used as a gene targeting vector for many applications. Infection markedly influenced the patterns of migratory behavior. We, therefore, believe that time-lapse videomicroscopy in combination with fractal analysis can contribute to differential characterization of distinct cellular migration patterns. This will be useful in situations of long-term alterations in cell culture systems.     

Dystroglycan is required for polarizing the epithelial cells and the oocyte in Drosophila

The transmembrane protein Dystroglycan is a central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin, agrin and perlecan, and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Using mosaic analysis and RNAi in the model organism Drosophila melanogaster, we show that Dystroglycan is required cell-autonomously for cellular polarity in two different cell types, the epithelial cells (apicobasal polarity) and the oocyte (anteroposterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and overexpression results in a reduced apical localization of these same markers. In Dystroglycan germline clones early oocyte polarity markers fail to be localized to the posterior, and oocyte cortical F-actin organization is abnormal. Dystroglycan is also required non-cell-autonomously to organize the planar polarity of basal actin in follicle cells, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity; and this study sets the stage for analyzing the Dystroglycan complex by using the power of Drosophila molecular genetics.     

Absence of the Lhcb1 and Lhcb2 proteins of the light-harvesting complex of photosystem II - effects on photosynthesis,grana stacking and fitness

We have constructed Arabidopsis thaliana plants that are virtually devoid of the major light-harvesting complex, LHC II. This was accomplished by introducing the Lhcb2.1 coding region in the antisense orientation into the genome by Agrobacterium-mediated transformation. Lhcb1 and Lhcb2 were absent, while Lhcb3, a protein present in LHC II associated with photosystem (PS) II, was retained. Plants had a pale green appearance and showed reduced chlorophyll content and an elevated chlorophyll a/b ratio. The content of PS II reaction centres was unchanged on a leaf area basis, but there was evidence for increases in the relative levels of other light harvesting proteins, notably CP26, associated with PS II, and Lhca4, associated with PS I. Electron microscopy showed the presence of grana. Photosynthetic rates at saturating irradiance were the same in wild-type and antisense plants, but there was a 10-15% reduction in quantum yield that reflected the decrease in light absorption by the leaf. The antisense plants were not able to perform state transitions, and their capacity for non-photochemical quenching was reduced. There was no difference in growth between wild-type and antisense plants under controlled climate conditions, but the antisense plants performed worse compared to the wild type in the field, with decreases in seed production of up to 70%.     

Bacteria as workers in the living factory: metal-accumulating bacteria and their potential for materials science

Metal micro-/nano-particles with suitable chemical modification can be organized into new ceramic-metal (cermet) or organic-metal (orgmet) composites or structured materials. These materials are attracting significant attention because of their unique structures and highly optimized properties. However, the synthesis of composite materials with inhomogeneities on the nanometer or sub-micrometer scale is a continuing challenge in materials science. Many industrial physical and chemical surface-coating processes using conventional techniques are both energy and cost inefficient and require sophisticated instrumentation. In the future, biology might offer a superior option.     

Progression from hypertrophic to dilated cardiomyopathy in mice that express a mutant myosin transgene

A mouse model of hypertrophic cardiomyopathy (HCM) was created by expression of a cardiac alpha-myosin transgene including the R(403)Q mutation and a deletion of a segment of the actin-binding domain. HCM mice show early histopathology and hypertrophy, with progressive hypertrophy in females and ventricular dilation in older males. To test the hypothesis that dilated cardiomyopathy (DCM) is part of the pathological spectrum of HCM, we studied chamber morphology, exercise tolerance, hemodynamics, isolated heart function, adrenergic sensitivity, and embryonic gene expression in 8- to 11-mo-old male transgenic animals. Significantly impaired exercise tolerance and both systolic and diastolic dysfunction were seen in vivo. Contraction and relaxation parameters of isolated hearts were also decreased, and lusitropic responsiveness to the beta-adrenergic agonist isoproterenol was modestly reduced. Myocardial levels of the G protein-coupled beta-adrenergic receptor kinase 1 (beta-ARK1) were increased by more than twofold over controls, and total beta-ARK1 activity was also significantly elevated. Induction of fetal gene expression was also observed in transgenic hearts. We conclude that transgenic male animals have undergone cardiac decompensation resulting in a DCM phenotype. This supports the idea that HCM and DCM may be part of a pathological continuum rather than independent diseases.     

A neural systems analysis of adaptive navigation

In the field of the neurobiology of learning, significant emphasis has been placed on understanding neural plasticity within a single structure (or synapse type) as it relates to a particular type of learning mediated by a particular brain area. To appreciate fully the breadth of the plasticity responsible for complex learning phenomena, it is imperative that we also examine the neural mechanisms of the behavioral instantiation of learned information, how motivational systems interact, and how past memories affect the learning process. To address this issue, we describe a model of complex learning (rodent adaptive navigation) that could be used to study dynamically interactive neural systems. Adaptive navigation depends on the efficient integration of external and internal sensory information with motivational systems to arrive at the most effective cognitive and/or behavioral strategies. We present evidence consistent with the view that during navigation: 1) the limbic thalamus and limbic cortex is primarily responsible for the integration of current and expected sensory information, 2) the hippocampal-septal-hypothalamic system provides a mechanism whereby motivational perspectives bias sensory processing, and 3) the amygdala-prefrontal-striatal circuit allows animals to evaluate the expected reinforcement consequences of context-dependent behavioral responses. Although much remains to be determined regarding the nature of the interactions among neural systems, new insights have emerged regarding the mechanisms that underlie flexible and adaptive behavioral responses.     

Uptake capacity of amino acids by ten grasses and forbs in relation to soil acidity and nitrogen availability

Uptake capacity of organic nitrogen was studied in solution experiments on eight grasses and two forbs growing in acid soils with relatively high nitrogen mineralisation in southern Sweden. Uptake of a mixture of amino acids (alanine, glutamine, glycine), that varied between 1.6 and 6.3 μmol g(-1) dw root h(-1), could not be explained by soil data from the species' field distributions (pH, total carbon and nitrogen, potential net mineralisation of ammonium and nitrate). The ratio between organic and inorganic nitrogen (methylamine) uptake was <0.05 for the forbs, higher for the grasses with a maximum of 1.42 for Deschampsia flexuosa. The ratio was negatively correlated with measures related to soil acidity (Ellenberg's R-value, soil nitrate and total carbon) but not, as hypothesised, with the total amount of mineralised nitrogen. The total demand on nitrogen by all components of the ecosystem would probably have described the extent to which competition among and between plants and microbes induced nitrogen limitation. In a methodological study two grasses were exposed to pH 3.8, 4.5 and 6.0 and to 50, 100 and 250 μmol l(-1) of three amino acids. Uptake was also compared between intact plants and excised roots. The treatment response varied considerably between the species which stresses the importance of studying intact plants at field-relevant pH and concentrations.     

Influence of high light and UV-B radiation on photosynthesis and D1 turnover in atrazine-tolerant and -sensitive cultivars of Brassica napus

An atrazine-tolerant mutant and an atrazine-sensitive cultivar of Brassica napus L. were grown under visible radiation (400 mumol m-2 s-1, photosynthetically active radiation, PAR) and then subjected to treatment conditions. These included short-term high PAR (1600 mumol m-2 s-1) which was given for 4 h either alone or in combination with an enhanced level of UV-BBE radiation (4.6 kJ m-2 h-1 biologically effective UV-B, 280-320 nm). Recovery from the radiation treatment was studied for 4 h under the light conditions for growth. Since it is known that the atrazine-tolerant mutant is susceptible to photoinhibition, one of the aims of the present study was to determine the effects of a supplemental, enhanced level of UV-B radiation with regard to the mutant. The results indicate an additive effect of UV-B radiation on Fv/Fm, photochemical yield and photosynthetic oxygen evolution during both exposure and recovery, and also a higher susceptibility of the mutant to photoinhibitory PAR conditions alone and in combination with UV-B, which may have implications in a changing environment. Both cultivars also showed a higher D1 turnover during the radiation stress than during recovery, as shown by immunoblotting and 35S-methionine incorporation measurements.