An amino-terminal signal sequence abrogates the intrinsic membrane-targeting information of mitochondrial uncoupling protein

Mitochondrial uncoupling protein, a polytopic integral protein of the inner membrane, is initially made in the cytoplasm as a soluble polypeptide (307 amino acids) lacking a cleavable targeting (signal) peptide. Earlier studies (Liu, X., Bell, A. W., Freeman, K. B., and Shore, G. C. (1988) J. Cell Biol. 107, 503-509) identified internal regions of the molecule that are critical for targeting and membrane insertion. Here, we demonstrate that the ability of uncoupling protein to insert into the inner membrane is abrogated when the molecule is fused behind the matrix-targeting signal of preornithine carbamyltransferase; the hybrid protein was imported across the inner membrane and deposited in the matrix where it was processed. In this context, however, the processed product remained in the matrix and was incapable of inserting into the inner membrane.     

Poisoning of reintroduced red kites (<Emphasis Type="Italic">Milvus Milvus</Emphasis>) in England

Programmes to reintroduce predatory birds are resource intensive and expensive, yet there are few long-term studies on the health of these reintroduced birds following release. A total of 326 red kites (Milvus milvus) were released at four sites in England between 1989 and 2006 as part of efforts to reintroduce this species to England and Scotland, resulting in the establishment of several rapidly expanding populations in the wild. Detailed post-mortem examinations were carried out on 162 individuals found dead between 1989 and 2007, involving both released and wild-fledged birds. Toxicological analysis of one or more compounds was performed on 110 of the 162 birds. Poisoning was diagnosed in 32 of these 110 kites, 19 from second-generation anticoagulant rodenticides, 9 from other pesticides and 6 from lead. Criteria for diagnosing anticoagulant rodenticide poisoning included visible haemorrhage on gross post-mortem examination and levels of anticoagulant rodenticide exceeding 100 ng/g, but levels were elevated above 100 ng/g in a further eight red kites without visible haemorrhages, suggesting poisoning may have occurred in more birds. The anticoagulant rodenticides difenacoum and bromadiolone were the most common vertebrate control agents involved during this period. Poisoning of red kites may be slowing their rate of population recovery and range expansion in England. Simple modifications of human activity, such as best practice in rodent control campaigns, tackling the illegal use of pesticides and the use of non-toxic alternatives to lead ammunition, can reduce our impact on red kites and probably other populations of predatory and scavenging species.     

Enhancing Identifications of Lipid-embedded Proteins by Mass Spectrometry for Improved Mapping of Endothelial Plasma Membranes in Vivo

Lipid membranes structurally define the outer surface and internal organelles of cells. The multitude of proteins embedded in lipid bilayers are clearly functionally important, yet they remain poorly defined. Even today, integral membrane proteins represent a special challenge for current large scale shotgun proteomics methods. Here we used endothelial cell plasma membranes isolated directly from lung tissue to test the effectiveness of four different mass spectrometry-based methods, each with multiple replicate measurements, to identify membrane proteins. In doing so, we substantially expanded this membranome to 1,833 proteins, including >500 lipid-embedded proteins. The best method combined SDS-PAGE prefractionation with trypsin digestion of gel slices to generate peptides for seamless and continuous two-dimensional LC/MS/MS analysis. This three-dimensional separation method outperformed current widely used two-dimensional methods by significantly enhancing protein identifications including single and multiple pass transmembrane proteins; >30% are lipid-embedded proteins. It also profoundly improved protein coverage, sensitivity, and dynamic range of detection and substantially reduced the amount of sample and the number of replicate mass spectrometry measurements required to achieve 95% analytical completeness. Such expansion in comprehensiveness requires a trade-off in heavy instrument time but bodes well for future advancements in truly defining the ever important membranome with its potential in network-based systems analysis and the discovery of disease biomarkers and therapeutic targets. This analytical strategy can be applied to other subcellular fractions and should extend the comprehensiveness of many future organellar proteomics pursuits.The plasma membrane provides a fundamental physical interface between the inside and outside of any cell. Beyond creating a protected compartment with a segregated, distinct, and well controlled internal milieu for the cell, it also mediates a wide variety of basic biological functions including signal transduction, molecular transport, membrane trafficking, cell migration, cell-cell interactions, intercellular communication, and even drug resistance. Plasma membrane-associated proteins, especially integral membrane proteins (IMPs)¹ that traverse the lipid bilayer, are key elements mediating these vital biological processes. Consistent with its fundamental importance in both normal cellular functions and pathophysiology, the plasma membrane has also been targeted extensively for biomarker discovery and drug development. In fact, more than two-thirds of known targets for existing drugs are plasma membrane proteins (1).Despite the potential benefits, profiling the proteome of plasma membranes comprehensively using standard large scale methods including MS-based strategies has been limited and technically quite challenging. Intrinsic hydrophobicity, a wide concentration range of proteins, and other factors have hampered IMP resolution and identification using conventional two-dimensional gel electrophoresis. Gel and gel-free protein separations, including combinations of both, have been reported as an alternative to two-dimensional gel electrophoresis (2–9). Yet most such efforts have focused predominantly on identifying rather soluble proteins from body fluids (i.e. plasma, serum, and cerebrospinal fluid), cell lysates, or cytoplasm. These proteins, unlike IMPs, are relatively abundant and readily susceptible to enzymatic digestion in solution. Various attempts have been made to solubilize and enrich for IMPs, including different detergents, solvents, high pH solutions, and affinity purification (10–22). Even when organellar membranes are enriched through isolation by subcellular fractionation, the yield of proteins identified has been below expectation, especially for multipass transmembrane proteins such as G-protein-coupled receptors.Here we systematically characterize four analytical approaches to enhance the identification of proteins, specifically those embedded in plasma membranes isolated directly from vascular endothelium in rat lung. Endothelial cells (ECs) constitute the tissue-blood interface that controls many important physiological functions, including tissue homeostasis, nutrition, vasomotion, and even drug delivery. In vivo mapping of the EC plasma membrane proteome provides unique opportunities for extending basic understanding in vascular biology and for directing the delivery of therapeutic and imaging agents in vivo (23–25). But it also presents distinct challenges beyond those generally associated with extraction, solubilization, and identification of IMPs in cells and tissues. ECs form a thin monolayer lining each blood vessel. They constitute a very small fraction of all the cells existing in tissue, thereby making it difficult to isolate sufficiently pure EC plasma membrane fractions for proteomics analysis using conventional subcellular fractionation techniques. Although relatively simple to isolate from tissue and grow in culture, ECs require cues from the tissue microenvironment to maintain their tissue-specific qualities and thus undergo rapid and considerable phenotypic drift after isolation (26).We have developed a specialized coating procedure using colloidal silica nanoparticles perfused through the blood vessels of the tissue to isolate luminal plasma membranes of the vascular endothelium as they exist natively in tissue (26–28). Our initial survey of these plasma membranes isolated directly from rat lungs used primarily three standard analytical techniques of the time: two-dimensional electrophoresis, Western analysis, and the shotgun method of two-dimensional liquid chromatography-tandem mass spectrometry (24, 26). We identified 450 proteins of which only ∼15% were IMPs. Although at the time this was a notable total number of proteins, more IMPs are expected. In fact, this large scale 2DC study did not identify several well known EC surface marker proteins, including specific enzymes, adhesion molecules, and growth factor receptors.Here we comparatively analyze four different MS-based strategies involving two- and three-dimensional separation by combining protein prefractionation via SDS-PAGE with in-gel digestion to produce peptides separated by one- and two-dimensional nano-HPLC before seamless and continuous MS analysis. Each method used multiple replicate measurements to comprehensively identify proteins, especially IMPs, and in doing so achieved a clear statistical definition of completeness that permits meaningful comparisons. Ultimately this analysis greatly expanded the EC plasma membranome to 1,833 proteins of which nearly 30% are membrane-embedded.     

Characterization of 22 microsatellite marker loci in the Madagascar rousette (Rousettus madagascariensis)

Twenty-two nuclear microsatellite loci were isolated from a genomic DNA library derived from Madagascar’s Rousettus madagascariensis. Marker characteristics were determined from a single population (37 individuals) from Fort Dauphin (southeastern Madagascar). Sixteen of the 22 loci were within Hardy–Weinberg expectations. These loci are highly informative with polymorphic information content values ranging between 0.757 and 0.916. These loci will provide valuable information for the study of population genetics and gene flow within this species of bats. Due to the dramatic reduction and alteration of their habitat, data generated utilizing this marker suite will potentially provide additional information for the effective long-term management of this near-threatened bat species.     

Telomeric chromatin: replicating and wrapping up chromosome ends

Recent advances in our understanding of the specialized chromatin structure at telomeres, the ends of eukaryotic chromosomes, have focused on three separate areas: replication of telomeres through the coordinated action of conventional DNA polymerases and the telomerase enzyme, protection of the chromosome end from DNA damage checkpoint sensors and DNA-repair processes, and the discovery of a novel deacetylase enzyme (Sir2p) required for the establishment and maintenance of telomeric heterochromatin. Although the number of proteins and the complexity of their interactions at telomeres continues to grow, a picture of at least some of the major players and mechanisms underlying telomere replication, end 'capping' and chromatin assembly is beginning to emerge.     

The Rationale for Optimal Combination Therapy With Sipuleucel-T for Patients With Castration-resistant Prostate Cancer

Immunotherapy encourages the recipient’s own immune response to destroy cancer cells, and current evidence suggests that immunotherapies may be most beneficial in early metastatic castration-resistant prostate cancer (mCRPC). Sipuleucel-T is the first therapeutic cancer vaccine to be approved by both the US Food and Drug Administration and European Medicines Agency for the treatment of asymptomatic or minimally symptomatic mCRPC. Combining immunotherapy with other treatments may have potent anticancer effects; cytoreductive therapies can release tumor antigens and promote a proinflammatory environment that could augment immunotherapies. However, some cytoreductive agents or coadministered drugs may be immunosuppressive. Understanding these interactions between different mCRPC treatment modalities may offer further potential to improve patient outcomes.Key words: Combination therapy, Prostate cancer, Sipuleucel-TImmunotherapy has emerged as a powerful tool against prostate cancer, in addition to surgery, radiotherapy, hormone therapy, and chemotherapy. For 30 years, investigators tried to rebalance the compromised immune system in patients with urologic cancers using a number of different agents.^1,2 In April 2010, the autologous cellular immunotherapy sipuleucel-T became the first therapeutic cancer vaccine to be approved by the US Food and Drug Administration (FDA).³ This therapy targets the prostatic acid phosphatase (PAP) and has been indicated for the treatment of asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC), based on results from three randomized, controlled, phase 3 studies.^3–6 Recently, sipuleucel-T was also approved by the European Medicines Agency (EMA) for the treatment of asymptomatic or minimally symptomatic mCRPC in men in whom chemotherapy is not yet clinically indicated.⁷Although this immunotherapy has been shown to extend overall survival (OS),⁵ sequencing or combining immunotherapy with other treatments for mCRPC has the potential to further improve outcomes.^8,9 However, before immunotherapy-based combination regimens can be integrated into clinical practice, it is critical to have a better understanding of the interactions between these different modalities.     

Early replication of short telomeres in budding yeast

The maintenance of an appropriate number of telomere repeats by telomerase is essential for proper chromosome protection. The action of telomerase at the telomere terminus is regulated by opposing activities that either recruit/activate the enzyme at shorter telomeres or inhibit it at longer ones, thus achieving a stable average telomere length. To elucidate the mechanistic details of telomerase regulation we engineered specific chromosome ends in yeast so that a single telomere could be suddenly shortened and, as a consequence of its reduced length, elongated by telomerase. We show that shortened telomeres replicate early in S phase, unlike normal-length telomeres, due to the early firing of origins of DNA replication in subtelomeric regions. Early telomere replication correlates with increased telomere length and telomerase activity. These data reveal an epigenetic effect of telomere length on the activity of nearby replication origins and an unanticipated link between telomere replication timing and telomerase action.     

Preparative size-exclusion chromatography of human serum apolipoproteins using an analytical liquid chromatograph

Apolipoproteins, extracted from human serum high-density lipoproteins, can be resolved and recovered with high yield from a preparative MicroPak TSK Type 3000SW size-exclusion column using Tris-buffered 6 m urea or 6 m guanidinium chloride mobile phases. Adequate resolution of some apolipoprotein pairs is only achieved at low flow velocities and low sample loads, necessitating repetitive injections of small amounts of material for preparative isolation. An analytical high-performance liquid chromatograph equipped with a simplified sample introduction scheme and low-pressure switching valves for fraction collection was used to isolate milligram quantities of HDL apolipoproteins.