Lectin binding as a probe of proliferative and differentiative phases in primary monolayer cultures of cutaneous keratinocytes

The surface of cells in the cutaneous epidermis of the newborn rat exhibits a discrete change in lectin-binding specificity from Griffonia simplicifolia I-B4 (GS I-B4), specific for alpha-D-galactosyl residues, to Ulex europeus agglutinin I (UEA), specific for alpha-L-fucose, as the cell leaves the basal layer and differentiates. Primary monolayer cultures of rat keratinocytes maintained in low Ca2+ medium (0.08 mM) exhibited a characteristic unimodal pattern in the ratio of bound UEA to bound GS I-B4 (UEA/B4 ratio) over a 7-day culture period as determined by a quantitative fluorometric assay. The UEA/B4 ratio was initially low between Days 1 and 2 (0.56 +/- 0.05), steadily increased to a maximum of 0.84 +/- 0.09 between Days 2 and 4, and then gradually decreased to 0.41 +/- 0.07 between Days 6 and 7. Estimation of DNA synthesis showed (a) a higher [3H]thymidine incorporation when the UEA/B4 ratio was low and (b) a steady but lower incorporation between Days 3 and 4, coincident with the higher UEA/B4 ratio. Autoradiographic results further showed that cells stained intensely with UEA failed to incorporate [3H]thymidine into their nuclei. Electrophoresis of [3H]fucose-labeled material isolated on UEA-Sepharose 4B revealed that the changes in labeling by [3H]fucose, bound UEA, and the UEA/B4 ratio in the monolayer were related in part to variable expression of "96K-associated UEA-bound" radioactivity corresponding to a major class of lectin-specific cell-surface glycoproteins (GP96 fraction) identified in situ. Overall, the results suggest that (a) the increase in the UEA/B4 ratio between Days 2 and 4 reflects the progression of a proportion of the cells in the monolayer to an early spinous cell stage, the ultimate fate of which is desquamation into the medium and (b) the decrease in the UEA/B4 ratio between Days 5 and 7 reflects a consequent proliferative response to this loss of cells. This system should be useful for studying environmental influences on the homeostasis of cell proliferation and differentiation in the cutaneous epidermis.     

Laser Doppler anemometer measurements of pulsatile flow in a model carotid bifurcation

Hemodynamics at the human carotid bifurcation is important to the understanding of atherosclerotic plaque initiation and progression as well as to the diagnosis of clinically important disease. Laser Doppler anemometry was performed in a large scale model of an average human carotid. Pulsatile waveforms and physiologic flow divisions were incorporated. Disturbance levels and shear stresses were computed from ensemble averages of the velocity waveform measurements. Flow in the common carotid was laminar and symmetric. Flow patterns in the sinus, however, were complex and varied considerably during the cycle. Strong helical patterns and outer wall flow separation waxed and waned during each systole. The changing flow patterns resulted in an oscillatory shear stress at the outer wall ranging from -13 to 9 dyn cm-2 during systole with a time-averaged mean of only -0.5 dyn cm-2. This contrasts markedly with an inner wall shear stress range of 17-50, (mean 26) dyn cm-2. The region of transient separation was confined to the carotid sinus outer wall with no reverse velocities detected in the distal internal carotid. Notable disturbance velocities were also time-dependent, occurring only during the deceleration phase of systole and the beginning of diastole. The present pulsatile flow studies have aided in identifying hemodynamic conditions which correlate with early intimal thickening and predict the physiologic level of flow disturbances in the bulb of undiseased internal carotid arteries.     

New monodisperse magnetic polymer microspheres biofunctionalized for enzyme catalysis and bioaffinity separations

Magnetic macroporous PGMA and PHEMA microspheres containing carboxyl groups are synthesized by multi-step swelling and polymerization followed by precipitation of iron oxide inside the pores. The microspheres are characterized by SEM, IR spectroscopy, AAS, and zeta-potential measurements. Their functional groups enable bioactive ligands of various sizes and chemical structures to couple covalently. The applicability of these monodisperse magnetic microspheres in biospecific catalysis and bioaffinity separation is confirmed by coupling with the enzyme trypsin and huIgG. Trypsin-modified magnetic PGMA-COOH and PHEMA-COOH microspheres are investigated in terms of their enzyme activity, operational and storage stability. The presence of IgG molecules on microspheres is confirmed.     

Purification of myosin from Ehrlich ascites tumour cells (phosphorylation of its light chain and heavy chain)

1. The myosin molecule from Ehrlich ascites tumour cells consists of heavy chains of about 200 kDa and three species of light chains of 20, 19 and 15 kDa. 2. The heavy chain can be phosphorylated in vitro either by endogenous Ca2+-independent kinase or by casein kinase II. 3. The 20 and 19 kDa light chains can be phosphorylated either by an endogenous kinase or by myosin light chain kinase from chicken gizzard. 4. The Ca2+-ATPase activity of the purified myosin was 0.3 mumol/min mg protein. The Mg2+-ATPase activity was activated 14-fold by actin upon the light chain phosphorylation.     

Proteases from the fungus Metarhizium anisopliae toxic for Galleria mellonella larvae

The high molecular fraction of the extract from Metarhizium anisopliae grown on wheat bran contains proteolytic enzymes which are toxic for Galleria mellonella larvae. The complex of proteases was fractionated using precipitation with ammonium sulfate, gel filtration, and electrofocusing. Two components have been found: one with the optimum of activity on hemoglobin at pH 6.5, and the second with the optimum around pH 9. The prevailing protease acting at pH 6.5 was inhibited by phenylmethylsulfonyl fluoride and the inhibition was followed by decrease of toxicity. The molecular weights of the enzymes are 35 × 10³ and 71 × 10³.     

Drivers of plant species composition of ecotonal vegetation in two fishpond management types

Wetlands Ecology and Management - Plants play an important role in fishpond littorals, but little is known about factors influencing their presence and growth patterns. We surveyed vegetation of...     

Effect of the fungal immunomodulatory protein FIP-fve on airway inflammation and cytokine production in mouse asthma model

The allergy is dependent on the balance between Th1 and Th2. The fungal immunodulatory protein (FIP-fve) was isolated from Flammulina velutipes. FIP-fve has been demonstrated to skew the response to Th1 cytokine production. We investigated whether oral administrations of FIP-fve inhibited allergen (OVA)-induced chronic airway inflammation in the mouse asthma model. After intranasal challenge with OVA, the airway inflammation and hyperresponsiveness were determined by bronchoalveolar lavage fluid (BALF) analysis and ELISA assay. Both pre-treated and post-treated with FIP-fve suppressed the airway hyperresponsiveness by methacholine challenge and significantly decreased the number of infiltrating inflammatory cells and Th2 cytokines in bronchoalveolar lavage fluid (BALF) and serum compared with the OVA sensitized mice. In addition, FIP-fve reduced OVA-specific IgE levels in serum. FIP-fve markedly alleviated the OVA-induced airway hyperresponsiveness (AHR) to inhaled methacholine. Based on lung histopathological studies using hematoxylin and Liu’s staining, FIP-fve inhibited inflammatory cell infiltration compared with the OVA-sensitized mice. Oral FIP-fve had an anti-inflammatory effect on OVA-induced airway inflammations and might posses the potential for alternative therapy for allergic airway diseases.     

Activity of the tick Ixodes ricinus monitored in a suburban park in Brno,Czech Republic,in association with the evaluation of selected repellents

The ever‐increasing number of Lyme borreliosis patients led us to consider more effective procedures for disease prevention. The aim of our study was to monitor the annual activity and infectivity of Ixodes ricinus ticks in the Pisárky region, City of Brno, CR, and to test the responses of the locally‐captured ticks to selected repellents. The result of regular one‐hour‐perweek monitoring in 2011 was the collection of ticks that directly reflected the highest number of Lyme disease patients (4,835) detected throughout the period of recording in the Czech Republic. The ticks were examined for spirochaetes by dark field microscopy. The positive samples were identified by PCR analysis, confirming that 76% of these were infected with Borrelia burgdorferi sensu lato. Ticks were most abundant in May and June, with August having the highest risk for spirochaetal infection. Tick activity was statistically correlated with temperature. The moving‐object‐bioassay was used to study repellent efficiency on the Ixodes ricinus nymphs captured in the above‐mentioned suburban park. Five selected commercial repellents based on DEET (N, N‐diethyl3methylbenzamide) showed statistically different effects on the non‐repellent control group.     

Chemotaxis of Bacillus cereus YL6 and its colonization of Chinese cabbage seedlings

Plant and Soil - Mycorrhizal type has been proposed as an effective trait integrator capturing varying biogeochemical syndromes in terrestrial ecosystems. However, for boreal peatlands, it is still...