18F-2-deoxy-2-fluoro-D-glucose positron emission tomography, computed tomography, and magnetic resonance imaging for the detection of experimental colorectal liver metastases

During the treatment of colorectal liver metastases, evaluation of treatment efficacy is of the utmost importance for decision making. The aim of the present study was to explore the ability of preclinical imaging modalities to detect experimental liver metastases. Nine male Wag/Rij rats underwent a laparotomy with intraportal injection of CC531 tumor cells. On days 7, 10, and 14 after tumor induction, sequential positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI) scans were acquired of each rat. At each time point, three rats were euthanized and the metastases in the liver were documented histologically. Topographically, the liver was divided into eight segments and the image findings were compared on a segment-by-segment basis with the histopathologic findings. Sixty-four liver segments were analyzed, 20 of which contained tumor deposits. The overall sensitivity of PET, CT, and MRI was 30%, 25%, and 20%, respectively. For the detection of tumors with a histologic diameter exceeding 1 mm (n = 8), the sensitivity of PET, CT, and MRI was 63%, 38%, and 38%, respectively. The overall specificity of PET, CT, and MRI was 98%, 100%, and 93%, respectively. This study showed encouraging detectability and sensitivity for preclinical imaging of small liver tumors and provides valuable information on the imaging techniques for designing future protocols.     

Human transformations of the Wadden Sea ecosystem through time: a synthesis

Todays Wadden Sea is a heavily human-altered ecosystem. Shaped by natural forces since its origin 7,500 years ago, humans gradually gained dominance in influencing ecosystem structure and functioning. Here, we reconstruct the timeline of human impacts and the history of ecological changes in the Wadden Sea. We then discuss the ecosystem and societal consequences of observed changes, and conclude with management implications. Human influences have intensified and multiplied over time. Large-scale habitat transformation over the last 1,000 years has eliminated diverse terrestrial, freshwater, brackish and marine habitats. Intensive exploitation of everything from oysters to whales has depleted most large predators and habitat-building species since medieval times. In the twentieth century, pollution, eutrophication, species invasions and, presumably, climate change have had marked impacts on the Wadden Sea flora and fauna. Yet habitat loss and overexploitation were the two main causes for the extinction or severe depletion of 144 species (~20% of total macrobiota). The loss of biodiversity, large predators, special habitats, filter and storage capacity, and degradation in water quality have led to a simplification and homogenisation of the food web structure and ecosystem functioning that has affected the Wadden Sea ecosystem and coastal societies alike. Recent conservation efforts have reversed some negative trends by enabling some birds and mammals to recover and by creating new economic options for society. The Wadden Sea history provides a unique long-term perspective on ecological change, new objectives for conservation, restoration and management, and an ecological baseline that allows us to envision a rich, productive and diverse Wadden Sea ecosystem and coastal society.     

Sequence Capture and Next Generation Resequencing of the MHC Region Highlights Potential Transplantation Determinants in HLA Identical Haematopoietic Stem Cell Transplantation

How cells coordinate the immune system activities is important for potentially life-saving organ or stem cell transplantations. Polymorphic immunoregulatory genes, many of them located in the human major histocompatibility complex, impact the process and assure the proper execution of tolerance-versus-activity mechanisms. In haematopoietic stem cell transplantation, on the basis of fully human leukocyte antigen (HLA)-matched donor–recipient pairs, adverse effects like graft versus leukaemia and graft versus host are observed and difficult to handle. So far, high-resolution HLA typing was performed with Sanger sequencing, but for methodological reasons information on additional immunocompetent major histocompatibility complex loci has not been revealed. Now, we have used microarray sequence capture and targeted enrichment combined with next generation pyrosequencing for 3.5 million base pair human major histocompatibility complex resequencing in a clinical transplant setting and describe 3025 variant single nucleotide polymorphisms, insertions and deletions among recipient and donor in a single sequencing experiment. Taken together, the presented data show that sequence capture and massively parallel pyrosequencing can be used as a new tool for risk assessment in the setting of allogeneic stem cell transplantation.     

Further Studies on a Virus Causing a Green Mosaic Disease of Eggplant in Nigeria

A virus reported earlier to cause a green mosaic disease of eggplant in Nigeria was studied in more detail. Its filamentous particles with a normal length of 820 nm reacted in immunoelectron microscopical tests strongly with the homologous antiserum and less strongly with antisera to dioscorea green banding mosaic, groundnut eyespot, zucchini yellow mosaic viruses and to a tomato potyvirus isolate from Taiwan. No reactions were seen with antisera to 25 other potyviruses. Several new host plants were identified. Infected cells contained cylindrical inclusions with scrolls and short curved laminated aggregates and clusters of small vesicles with electron-dense content. Host range and serological reactivities differentiate the virus for which the name eggplant green mosaic virus is suggested from all potyviruses so far known.     

Replication of simian virus 40 chromatin in vitro depends on the amount of DNA polymerase alpha associated with replicating chromatin

Replication of simian virus 40 (SV40) chromatin in vitro is inhibited by chloride but stimulated by acetate anions even at physiological concentrations of 100-200 mM. In a similar fashion DNA polymerase alpha is affected with respect to the activity with activated DNA as primer template. Furthermore, at concentrations of 100-200 mM acetate DNA polymerase alpha remains associated with replicating chromatin, whereas association is strongly reduced when chloride anions are used at the corresponding concentrations. Thus the salt behaviour of DNA polymerase alpha explains the salt sensitivity of the replication system. Our results confirm the importance of this enzyme for DNA replication.     

Preparation and properties of metal ion derivatives of the lentil and pea lectins

Lentil lectin (LcH) and pea lectin (PSA) belong to the class of D-glucose/D-mannose binding lectins and resemble concanavalin A (Con A) closely in physicochemical, structural, and biological properties. LcH and PSA, like Con A, are Ca2+-Mn2+ metalloproteins that require the metal ions for their saccharide binding and biological activities. Studies of the relationship between the metal ions binding and saccharide binding activity in LcH and PSA have been difficult due to the problem of metal ion replacement in these proteins. We now report a method of metal ion replacement in both lectins that allows substitution of the Mn2+ in the native proteins with a variety of transition metal ions, as well as substitution of the Ca2+ with Cd2+ in a particular complex. The following metal ion derivatives of both LcH and PSA have been prepared: Ca2+-Zn2+, Ca2+-Co2+, Ca2+-Ni2+, and Cd2+-Cd2+. All of these derivatives are as active as the native lectins, as demonstrated by precipitation with specific polysaccharides, saccharide inhibition of precipitation, and hemagglutination assays. The yields of these derivatives are good (generally greater than 70%), and the degree of metal ion incorporation is high (generally greater than 90%). The method of preparation is quite different from that for metal ion substitution in Con A, which proceeds via the apoprotein. In contrast, the apoproteins of LcH and PSA are unstable, aggregate above pH 4.0, and cannot be remetallized once formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation.

To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.     

Heterogeneity of autoantibodies in 100 patients with autoimmune myositis: insights into clinical features and outcomes

The objective of this study was to determine the prevalence, mutual associations, clinical manifestations, and diagnoses associated with serum autoantibodies, as detected using recently available immunoassays, in patients with autoimmune myositis (AIM). Sera and clinical data were collected from 100 patients with AIM followed longitudinally. Sera were screened cross-sectionally for 21 autoantibodies by multiplex addressable laser bead immunoassay, line blot immunoassay, immunoprecipitation of in vitro translated recombinant protein, protein A assisted immunoprecipitation, and enzyme-linked immunosorbent assay. Diagnoses were determined using the Bohan and Peter classification as well as recently proposed classifications. Relationships between autoantibodies and clinical manifestations were analyzed by multiple logistic regression. One or more autoantibodies encompassing 19 specificities were present in 80% of the patients. The most common autoantibodies were anti-Ro52 (30% of patients), anti-Ku (23%), anti-synthetases (22%), anti-U1RNP (15%), and anti-fibrillarin (14%). In the presence of autoantibodies to Ku, synthetases, U1RNP, fibrillarin, PM-Scl, or scleroderma autoantigens, at least one more autoantibody was detected in the majority of sera and at least two more autoantibodies in over one-third of sera. The largest number of concurrent autoantibodies was six autoantibodies. Overall, 44 distinct combinations of autoantibodies were counted. Most autoantibodies were unrestricted to any AIM diagnostic category. Distinct clinical syndromes and therapeutic responses were associated with anti-Jo-1, anti-fibrillarin, anti-U1RNP, anti-Ro, anti-Ro52, and autoantibodies to scleroderma autoantigens. We conclude that a significant proportion of AIM patients are characterized by complex associations of autoantibodies. Certain myositis autoantibodies are markers for distinct overlap syndromes and predict therapeutic outcomes. The ultimate clinical features, disease course, and response to therapy in a given AIM patient may be linked to the particular set of associated autoantibodies. These results provide a rationale for patient profiling and its application to therapeutics, because it cannot be assumed that the B-cell response is the same even in the majority of patients in a given diagnostic category.     

Identification of blottin: a novel gastric trefoil factor family-2 binding protein

The trefoil factor family (TFF) peptides are important in gastro-intestinal mucosal protection and repair. Their mechanism of action remains unclear and receptors are sought. We aimed to identify and characterise proteins binding to TFF2. A fusion protein of mouse TFF2 with alkaline phosphatase was generated and used to probe 2-D protein blots of mouse stomach. The resulting spots were analysed by MS. The protein identified was characterised by bioinformatics, rapid amplification of cDNA ends, in situ hybridisation (ISH) and immunohistochemistry (IHC). Functional assays were performed in gastrointestinal cell lines. A single major murine protein was identified and named blottin. It was previously unknown as a translated product. Blottin is also present in rat and human; the latter gene is also known as GDDR. The predicted full-length proteins are 184 amino acids long (20 kDa), reducing to 164 amino acids (18 kDa) after signal peptide cleavage. ISH of gastrointestinal tissues shows abundant blottin mRNA in gastric surface and foveolar epithelium. IHC shows cytoplasmic staining for blottin protein, and by immunoelectron microscopy in mucus granules and Golgi stacks. Previous work showed that blottin is down-regulated in gastric cancers. Blottin contains a BRICHOS domain, and has 56% similarity with gastrokine-1. Cultured HT-29 cells express blottin and show increased DNA synthesis with antiblottin antibody; however, this effect is reversed by the immunising peptide. We have identified and characterised a TFF2-binding protein produced by gastric epithelium. Blottin may play a role in gastrointestinal mucosal protection and modulate gut epithelial cell proliferation.