A Novel, Drug-based, Cellular Assay for the Activity of Neurotoxic Mutants of the Prion Protein

In prion diseases, the infectious isoform of the prion protein (PrP^Sc) may subvert a normal, physiological activity of the cellular isoform (PrP^C). A deletion mutant of the prion protein (Δ105–125) that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrP^C and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing Δ105–125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines. This unusual phenomenon mimics several essential features of Δ105–125 PrP toxicity seen in transgenic mice, including rescue by co-expression of wild type PrP. Cells expressing Δ105–125 PrP are susceptible to drug toxicity within minutes, suggesting that the mutant protein enhances cellular accumulation of these cationic compounds. Our results establish a screenable cellular phenotype for the activity of neurotoxic forms of PrP, and they suggest possible mechanisms by which these molecules could produce their pathological effects in vivo.     

Dominant role of thyrotropin-releasing hormone in the hypothalamic-pituitary-thyroid axis

Hypothalamic thyrotropin-releasing hormone (TRH) stimulates thyroid-stimulating hormone (TSH) secretion from the anterior pituitary. TSH then initiates thyroid hormone (TH) synthesis and release from the thyroid gland. Although opposing TRH and TH inputs regulate the hypothalamic-pituitary-thyroid axis, TH negative feedback is thought to be the primary regulator. This hypothesis, however, has yet to be proven in vivo. To elucidate the relative importance of TRH and TH in regulating the hypothalamic-pituitary-thyroid axis, we have generated mice that lack either TRH, the beta isoforms of TH receptors (TRbeta KO), or both (double KO). TRbeta knock-out (KO) mice have significantly higher TH and TSH levels compared with wild-type mice, in contrast to double KO mice, which have reduced TH and TSH levels. Unexpectedly, hypothyroid double KO mice also failed to mount a significant rise in serum TSH levels, and pituitary TSH immunostaining was markedly reduced compared with all other hypothyroid mouse genotypes. This impaired TSH response, however, was not due to a reduced number of pituitary thyrotrophs because thyrotroph cell number, as assessed by counting TSH immunopositive cells, was restored after chronic TRH treatment. Thus, TRH is absolutely required for both TSH and TH synthesis but is not necessary for thyrotroph cell development.     

Peripheral blood neutrophil activation patterns are associated with pulmonary inflammatory responses to lipopolysaccharide in humans

Increased nuclear accumulation of NF-kappaB in LPS-stimulated peripheral blood neutrophils has been shown to be associated with more severe clinical course in patients with infection associated acute lung injury. Such observations suggest that differences in neutrophil response may contribute to the pulmonary inflammation induced by bacterial infection. To examine this question, we sequentially measured LPS-induced DNA binding of NF-kappaB in neutrophils collected from healthy humans on at least three occasions, each separated by at least 2 wk, and then determined pulmonary inflammatory responses after instillation of LPS into the lungs. Consistent patterns of peripheral blood neutrophil responses, as determined by LPS-induced NF-kappaB DNA binding, were present in volunteers, with a >80-fold difference between individuals in the mean area under the curve for NF-kappaB activation. The number of neutrophils recovered from bronchoalveolar lavage after exposure to pulmonary LPS was significantly correlated with NF-kappaB activation in peripheral blood neutrophils obtained over the pre-LPS exposure period (r = 0.65, p = 0.009). DNA binding of NF-kappaB in pulmonary neutrophils also was associated with the mean NF-kappaB area under the curve for LPS-stimulated peripheral blood neutrophils (r = 0.63, p = 0.01). Bronchoalveolar lavage levels of IL-6 and TNFRII were significantly correlated with peripheral blood neutrophil activation patterns (r = 0.75, p = 0.001 for IL-6; and r = 0.48, p = 0.049 for TNFRII. These results demonstrate that stable patterns in the response of peripheral blood neutrophils to LPS exist in the human population and correlate with inflammatory response following direct exposure to LPS in the lung.     

Breast cancer resistance protein (BCRP/ABCG2) is expressed by progenitor cells/reactive ductules and hepatocytes and its expression pattern is influenced by disease etiology and species type: possible functional consequences.

Breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette transport protein that is expressed in several organs including the liver. Previous studies have shown that ABC transport proteins play an important pathophysiological role in several liver diseases. However, to date, expression pattern and possible role of BCRP in human liver diseases and animal models have not been studied in detail. Here we investigated the expression pattern of BCRP in normal liver, chronic parenchymal and biliary human liver diseases, and parallel in different rat models of liver diseases. Expression was studied by immunohistochemistry and additionally by RT-PCR analysis in Thy-1-positive rat oval cells. Bile ducts, hepatic progenitor cells, reactive bile ductules, and blood vessel endothelium were immunoreactive for BCRP in normal liver and all types of human liver diseases and in rat models. BCRP was expressed by the canalicular membrane of hepatocytes in normal and diseased human liver, but never in rat liver. Remarkably, there was also expression of BCRP at the basolateral pole of human hepatocytes, and this was most pronounced in chronic biliary diseases. In conclusion, BCRP positivity in the progenitor cells/reactive ductules could contribute to the resistance of these cells to cytotoxic agents and xenotoxins. Basolateral hepatocytic expression in chronic biliary diseases may be an adaptive mechanism to pump bile constituents back into the sinusoidal blood. Strong differences between human and rat liver must be taken into account in future studies with animal models.     

Micro- and nanofabrication methods in nanotechnological medical and pharmaceutical devices

Micro- and nanofabrication techniques have revolutionized the pharmaceutical and medical fields as they offer the possibility for highly reproducible mass-fabrication of systems with complex geometries and functionalities, including novel drug delivery systems and bionsensors. The principal micro- and nanofabrication techniques are described, including photolithography, soft lithography, film deposition, etching, bonding, molecular self assembly, electrically induced nanopatterning, rapid prototyping, and electron, X-ray, colloidal monolayer, and focused ion beam lithography. Application of these techniques for the fabrication of drug delivery and biosensing systems including injectable, implantable, transdermal, and mucoadhesive devices is described.     

Phospholipase B activity enhances adhesion of Cryptococcus neoformans to a human lung epithelial cell line

Secreted phospholipase B (PLB1), which contains three enzyme activities in the one protein, is necessary for the initiation of pulmonary infection by Cryptococcus neoformans and for dissemination from the lung via the lymphatics and blood. Adhesion to lung epithelium is the first step in this process, therefore we investigated the role of PLB1 in adhesion to a human lung epithelial cell line, A549, using C. neoformans var. grubii wild-type strain H99, a PLB1 deletion mutant (deltaplb1), and a reconstituted strain (deltaplb1rec). Adhesion of H99 and deltaplb1rec was approximately 69% greater than deltaplb1 at 4 h. Adhesion of deltaplb1 significantly increased after killing by chemicals or heat, and Fourier-transformed analysis by FTIR spectroscopy indicated this was due to changes in capsular and/or cell wall polysaccharides and proteins. Inhibition by specific PLB1 antibodies, or inhibitors of phospholipase B (PLB), but not lysophospholipase (LPL) or lysophospholipase transacylase (LPTA) activities decreased the adhesion of H99 and deltaplb1rec by 33-58%. Growth under conditions of osmotic stress and high glucose concentration increased both PLB secretion and subsequent cryptococcal adhesion. Dose-dependent increases (to 67%) in adhesion of live deltaplb1 were observed in the presence of 0.1-2 mM palmitic acid. We conclude that PLB1 plays a role in the binding of C. neoformans to host lung epithelial cells, possibly due to production of fatty acids from plasma membranes and/or surfactant by PLB activity.     

Inhibition of apoptosis by p26: implications for small heat shock protein function during Artemia development

p26, an abundantly expressed small heat shock protein, is thought to establish stress resistance in oviparously developing embryos of the crustacean Artemia franciscana by preventing irreversible protein denaturation, but it might also promote survival by inhibiting apoptosis. To test this possibility, stably transfected mammalian cells producing p26 were generated and their ability to resist apoptosis induction determined. Examination of immunofluorescently stained transfected 293H cells by confocal microscopy demonstrated p26 is diffusely distributed in the cytoplasm with a minor amount of the protein in nuclei. As shown by immunoprobing of Western blots, p26 constituted approximately 0.6% of soluble cell protein. p26 localization and quantity were unchanged during prolonged culture, and the protein had no apparent ill effects on transfected cells. Molecular sieve chromatography in Sepharose 6B revealed p26 oligomers of about 20 monomers, with a second fraction occurring as larger aggregates. A similar pattern was observed in sucrose gradients, but overall oligomer size was smaller. Mammalian cells containing p26 were more thermotolerant than cells transfected with the expression vector only, and as measured by annexin V labeling, Hoescht 33342 nuclear staining and procaspase-3 activation, transfected cells effectively resisted apoptosis induction by heat and staurosporine. The ability to confer thermotolerance and limit heat-induced apoptosis is important because Artemia embryos are frequently exposed to high temperature in their natural habitat. p26 also blocked apoptosis in transfected cells during drying and rehydration, findings with direct relevance to Artemia life history characteristics because desiccation terminates cyst diapause. Thus, in addition to functioning as a molecular chaperone, p26 inhibits apoptosis, an activity shared by other small heat shock proteins and with the potential to play an important role during Artemia embryo development.     

Comparison of three ELISA protocols to measure antibody responses elicited against serogroup C meningococcal polysaccharide in mouse, monkey and human sera

In this study we compared the following ELISA protocols to measure antibody levels against serogroup C meningococcal polysaccharide: a traditional protocol using poly-L-Lysine mixed with the polysaccharide as coating antigen, a second protocol coating with a mixture of methylated human serum albumin with the C polysaccharide, a modified protocol coating with derivatized polysaccharide and a modification to the last one, specifically without adding ammonium thiocyanate to the sample buffer. Serum bactericidal activity of mouse, monkey and human sera were measured and correlation coefficients were calculated. For all serum types the modified ELISA protocol showed the highest correlation coefficients while the traditional protocol showed the lower ones. We demonstrated that the traditional protocol measures non-specific antibodies to the C polysaccharide, because no differences were detected between pre-immune and post-immune human sera (P>0.05), while the modified protocol detected the highest difference (P<0.01).     

Current state and perspectives of truffle genetics and sustainable biotechnology

Mycorrhizal fungi belonging to the genus Tuber produce, after the establishment of a productive interaction with a plant host, hypogeous fruitbodies of great economic value known as ‘‘truffles’’. This review summarizes the state of art on life cycle, genetic, and biotechnological investigations of Tuber spp. The ascocarp formation in truffles is a consequence of the activation of the sexual phase of the biological cycle. The formation of a dikaryotic secondary mycelium and the karyogamy in the ascal cell (followed by meiosis with ascospores formation) have been hypothesized by several authors but some doubts yet arise from the Tuber cycle by considering that a series of abnormalities have been pointed out in respect to other Ascomycetes. It is unclear if binucleated hyphal cells are derived from the fusion of mononucleated cells belonging to mycelia from different mating types or from one only. According to the karyotypes of Tuber melanosporum, Tuber magnatum, and Tuber borchii, the numbers of hyphal chromosomes suggest a chromosome number of eight (2n); these values are in the range of those of several Ascomycetes and observed for Tuber aestivum (2n=10). The importance and growth in interest during the last years in the fungi protoplasts isolation and transformation techniques can be related to current developments in Tuber genetics and biotechnology. T. borchii could be transformed through liposome-mediated delivery of genetic material as mycelial protoplasts isolation and fusion with liposomes has already been established. On the other hand, Agrobacterium-mediated transformation has been successfully established for T. borchii.     

A 36,000-yr vegetation history from the Peloncillo Mountains, southeastern Arizona, USA

A 36,200 cal yr B.P. vegetation history was developed from macrofossils and pollen from 55 packrat middens from 1287 to 1442 m elevation in the Peloncillo Mountains of southeasternmost Arizona, USA. Today, these elevations are dominated by semidesert grassland with a mixture of Chihuahuan and Sonoran Desert shrubs, including an eastern disjunct population of jojoba (Simmondsia chinensis). From 36,200 to 15,410 cal yr B.P., rocky areas just above large, pluvial lakes that occupied what are now dry playas supported Pinus edulis, Juniperus osteosperma, Juniperus cf. coahuilensis, Quercus cf. turbinella and a rich understory of summer-flowering C₄ annuals and grasses, indicating abundant summer rains and mild winters. After  15,410 cal yr B.P., P. edulis declined in abundance and disappeared briefly at 13,925 cal yr B.P., coincident with expansion of more xeric species and paleohydrological evidence for regional aridity during the Bølling–Allerød warm interval. P. edulis rebounded briefly during the Younger Dryas at 12,405 cal yr B.P. before disappearing along with other mesic woodland species sometime after 12,100 cal yr B.P. The few middens dating from the early to middle Holocene (10,000–4000 yr B.P.) indicate wetter conditions than today at 7790 cal yr B.P. followed by a general drying trend. The 35 middens from the late Holocene detail the sequential arrival of desertscrub species as vegetation became more modern in character.