A modified nick translation method used with FISH that produces reliable results with archival tissue sections

Nick translation is used to label DNA and RNA to produce probes for in situ hybridization and Northern and Southern blotting. Fluorescence in situ hybridization (FISH) is a widely applied technique used to determine chromosomal and genetic anomalies in many biological samples. Initially the technique was applied to metaphase preparations, but the usefulness of detecting genetic anomalies in solid tumors in situ has resulted in the development of modified protocols. Formalin fixed paraffin processed tissue sections present novel challenges when applying FISH; the probes must be small (between 200 and 600 base pairs) and pretreatment is necessary before the probes can be applied to tissue sections, to promote probe access to target DNA. Here we report on a modification of a nick translation method to produce a probe that can reliably be used with FISH in paraffin processed tissue sections.     

Induction and patterning of the cardiac conduction system

The cardiac conduction system (CCS) is the component of the heart that initiates and maintains a rhythmic heartbeat. As the embryonic heart forms, the CCS must continue to develop and mature in a coordinated manner to ensure that proper pace making potential and distribution of action potential is maintained at all stages. This requires not only the formation of distinct and disparate components of the CCS, but the integration of these components into a functioning whole as the heart matures. Though research in this area of development may have lagged behind other areas of heart development, in recent years there has been much progress in understanding the ontogeny of the CCS and the developmental cues that drive its formation. This is largely due to studies on the avian heart as well as the use of molecular biology approaches. This review gives a perspective on advances in understanding the development of the vertebrate CCS, and reports new data illuminating the mechanism of conduction cell determination and maintenance in the mammalian heart. As much of our knowledge about the development of the CCS has been derived from the chick embryo, one important area facing the field is the relationship and similarities between the structure and development of avian and mammalian conduction systems. Specifically, the morphology of the distal elements of the mammalian CCS and the manner in which its components are recruited from working cardiomyocytes are areas of research that will, hopefully, receive more attention in the near future. A more general and outstanding question is how the disparate components of all vertebrate conduction systems integrate into a functional entity during embryogenesis. There is mounting evidence linking the patterning and formation of the CCS to instructive cues derived from the cardiac vasculature and, more specifically, to hemodynamic-responsive factors produced by cardiac endothelia. This highlights the need for a greater understanding of the biophysical forces acting on, and created by, the cardiovascular system during embryonic development. A better understanding of these processes will be necessary if therapeutics are to be developed that allow the regeneration of damaged cardiac tissues or the construction of biologically engineered heart tissues.     

Influence of temperature on pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae) resistance to natural enemy attack

The ability to resist or avoid natural enemy attack is a critically important insect life history trait, yet little is understood of how these traits may be affected by temperature. This study investigated how different genotypes of the pea aphid Acyrthosiphon pisum Harris, a pest of leguminous crops, varied in resistance to three different natural enemies (a fungal pathogen, two species of parasitoid wasp and a coccinellid beetle), and whether expression of resistance was influenced by temperature. Substantial clonal variation in resistance to the three natural enemies was found. Temperature influenced the number of aphids succumbing to the fungal pathogen Erynia neoaphidis Remaudière & Hennebert, with resistance increasing at higher temperatures (18 vs. 28 degrees C). A temperature difference of 5 degrees C (18 vs. 23 degrees C) did not affect the ability of A. pisum to resist attack by the parasitoids Aphidius ervi Haliday and A. eadyi Stary, González & Hall. Escape behaviour from foraging coccinellid beetles (Hippodamia convergens Guerin-Meneville) was not directly influenced by aphid clone or temperature (16 vs. 21 degrees C). However, there were significant interactions between clone and temperature (while most clones did not respond to temperature, one was less likely to escape at 16 degrees C), and between aphid clone and ladybird presence (some clones showed greater changes in escape behaviour in response to the presence of foraging coccinellids than others). Therefore, while larger temperature differences may alter interactions between Acyrthosiphon pisum and an entomopathogen, there is little evidence to suggest that smaller changes in temperature will alter pea aphid-natural enemy interactions.     

AGAP1, an endosome-associated,phosphoinositide-dependent ADP-ribosylation factor GTPase-activating protein that affects actin cytoskeleton

We have identified three members of the AGAP subfamily of ASAP family ADP-ribosylation factor GTPase-activating proteins (Arf GAPs). In addition to the Arf GAP domain, these proteins contain GTP-binding protein-like, ankyrin repeat and pleckstrin homology domains. Here, we have characterized the ubiquitously expressed AGAP1/KIAA1099. AGAP1 had Arf GAP activity toward Arf1>Arf5>Arf6. Phosphatidylinositol 4,5-bisphosphate and phosphatidic acid synergistically stimulated GAP activity. As found for other ASAP family Arf GAPs, the pleckstrin homology domain was necessary for activity. Deletion of the GTP-binding protein-like domain affected lipid dependence of Arf GAP activity. In vivo effects of AGAP1 were distinct from other ASAP family Arf GAPs. Overexpressed AGAP1 induced the formation of and was associated with punctate structures containing the endocytic markers transferrin and Rab4. AP1 was redistributed from the trans-Golgi to the punctate structures. Like other ASAP family members, AGAP1 overexpression inhibited the formation of PDGF-induced ruffles. However, distinct from other ASAP family members, AGAP1 also induced the loss of actin stress fibers. Thus, AGAP1 is a phosphoinositide-dependent Arf GAP that impacts both the endocytic compartment and actin.     

Dok-related protein negatively regulates T cell development via its RasGTPase-activating protein and Nck docking sites

Downstream of kinase (Dok)-related protein (DokR, also known as p56(dok)/FRIP/Dok-R) is implicated in cytokine and immunoreceptor signaling in myeloid and T cells. Tyrosine phosphorylation induces DokR to bind the signal relay molecules, RasGTPase-activating protein (RasGAP) and Nck. Here, we have examined the function of DokR during hematopoietic development and the requirement for RasGAP and Nck binding sites in its biological function. Retroviral-mediated expression of DokR in bone marrow cells dramatically inhibited their capacity to form colonies in vitro in response to the cytokines macrophage colony-stimulating factor and stem cell factor, whereas responses to interleukin-3 and granulocyte macrophage colony-stimulating factor were only weakly affected. When introduced into lethally irradiated mice, hematopoietic cells expressing DokR showed a drastically reduced capacity to repopulate lymphoid tissues. Most notably, DokR dramatically reduced repopulation of the thymus, in part by reducing the number of T cell precursors seeding in the thymus, but equally, through inhibiting the transition of CD4(-)CD8(-) to CD4(+)CD8(+) T cells. Consequently, the number of mature peripheral T cells was markedly reduced. In contrast, a minimal effect on B cell and myeloid lineage development was observed. Importantly, functional RasGAP and Nck binding sites were found to be essential for the biological effects of DokR in vitro and in vivo.     

Genome-wide detection of LOH in prostate cancer using human SNP microarray technology

Loss of heterozygosity (LOH) of chromosomal regions is crucial in tumor progression. In this study we assessed the potential of the Affymetrix GeneChip HuSNP mapping assay for detecting genome-wide LOH in prostate tumors. We analyzed two human prostate cell lines, P69SV40Tag (P69) and its tumorigenic subline, M12, and 11 prostate cancer cases. The M12 cells showed LOH in chromosomes 3p12.1-p22.1, 11q22.1-q24.2, 19p13.12, and 19q13.42. All of the prostate cases with informative single-nucleotide polymorphism (SNP) markers showed LOH in 1p31.2, 10q11.21, 12p13.1, 16q23.1-q23.2, 17p13.3, 17q21.31, and 21q21.2. Additionally, a high percentage of cases showed LOH at 6p25.1-p25.3 (75%), 8p22-p23.2, and 10q22.1 (70%). Several tumor suppressor genes (TSGs) have been mapped in these loci. These results demonstrate that the HuSNP mapping assay can serve as an alternative to comparative genomic hybridization for assessing genome-wide LOH and can identify chromosomal regions harboring candidate TSGs implicated in prostate cancer.     

A "dock, lock, and latch" structural model for a staphylococcal adhesin binding to fibrinogen

Gram-positive pathogens such as staphylococci contain multiple cell wall-anchored proteins that serve as an interface between the microbe and its environment. Some of these proteins act as adhesins and mediate bacterial attachment to host tissues. SdrG is a cell wall-anchored adhesin from Staphylococcus epidermidis that binds to the Bbeta chain of human fibrinogen (Fg) and is necessary and sufficient for bacterial attachment to Fg-coated biomaterials. Here, we present the crystal structures of the ligand binding region of SdrG as an apoprotein and in complex with a synthetic peptide analogous to its binding site in Fg. Analysis of the crystal structures, along with mutational studies of both the protein and of the peptide, reveals that SdrG binds to its ligand with a dynamic "dock, lock, and latch" mechanism. We propose that this mechanism represents a general mode of ligand binding for structurally related cell wall-anchored proteins of gram-positive bacteria.     

Postactivation potentiation response in athletic and recreationally trained individuals

To determine if training status directly impacted the response to postactivation potentiation, athletes in sports requiring explosive strength (ATH; n = 7) were compared to recreationally trained (RT; n = 17) individuals. Over the course of 4 sessions, subjects performed rebound and concentric-only jump squats with 30%, 50%, and 70% 1 RM loads. Jump squats were performed 5 minutes and 18.5 minutes following control or heavy load warm-ups. Heavy load warm-up consisted of 5 sets of 1 repetition at 90% 1 RM back squat. Jump squat performance was assessed with a force platform and position transducer. Heavy load warm-up did not have an effect on the subjects as a single sample. However, when percent potentiation was compared between ATH and RT groups, force and power parameters were significantly greater for ATH (p < 0.05). Postactivation potentiation may be a viable method of acutely enhancing explosive strength performance in athletic but not recreationally trained individuals. Reference Data: Chiu, L.Z.F., A.C. Fry, L.W. Weiss, B.K. Schilling, L.E. Brown, and S.L. Smith. Postactivation potentiation response in athletic and recreationally trained individuals.     

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector. Received: 27 December 1999 / Accepted: 27 January 2000     

Molecular characterization of a gene region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum: cloning, sequencing and expression of rfaf gene

A 3.0-kb region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum was sequenced. One complete open reading frame was identified which encodes a polypeptide of 354 amino acid residues with a predicted molecular mass of 38 209 Da. Expression of the protein using a T7 gene expression system revealed a band of similar molecular mass after sodium dodecyl sulfate polyacrylamide gel electrophoresis. A database search against known gene sequences revealed a significant sequence similarity to the rfaF gene cloned from several Gram-negative bacteria. The rfaF gene is known to encode heptosyltransferase II that transfers a second heptose to the inner core of lipopolysaccharide. The cloned B. japonicum open reading frame was able to functionally complement a rfaF mutant of Salmonella typhimurium SL3789. Transformation of this mutant with the B. japonicum gene restored production of an intact lipopolysaccharide and resistance to the hydrophobic antibiotic, novobiocin. An additional open reading frame having a significant sequence similarity to the rfaD gene was found to be divergently oriented to the rfaF gene.