Drosophila Kelch functions with Cullin-3 to organize the ring canal actin cytoskeleton

Drosophila melanogaster Kelch (KEL) is the founding member of a diverse protein family defined by a repeated sequence motif known as the KEL repeat (KREP). Several KREP proteins, including Drosophila KEL, bind filamentous actin (F-actin) and contribute to its organization. Recently, a subset of KREP proteins has been shown to function as substrate adaptor proteins for cullin-RING (really interesting new gene) ubiquitin E3 ligases. In this study, we demonstrate that association of Drosophila KEL with Cullin-3, likely in a cullin-RING ligase, is essential for the growth of Drosophila female germline ring canals. These results suggest a role for protein ubiquitylation in the remodeling of a complex F-actin cytoskeletal structure.     

The Constitutive Activity of Epidermal Growth Factor Receptor vIII Leads to Activation and Differential Trafficking of Wild-type Epidermal Growth Factor Receptor and erbB2

A constitutively active epidermal growth factor receptor (EGFR) mutant, EGFR variant III (EGFRvIII), has been detected at high frequencies in certain human cancers. This study evaluated transactivation and trafficking of erbB family members as a result of constitutive EGFR activity in a cancer cell line. Expression of EGFRvIII modulated erbB family members through different mechanisms; the erbB3 mRNA level was reduced, whereas wild-type EGFR (wtEGFR) and erbB2 protein levels were diminished, with no change in their mRNA levels, and there was no change in the erbB4 expression level. Both EGFR and erbB2 were internalized as a result of EGFRvIII''s activity and redistributed to the cell surface upon addition of AG1478, an inhibitor of wtEGFR/EGFRvIII catalytic activity. Acute activation of EGFRvIII by removing AG1478 from cells increased phosphorylation of both wtEGFR and erbB2 and caused differential trafficking of EGFRvIII''s activation partners; wtEGFR was directed primarily to lysosomal compartments and partially to recycling compartments, whereas erbB2 was directed primarily to recycling compartments and partially to lysosomal compartments. Our data demonstrate that the constitutive activity of EGFRvIII is sufficient to trigger endocytosis and trafficking of wtEGFR and erbB2, which may play a role in activating signaling pathways that are triggered during receptor endocytosis. (J Histochem Cytochem 58:529–541, 2010)     

Geographical variation in the response of visceral leishmaniasis to paromomycin in East Africa: a multicentre, open-label, randomized trial

Background

Visceral leishmaniasis (VL) is a major health problem in developing countries. The untreated disease is fatal, available treatment is expensive and often toxic, and drug resistance is increasing. Improved treatment options are needed. Paromomycin was shown to be an efficacious first-line treatment with low toxicity in India.

Methods

This was a 3-arm multicentre, open-label, randomized, controlled clinical trial to compare three treatment regimens for VL in East Africa: paromomycin sulphate (PM) at 15 mg/kg/day for 21 days versus sodium stibogluconate (SSG) at 20 mg/kg/day for 30 days; and the combination of both dose regimens for 17 days. The primary efficacy endpoint was cure based on parasite-free tissue aspirates taken 6 months after treatment.

Findings

Overall, 135 patients per arm were enrolled at five centres in Sudan (2 sites), Kenya (1) and Ethiopia (2), when the PM arm had to be discontinued due to poor efficacy. The trial has continued with the higher dose of PM as well as the combination of PM and SSG arms. These results will be reported later. Baseline patient characteristics were similar among treatment arms. The overall cure with PM was significantly inferior to that with SSG (63.8% versus 92.2%; difference 28.5%, 95%CI 18.8% to 38.8%, p<0.001). The efficacy of PM varied among centres and was significantly lower in Sudan (14.3% and 46.7%) than in Kenya (80.0%) and Ethiopia (75.0% and 96.6%). No major safety issues with PM were identified.

Conclusion

The efficacy of PM at 15 mg/kg/day for 21 days was inadequate, particularly in Sudan. The efficacy of higher doses and the combination treatment warrant further studies.     

Regulation of avoidant behaviors and pain by the anti-inflammatory tyrosine phosphatase SHP-1

The protein tyrosine phosphatase SHP-1 is a critical regulator of cytokine signaling and inflammation. Mice homozygous for a null allele at the SHP-1 locus have a phenotype of severe inflammation and are hyper-responsive to the TLR4 ligand LPS. TLR4 stimulation in the CNS has been linked to both neuropathic pain and sickness behaviors. To determine if reduction in SHP-1 expression affects LPS-induced behaviors, responses of heterozygous SHP-1-deficient (me/+) and wild-type (+/+) mice to LPS were measured. Chronic (4-week) treatment with LPS induced avoidant behaviors indicative of fear/anxiety in me/+, but not +/+, mice. These behaviors were correlated with a LPS-induced type 2 cytokine, cytokine receptor, and immune effector arginase profile in the brains of me/+ mice not found in +/+ mice. Me/+ mice also had a constitutively greater level of TLR4 in the CNS than +/+ mice. Additionally, me/+ mice displayed constitutively increased thermal sensitivity compared to +/+ mice, measured by the tail-flick test. Moreover, me/+ glial cultures were more responsive to LPS than +/+ glia. Therefore, the reduced expression of SHP-1 in me/+ imparts haploinsufficiency with respect to the control of CNS TLR4 and pain signaling. Furthermore, type 2 cytokines become prevalent during chronic TLR4 hyperstimulation in the CNS and are associated positively with behaviors that are usually linked to type 1 pro-inflammatory cytokines. These findings question the notion that type 2 immunity is solely anti-inflammatory in the CNS and indicate that type 2 immunity induces/potentiates CNS inflammatory processes.     

Molecular mimicry and horror autotoxicus: do chlamydial infections elicit autoimmunity?

All species of the order Chlamydiales are obligate intracellular eubacterial pathogens of their various hosts. Two chlamydial species, Chlamydia trachomatis and Chlamydia pneumoniae, are primarily human pathogens, and each is known to cause important diseases. Some strains of C. trachomatis are sexually transmitted and frequently cause severe reproductive problems, primarily in women. Other strains of the organism serve as the aetiological agents for blinding trachoma, still the leading cause of preventable blindness in underdeveloped nations. C. pneumoniae is a respiratory pathogen known to cause community-acquired pneumonia. Importantly, both organisms engender an immunopathogenic response in the human host, and both have been associated with widely diverse, relatively common and currently idiopathic chronic diseases, most of which include an important autoimmune component. In this article, we explore the available experimental data regarding the possible elicitation of autoimmunity in various contexts by chlamydial infection, and we suggest several avenues for research to explore this potentially important issue further.     

Evaluation of methods for oligonucleotide array data via quantitative real-time PCR

Background  

There are currently many different methods for processing and summarizing probe-level data from Affymetrix oligonucleotide arrays. It is of great interest to validate these methods and identify those that are most effective. There is no single best way to do this validation, and a variety of approaches is needed. Moreover, gene expression data are collected to answer a variety of scientific questions, and the same method may not be best for all questions. Only a handful of validation studies have been done so far, most of which rely on spike-in datasets and focus on the question of detecting differential expression. Here we seek methods that excel at estimating relative expression. We evaluate methods by identifying those that give the strongest linear association between expression measurements by array and the "gold-standard" assay.     

Formative assessment can be fun as well as educational

An understanding of the hormonal basis of normal growth and development, including the changes occurring at puberty, is important foundation knowledge for contemporary medical practice in most fields of medicine. A quiz, testing the important physiological concepts of growth and puberty, was designed using the format of the well-known television game "Who Wants to Be a Millionaire." An evaluation of this formative assessment activity revealed that a cohort of first-year undergraduate medical students valued learning with peers in an enjoyable, interactive environment, where they were able to admit to uncertainties and clarify answers. It also showed that making an educational activity fun need not detract from the focus of giving feedback on learning. Formative assessment, known to produce learning gains in a range of educational settings, is an important activity in contemporary medical education. With a greater emphasis on self-directed learning and less well-defined curriculum boundaries, feedback helps students to understand and apply the important physiological concepts that underpin the practice of medicine.