Characterization of different microenvironments at the surface of the frog's taste organ

We used a panel of histochemical techniques to identify and characterize the cell-associated extracellular material at the surface of the frog's taste organ. We employed morphological and histochemical techniques using both the light microscope and the electron microscope. Results show that the apical, external aspect of cells reaching the surface of the taste organ is in close contact with a layer of amorphous material. The histochemical characteristics of this material vary according to the cell type with which it is in contact. Three different microenvironments can be identified at the surface of the frog's taste organ: type 1 microenvironment is associated with the superficial layer of mucus (secretory) cells; type 2 microenvironment characterizes the surface of the so-called wing cells, which reach the surface of the taste organ as thin laminae running among mucus cells; and type 3 microenvironment shrouds the free endings of putative taste cells and is rich in calcium and lipids. Type 2 and type 3 microenvironments fix peroxidase (a sapid macromolecule) with increasing affinity. We conclude that highly differentiated microenvironments exist at the surface of the frog's taste organ, and these could play a role in the chain of biological events leading to the taste sensation. Furthermore, characterization of the cellassociated, specific microenvironments can help clarify the role of the different cell types in the frog's taste organ.     

Interaction of cefotetan and the metallo-beta-lactamases produced in Aeromonas spp. and in vitro activity

Aeromonas spp. are increasingly being recognized as human pathogens. The presence of metallo-beta-lactamases in these organisms represents a potential problem in antimicrobial therapy. Mechanism-based inactivators of beta-lactamases are used to overcome the resistance of clinical pathogens to beta-lactam antibiotics, but no clinical useful inhibitors of the metallo-beta-lactamases are presently known. Studying the interaction between cefotetan and Aeromonas spp. producing metallo-beta-lactamase activity, we observed that cefotetan behaved as a transient inactivator for both the crude extracts of Aeromonas strains and the purified enzymes from Aeromonas hydrophila AE036 and Aeromonas schubertii MNSA20. The direct hydrolysis of cefotetan showed that it was a poor substrate for both purified enzymes. In view of the minimum inhibitory concentrations, cefotetan shows to be a useful antimicrobial agent against Aeromonas spp.     

Overproduction and biochemical characterization of the Chryseobacterium meningosepticum BlaB metallo-beta-lactamase

The BlaB metallo-beta-lactamase of Chryseobacterium meningosepticum CCUG4310 was overproduced in Escherichia coli by means of a T7 promoter-based expression system. The overproducing system, scaled up in a 15-liter fermentor, yielded approximately 10 mg of BlaB protein per liter, mostly released in the culture supernatant. The enzyme was purified by two ion-exchange chromatographic steps with an overall yield of 66%. Analysis of the kinetic parameters revealed efficient activities (k(cat)/K(m) ratios of >10(6) M(-1) s(-1)) toward most penam and carbapenem compounds, with the exception of the 6-alpha-methoxypenam derivative temocillin and of biapenem, which were poorer substrates. Hydrolysis of cephalosporins was overall less efficient, with a remarkable variability that was largely due to variable affinities of the BlaB enzyme for different compounds. BlaB was also able to hydrolyze serine-beta-lactamase inhibitors, including beta-iodopenicillanate, sulbactam and, although less efficiently, tazobactam.     

Beta-lactamase of Mycobacterium fortuitum: kinetics of production and relationship with resistance to beta-lactam antibiotics.

The kinetics of both intracellular and extracellular beta-lactamase production and the relationship between extracellular enzyme and in vitro susceptibility of Mycobacterium fortuitum to beta-lactam antibiotics have been studied. To this end we used a panel of stable nitrosoguanidine-induced mutants of M. fortuitum derived from the parental strain ATCC 19542 and differing in beta-lactamase production from 0.0001 to 278 U/liter in Mueller-Hinton broth. For overproducers of beta-lactamase (mutants A188, B180, C207, D316, and E31), MICs of benzylpenicillin, amoxicillin, ampicillin, and cephaloridine progressively increased with the amount of enzyme released into the medium, whereas MICs of imipenem and cefoxitin did not. The resistance of the mutants to amoxicillin was reduced up to 32-fold by clavulanic acid, whereas that to ampicillin was reduced 8-fold by sulbactam. These data suggest that the enzyme participated in the mechanisms of resistance to the beta-lactam antibiotics. However, for a mutant of M. fortuitum (gamma 27) with virtually nonexistent beta-lactamase production, the antibiotics still had relatively high MICs (for instance, benzylpenicillin and cephaloridine had MICs of 64 and 32 micrograms/ml, respectively). This suggests that, aside from beta-lactamase production, other mechanisms such as cell wall permeability and/or affinity for penicillin-binding proteins could coexist in M. fortuitum and explain its natural resistance to beta-lactam antibiotics.     

Characterization of the metallo-beta-lactamase determinant of Acinetobacter baumannii AC-54/97 reveals the existence of bla(IMP) allelic variants carried by gene cassettes of different phylogeny

The metallo-beta-lactamase determinant of Acinetobacter baumannii AC-54/97, a clinical isolate from Italy that was previously shown to produce an enzyme related to IMP-1, was isolated by means of a PCR methodology which targets amplification of gene cassette arrays inserted into class 1 integrons. Sequencing revealed that this determinant was an allelic variant (named bla(IMP-2)) of bla(IMP) found in Japanese isolates and that it was divergent from the latter by 12% of its nucleotide sequence, which evidently had been acquired independently. Similar to bla(IMP), bla(IMP-2) was also carried by an integron-borne gene cassette. However, the 59-base element of the bla(IMP-2) cassette was unrelated to those of the bla(IMP) cassettes found in Japanese isolates, indicating a different phylogeny for the gene cassettes carrying the two allelic variants. Expression of the integron-borne bla(IMP-2) gene in Escherichia coli resulted in a significant decrease in susceptibility to a broad array of beta-lactams (ampicillin, carbenicillin, cephalothin, cefoxitin, ceftazidime, cefepime, and carbapenems). The IMP-2 enzyme was purified from an Escherichia coli strain carrying the cloned determinant, and kinetic parameters were determined with several beta-lactam substrates. Compared to IMP-1, the kinetic parameters of IMP-2 were similar overall with some beta-lactam substrates (cefoxitin, ceftazidime, cefepime, and imipenem) but remarkably different with others (ampicillin, carbenicillin, cephaloridine, and meropenem), revealing a functional significance of at least some of the mutations that differentiate the two IMP variants. Present findings suggest that the environmental reservoir of bla(IMP) alleles could be widespread and raise a question about the global risk of their transfer to clinically relevant species.     

Evidence that cerebral blood volume can provide brain activation maps with better spatial resolution than deoxygenated hemoglobin

With the aim of evaluating the relative performance of hemodynamic contrasts for mapping brain activity, the spatio-temporal response of oxy-, deoxy-, and total-hemoglobin concentrations were imaged with diffuse optical tomography during electrical stimulation of the rat somatosensory cortex. For both 6-s and 30-s stimulus durations, total hemoglobin images provided smaller activation areas than oxy- or deoxy-hemoglobin images. In addition, analysis of regions of interest near the sagittal sinus vein show significantly greater contrast in both oxy- and deoxy-relative to total hemoglobin, suggesting that oximetric contrasts have larger draining vein contributions compared to total hemoglobin contrasts under the given stimulus conditions. These results indicate that total hemoglobin and cerebral blood volume may have advantages as hemodynamic mapping contrasts, particularly for large amplitude, longer duration stimulus paradigms.     

Distribution of neurochemical abnormalities in patients with narcolepsy with cataplexy: An in vivo brain proton MR spectroscopy study

Narcolepsy with cataplexy is characterised by excessive daytime sleepiness, sudden drops of muscle tone triggered by emotions, termed cataplexy, disrupted nocturnal sleep and other dissociated rapid eye movement (REM) sleep phenomena. Narcolepsy has been linked to a loss of hypothalamic neurons producing hypocretins, neuropeptides implicated in the regulation of the arousal system. Neuroimaging and neurometabolic studies have shown the pathophysiological involvement of other brain structures such as cerebral cortex and thalamus, but, overall with inconsistent results.We investigated, by using an advanced quantitative MR technique, proton MR spectroscopy (¹H-MRS), the distribution of brain neurochemical abnormalities in narcolepsy with cataplexy patients. Single voxel ¹H-MRS study was performed in the thalamus, hypothalamus, and parietal–occipital cortex of hypocretin deficient, narcolepsy with cataplexy patients, HLA-DQB1*0602-positive, drug free. No significant changes were detected in the thalamus and parietal–occipital cortex of the patients. On the other hand, the neuronal marker N-acetyl-aspartate was reduced in the hypothalamus of narcolepsy with cataplexy patients compared to controls.These ¹H-MRS findings further support that in narcolepsy with cataplexy patients, the hypothalamus is the primary site of neural lesions. The absence of ¹H-MRS neurodegenerative changes in the thalamus and cerebral cortex suggests that the abnormalities detected in these brain regions by other neuroimaging techniques are likely of functional nature.