Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

Purification and preliminary characterization of brain aspartoacylase

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) in the brain to produce acetate and L-aspartate. An aspartoacylase deficiency, with concomitant accumulation of NAA, is responsible for Canavan disease, a lethal autosomal recessive disorder. To examine the mechanism of this enzyme the genes encoding murine and human aspartoacylase were cloned and expressed in Escherichia coli. A significant portion of the enzyme is expressed as soluble protein, with the remainder found as inclusion bodies. A convenient enzyme-coupled continuous spectrophotometric assay has been developed for measuring aspartoacylase activity. Kinetic parameters were determined with the human enzyme for NAA and for selected N-acyl analogs that demonstrate relaxed substrate specificity with regard to the nature of the acyl group. The clinically relevant E285A mutant reveals an altered enzyme with poor stability and barely detectable activity, while a more conservative E285D substitution leads to only fivefold lower activity than native aspartoacylase.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Issues in using whole slide imaging for diagnostic pathology: “routine” stains,immunohistochemistry and predictive markers

The traditional microscope, together with the “routine” hematoxylin and eosin (H & E) stain, remains the “gold standard” for diagnosis of cancer and other diseases; remarkably, it and the majority of associated biological stains are more than 150 years old. Immunohistochemistry has added to the repertoire of “stains” available. Because of the need for specific identification and even measurement of “biomarkers,” immunohistochemistry has increased the demand for consistency of performance and interpretation of staining results. Rapid advances in the capabilities of digital imaging hardware and software now offer a realistic route to improved reproducibility, accuracy and quantification by utilizing whole slide digital images for diagnosis, education and research. There also are potential efficiencies in work flow and the promise of powerful new analytical methods; however, there also are challenges with respect to validation of the quality and fidelity of digital images, including the standard H & E stain, so that diagnostic performance by pathologists is not compromised when they rely on whole slide images instead of traditional stained tissues on glass slides.     

Gene structure,transcripts and calciotropic effects of the PTH family of peptides in <Emphasis Type="Italic">Xenopus</Emphasis> and chicken

Background  

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken.     

Morphological and molecular characterization of populations of plains bristlegrass (<i>Setaria macrostachya</i> Kunth) in Chihuahua,México

Plains bristlegrass (Setaria macrostachya Kunth) is a native grass with forage value. However, due to the lack of grazing management practices, populations and thus genetic diversity, have been reduced. Morphological and genetic variability were analyzed on 44 populations of plains bristlegrass in the State of Chihuahua. Plants were transplanted in a common area under natural conditions. Two years later, morphological characterization was evaluated measuring nine variables, and genetic variability using AFLP molecular markers. The principal components analysis (PC) showed that the three first principal components explained 73.74% of the variation. The variables with the greatest contribution to the variance in PC1 were plant height and inflorescence length; in CP2, tiller number and leaf width; and in PC3, tiller thickness. Application of four pairs of primers, presented 186 total bands, from which 87.10% showed polymorphism and 12.90% monomorphism. The combination of EcoRI-AGG MseI-CAG primers detected the highest percentage (93%) of polymorphism with 40 polymorphic bands. The cluster analysis and Dice coefficient indicated that populations clump into two groups. The wide genetic variability and morphological characteristics detected among populations represent the basis for the selection of populations that could be used with different purposes in the rehabilitation of ecosystems. In addition, this study will allow establishment of in situ conservation strategies.     

Identification of DNA sequence variation in Campylobacter jejuni strains associated with the Guillain-Barré syndrome by high-throughput AFLP analysis

Background  

Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular.     

Argonaute MID domain takes centre stage

Two recent papers, one in EMBO reports and one in Nature give us the first eukaryotic structures of Argonaute MID domains; providing a structural basis for the 5′-nucleotide recognition of the guide strand and a possible explanation for the allosteric regulation of RNA binding.EMBO Rep (2010) advance online publication. doi: 10.1038/embor.2010.81Argonaute (AGO) proteins are the central component of small RNA-mediated gene silencing in eukaryotes. Functional AGO complexes are loaded with single-stranded small RNAs, which guide AGO to a messenger RNA (mRNA) target through base pairing. Although the structure of a full-length eukaryotic AGO has yet to be described, insights into the mechanism of guide RNA binding and target recognition have been revealed by the structures of distantly related AGO homologues from archaea and eubacteria (Song et al, 2004; Wang et al, 2008, 2009). These studies show that AGO proteins are composed of amino-terminal, PAZ (PIWI/Argonaute/Zwille), MID (middle) and PIWI (P-element-induced whimpy testes) domains. The phosphorylated 5′-end of the guide strand RNA is localized in the MID–PIWI domain interface with the 3′-end anchored to the PAZ domain. On binding to mRNA the catalytic RNase H-like active site located in the PIWI domain is in position to cleave the targeted mRNA.Two recent papers, one in EMBO reports (Boland et al, 2010) and one in Nature (Frank et al, 2010), give us the first eukaryotic structures of AGO MID domains. The human AGO MID domain structure provides a structural basis for the 5′-nucleotide recognition of the guide strand observed in eukaryotic AGOs, and the structure of the MID domain of QDE-2 from Neurospora crassa published in this journal offers a possible explanation for the allosteric regulation of RNA binding discovered earlier this year by Rachel Green''s group (Djuranovic et al, 2010)The two structures have a similar topology resembling a Rossmann-like fold with four β-strands forming a central β-sheet flanked by α-helices. Superposition of the two structures, which are 24% identical in sequence, shows that they are also similar in three dimensions, with a root mean square deviation (r.m.s.d.) of 2.1 Å. The archaeal and eubacterial AGO MID domains solved previously share less than 20% sequence identity and have a greater than 2.5 Å r.m.s.d. for backbone atoms from both QDE-2 and human AGO2, despite having a similar overall fold.Crystals of the QDE-2 MID domain contain two sulphate ions. The first sulphate (sulphate I) is coordinated by the highly conserved amino acids Tyr 595, Lys 599 and Lys 638, and is in the same position as the 5′-phosphate of UMP observed in the human AGO2 MID domain structure (Fig 1A). These interactions are similar to those observed for the 5′-phosphate of the guide strand of the previously solved archaeal and eubacterial structures (Ma et al, 2005; Parker et al, 2005; Wang et al, 2008). Thus, sulphate I bound in the QDE-2 MID domain structure likely represents the 5′-nucleotide-binding site. Most intriguing is the position of the second sulphate (sulphate II), located in an adjacent but partly overlapping binding site with sulphate I. Sulphate II is 6.3 Å from sulphate I, shares coordination with Lys 599 and Lys 638, and is further coordinated by Thr 610. Sulphate II can be excluded from representing the phosphate backbone of a microRNA (miRNA) or target because it is bound in the side of the MID domain opposite from where the guide RNA extends from the 5′-nucleotide-binding site. Although the presence of sulphate II does not guarantee a biologically relevant ligand-binding site, it is tempting to speculate, in the light of a recent study by Djuranovic et al, that sulphate II occupies an allosteric ligand-binding site.Open in a separate windowFigure 1Structural comparison of Neurospora crassa QDE-2 MID domain and human Argonaute 2 MID domain. (A) The N. crassa QDE-2 MID domain structure (green ribbon). UMP is modelled from a superposition of the human AGO2 MID domain structure in a complex with UMP (Protein Data Bank code 3LUJ). Sulphate I and II (S I and S II) are shown as observed in the QDE-2 structure. Conserved QDE-2 amino acids involved in binding sulphate I and II are shown as sticks. (B) Human AGO2 MID domain structure (blue ribbon) in complex with UMP (sticks). The nucleotide specificity loop is coloured in yellow. Sulphate I and II are modelled from a superposition of the N. crassa QDE-2 MID domain. AGO, Argonaute; MID, middle.Djuranovic et al describe a second ligand-binding site in the Drosophila melanogaster AGO1 MID domain that is separate and distinct from the 5′-nucleotide-binding site. They demonstrate that free nucleotides, including the cap analogue m⁷GpppG, bind to an allosteric site, which in turn enhances the binding of miRNA. Cap binding was reported previously for human AGO2 (Kiriakidou et al, 2007), leading to the proposal that two phenylalanine residues in the MID domain make stacking interactions with the m⁷GpppG cap structure, analogous to eukaryotic initiation factor 4E. However, in the human AGO2 MID domain structure it is clear that these phenylalanine residues are on opposite sides of the MID domain and are located in the hydrophobic core. In the QDE-2 MID domain only one of these phenylalanines is conserved, but a similar conclusion is drawn on the basis of the positions of the two residues being more than 25 Å apart. This strongly argues that an alternative mechanism exists for cap binding by eukaryotic AGO proteins.The data presented by Djuranovic et al might be explained by the structure of the QDE-2 MID domain, with sulphate I representing the 5′-binding site of a miRNA and sulphate II representing the allosteric site. This argument is strengthened by the fact that the two binding sites are partly shared, namely by interactions with the side chains of Lys 599 and Lys 638, so it would not be surprising that binding of a ligand to one site would have a positive effect on ligand binding at the other site. To identify the location of the potential allosteric site, Djuranovic et al mutated Asp 627—a conserved residue located in a loop 15 Å away from the 5′-nucleotide binding site—to a lysine in D. melanogaster AGO1. The D627K mutant failed to bind cap analogues, indicating the importance of Asp 627 for binding ligands in the allosteric site. When mapped onto the new structures of the QDE-2 and human AGO2 MID domains, this loop and Asp 627 (Asp 603 in QDE-2 and Asp 537 in human AGO2) are in the vicinity of sulphate II, thus Asp 627 is probably a part of the allosteric binding site (Fig 1A,B). The most significant finding in the Djuranovic et al study is that the D627K mutant located in the allosteric site fails to bind to miRNA in the 5′-nucleotide-binding site and no longer associates with GW182, an essential factor in miRNA-induced gene silencing.Almost all miRNA sequences and RNA sequencing data obtained from immunopurified AGO proteins show a marked bias for uridine and adenosine nucleotides at the 5′-end of miRNA guide strands. The structure of human AGO2 MID domain alone and in a complex with UMP, AMP, GMP and CMP provides the first explanation for the observed 5′-nucleotide bias in eukaryotic AGO proteins (Frank et al, 2010). There is little movement induced on nucleotide binding in the overall fold of the MID domain. Electron density is observed for the entire nucleotide in the case of UMP and AMP. The 5′-phosphate in the UMP and AMP complexes is hydrogen bonded to the highly conserved side chains of Tyr 529, Lys 533, Gln 545 and Lys 570. The base of each nucleotide stacks with Tyr 529, completing a nonspecific recognition pocket for the 5′-nucleotide (Fig 1B). A similar pocket is formed in the N. crassa MID domain structure to recognize the 5′-nucleotide. Interestingly, clear electron density for only the phosphate and ribose is observed for GMP and CMP, with density for the GMP and CMP bases missing. These results are consistent with the preference for UMP and AMP binding to human AGO2, but where does this specificity originate?A closer look at the UMP and AMP complex structures show that base-specific contacts are formed with backbone atoms of a loop spanning residues Pro 523 through Pro 527, appropriately termed the nucleotide specificity loop. In the case of GMP and CMP, the hydrogen-bonding partners are in the opposite orientation, resulting in charge repulsion from backbone atoms in the nucleotide specificity loop, thus explaining the observed bias in the 5′-position of the guide strand. In the nucleotide-free structure, the conformation of the nucleotide specificity loop is merely unchanged, suggesting that the loop is rather rigid. When the length of the nucleotide specificity loop is increased by the insertion of a single glycine residue, the specificity for uridine and adenosine nucleotides is lost, further endorsing the idea that the particular conformation and rigid nature of the loop is essential for specific base recognition. Interestingly, the QDE-2 MID domain deviates from the human AGO2 MID domain in the nucleotide specificity loop. An insertion of an aspartate residue in QDE-2 makes the nucleotide specificity loop one amino acid longer, suggesting that QDE-2 might have lost its specificity for nucleotides at the 5′-end of miRNAs, although this is yet to be tested.A complete understanding of miRNA loading and the allosteric mechanism will have to await structures of full-length eukaryotic AGO proteins, as the PIWI domain contributes numerous contacts with the MID domain, encompassing both the 5′-nucleotide-binding site and the putative allosteric site. However, the structures of N. crassa QDE-2 MID and human AGO2 MID domains together are important pieces of the puzzle in our understanding of the mechanism of RNA interference. Specific recognition of the 5′-nucleotide of the guide strand might be a quality control mechanism for some eukaryotic AGOs, ensuring that after primary processing the correct miRNA guide sequence is loaded. Once loaded with a proper guide strand, AGO might trigger the adjacent allosteric site to bind to m⁷GpppG-capped mRNA, GW182 or other unknown ligands. Together, these events ultimately lead to effective gene silencing.