Random screening for dominant-negative mutants of the cytomegalovirus nuclear egress protein M50

Inactivation of gene products by dominant-negative (DN) mutants is a powerful tool to assign functions to proteins. Here, we present a two-step procedure to establish a random screen for DN alleles, using the essential murine cytomegalovirus gene M50 as an example. First, loss-of-function mutants from a linker-scanning library were tested for inhibition of virus reconstitution with the help of FLP-mediated ectopic insertion of the mutants into the viral genome. Second, DN candidates were confirmed by conditional expression of the inhibitory proteins in the virus context. This allowed the quantification of the inhibitory effect, the identification of the morphogenesis block, and the construction of DN mutants with improved activity. Based on these observations a DN mutant of the homologous gene (UL50) in human cytomegalovirus was predicted and constructed. Our data suggest that a proline-rich sequence motif in the variable region of M50/UL50 represents a new functional site which is essential for nuclear egress of cytomegalovirus capsids.     

Automated robotic harvesting of protein crystals—addressing a critical bottleneck or instrumentation overkill?

One of the critical steps in high throughput crystallography that so far has evaded automation is the actual harvesting of the delicate crystals from the mother liquor in which they are growing. The late-stage operation of harvesting is presently a most risky and loss-intensive procedure, compounded by its tight integration with the critical steps of cryo-protection and cryo-quenching. Recent advances in micromanipulation robotics and micro-fabrication have made it possible to seriously consider automation of protein crystal harvesting. Based on the experience gained during the development of an operator-assisted (and now operator-assisting) universal micromanipulation robot (UMR) prototype, we discuss the challenges ahead for the design of a fully autonomous, integrated system capable of the reliable harvesting of protein microcrystals. Experience from participation in NIH structural genomics projects and feedback from bottleneck workshops indicates that genuine demand exists in the high throughput community as well as in pharmaceutical production pipelines, justifying the effort and resources to develop autonomous harvesting robotics.     

Prevalence, genetic conservation and transmissibility of the Chlamydia pneumoniae bacteriophage (phiCpn1)

The Chlamydia pneumoniae bacteriophage was first identified in isolate AR-39. Its relevance for chlamydial biology and pathogenicity remains unknown. In this study, a collection of 36 C. pneumoniae isolates was screened and the phage was detected in eight. As the positive isolates differed by several polymorphisms, they presumably belonged to different genetic lineages. It was investigated whether different genotypes of the phage also existed and whether they could be assigned to chlamydial genotypes as evidence of coevolution. Sequencing of >3000 bp of the 4524 bp phage genome revealed complete identity to the published sequences. Thus, it was hypothesized that the genetic conservation was related to easy transmissibility of the phage between C. pneumoniae isolates. Cocultivation of phage positive and negative isolates followed by cloning and identification of different C. pneumoniae genotypes demonstrated for the first time transmissibility of the bacteriophage from one isolate to the other. These observations indicate that the phage is capable of infecting C. pneumoniae isolates of different genetic backgrounds and suggest that all C. pneumoniae strains might be susceptible. The successful in vitro infection of C. pneumoniae with the phage provides the basis for studying its pathogenetic relevance in isolates of identical genetic background and provides a potential tool for genetic manipulation of C. pneumoniae.     

Temporal progression and extent of the return of sensation in the foot provided by the saphenous nerve after sciatic nerve transection and repair in the rat - implications for nociceptive assessments

Sensory testing, by providing stimuli for nociceptors of the foot, is a popular method of evaluating sensory regeneration after damage to the sciatic nerve in the rat. In the following study, 20 rats were submitted to double transection of the sciatic nerve. The subsequent 14 mm gap was repaired through guidance interponation. In order to evaluate nerve regeneration, sensory testing was performed additionally to other methods, which included motor testing, morphometry, and electron microscopic assessments of nerves. Somatosensory testing revealed that all animals exhibited next to the same amount of sensory reinnervation on their foot regardless of their experimental group. In motor tests, however, two out of the three experimental groups did not improve at all. These groups also failed to show neural regrowth in morphometric and electron microscopic assessments of the associated nerve. Retrograde tracing was able to prove the saphenous nerve as an alternative source of sensory reinnervation in animals with failed sciatic regeneration. This means that results of sensory testing in the rat should be treated with caution, taking into account the areas tested and the likelihood that in these areas saphenous sprouting could have taken place. Furthermore, it is strongly advised that somatosensory testing should be conducted only on toe 5.     

SorCS1 variants and amyloid precursor protein (APP) are co‐transported in neurons but only SorCS1c modulates anterograde APP transport

Processing of amyloid precursor protein (APP) into amyloid‐β peptide (Aβ) is crucial for the development of Alzheimer's disease (AD). Because this processing is highly dependent on its intracellular itinerary, altered subcellular targeting of APP is thought to directly affect the degree to which Aβ is generated. The sorting receptor SorCS1 has been genetically linked to AD, but the underlying molecular mechanisms are poorly understood. We analyze two SorCS1 variants; one, SorCS1c, conveys internalization of surface‐bound ligands whereas the other, SorCS1b, does not. In agreement with previous studies, we demonstrate co‐immunoprecipitation and co‐localization of both SorCS1 variants with APP. Our results suggest that SorCS1c and APP are internalized independently, although they mostly share a common post‐endocytic pathway. We introduce functional Venus‐tagged constructs to study SorCS1b and SorCS1c in living cells. Both variants are transported by fast anterograde axonal transport machinery and about 30% of anterograde APP‐positive transport vesicles contain SorCS1. Co‐expression of SorCS1b caused no change of APP transport kinetics, but SorCS1c reduced the anterograde transport rate of APP and increased the number of APP‐positive stationary vesicles. These data suggest that SorCS1 and APP share trafficking pathways and that SorCS1c can retain APP from insertion into anterograde transport vesicles.

     

RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

Background

The combinatorial library strategy of using multiple candidate ligands in mixtures as library members is ideal in terms of cost and efficiency, but needs special screening methods to estimate the affinities of candidate ligands in such mixtures. Herein, a new method to screen candidate ligands present in unknown molar quantities in mixtures was investigated.

Results

The proposed method involves preparing a processed-mixture-for-screening (PMFS) with each mixture sample and an exogenous reference ligand, initiating competitive binding among ligands from the PMFS to a target immobilized on magnetic particles, recovering target-ligand complexes in equilibrium by magnetic force, extracting and concentrating bound ligands, and analyzing ligands in the PMFS and the concentrated extract by chromatography. The relative affinity of each candidate ligand to its reference ligand is estimated via an approximation equation assuming (a) the candidate ligand and its reference ligand bind to the same site(s) on the target, (b) their chromatographic peak areas are over five times their intercepts of linear response but within their linear ranges, (c) their binding ratios are below 10%. These prerequisites are met by optimizing primarily the quantity of the target used and the PMFS composition ratio. The new method was tested using the competitive binding of biotin derivatives from mixtures to streptavidin immobilized on magnetic particles as a model. Each mixture sample containing a limited number of candidate biotin derivatives with moderate differences in their molar quantities were prepared via parallel-combinatorial-synthesis (PCS) without purification, or via the pooling of individual compounds. Some purified biotin derivatives were used as reference ligands. This method showed resistance to variations in chromatographic quantification sensitivity and concentration ratios; optimized conditions to validate the approximation equation could be applied to different mixture samples. Relative affinities of candidate biotin derivatives with unknown molar quantities in each mixture sample were consistent with those estimated by a homogenous method using their purified counterparts as samples.

Conclusions

This new method is robust and effective for each mixture possessing a limited number of candidate ligands whose molar quantities have moderate differences, and its integration with PCS has promise to routinely practice the mixture-based library strategy.     

Proteolytic cleavage of covalently linked cell wall proteins by Candida albicans Sap9 and Sap10

The cell wall of the human-pathogenic fungus Candida albicans is a robust but also dynamic structure which mediates adaptation to changing environmental conditions during infection. Sap9 and Sap10 are cell surface-associated proteases which function in C. albicans cell wall integrity and interaction with human epithelial cells and neutrophils. In this study, we have analyzed the enzymatic properties of Sap9 and Sap10 and investigated whether these proteases cleave proteins on the fungal cell surface. We show that Sap9 and Sap10, in contrast to other aspartic proteases, exhibit a near-neutral pH optimum of proteolytic activity and prefer the processing of peptides containing basic or dibasic residues. However, both proteases also cleaved at nonbasic sites, and not all tested peptides with dibasic residues were processed. By digesting isolated cell walls with Sap9 or Sap10, we identified the covalently linked cell wall proteins (CWPs) Cht2, Ywp1, Als2, Rhd3, Rbt5, Ecm33, and Pga4 as in vitro protease substrates. Proteolytic cleavage of the chitinase Cht2 and the glucan-cross-linking protein Pir1 by Sap9 was verified using hemagglutinin (HA) epitope-tagged versions of both proteins. Deletion of the SAP9 and SAP10 genes resulted in a reduction of cell-associated chitinase activity similar to that upon deletion of CHT2, suggesting a direct influence of Sap9 and Sap10 on Cht2 function. In contrast, cell surface changes elicited by SAP9 and SAP10 deletion had no major impact on the phagocytosis and killing of C. albicans by human macrophages. We propose that Sap9 and Sap10 influence distinct cell wall functions by proteolytic cleavage of covalently linked cell wall proteins.     

First experiences with semi-autonomous robotic harvesting of protein crystals

The demonstration unit of the Universal Micromanipulation Robot (UMR) capable of semi-autonomous protein crystal harvesting has been tested and evaluated by independent users. We report the status and capabilities of the present unit scheduled for deployment in a high-throughput protein crystallization center. We discuss operational aspects as well as novel features such as micro-crystal handling and drip-cryoprotection, and we extrapolate towards the design of a fully autonomous, integrated system capable of reliable crystal harvesting. The positive to enthusiastic feedback from the participants in an evaluation workshop indicates that genuine demand exists and the effort and resources to develop autonomous protein crystal harvesting robotics are justified.