The human immunodeficiency virus (HIV) protease inhibitor indinavir directly affects the opportunistic fungal pathogen Cryptococcus neoformans

Highly active antiretroviral therapy (HAART), that includes human immunodeficiency virus (HIV) protease inhibitors (PIs), has been remarkably efficacious including against some opportunistic infections. In this report we investigated the effect(s) of the PI indinavir on protease activity by Cryptococcus neoformans, an opportunistic fungal pathogen responsible for recurrent meningoencephalitis in AIDS patients. Indinavir was also tested for potential effects on other parameters, such as fungal viability, growth ability and susceptibility to immune effector cells. It was found that indinavir impaired cryptococcal protease activity in a time- and dose-dependent fashion. The phenomenon was similarly detectable in ATCC/laboratory strains and clinical isolates. C. neoformans growth rate was also significantly reduced upon exposure to indinavir, while fungal viability was not affected and mitochondrial toxicity not detected. Furthermore, as assessed by an in vitro infection model, indinavir significantly and consistently augmented C. neoformans susceptibility to microglial cell-mediated phagocytosis and killing. Overall, by providing the first evidence that indinavir directly affects C. neoformans, these data add new in vitro insights on the wide-spectrum efficacy of PIs, further arguing for the clinical relevance of HAART against opportunistic infections in AIDS.     

Ezrin is essential for epithelial organization and villus morphogenesis in the developing intestine

Ezrin, Radixin, and Moesin (the ERM proteins) supply regulated linkage between membrane proteins and the actin cytoskeleton. The study of mammalian ERM proteins has been hampered by presumed functional overlap. We have found that Ezrin, the only ERM detected in epithelial cells of the developing intestine, provides an essential role in configuring the mouse intestinal epithelium. Surprisingly, Ezrin is not absolutely required for the formation of brush border microvilli or for the establishment or maintenance of epithelial polarity. Instead, Ezrin organizes the apical terminal web region, which is critical for the poorly understood process of de novo lumen formation and expansion during villus morphogenesis. Our data also suggest that Ezrin controls the localization and/or function of certain apical membrane proteins that support normal intestinal function. These in vivo studies highlight the critical function of Ezrin in the formation of a multicellular epithelium rather than an individual epithelial cell.     

Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project

One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.     

Organic tissues in rotating bioreactors: fluid-mechanical aspects, dynamic growth models, and morphological evolution

This analysis deals with advances in tissue-engineering models and computational methods as well as with novel results on the relative importance of "controlling forces" in the growth of organic constructs. Specifically, attention is focused on the rotary culture system, because this technique has proven to be the most practical solution for providing a suitable culture environment supporting three-dimensional tissue assemblies. From a numerical point of view, the growing biological specimen gives rise to a moving boundary problem. A "volume-of-fraction" method is specifically and carefully developed according to the complex properties and mechanisms of organic tissue growth and, in particular, taking into account the sensitivity of the construct/liquid interface to the effect of the fluid-dynamic shear stress (it induces changes in tissue metabolism and function that elicit a physiological response from the biological cells). The present study uses available data to introduce a set of growth models. The surface conditions are coupled to the transfer of mass and momentum at the specimen/culture-medium interface and lead to the introduction of a group of differential equations for the nutrient concentration around the sample and for the evolution of tissue mass displacement. The models are then used to show how the proposed surface kinetic laws can predict (through sophisticated numerical simulations) many of the known characteristics of biological tissues grown using rotating-wall perfused vessel bioreactors. This procedure provides a validation of the models and associated numerical method and also gives insight into the mechanisms of the phenomena. The interplay between the increasing size of the tissue and the structure of the convective field is investigated. It is shown that this interaction is essential in determining the time evolution of the tissue shape. The size of the growing specimen plays a critical role with regard to the intensity of convection and the related shear stresses. Convective effects, in turn, are found to impact growth rates, tissue size, and morphology, as well as the mechanisms driving growth. The method exhibits novel capabilities to predict and elucidate experimental observations and to identify cause-and-effect relationships.     

A (1-->6)-linked beta-mannopyrananan, pseudonigeran, and a (1-->4)-linked beta-xylan, isolated from the lichenised basidiomycete Dictyonema glabratum

Extraction of Dictyonema glabratum with hot 2% (w/v) aqueous KOH at 100 degrees C, followed by neutralisation and freeze-thawing, gave an insoluble glucan. The residue was further extracted by a similar process, but with hot 10% (w/v) aqueous KOH, furnishing a mixture of glucan, mannan and xylan. The mannan and xylan were obtained via precipitation of its copper complex with Fehling's solution, leaving the glucan in the supernatant. The insoluble complex was finally purified through gel permeation chromatography. Methylation analysis, one- and two-dimensional nuclear magnetic resonance examination showed the polysaccharides to be a (1-->3)-linked alpha-glucan (pseudonigeran) and a (1-->4)-linked beta-xylan, both not previously encountered in lichens, and a newly discovered (1-->6)-linked beta-mannan.     

Novel paralogy relations among human chromosomes support a link between the phylogeny of doublesex-related genes and the evolution of sex determination

Recent advances in the evolutionary genetics of sex determination indicate that DMRT1 may be a vertebrate equivalent of the Drosophila melanogaster master sex regulator gene, doublesex. The role of DMRT1 seems to be confined to some aspects of male sex differentiation, whereas in Drosophila, doublesex has wider developmental effects in both sexes. This suggests other homologs of doublesex may exist in the vertebrate genome and encode sex-specific functions not displayed by DMRT1. We identified and characterized five novel human DM genes, distinct from previously described family members. Human DM genes map to three well-defined regions of chromosomes 1, 9, and 19 (one gene on chromosome 19 having an additional homolog on chromosome X). We collated data indicating these chromosomal regions harbor multiple syntenic genes sharing highly specific paralogy relations, suggesting that they arose early during vertebrate evolution. The 9p21-p24.3 bands represent the ancestral copy and harbor closely linked DM genes that may reflect the overall diversity of the fruit fly DM gene family. The human genome contains a small number of potential doublesex homologs that may be involved in human sexual development. Identifying highly conserved chromosomal regions, such as distal 9p, is an important tool to trace complex ancient evolutionary processes inaccessible by other approaches.     

Karyopherins in nuclear pore biogenesis: a role for Kap121p in the assembly of Nup53p into nuclear pore complexes

The mechanisms that govern the assembly of nuclear pore complexes (NPCs) remain largely unknown. Here, we have established a role for karyopherins in this process. We show that the yeast karyopherin Kap121p functions in the targeting and assembly of the nucleoporin Nup53p into NPCs by recognizing a nuclear localization signal (NLS) in Nup53p. This karyopherin-mediated function can also be performed by the Kap95p-Kap60p complex if the Kap121p-binding domain of Nup53p is replaced by a classical NLS, suggesting a more general role for karyopherins in NPC assembly. At the NPC, neighboring nucleoporins bind to two regions in Nup53p. One nucleoporin, Nup170p, associates with a region of Nup53p that overlaps with the Kap121p binding site and we show that they compete for binding to Nup53p. We propose that once targeted to the NPC, dissociation of the Kap121p-Nup53p complex is driven by the interaction of Nup53p with Nup170p. At the NPC, Nup53p exists in two separate complexes, one of which is capable of interacting with Kap121p and another that is bound to Nup170p. We propose that fluctuations between these two states drive the binding and release of Kap121p from Nup53p, thus facilitating Kap121p's movement through the NPC.     

Carbocysteine lysine salt monohydrate (SCMC-LYS) is a selective scavenger of reactive oxygen intermediates (ROIs)

Carbocysteine lysine salt monohydrate (SCMC-Lys) is a well-known mucoactive drug whose therapeutic efficacy is commonly related to the ability of SCMC-Lys to replace fucomucins by sialomucins. The aim of this study was to determine if SCMC-Lys could exert an anti-oxidant action by scavenging reactive oxygen intermediates (ROIs). Our results show that SCMC-Lys proved effective as a selective scavenger of hypochlorous acid (HOCl) and hydroxyl radical (OH.), this effect being related to the reactivity of the SCMC tioether group. The scavenger activity of SCMC-Lys was observed in free cellular system as well as in activated human polymorphonuclear neutrophils (PMNs). SCMC-Lys scavenger activity on HOCl was paralleled by a powerful protection from HOCl-mediated inactivation of alpha1-antitripsin (alpha1-AT) inhibitor, the main serum protease inhibitor. Production of interleukin-(IL-)8, a major mediator of PMN recruitment in inflammatory diseases, is known to be mediated by intracellular OH. SCMC-Lys significantly reduced IL-8 production on stimulated human peripheral blood mononuclear cells (PBMCs) in the same range of concentrations affecting OH. activity. It is concluded that SCMC-Lys could exert, in addition to its mucoactive capacity, an anti-oxidant action, thus contributing to the therapeutic efficacy of SCMC-Lys.