On the flexibility of myosin in solution.

Rabbit skeletal muscle myosin from the same rabbit was prepared by two different methods, and then purified by either Sephadex or hydroxylapatite chromatography. The resulting myosin samples were analyzed in 2-10 mM sodium pyrophosphate solutions at pH 9 using transient electric birefringence. The birefringence decay signals were fitted using a Fortran program called DISCRETE and two relaxation times, 49.7 +/- 5.6 and 11.2 +/- 2.5 microseconds, were determined. These relaxation times were independent of the method of myosin preparation, the method of myosin purification, the concentration of sodium pyrophosphate between 2 and 10 mM, the concentration of myosin between 0.08 and 1.59 mg/mL, and the temperature between 4.0 and 20.0 degrees C, after correction to 20.0 degrees C. The longer relaxation time is consistent with a rigid, linear myosin molecule. The shorter relaxation time is consistent with myosin that has a completely flexible hinge region in the myosin tail. Both relaxation times are inconsistent with the previously reported single relaxation time of myosin obtained by fitting the birefringence decay data to only 90% of the decay signal. By forcing some of the birefringence decay data in the presence work to fit 90% of the decay signal with a single relaxation time, approximately the same relaxation time as previously reported was obtained.     

On the use of 2,1,3-benzothiadiazole derivatives as selective live cell fluorescence imaging probes

Newly designed 2,1,3-benzothiadiazole-containing fluorescent probes with four excited state intramolecular proton transfer (ESIPT) sites were successfully tested in live cell-imaging assays using a confluent monolayer of human stem-cells (tissue). All tested dyes were compared with the commercially available DAPI and gave far better results.     

Sequence diversity of the peptaibol antibiotic suzukacillin-A from the mold Trichoderma viride.

From the culture broth of the mold Trichoderma viride, strain 63 C-I, the polypeptide antibiotic suzukacillin (SZ) was isolated. A peptide mixture named SZ-A was obtained by crystallization from crude SZ. Individual peptides from SZ-A were isolated by semipreparative HPLC and sequences were determined by HPLC-ESI-MS. The data confirm a general sequence of SZ-A published previously and in addition establish the individual sequences of 15 acetylated eicosa peptides with C-terminal alcohols. The major peptide SZ-A4 (21% of all peptides) shows the sequence:Ac-Aib-Ala-Aib-Ala-Aib-Ala(6)-Gln-Aib-Lx(9)-Aib-Gly-Aib(12)-Aib-Pro-Vx(15)-Aib-Vx(17)-Gln-Gln-Fol. Amino acid exchanges of the peptaibol are located in position 6 (Ala/Aib), 9 (Vx/Lx), 12 (Aib/Lx), 17 (Aib/Vx) and possibly at position15 (Val/Iva) (uncommon abbreviations: Aib (alpha-aminoisobutyric acid); Iva (D-isovaline); Lx (L-leucine or L-isoleucine); Vx (L-valine or D-isovaline); Fol (L-phenylalaninol)).     

Characterization of some previously unclassified Pasteurellaceae isolated from hamsters

Bacteria isolated from purulent processes on the jaws of European hamsters ( Cricetus cricetus ) and from intestinal inflammatory processes in Syrian hamsters ( Mesocricetus auratus ), bred as laboratory animals have been shown to be phenotypically similar but not identical with Pasteurella pneumotropica . Deoxyribonucleic acid (DNA)–DNA hybridization studies indicate that with one exception, the strains represent two new species of the family Pasteurellaceae. In the absence of a close genomic relatedness to members of the genera Actinobacillus or Pasteurella or allied organisms, however, the two new taxa are described without any formal designation. The one exception was identified as Actinobacillus capsulatus , a species not previously isolated from hamsters.     

Ca2+-induced exocytosis in individual human neutrophils: high- and low-affinity granule populations and submaximal responses.

We have investigated Ca2+-induced exocytosis from human neutrophils using the whole cell patch-clamp capacitance technique. Microperfusion of Ca2+ buffer solutions (<30 nM to 5 mM free Ca2+) through the patch-clamp pipette revealed a biphasic activation of exocytosis by Ca2+. The first phase was characterized by high affinity (1.5-5 microM) and low apparent cooperativity (<=2) for Ca2+, and the second phase by low affinity (approximately 100 microM) and high cooperativity (>6). Only the second phase was accompanied by loss of myeloperoxidase, suggesting that the low-affinity exocytosis reflected release of peroxidase-positive (primary) granules, while the high-affinity exocytosis reflected release of peroxidase-negative (secondary and tertiary) granules. At submaximal Ca2+ concentrations, only a fraction of a given granule population was released. This submaximal release cannot simply be explained by Ca2+ modulation of the rate of exocytosis, and it suggests that the secretory response of individual cells is adjusted to the strength of the stimulus. The Ca2+ dependence of the high- and low-affinity phases of neutrophil exocytosis bears a resemblance to endocrine and neuronal exocytosis, respectively. The occurrence of such high- and low-affinity exocytosis in the same cell is novel, and suggests that the Ca2+ sensitivity of secretion is granule-, rather than cell-specific.     

Intertidal rocky shore biotopes of the Azores: a quantitative approach

Rocky littoral communities of the Azores are dominated by macroalgae where turf formations represent the main life form as on many subtropical and tropical shores. The present study aimed at describing and classifying benthic intertidal biotopes of the Azores using clear quantification and data analysis methodologies to be used in similar studies for spatial and/or temporal comparison. This numerical approach intends to have management and conservation applicability. Thirty study locations along the coast of São Miguel island were characterised by substratum type (cobbles, boulders or bedrock – different stability levels) and exposure to wave action (high, medium or low exposure), from June to August 2002. Algae, sessile and sedentary invertebrates were recorded along transects, and the more conspicuous taxa quantified. Community structure is mainly shaped by substratum stability – unstable cobble communities are separate from those of more stable boulders and bedrock. Boulders present an intermediate community composition between cobbles and bedrock. Exposure to wave action induces smaller variation in community composition. Sixteen biotopes have been recognised. Ultimately the present paper provides an objective set of biotope definition tools for a broad public whose interest focuses on spatial and temporal comparison of coastal communities and associated habitats.     

Planar microfluidic chamber for generation of stable and steep chemoattractant gradients

The extracellular availability of growth factors, hormones, chemokines, and neurotransmitters under gradient conditions is required for directional cellular responses such as migration, axonal pathfinding, and tissue patterning. These responses are, in turn, important in disease and developmental processes. This article addresses critical barriers toward devising a chemotaxis assay that is broadly applicable for different kinds of cancer cells through the design of a microfluidic chamber that produces a steep gradient of chemoattractant. Photolithography was used to create microchannels for chemoattractant delivery, flow diversion barriers/conduits, and small outlets in the form of apertures. The 1-μm apertures were made at the active surface by uncapping a thin (1.5 μm) layer of AZ1518. This process also created a vertical conduit that diverted the flow such that it occurred perpendicularly to the active, experimental surface where the gradients were measured. The other side of the vertical conduit opened to underlying 20-μm deep channels that carried microfluidic flows of tracer dyes/growth factors. Modeled data using computational fluid dynamics produced gradients that were steep along the horizontal, active surface. This simulation mirrors empirically derived gradients obtained from the flow analyses of fluorescent compounds. The open chamber contains a large buffer volume, which prevents chemoattractant saturation and permits easy cell and compound manipulation. The technique obviates the use of membranes or laminar flow that may hinder imaging, rinsing steps, cell seeding, and treatment. The utility of the chamber in the study of cell protrusion, an early step during chemotaxis, was demonstrated by growing cancer cells in the chamber, inducing a chemoattractant gradient using compressed air at 0.7 bar, and performing time-lapse microscopy. Breast cancer cells responded to the rapidly developed and stable gradient of epidermal growth factor by directing centroid positions toward the gradient and by forming a leading edge at a speed of 0.45 μm/min.