Levels of hypoxanthine phosphoribosyltransferase RNA in human cells

The gene for the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT) is expressed at a low level in many cells. As is the case with several other “housekeeping genes,” thorough studies of hprt gene regulation have been hampered by the low levels of its mRNA. We have used RNA/RNA hybridization in solution to determine the concentration of hprt-RNA in human cells. The sensitivity and specificity of the method have been validated, and it is shown that hprt-RNA can be accurately determined at a level of a few mRNA molecules per cell. As expected for a housekeeping gene, low and relatively constant hprt-RNA levels (0.3–0.8 pg/μg DNA) were found in primary cultures of normal amnion cells and fibroblasts, EBV-transformed lymphoblastoid cell lines, neuroblastoma, glioblastoma, and melanoma cell cultures. While resting lymphocytes were found to contain very low amounts of hprt-RNA, lymphocytes stimulated with phytohemagglutinin (PHA) showed a 10-fold increase to about 0.8–1.2 pg/μg DNA, which corresponds to 6–10 hprt-RNA molecules per cell. The level started to increase about 20 h after PHA stimulation, 5–10 h before the onset of DNA synthesis, and a steady-state level was reached after 2–3 days in culture. In PHA-stimulated lymphocytes from two brothers with inherited HPRT deficiency (LeschNyhans syndrome), the hprt-RNA level in PHA-stimulated lymphocytes was only about 25% of that in normal subjects. In T-cells selected for HPRT deficiency by growth in 6-thioguanine medium, the levels of hprt-RNA were either normal or very low, which probably reflects the different nature of the mutations involved. These results demonstrate the sensitivity of this method for determinations of low levels of RNA and clearly show induction of hprt-RNA after mitogenic stimulation of human lymphocytes.     

ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking

COPI‐coated vesicles mediate retrograde membrane traffic from the cis‐Golgi to the endoplasmic reticulum (ER) in all eukaryotic cells. However, it is still unknown whether COPI vesicles fuse everywhere or at specific sites with the ER membrane. Taking advantage of the circumstance that the vesicles still carry their coat when they arrive at the ER, we have visualized active ER arrival sites (ERAS) by monitoring contact between COPI coat components and the ER‐resident Dsl tethering complex using bimolecular fluorescence complementation (BiFC). ERAS form punctate structures near Golgi compartments, clearly distinct from ER exit sites. Furthermore, ERAS are highly polarized in an actin and myosin V‐dependent manner and are localized near hotspots of plasma membrane expansion. Genetic experiments suggest that the COPI?Dsl BiFC complexes recapitulate the physiological interaction between COPI and the Dsl complex and that COPI vesicles are mistargeted in dsl1 mutants. We conclude that the Dsl complex functions in confining COPI vesicle fusion sites.     

Discovery of the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine scaffold as a novel,potent and selective GABAA α5 inverse agonist series

Through iterative design cycles we have discovered a number of novel new classes where the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine was deemed the most promising GABA_A α5 inverse agonist class with potential for cognitive enhancement. This class combines a modest subtype binding selectivity with inverse agonism and has the most favourable molecular properties for further lead optimisation towards a central nervous system (CNS) acting medicine.     

Streptomyces lividans inhibits the proliferation of the fungus Verticillium dahliae on seeds and roots of Arabidopsis thaliana

Verticillium wilt, a vascular disease in more than 200 dicotyledonous plants, is due to the ascomycete fungus Verticillium dahliae. As documented by video-microscopy, the soil bacterium Streptomyces lividans strongly reduces the germination of V. dahliae conidia, and the subsequent growth of hyphae. Quantification by the use of DNA-intercalating dyes and Calcofluor-staining revealed that during prolonged co-cultivation, bacterial hyphae proliferate to a dense network, provoke a poor development of V. dahliae vegetative hyphae and lead to an enormous reduction of conidia and microsclerotia. Upon individual application to seeds of the model plant Arabidopsis thaliana, either the bacterial spores or the fungal conidia germinate at or within the mucilage, including its volcano-shaped structures. The extension of hyphae from each individual strain correlates with the reduction of the pectin-containing mucilage-layer. Proliferating hyphae then spread to roots of the emerging seedlings. Plants, which arise in the presence of V. dahliae within agar or soil, have damaged root cells, an atrophied stem and root, as well as poorly developed leaves with chlorosis symptoms. In contrast, S. lividans hyphae settle in bunches preferentially at the outer layer near tips and alongside roots. Resulting plants have a healthy appearance including an intact root system. Arabidopsis thaliana seeds, which are co-inoculated with V. dahliae and S. lividans, have preferentially proliferating bacterial hyphae within the mucilage, and at roots of the outgrowing seedlings. As a result, plants have considerably reduced disease symptoms. As spores of the beneficial S. lividans strain are obtainable in large quantity, its application is highly attractive.     

Molecular Modeling of the Misfolded Insulin Subunit and Amyloid Fibril

Insulin, a small hormone protein comprising 51 residues in two disulfide-linked polypeptide chains, adopts a predominantly α-helical conformation in its native state. It readily undergoes protein misfolding and aggregates into amyloid fibrils under a variety of conditions. Insulin is a unique model system in which to study protein fibrillization, since its three disulfide bridges are retained in the fibrillar state and thus limit the conformational space available to the polypeptide chains during misfolding and fibrillization. Taking into account this unique conformational restriction, we modeled possible monomeric subunits of the insulin amyloid fibrils using β-solenoid folds, namely, the β-helix and β-roll. Both models agreed with currently available biophysical data. We performed molecular dynamics simulations, which allowed some limited insights into the relative structural stability, suggesting that the β-roll subunit model may be more stable than the β-helix subunit model. We also constructed β-solenoid-based insulin fibril models and conducted fiber diffraction simulation to identify plausible fibril architectures of insulin amyloid. A comparison of simulated fiber diffraction patterns of the fibril models to the experimental insulin x-ray fiber diffraction data suggests that the model fibers composed of six twisted β-roll protofilaments provide the most reasonable fit to available experimental diffraction patterns and previous biophysical studies.     

Dimeric chlorite dismutase from the nitrogen‐fixing cyanobacterium Cyanothece sp. PCC7425

It is demonstrated that cyanobacteria (both azotrophic and non‐azotrophic) contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite ‘dismutase’, Cld). Beside the water‐splitting manganese complex of photosystem II, this metalloenzyme is the second known enzyme that catalyses the formation of a covalent oxygen–oxygen bond. All cyanobacterial Clds have a truncated N‐terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in Escherichia coli and shown to efficiently degrade chlorite with an activity optimum at pH 5.0 [k_cat 1144 ± 23.8 s^?1, K_M 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 10⁶ M^?1 s^?1]. The resting ferric high‐spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of ?126 ± 1.9 mV at pH 7.0. Cyanide mediates the formation of a low‐spin complex with k_on = (1.6 ± 0.1) × 10⁵ M^?1 s^?1 and k_off = 1.4 ± 2.9 s^?1 (K_D ～ 8.6 μM). Both, thermal and chemical unfolding follows a non‐two‐state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure–function relationships of Clds. We ask for the physiological substrate and putative function of these O₂‐producing proteins in (nitrogen‐fixing) cyanobacteria.     

Prevalence of Neurocysticercosis in People with Epilepsy in the Eastern Province of Zambia

Zambia is endemic for Taenia solium taeniosis and cysticercosis. In this single-centered, cross-sectional, community-based study, the role of neurocysticercosis (NCC) as a cause of epilepsy was examined. People with epilepsy (PWE, n = 56) were identified in an endemic area using a screening questionnaire followed by in-depth interviews and neurological examination. Computed tomography (CT) was performed on 49 people with active epilepsy (PWAE) and their sera (specific antibody and antigen detection, n = 56) and stools (copro-antigen detection, n = 54) were analyzed. The CT scan findings were compared to a group of 40 CT scan controls. Of the PWE, 39.3% and 23.2% were positive for cysticercal antibodies and antigens, respectively, and 14.8% for coproantigens (taeniosis). Lesions highly suggestive of NCC were detected in 24.5% and definite NCC lesions in 4.1% of CT scans of PWAE. This compares to 2.5% and 0%, respectively, in the control CT scans. Using the Del Brutto diagnostic criteria, 51.8% of the PWAE were diagnosed with probable or definitive NCC and this rose to 57.1% when the adapted criteria, as proposed by Gabriël et al. (adding the sero-antigen ELISA test as a major criterion), were used. There was no statistically significant relationship between NCC, current age, age at first seizure and gender. This study suggests that NCC is the single most important cause of epilepsy in the study area. Additional large-scale studies, combining a community based prevalence study for epilepsy with neuroimaging and serological analysis in different areas are needed to estimate the true impact of neurocysticercosis in endemic regions and efforts should be instituted to the control of T. solium.     

The F-box protein SKP2 mediates androgen control of p27 stability in LNCaP human prostate cancer cells

Background

The cyclin-dependent kinase inhibitor p27 is a putative tumor suppressor that is downregulated in the majority of human prostate cancers. The mechanism of p27 down-regulation in prostate cancers in unknown, but presumably involves increased proteolysis mediated by the SCF^SKP2 ubiquitin ligase complex. Here we used the human prostate cancer cell line LNCaP, which undergoes G1 cell cycle arrest in response to androgen, to examine the role of the SKP2 F-box protein in p27 regulation in prostate cancer.

Results

We show that androgen-induced G1 cell cycle arrest of LNCaP cells coincides with inhibition of cyclin-dependent kinase 2 activity and p27 accumulation caused by reduced p27 ubiquitylation activity. At the same time, androgen decreased expression of SKP2, but did not affect other components of SCF^SKP2. Adenovirus-mediated overexpression of SKP2 led to ectopic down-regulation of p27 in asynchronous cells. Furthermore, SKP2 overexpression was sufficient to overcome p27 accumulation in androgen arrested cells by stimulating cellular p27 ubiquitylation activity. This resulted in transient activation of CDK2 activity, but was insufficient to override the androgen-induced G1 block.

Conclusions

Our studies suggest that SKP2 is a major determinant of p27 levels in human prostate cancer cells. Based on our in vitro studies, we suggest that overexpression of SKP2 may be one of the mechanisms that allow prostate cancer cells to escape growth control mediated by p27. Consequently, the SKP2 pathway may be a suitable target for novel prostate cancer therapies.     

Small-molecule aggregates inhibit amyloid polymerization

Many amyloid inhibitors resemble molecules that form chemical aggregates, which are known to inhibit many proteins. Eight known chemical aggregators inhibited amyloid formation of the yeast and mouse prion proteins Sup35 and recMoPrP in a manner characteristic of colloidal inhibition. Similarly, three known anti-amyloid molecules inhibited beta-lactamase in a detergent-dependent manner, which suggests that they too form colloidal aggregates. The colloids localized to preformed fibers and prevented new fiber formation in electron micrographs. They also blocked infection of yeast cells with Sup35 prions, which suggests that colloidal inhibition may be relevant in more biological milieus.