Substance P receptor antagonist reverses intestinal pathophysiological alterations occurring in a novel ex-vivo model of Cryptosporidium parvum infection of intestinal tissues derived from SIV-infected macaques

BACKGROUND: Cryptosporidium infection leads to life-threatening diarrhea in AIDS patients. Pathogenesis of cryptosporidiosis is due to intestinal physiological alterations. We devised an ex-vivo model using ex-vivo Cryptosporidium parvum infection of jejunal tissues derived from SIV-infected macaques and studied the role of substance P (SP) in the pathogenesis of cryptosporidiosis. METHODS: We measured jejunal SP protein levels using ELISA, and electrophysiological alterations using the Ussing chamber technique in an ex vivo model of Cryptosporidium infection. Paraformaldehyde-fixed jejunum from SIV-infected macaques with and without naturally occurring cryptosporidiosis was studied for SP protein expression by immunohistochemistry and fluorescence deconvolution microscopy. RESULTS: Ex-vivo Cryptosporidium-infected tissues and tissues from SIV-infected macaques with naturally occurring cryptosporidiosis demonstrated elevated SP protein levels compared with tissues from SIV-infected animals without ex-vivo C. parvum infection or tissues from SIV-infected animals that have no evidence of cryptosporidiosis. In our ex-vivo model of Cryptosporidium infection, we demonstrated pathophysiological alterations that were blocked by SP-receptor antagonist treatment. CONCLUSIONS: These studies suggest that SP-receptor antagonists could prove useful for treatment of AIDS-related cryptosporidiosis.     

Ty3 requires yeast La homologous protein for wild-type frequencies of transposition

The Saccharomyces cerevisiae retrovirus-like element Ty3 inserts specifically into the initiation sites of genes transcribed by RNA polymerase III (pol III). A strain with a disruption of LHP1, which encodes the homologue of autoantigen La protein, was recovered in a screen for mutants defective in Ty3 transposition. Transposition into a target composed of divergent tRNA genes was decreased eightfold. In lhp1 mutants, Ty3 polyproteins were produced at wild-type levels, assembled into virus-like particles (VLPs) and processed efficiently. The amount of cDNA associated with these particles was about half the amount in a wild-type control at early times, but approached the wild-type level after 48 h of induction. Ty3 integration was examined at two genomic tRNA gene families and two plasmid-borne tRNA promoters. Integration was significantly decreased at one of the tRNA gene families, but was only slightly decreased at the second tRNA gene family. These findings suggest that Lhp1p contributes to Ty3 cDNA synthesis, but might also act at a target-specific step, such as integration.     

The upper dentition and face of Pondaungia cotteri from central Myanmar

A specimen of Pondaungia from the late middle Eocene Pondaung Formation in central Myanmar includes maxillary fragments and parts of the dentition, some hitherto undocumented, including the upper central incisor, canine, premolars and molars. Pondaungia has a large spatulate I1 closely resembling that of crown anthropoids. It possesses a stout projecting upper canine (like anthropoids) but differs from that tooth of crown anthropoids in lacking a strong mesial groove. There are three upper premolars of which P2 is distinctly smaller than P3 or P4. P3 has a buccolingually oriented mesial profile and an inflated distal profile resembling that of parapithecids and crown anthropoids. The distolingual molar cusp is a hypocone and is not homologus with the "pseudohypocone" of notharctines because the cusp is neither twinned with the protocone nor attached to a Nannopithex-fold. Pondaungia has a stout zygomatic root with a strongly demarcated muscle scar for the superficial masseter situated well above the occlusal plane. The inferior orbital margin is not preserved but the inflated suborbital region allows for the inference that the orbit was small. This specimen is not sufficiently well preserved to identify if there was postorbital closure. However, a specimen of the frontal bone of Amphipithecus shows that its orbital septum was absent or poorly developed. If, as commonly supposed, Pondaungia andAmphipithecus are sister taxa, postorbital closure was probably absent in Pondaungia. The large incisors, molars with poorly developed crests and thick enamel, together with the stoutly developed and strong dorsal component of the force vector of the superficial masseter muscle suggest that Pondaungia had a diet low in fiber, but that included hard food objects like nuts or seeds. The present material adds to the structural similarities between Pondaungia and anthropoids, but whether these similarities are due to shared descent or functional and adaptive convergence remains unresolved.     

A rapid cellular FRET assay of polyglutamine aggregation identifies a novel inhibitor

Many neurodegenerative diseases, including tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, and the polyglutamine diseases, are characterized by intracellular aggregation of pathogenic proteins. It is difficult to study modifiers of this process in intact cells in a high-throughput and quantitative manner, although this could facilitate molecular insights into disease pathogenesis. Here we introduce a high-throughput assay to measure intracellular polyglutamine protein aggregation using fluorescence resonance energy transfer (FRET). We screened over 2800 biologically active small molecules for inhibitory activity and have characterized one lead compound in detail. Y-27632, an inhibitor of the Rho-associated kinase p160ROCK, diminished polyglutamine protein aggregation (EC(50) congruent with 5 microM) and reduced neurodegeneration in a Drosophila model of polyglutamine disease. This establishes a novel high-throughput approach to study protein misfolding and aggregation associated with neurodegenerative diseases and implicates a signaling pathway of previously unrecognized importance in polyglutamine protein processing.     

An Integrated Global Analysis of Compartmentalized HRAS Signaling

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A cellular trafficking signal in the SIV envelope protein cytoplasmic domain is strongly selected for in pathogenic infection

The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-based trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting. Despite extensive analysis, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which this signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY), replicates acutely to high levels in pigtail macaques (PTM) but is rapidly controlled. However, we previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion encoding amino acids 734–736 (ΔQTH) that overlaps the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected that generated a new signal for polarized sorting but not endocytosis. This genotype, together with the ΔGY mutation, was conserved in association with high viral loads for several months when introduced into naïve PTMs. For the first time, our findings reveal strong selection pressure for Env endocytosis and particularly for polarized sorting during pathogenic SIV infection in vivo.     

Effects of Disrupting Calcium Homeostasis on Neuronal Maturation: Early Inhibition and Later Recovery

It has become increasingly clear that agents that disrupt calcium homeostasis may also be toxic to developing neurons. Using isolated primary neurons, we sought to understand the neurotoxicity of agents such as MK801 (which blocks ligand-gated calcium entry), BAPTA (which chelates intracellular calcium), and thapsigargin (TG; which inhibits the endoplasmic reticulum Ca²⁺-ATPase pump). Thus, E18 rat cortical neurons were grown for 1 day in vitro (DIV) and then exposed to vehicle (0.1% DMSO), MK801 (0.01–20 μM), BAPTA (0.1–20 μM), or TG (0.001–1 μM) for 24 h. We found that all three agents could profoundly influence early neuronal maturation (growth cone expansion, neurite length, neurite complexity), with the order of potency being MK801 < BAPTA < TG. We next asked if cultures exposed to these agents were able to re-establish their developmental program once the agent was removed. When we examined network maturity at 4 and 7 DIV, the order of recovery was MK801 > BAPTA > TG. Thus, mechanistically distinct ways of disrupting calcium homeostasis differentially influenced both short-term and long-term neuronal maturation. These observations suggest that agents that act by altering intracellular calcium and are used in obstetrics or neonatology may be quite harmful to the still-developing human brain.     

Chemotaxonomic characterization of rice seedling blight complex using fatty acid methyl ester (FAME) profiles

Rice seedling blight is an important disease caused by a complex of fungi that include Fusarium, Rhizopus, Pythium, and Trichoderma species. A modified MIDI method was used for extraction of fatty acids from these causal pathogens, and fatty acid methyl ester (FAME) profiles were characterized. Factors that might affect fatty acid production, such as period of culture and saponification in extraction, were also evaluated. A total of 14 fatty acids were detected, and FAME profiles showed quantitative and qualitative variations by discriminant analysis and principal component analysis. Genus-specific FAME profiles consisting of the types of fatty acid produced and remarkable components of individual fatty acids were observed. The possibility of application as chemotaxonomic methods based on the FAME profiles for diagnosis of the rice seedling blight complex is also discussed.     

Reversal of thymic stromal lymphopoietin-induced airway inflammation through inhibition of Th2 responses

Lung-specific thymic stromal lymphopoietin (TSLP) expression is sufficient for the development of an asthma-like chronic airway inflammatory disease. However, the nature of the downstream pathways that regulate disease development are not known. In this study, we used IL-4- and Stat6-deficient mice to establish the role of Th2-type responses downstream of TSLP. IL-4 deficiency greatly reduced, but did not eliminate, TSLP-induced airway hyperresponsiveness, airway inflammation, eosinophilia, and goblet cell metaplasia, while Stat6 deficiency eliminated these asthma-like symptoms. We further demonstrate, using the chronic model of TSLP-mediated airway inflammation, that blockade of both IL-4 and IL-13 responses, through administration of an anti-IL-4R alpha mAb, reversed asthma-like symptoms, when given to mice with established disease. Collectively these data provide insight into the pathways engaged in TSLP-driven airway inflammation and demonstrate that simultaneous blockade of IL-4 and IL-13 can reverse established airway disease, suggesting that this may be an effective approach for the therapy of Th2-mediated inflammatory respiratory disease.