Reversible immobilization of invertase on Sepabeads coated with polyethyleneimine: optimization of the biocatalyst's stability

Invertase from S. cerevisiae has been immobilized by ionic adsorption on Sepabeads fully coated with PEI. The enzyme was strongly adsorbed on the support (no desorption of the invertase was found under conditions in which all of the enzyme was released from conventional anionic exchanger supports (e.g., DEAE-agarose)). Nevertheless, the enzyme could still be desorbed after its inactivation, and new fresh enzyme could be adsorbed on the supports without detrimental effects on enzyme loading. This is a multimeric enzyme, its minimal oligomerization active state being the dimer, but under certain conditions of pH and concentration it may give larger multimers. Very interestingly, results suggested that the adsorption of the enzyme on this large and flexible polymeric bed was able to freeze some of the different oligomeric structures of the enzyme. Thus, we have found that the enzyme immobilized at certain pH values (pH 8.5) and high enzyme concentration, in which the main enzyme structure is the tetramer, was more stable than immobilized preparations produced in conditions under which oligomerization was not favorable (dimers at low enzyme concentration) or it was too high (e.g., hexamers-octamers at low pH value). The optimal enzyme preparation remained fully active after a 15-day incubation at 50 degrees C and pH 4.5 (conditions of standard industrial use) and presented an optimal temperature approximately 5 degrees C higher than that of soluble enzyme.     

Synthesis and in vivo biodisposition of [14C]-quaternary ammonium-melphalan conjugate,a potential cartilage-targeted alkylating drug

For the purpose of developing more selective anticancer drugs that would concentrate in the malignant cartilaginous tumors (chondrosarcomas), and so improve therapeutic index through a reduction of side effects, a quaternary ammonium (QA) conjugate of melphalan was synthesized and labeled with (14)C by linking the QA moiety to nitrogen mustard via an amide bond. Comparative pharmacokinetic study of [(14)C]-melphalan and its [(14)C]-QA conjugate conducted on rats showed that the two compounds were principally excreted by the urinary way. The blood elimination of the QA conjugate was faster than that of the melphalan. In the other hand a higher rate of radioactivity derived of [(14)C]-MQA was found in feces. In the biodisposition for most organs, no striking differences were found between melphalan and its QA conjugate except for cartilages which exhibited more higher radioactivity level. Amounts of radioactivity derived from [(14)C]-QA conjugates measured in cartilaginous tissues until 1 h after injection demonstrate that the introduction of a QA moiety on melphalan allows the molecule to be carried selectively to cartilaginous tissues. As the [(14)C]-QA conjugate is radiolabeled on the chloroethyl alkylating moiety, levels of radioactivity measured in the cartilaginous tissues results from unchanged compound or metabolite having kept the active group.     

First record of anomuran and brachyuran larvae (Crustacea: Decapoda) from Antarctic waters

Two decapod crustacean larval morphotypes belonging to the Anomura and Brachyura were found for the first time in Antarctic waters. Nine specimens were obtained from qualitative plankton hauls in Maxwell Bay (Bransfield Strait) (62°14'33S; 58°43'81W) off King George Island, Antarctic Peninsula. The anomuran morphotype belonged to the Hippidae, and apparently to the genus Emerita, whereas the brachyuran representative was assigned to the genus Pinnotheres (Pinnotheridae). At present, species determination is not possible due to lack of knowledge of larval morphology in both families. Adult forms of these reptant decapods are not known from Antarctic waters; the occurrence of the present larval forms is considered as a possible intrusion of Subantarctic water masses into the Antarctic environment. This hypothesis is supported by the additional presence of the copepod genus Acartia in the same sample material, which is exclusively known from Subantarctic waters.     

The PATCHED/Sonic Hedgehog signalling pathway in superficial bladder cancer

Superficial bladder cancer shows a high frequency of total or partial chromosome 9 losses. Loss of heterozygosity at position 9q22.3 is one of the most frequent and is associated with highly recurrent tumours. The PATCHED gene, ortholog of a gene first described in the drosophila as a segment polarity gene, is located at 9q22.3. It is a member of a signal transduction pathway and a tumour suppressor gene (TSG), involved in basal cell carcinoma. We propose PATCHED as a TSG candidate in superficial bladder cancer.     

Interference of a short-term exposure to nitrogen dioxide with allergic airways responses to allergenic challenges in BALB/c mice

Nitrogen dioxide (NO(2)) is a common indoor and outdoor air pollutant whose role in the induction of asthma is unclear. We investigated the effects of NO(2) on the development of asthma-like responses to allergenic challenge in BALB/c mice. Ovalbumin (OVA)-immunized mice were intranasally challenged with OVA or saline solution just before starting a 3 h exposure to 5 or 20 ppm NO(2) or air. Twenty parts per million of NO(2) induced a significant increase of bronchopulmonary hyperreactivity in OVA-challenged mice and of permeability according to the fibronectin content of the bronchoalveolar lavage fluid (BALF) 24 h after exposure, as compared with air or 5 ppm NO(2). Eosinophilia (cell counts in the BALF and eosinophil peroxidase of lung tissue) was detected at 24 and 72 h with similar levels for air and 20 ppm NO(2), whereas a marked reduction was unexpectedly observed for 5 ppm NO(2). At 24 h, interleukin-5 in the BALF was markedly reduced at 5 ppm compared with 20 ppm NO(2) and was also more intense for 20 ppm NO(2) than for the air group. In contrast to specific IgG1 titers, anti-OVA IgE titers and interleukin-4 in the BALF were not affected by NO(2) exposure. Irrespective of the concentration of NO(2), OVA-challenged mice did not develop late mucosal metaplasia compared with those exposed to OVA-air. These results indicate that a short exposure to NO(2) can exacerbate or inhibit some features of the development of allergic disease in mice and may depend on the concentration of pollutant.     

Conformational changes in azurin from Pseudomona aeruginosa induced through chemical and physical protocols

Azurin from Pseudomona aeruginosa is a small copper protein with a single tryptophan (Trp) buried in the structure. The Gibbs free energies associated with the folding of holo azurin, calculated monitoring Trp fluorescence and changes in absorbance on the ligand-to-metal band, are different because these techniques probe their local environments, thereby being able to probe different conformational changes. The presence of an intermediate state was observed during the chemical denaturation of the protein. Upon denaturation, a 30-fold increase is observed in the magnitude of the quenching constant of the tryptophan fluorescence by acrylamide, because this residue becomes more accessible to the quencher. Entrapping the protein in sol-gel materials lowers its stability possibly because the solvation properties of the macromolecule are changed. The thermal denaturation of azurin immobilized in a sol-gel monolith is irreversible, which tends to rule out an aggregation mechanism to account for the irreversibility of the denaturation of the protein free in solution. Unlike the Cu(II) ion, the Gd(III) ion accommodates in site B of azurin with high affinity and the folding free energy of Gd-azurin is larger than that of apo azurin.     

A functional neuroimaging study of sound localization: visual cortex activity predicts performance in early-blind individuals

Blind individuals often demonstrate enhanced nonvisual perceptual abilities. However, the neural substrate that underlies this improved performance remains to be fully understood. An earlier behavioral study demonstrated that some early-blind people localize sounds more accurately than sighted controls using monaural cues. In order to investigate the neural basis of these behavioral differences in humans, we carried out functional imaging studies using positron emission tomography and a speaker array that permitted pseudo-free-field presentations within the scanner. During binaural sound localization, a sighted control group showed decreased cerebral blood flow in the occipital lobe, which was not seen in early-blind individuals. During monaural sound localization (one ear plugged), the subgroup of early-blind subjects who were behaviorally superior at sound localization displayed two activation foci in the occipital cortex. This effect was not seen in blind persons who did not have superior monaural sound localization abilities, nor in sighted individuals. The degree of activation of one of these foci was strongly correlated with sound localization accuracy across the entire group of blind subjects. The results show that those blind persons who perform better than sighted persons recruit occipital areas to carry out auditory localization under monaural conditions. We therefore conclude that computations carried out in the occipital cortex specifically underlie the enhanced capacity to use monaural cues. Our findings shed light not only on intermodal compensatory mechanisms, but also on individual differences in these mechanisms and on inhibitory patterns that differ between sighted individuals and those deprived of vision early in life.