Rab11-family interacting protein 2 and myosin Vb are required for CXCR2 recycling and receptor-mediated chemotaxis

Agonist-stimulated internalization followed by recycling to the cell membrane play an important role in fine-tuning the activity of chemokine receptors. Because the recycling of chemokine receptors is critical for the reestablishment of the cellular responsiveness to ligand, it is crucial to understand the mechanisms underlying the receptor recycling and resensitization. In the present study, we have demonstrated that the chemokine receptor CXCR2 associated with myosin Vb and Rab11-family interacting protein 2 (FIP2) in a ligand-dependent manner. Truncation of the C-terminal domain of the receptor did not affect the association, suggesting that the interactions occur upstream of the C terminus of CXCR2. After ligand stimulation, the internalized CXCR2 colocalized with myosin Vb and Rab11-FIP2 in Rab11a-positive vesicles. The colocalization lasted for approximately 2 h, and little colocalization was observed after 4 h of ligand stimulation. CXCR2 also colocalized with myosin Vb tail or Rab11-FIP2 (129-512), the N-terminal-truncated mutants of myosin Vb and Rab11-FIP2, respectively, but in a highly condensed manner. Expression of the enhanced green fluorescent protein-tagged myosin Vb tail significantly retarded the recycling and resensitization of CXCR2. CXCR2 recycling was also reduced by the expression Rab11-FIP2 (129-512). Moreover, expression of the myosin Vb tail reduced CXCR2- and CXCR4-mediated chemotaxis. These data indicate that Rab11-FIP2 and myosin Vb regulate CXCR2 recycling and receptor-mediated chemotaxis and that passage of internalized CXCR2 through Rab11a-positive recycling system is critical for physiological response to a chemokine.     

Light-directed 5'-->3' synthesis of complex oligonucleotide microarrays

Light-directed synthesis of high-density microarrays is currently performed in the 3'-->5' direction due to constraints in existing synthesis chemistry. This results in the probes being unavailable for many common types of enzymatic modification. Arrays that are synthesized in the 5'-->3' direction could be utilized to perform parallel genotyping and resequencing directly on the array surface, dramatically increasing the throughput and reducing the cost relative to existing techniques. In this report we demonstrate the use of photoprotected phosphoramidite monomers for light-directed array synthesis in the 5'-->3' direction, using maskless array synthesis technology. These arrays have a dynamic range of >2.5 orders of magnitude, sensitivity below 1 pM and a coefficient of variance of <10% across the array surface. Arrays containing >150,000 probe sequences were hybridized to labeled mouse cRNA producing highly concordant data (average R(2) = 0.998). We have also shown that the 3' ends of array probes are available for sequence-specific primer extension and ligation reactions.     

Morphometry of Macaca mulatta forelimb. II. Fiber-type composition in shoulder and elbow muscles

The present study examined the fiber-type proportions of 22 muscles spanning the shoulder and/or elbow joints of three Macaca mulatta. Fibers were classified as one of three types: fast-glycolytic (FG), fast-oxidative-glycolytic (FOG), or slow-oxidative (SO). In most muscles, the FG fibers predominated, but proportions ranged from 25-67% in different muscles. SO fibers were less abundant except in a few deep, small muscles where they comprised as much as 56% of the fibers. Cross-sectional area (CSA) of the three fiber types was measured in six different muscles. FG fibers tended to be the largest, whereas SO fibers were the smallest. While fiber-type size was not always consistent between muscles, the relative size of FG fibers was generally larger than FOG and SO fibers within the same muscle. When fiber CSA was taken into consideration, FG fibers were found to comprise over 50% of the muscle's CSA in almost all muscles.     

The synaptic vesicle cycle: exocytosis and endocytosis in Drosophila and C. elegans

Advances in the study of Drosophila melanogaster and Caenorhabditis elegans have provided key insights into the processes of neurotransmission and neuromodulation. Work in the past year has revealed that Unc-13 and Rab3a-interacting molecule regulate the conformational state of syntaxin to prime synaptic vesicle fusion. Analyses of synaptotagmin support its role as a putative calcium sensor triggering vesicular fusion and highlight the possible role of SNARE complex oligomerization in the fusion mechanism. Characterization of endophilin mutants demonstrates that kiss-and-run endocytosis is a major component of synaptic vesicle recycling. In neuromodulation, dcaps mutants provide the first genetic insight into possible roles of the CAPS protein in mediating dense core vesicle fusion and modulating synaptic vesicle fusion.     

Control of coronary blood flow during exercise

Under normal physiological conditions, coronary blood flow is closely matched with the rate of myocardial oxygen consumption. This matching of flow and metabolism is physiologically important due to the limited oxygen extraction reserve of the heart. Thus, when myocardial oxygen consumption is increased, as during exercise, coronary vasodilation and increased oxygen delivery are critical to preventing myocardial underperfusion and ischemia. Exercise coronary vasodilation is thought to be mediated primarily by the production of local metabolic vasodilators released from cardiomyocytes secondary to an increase in myocardial oxygen consumption. However, despite various investigations into this mechanism, the mediator(s) of metabolic coronary vasodilation remain unknown. As will be seen in this review, the adenosine, K(+)(ATP) channel and nitric oxide hypotheses have been found to be inadequate, either alone or in combination as multiple redundant compensatory mechanisms. Prostaglandins and potassium are also not important in steady-state coronary flow regulation. Other factors such as ATP and endothelium-derived hyperpolarizing factors have been proposed as potential local metabolic factors, but have not been examined during exercise coronary vasodilation. In contrast, norepinephrine released from sympathetic nerve endings mediates a feed-forward betaadrenoceptor coronary vasodilation that accounts for approximately 25% of coronary vasodilation observed during exercise. There is also a feed-forward alpha-adrenoceptor-mediated vasoconstriction that helps maintain blood flow to the vulnerable subendocardium when heart rate, myocardial contractility, and oxygen consumption are elevated during exercise. Control of coronary blood flow during pathophysiological conditions such as hypertension, diabetes mellitus, and heart failure is also addressed.     

Model based decoding of spike trains

Reliably decoding neuronal responses requires knowing what aspects of neuronal responses are stimulus related, and which aspects act as noise. Recent work shows that spike trains can be viewed as stochastic samples from the rate variation function, as estimated by the time dependent spike density function (or normalized peristimulus time histogram). Such spike trains are exactly described by order statistics, and can be decoded millisecond-by-millisecond by iterative application of order statistics.     

Sustainable, High-Yielding Outdoor Mass Cultures of Chaetoceros muelleri var. subsalsum and Isochrysis galbana in Vertical Plate Reactors

Continuous cultures of Chaetoceros muelleri and Isochrysis galbana were grown outdoors in flat plate-glass reactors in which light-path length (LPL) varied from 5 to 30 cm. High daily productivity (13 to 16 g cell mass per square meter of irradiated reactor surface) for long periods of time was obtained in reactors in which the optical path as well as cell density were optimized. 'Twenty centimeters was the optimal LPL, yielding the highest areal productivity of cell mass (g m^–2d^–1), eicosapentaenoic acid, and docosahexaenoic acid, which was identical with that previously found for polysaccharide production of Porphyridium and not far from the optimal LPL affecting maximal productivity in Nannochloropsis species. Relating the energy impinging on a given reactor surface area to the appropriate number of cells showed that the most efficient light dose per cell, obtained with the 20-cm LPL reactor, was approximately 2.5 times lower than the light dose available per cell in the 5-cm LPL reactor, in which a significant decline in areal cell density accompanied the lowest areal output of cell mass. The most effective harvesting regimen was in the range of 10% to 15% of culture volume harvested daily and replaced with fresh growth medium, resulting in a sustainable culture density of 24 × 10⁶ and 28 × 10⁶ cells/ml of C. muelleri and I. galbana, respectively.     

What's hot in animal biosafety?

In recent years, the emergence or re-emergence of critical issues in infectious disease and public health has presented new challenges and opportunities for laboratory animal care professionals. The re-emergence of bioterrorism as a threat activity of individuals or small groups has caused a heightened awareness of biosecurity and improved biosafety. The need for animal work involving high-risk or high-consequence pathogens and for arthropod-borne diseases has stimulated renewed interest in animal biosafety matters, particularly for work in containment. Application of these principles to animals retained in outdoor environments has been a consequence of disease eradication programs. The anticipated global eradication of wild poliovirus has prompted the promulgation of new biosafety guidelines for future laboratory and animal work. Increased concern regarding the use of biologically derived toxins and hazardous chemicals has stimulated a new categorization of facility containment based on risk assessment. Recognition that prion disease agents and other high-consequence pathogens require safe handling and thorough destruction during terminal decontamination treatment has led to the development of new biosafety guidelines and technologies. The implementation of these guidelines and technologies will promote state-of-the-art research while minimizing risk to laboratory animals, researchers, and the environment.