Purification and Some Properties of Lignostilbene-a,/i-dioxygenase Responsible for the Ca—Cp Cleavage of a Diarylpropane Type Lignin Model Compound from Pseudomonas sp. TMY1009

A novel dioxygenase, lignostilbene-a,β-dioxygenase (LSD), which catalyzes cleavage of the interphenyl double bond of lignin-derived stilbenes, was isolated. Four isozymes of LSD were separated from cell-free extracts of Pseudomonas sp. TMY1009 by ion-exchange chromatography on a DEAE- Toyopearl column. The major isozyme, LSD-I, was purified to electrophoretic homogeneity and characterized.

LSD-I cleaved the interphenyl double bond of l,2-bis(4′-hydroxy-3′-methoxyphenyl)ethylene with the optimum pH at 8.5. The Km of LSD-I was 11 μm for the stilbene and 110/iM for oxygen. The molecular weight of LSD-I, which is composed of two identical subunits, was estimated to be 94,000. LSD-I contained 1 g atom of iron per 1 mol of enzyme protein.     

The Open Microscopy Environment (OME) Data Model and XML file: open tools for informatics and quantitative analysis in biological imaging

The Open Microscopy Environment (OME) defines a data model and a software implementation to serve as an informatics framework for imaging in biological microscopy experiments, including representation of acquisition parameters, annotations and image analysis results. OME is designed to support high-content cell-based screening as well as traditional image analysis applications. The OME Data Model, expressed in Extensible Markup Language (XML) and realized in a traditional database, is both extensible and self-describing, allowing it to meet emerging imaging and analysis needs.     

Design of Deinococcus radiodurans thioredoxin reductase with altered thioredoxin specificity using computational alanine mutagenesis

In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs have higher affinity for cognate Trxs (same species) than that for Trxs from different species. Computational alanine scanning mutagenesis and visual inspection of the EC TrxR-Trx interface suggested that only four residues (F81, R130, F141, and F142) account for the majority of the EC TrxR-Trx interface stability. Individual replacement of equivalent residues in DR TrxR (M84, K137, F148, and F149) with alanine resulted in drastic changes in binding affinity, confirming that the four residues account for most of TrxR-Trx interface stability. When M84 and K137 were changed to match equivalent EC TrxR residues (K137R and M84F), the DR TrxR substrate specificity was altered from its own Trx to that of EC Trx. The results suggest that a small subset of the TrxR-Trx interface residues is responsible for the majority of Trx binding affinity and species-specific recognition.     

Studies on the generation of B lymphocytes in fetal liver and bone marrow.

With the use of immunofluorescence techniques, cells containing cytoplasmic IgM (cIgM+), but lacking detectable surface IgM (sIgM+), have been identified in mouse fetal liver and adult bone marrow as a distinct cell population to sIgM+ B lymphocytes. We have shown that there is a considerable difference in the rate of entry of cIgM+ and sIgM+ cells into DNA synthesis in these locations. Moreover, within the cIgM+ population, the largest cells are the main group entering DNA synthesis. Our results are compatible with the notion that a pool of rapidly proliferating, large cIgM+ cells is present in fetal liver and adult bone marrow and that these cells give rise to populations of smaller cIgM+ cells, which move out of cell cycle, and convert to sIgM+ B lymphocytes. However, we recognize that this interpretation is speculative. Finally, we have shown that fetal bone marrow is a site of generation of sIgM+ B lymphocytes, but the question as to whether these cells are derived from Ig- precursors within marrow itself remains open.     

Identification of a MAD2-binding protein,CMT2, and its role in mitosis

MAD2 is a key component of the spindle checkpoint that delays the onset of anaphase until all the kinetochores are attached to the spindle. It binds to human p55CDC and prevents it from promoting destruction of an anaphase inhibitor, securin. Here we report the characterization of a novel MAD2-binding protein, CMT2. Upon the completion of spindle attachment, formation of the CMT2-MAD2 complex coincides with dissociation of the p55CDC-MAD2 complex. Overexpression of CMT2 in cells arrested by the spindle checkpoint causes premature destruction of securin and allows exit from mitosis without chromosome segregation. Depletion of CMT2 induces cell death following a transient delay in the onset of anaphase. These results indicate that CMT2 interacts with the spindle checkpoint and coordinates cell cycle events in late mitosis.     

Interaction with heparin and matrix metalloproteinase 2 cleavage expose a cryptic anti-adhesive site of fibronectin

We recently found that fibronectin (FN) had a functional site [YTIYVIAL sequence in the heparin-binding domain 2 (Hep 2)] that was capable of suppressing the integrin-mediated cell adhesion to extracellular matrix. However, our results also indicated that this anti-adhesive site seemed to be usually buried within the Hep 2 domain structure because of its hydrophobic nature, raising a question as to the physiological significance of the cryptic anti-adhesive activity of FN. The present study demonstrates that the cryptic anti-adhesive activity can be exposed through the physiological processes. A 30-kDa chymotryptic FN fragment derived from Hep 2 domain (Hep 2 fragment), which had no effect on adhesion of MSV-transformed nonproducer 3T3 cell line (KN(7)8) to FN, expressed the anti-adhesive activity after treatment with 6 M urea. Light scattering and circular dichroism measurements showed that the urea treatment induced the conformational change of the Hep 2 fragment from a more compact form to an unfolded one. Incubation of the Hep 2 fragment with heparin also induced similar conformational changes and expression of anti-adhesive activity. Additionally, both the urea and heparin treatments made the Hep 2 fragment and intact FN much more accessible to the polyclonal antibody (alphaIII14A), with a recognition site near the anti-adhesive site of FN. Specific cleavage of either the Hep 2 fragment or intact FN by matrix metalloproteinase 2 (MMP-2) released a 10-kDa fragment with the anti-adhesive activity, which was shown to have the exposed anti-adhesive site on the amino-terminal region. Thus, the cryptic anti-adhesive activity of FN can be expressed upon conformational change and proteolytic cleavage of Hep 2 domain.     

Mouse CD8+ CD122+ T cells with intermediate TCR increasing with age provide a source of early IFN-gamma production

Although CD8+ IL-2Rbeta (CD122)+ T cells with intermediate TCR reportedly develop extrathymically, their functions still remain largely unknown. In the present study, we characterized the function of CD8+ CD122+ T cells with intermediate TCR of C57BL/6 mice. The proportion of CD8+ CD122+ T cells in splenocytes gradually increased with age, whereas CD8+ IL-2Rbeta-negative or -low (CD122-) T cells conversely decreased. The IFN-gamma production from splenocytes stimulated with immobilized anti-CD3 Ab in vitro increased with age, whereas the IL-4 production decreased. When sorted CD8+ CD122+ T cells were stimulated in vitro by the anti-CD3 Ab, they promptly produced a much larger amount of IFN-gamma than did CD8+ CD122- T cells or CD4+ T cells, whereas only CD4+ T cells produced IL-4. The depletion of CD8+ CD122+ T cells from whole splenocytes greatly decreased the CD3-stimulated IFN-gamma production and increased the IL-4 production, whereas the addition of sorted CD8+ CD122+ T cells to CD8+ CD122+ T cell-depleted splenocytes restored the IFN-gamma production and partially decreased IL-4 production. It is of interest that CD8+ CD122+ T cells stimulated CD4+ T cells to produce IFN-gamma. The CD3-stimulated IFN-gamma production from each T cell subset was augmented by macrophages. Furthermore, CD3-stimulated CD8+ CD122+ T cells produced an even greater amount of IFN-gamma than did liver NK1.1+ T cells and also showed antitumor cytotoxicity. These results show that CD8+ CD122+ T cells may thus be an important source of early IFN-gamma production and are suggested to be involved in the immunological changes with aging.     

FAT AND CARBOHYDRATE METABOLISM IN HUMANS—A Study of Nutritional and Hormonal Effects

As an index to the rate of fat utilization in human subjects, the recovery of all radioactive carbon dioxide in the expired air was measured for one hour following intravenous injection of palmitate-1-C¹⁴. In the normal fasted subject, about 10 per cent of the injected dose was recovered, and the proportion was lowered to about 5 per cent by administration of glucose. With prolonged fasting, the recovery of radioactive carbon dioxide did not change, despite a rising concentration of fatty acids in the serum. This was interpreted as due to the development of a balance between increasing mobilization and oxidation and was thought to indicate increasing fatty acid oxidation.In chronic undernutrition and diabetes mellitus there was increased fatty acid oxidation due presumably to adaptation to a chronic increase in fat utilization for energy.Administration of human growth hormone did not increase fat oxidation but prevented the usual inhibition produced by glucose. This was interpreted to mean that growth hormone increases fat utilization only indirectly by inhibiting the usual preferential utilization of glucose over fat.     

Deaths of detainees in the custody of US forces in Iraq and Afghanistan from 2002 to 2005

In light of the large number of detainees who continue to be taken and held in US custody in settings with limited judicial or public oversight, deaths of detainees warrant scrutiny. We have undertaken the task of reviewing all known detainee deaths between 2002 and early 2005 based on reports available in the public domain. Using documents obtained from the Department of Defense through a Freedom of Information Act request, combined with a review of anecdotal published press accounts, 112 cases of death of detainees in United States custody (105 in Iraq, 7 in Afghanistan) during the period from 2002 to early 2005 were identified. Homicide accounted for the largest number of deaths (43) followed by enemy mortar attacks against the detention facility (36). Deaths attributed to natural causes numbered 20. Nine were listed as unknown cause of death, and 4 were reported as accidental or natural. A clustering of 8 deaths ascribed to natural causes in Iraq in August 2003 raises questions about the adequacy and availability of medical care, as well as other conditions of confinement that may have had an impact on the mortality rate.