Discrepancies between MIC and MLC values of amphotericin B against isolates ofAspergillus species

There is little information addressing the phenomena of discrepancy between minimal inhibitory concentrations (MIC) and minimal lethal concentrations (MLC) values of amphotericin B (AMB) to clinical isolates of fungi. This study assessed in vitro activity of AMB against 70 clinical isolates of aspergilli: 30 strains ofAspergillus fumigatus, 20 strains ofAspergillus flavus and 20 strains ofAspergillus niger. Susceptibility tests were accomplished using a macro broth dilution procedure, with special emphasis on ascertainment of MLCs. AMB exhibited low MIC values against all clinical isolates. While we did not identify any AMB resistant isolates among 70Aspergillus spp. studied as judged by MIC levels, analysis of the data demonstrated a clear discrepancy between the MIC and MLC levels of AMB obtained against clinical isolates ofAspergillus spp. The MLC values of AMB were significantly higher than the MIC values with MIC 50 and MIC 90 of 0.29 and 0.5 µg/ml, respectively, at the second reading time, and MLC 50 and MLC 90 of 2.31 and 9.24 µg/ml, respectively (p<0.001). Additionally, minimal lethal concentrations in 36/70 (51%) of aspergillal isolates studied produced drug concentrations above those which can usually be sustained in patient plasma or tissue.     

Chiral discrimination by ligand-exchange chromatography: A comparison between phenylalaninamide-based stationary and mobile phases

The copper(II) complexes of two new diastereomeric ligands, N²-(R)- and N²-(S)-2′-hydroxypropyl-(S)-phenylalaninamide [(R, S)-1 and (S, S)-1], have been used as additives to the eluent in high-performance liquid chromatography (HPLC) reversed phase for the chiral separation of DNS-amino acids. The aim was that of comparing the separation process obtained by the chiral eluent with that obtained by an analogous bonded stationary phase containing (S)-phenylalaninamide, previously studied [CSP-(S)-Phe-NH₂]. The affinity of the ternary complexes for the C₁₈ column was determined by adsorption experiments in HPLC. It was shown that the two systems (chiral eluent, chiral stationary phase) work according to different mechanisms. Ternary complex formation in solution was studied by fluorescence spectroscopy. It was shown that chiral separation with the Cu(II) complexes added to the eluent was determined by the relative affinities of the ternary complexes for the column-stationary phase rather than by their stabilities in solution. With CSP-(S)-Phe-NH₂ the separation is accounted for by the relative stabilities of the ternary complexes, which depends mainly on the “allowed” geometry of the complex and on the steric repulsion of the amino acid side chain with the spacer. © 1996 Wiley-Liss, Inc.     

Brazilian Angiostrongylus cantonensis haplotypes,ac8 and ac9, have two different biological and morphological profiles

Angiostrongylus cantonensis is the etiologic agent of eosinophilic meningoencephalitis in humans. Cases have been recorded in many parts of the world, including Brazil. The aim of this study was to compare the differences in the biology and morphology of two different Brazilian haplotypes of A. : ac8 and ac9. A significantly larger number of L1 larvae eliminated in the faeces of rodents at the beginning of the patent period was observed for ac9 haplotype and compared to the total of L1 larvae eliminated, there was a significant difference between the two haplotypes. The ac9 haplotype showed a significant difference in the proportion of female and male specimens (0.6:1), but the same was not observed for ac8 (1.2:1). The morphometric analysis showed that male and female specimens isolated from ac8 haplotype were significantly larger with respect to body length, oesophagus length, spicule length (male) and distance from the anus to the rear end (female) compared to specimens from ac9. The morphological analysis by light microscopy showed little variation in the level of bifurcations at the lateral rays in the right lobe of the copulatory bursa between the two haplotypes. The biological, morphological and morphometric variations observed between the two haplotypes agree with the observed variation at the molecular level using the cytochrome oxidase subunit I marker and reinforce the possible influence of geographical isolation on the development of these haplotypes.     

The 29.9 kDa subunit of mitochondrial complex I is involved in the enzyme active/de-active transitions

Mitochondrial respiratory chain complex I undergoes transitions from active to de-activated forms. We have investigated the phenomenon in sub-mitochondrial particles from Neurospora crassa wild-type and a null-mutant lacking the 29.9 kDa nuclear-coded subunit of complex I. Based on enzymatic activities, genetic crosses and analysis of mitochondrial proteins in sucrose gradients, we found that about one-fifth of complex I with catalytic properties similar to the wild-type enzyme is assembled in the mutant. Mutant complex I still displays active/de-active transitions, indicating that other proteins are involved in the phenomenon. However, the kinetic characteristics of complex I active/de-active transitions in nuo29.9 differ from wild-type. The spontaneous de-activation of the mutant enzyme is much slower, implicating the 29.9 kDa polypeptide in this event. We suggest that the fungal 29.9 kDa protein and its homologues in other organisms may modulate the active/de-active transitions of complex I.     

Echinococcus granulosus antigen B hydrophobic ligand binding properties

Antigen B (AgB), an immunodominant component of the cestode parasite Echinococcus granulosus, presents homology to and shares apparent structural similarities with helix-rich hydrophobic ligand binding proteins (HLBPs) from other cestodes. In order to investigate the fatty acid binding properties of AgB, two of its subunit components (rAgB8/1 and rAgB8/2) were expressed in Escherichia coli and purified, and the native antigen was purified from the hydatid cyst fluid by affinity chromatography using a monoclonal antibody raised against rAgB8/1. The interaction of the purified native and recombinant proteins with the fluorescent ligands DAUDA, ANS, DACA and 16-AP was investigated. The palmitic acid derived fluorescent ligand, 16-AP, showed the greatest enhancement in fluorescence when bound to native AgB or to its recombinant subunits, and the dissociation constants for 16-AP binding were determined. Surprisingly, in contrast to HLBPs from other cestodes, interactions with other fatty acids, including palmitic acid, caused an increase in fluorescence instead of competing with 16-AP. Our results suggest that AgB might have evolved different functions in the binding of hydrophobic compounds, dependent on cestode environment.     

Praziquantel and albendazole damaging action on in vitro developing Mesocestoides corti (Platyhelminthes: Cestoda)

Parasitic flatworms present several steps of body architecture rearrangement during their fast transition from one developmental stage to another, which are, at least in part, responsible for their evasion from host immune response. Besides, different developmental stages present different degrees of susceptibility to drug action, and the identification of more susceptible stages is of importance for the definition of therapeutical approaches. Mesocestoides corti (syn. Mesocestoides vogae) is considered a good model to study cestode biology because it can be easily manipulated both in vivo and in vitro and due to its relatively close relationship to cestodes of medical relevance, such as those from genera Echinococcus or Taenia. We have analyzed the damaging action of two broad spectrum anthelmintic drugs (praziquantel and albendazole) throughout the in vitro strobilization process of M. corti in order to identify developmental stages or body structures more susceptible to these drugs. Tetrathyridia (larval stage) and segmented-induced worms were cultivated and treated with praziquantel and albendazole. Whole mounted samples, taken from different developmental stages, were fixed and stained with fluorophore-labeled WGA lectin and phalloidin for the analysis of tegument and muscles, respectively. Confocal laser scanning microscopy was used to identify anatomical changes and lesions caused by each anthelmintic drug in a 3D view. We demonstrated that both praziquantel and albendazole cause extensive tissue damage, especially on tegument, and that adult forms were the most susceptible to drug exposure.     

Up-regulation of Skp2 after prostate cancer cell adhesion to basement membranes results in BRCA2 degradation and cell proliferation

Aberrant interaction of carcinoma cells with basement membranes (BM) is a fundamental pathophysiological process that initiates a series of events resulting in cancer cell invasion and metastasis. In this report, we describe the results of our investigations pertaining to the events triggered by the adhesion of normal (PNT1A) and highly metastatic (PC-3) prostate cells onto BM proteins. Unlike PNT1A, PC-3 cells adhered avidly to Matrigel BM matrix as well as to isolated collagen type IV, laminin, and heparan sulfate proteoglycan perlecan, main BM components. This aberrantly increased cancer cell adhesion resulted in sustained BRCA2 protein depletion and vigorous cell proliferation, a cascade triggered by beta1 integrin-mediated phosphatidylinositol 3-kinase activation leading to BRCA2 degradation in the proteasome. This latter effect was orchestrated by phosphatidylinositol 3-kinase-dependent up-regulation of Skp2, a subunit of the Skp1-Cul1-F-box protein ubiquitin complex that directly associates with BRCA2 as demonstrated by coimmunoprecipitation assays, determines its ubiquitination, and ultimately targets it for proteasomal degradation. Inhibition of Skp2 expression by small interference RNA prevented BRCA2 depletion and inhibited the trophic effect upon cell proliferation. These results provide additional evidence on the role of BRCA2 as a modulator of cancer cell growth and elucidate the molecular mechanisms involved in its down-regulation in cancer cells when interacting with BM, a crucial step in the biology of metastasis. Furthering the understanding of this molecular pathway may prove valuable in designing new therapeutic strategies aimed at modifying the natural history of prostate carcinoma.     

Characterization of apoptosis-related oxidoreductases from Neurospora crassa

The genome from Neurospora crassa presented three open reading frames homologous to the genes coding for human AIF and AMID proteins, which are flavoproteins with oxidoreductase activities implicated in caspase-independent apoptosis. To investigate the role of these proteins, namely within the mitochondrial respiratory chain, we studied their cellular localization and characterized the respective null mutant strains. Efficiency of the respiratory chain was analyzed by oxygen consumption studies and supramolecular organization of the OXPHOS system was assessed through BN-PAGE analysis in the respective null mutant strains. The results demonstrate that, unlike in mammalian systems, disruption of AIF in Neurospora does not affect either complex I assembly or function. Furthermore, the mitochondrial respiratory chain complexes of the mutant strains display a similar supramolecular organization to that observed in the wild type strain. Further characterization revealed that N. crassa AIF appears localized to both the mitochondria and the cytoplasm, whereas AMID was found exclusively in the cytoplasm. AMID2 was detected in both mitochondria and cytoplasm of the amid mutant strain, but was barely discernible in wild type extracts, suggesting overlapping functions for the two proteins.