Optimization of the green fluorescent protein (GFP) expression from a lactose-inducible promoter in Lactobacillus casei

An expression vector for Lactobacillus casei has been constructed containing the inducible lac promoter and the gene encoding ultraviolet visible green fluorescent protein (GFP(UV)) as reporter. Different conditions to grow L. casei were assayed and fluorescence as well as total protein synthesized were quantified. The maintenance of neutral pH had the greatest incidence on GFP(UV) expression, followed by aeration and a temperature of 30 degrees C. Environmental factors favoring GFP(UV) accumulation did not exactly correlate with those enhancing fluorescence. Therefore, oxygenation, by stirring the culture, had the greatest influence on the proportion of fluorescent protein, which is in accordance with the structural requirements of this protein. The highest yield obtained was 1.3 microg of GFP per mg of total protein, from which 55% was fluorescent.     

Low activity of amine-oxidases and accumulation of conjugated polyamines in disfavour of organogenic programs in Chrysanthemum leaf disc explants

Foliar discs (8 mm diameter) from expanding leaves of the middle part of vegetative shoots of Chrysanthemum morifolium Ramat raised in vitro were induced to form directly on specific media in vitro either roots or vegetative buds, or callus. The budding programme, on its specific medium, was deviated to callus formation by the addition of 2 mM β-OH-E (β-OH-ethyldrazine, an inhibitor of diamine oxidase). Conversely vegetative buds instead of callus were formed on the callus medium in the presence of 2 mM DFMO (difluoromethylornithine, an inhibitor of ornithine decarboxylase). Callus formation was characterized by high accumulation of free and particularly conjugated polyamines (PA), very low or undetectable activities of diamine- and polyamine oxidases, and transglutaminase. DFMO-deviation of callus initiation in favour of bud formation lowered the accumulation of PA and increased the activity of amine-oxidases. The high catabolism of PA in the organogenic (rooting, budding) programs was questioned as to its role in developmental processes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Apyrase Activity and Platelet Aggregation Inhibitors in the Tick Ornithodoros Savignyi (Acari: Argasidae)

Ticks are ectoparasites that cause considerable damage to their hosts while feeding. The feeding process is facilitated by anti-haemostatic factors present in the tick saliva. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is a platelet aggregation inhibitor found in most haematophagous organisms studied. The present study describes the identification and characterization of such an activity in the tick Ornithodoros savignyi. The enzyme conformed to many properties common to apyrases. These included a low substrate specificity, dependence on bivalent metal ions for activity and insensitivity to the classical ATPase inhibitors. Heat denaturation studies, pH optima and similar effects of inhibitors on the enzyme's ATP and ADP hydrolysing activities supported its classification as an apyrase. Salivary gland extracts inhibited the platelet aggregation induced by ADP, collagen and thrombin and disaggregated aggregated platelets. The results suggest the presence of two or more anti-platelet factors present in the salivary glands of this tick species.     

Identification and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase.

p85/p110 phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85-regulatory and a p110-catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65-PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85alpha-protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.     

Characterization of Trypanosoma cruzil-cysteine transport mechanisms and their adaptive regulation

l -Cysteine and methionine are unique amino acids that act as sulfur donors in all organisms. In the specific case of Trypanosomatids, l -cysteine is particularly relevant as a substrate in the synthesis of trypanothione. Although it can be synthesized de novo , l -cysteine is actively transported in Trypanosoma cruzi epimastigote cells. l -Cysteine uptake is highly specific; none of the amino acids assayed yield significant differences in terms of transport rates. l -Cysteine is transported by epimastigote cells with a calculated apparent K _m of 49.5 μM and a V _max of about 13 pmol min⁻¹ per 10⁷ cells. This transport is finely regulated by amino acid starvation, extracellular pH, and between the parasite growth phases. In addition, l -cysteine is incorporated post-translationally into proteins, suggesting its role in iron–sulfur core formation. Finally, the metabolic fates of l -cysteine were predicted in silico .     

Relative Pigment Composition and Remote Sensing Reflectance of Caribbean Shallow-Water Corals

Reef corals typically contain a number of pigments, mostly due to their symbiotic relationship with photosynthetic dinoflagellates. These pigments usually vary in presence and concentration and influence the spectral characteristics of corals. We studied the variations in pigment composition among seven Caribbean shallow-water Scleractinian corals by means of High Performance Liquid Chromatography (HPLC) analysis to further resolve the discrimination of corals. We found a total of 27 different pigments among the coral species, including some alteration products of the main pigments. Additionally, pigments typically found in endolithic algae were also identified. A Principal Components Analysis and a Hierarchical Cluster Analysis showed the separation of coral species based on pigment composition. All the corals were collected under the same physical environmental conditions. This suggests that pigment in the coral’s symbionts might be more genetically-determined than influenced by prevailing physical conditions of the reef. We further investigated the use of remote sensing reflectance (Rrs) as a tool for estimating the total pigment concentration of reef corals. Depending on the coral species, the Rrs and the total symbiont pigment concentration per coral tissue area correlation showed 79.5–98.5% confidence levels demonstrating its use as a non-invasive robust technique to estimate pigment concentration in studies of coral reef biodiversity and health.     

Orthotic effect of a stabilising mechanism in the surface of gymnastic mats on foot motion during landings.

The purpose of this study was to examine two hypotheses: (a) mat hardness affects foot motion during landing; (b) the influence of a surface stabilising interface integrated in a mat on foot motion is detectable. Two studies were carried out: In the first one, six female gymnasts performed barefoot landings from different falling heights onto three mats having different hardness. In the second study, a stabilising mechanism was integrated in the surface of three new mats with different hardness. Three high speed video cameras (250Hz) captured the motion of the left leg and foot. These were modelled by means of a four rigid body system. The maximal eversion at the ankle joint was not influenced by the different mats (hard: 4.6 degrees +/-1.9 to 9.3 degrees +/-3.4, medium: 3.1 degrees +/-2.7 to 7.4 degrees +/-3.5, soft: 4.8 degrees +/-2.1 to 8.4 degrees +/-3.5). The soft mat without the stabilised surface showed higher eversion values (p<0.05) between forefoot and rearfoot (medial joint: hard: 5.1 degrees +/-3.2 to 7.3 degrees +/-3.3, medium: 6.9 degrees +/-3.1 to 7.5 degrees +/-2.9, soft: 12.7 degrees +/-4.1 to 13.4 degrees +/-3.3; lateral joint: hard: 8.5 degrees +/-3.1 to 9.7 degrees +/-1.1, medium: 9.5 degrees +/-2.6 to 11.2 degrees +/-3.3, soft: 12.1 degrees +/-2.3 to 15.7 degrees +/-3.3). For the mats with the surface stabilising interface, the different hardness did not cause any significant differences in maximal eversion values at the medial (hard: 1.5 degrees +/-3.3 to 5.5 degrees +/-4.5, medium: 1.3 degrees +/-3.5 to 5.1 degrees +/-3.6, soft: 0.7 degrees +/-4.9 to 5.4 degrees +/-4.2) nor at the lateral (hard: 11.3 degrees +/-4.2 to 17.3 degrees +/-4.2, medium: 12.3 degrees +/-4.8 to 17.1 degrees +/-3.7, soft: 11.5 degrees +/-4.6 to 17.1 degrees +/-4.3) forefoot joints. The structure of the mat and the consequent deformation hollow did not influence the kinematics of the ankle joint during landings, but it influenced the motion at the medial and the lateral forefoot joints. By means of a stabilised surface, it is possible to reduce the influence of mat deformation on the maximal eversion between forefoot and rearfoot.     

Effect of contraction form and contraction velocity on the differences between resultant and measured ankle joint moments

During a maximal isometric plantar flexion effort the moment measured at the dynamometer differs from the resultant ankle joint moment. The present study investigated the effects of contraction form and contraction velocity during isokinetic plantar/dorsal flexion efforts on the differences between resultant and measured moments due to the misalignment between ankle and dynamometer axes. Eleven male subjects (age: 31+/-6 years, mass: 80.6+/-9.6 kg, height: 178.4+/-7.4 cm) participated in this study. All subjects performed isometric-shortening-stretch-isometric contractions induced by electrical stimulation at three different angular velocities (25 degrees /s, 50 degrees /s and 100 degrees /s) on a customised dynamometer. The kinematics of the leg were recorded using the vicon 624 system with eight cameras operating at 250 Hz. The resultant moments at the ankle joint were calculated through inverse dynamics. The relative differences between resultant and measured ankle joint moments due to axis misalignment were fairly similar in all phases of the isometric-shortening-stretch-isometric contraction (in average 5-9% of the measured moment). Furthermore these findings were independent of the contraction velocity. During dynamic plantar/dorsal flexion contractions the differences between measured and resultant joint moment are high enough to influence conclusions regarding the mechanical response of ankle extensor muscles. However the relative differences were not increased during dynamic contractions as compared to isometric contractions.