Functional domains of Escherichia coli recA protein deduced from the mutational sites in the gene

Summary The sites of recA mutations of Escherichia coli, recA441 (tif-1), recA1, recA430 (lexB30) and recA44, were determined by analyses of the nucleotide sequences. All mutations are single point missense mutations within the coding region of the recA gene. In the recA441, recA1, recA430 and recA44 proteins, the 38th, 160th, 204th, and 246th amino acids, respectively, from the amino terminal ends are altered. Based on the properties of these mutant proteins and modified forms of recA protein, the locations of various regions of the recA protein that are involved in binding with ATP, binding with single-stranded DNA, hydrolysis of ATP, interaction between the recA protein molecules and interaction with the cI or lexA repressors are mapped on the primary structure of the protein.     

Morphological variety of intracellular microcolonies of Legionella species in Vero cells

Intracellular microcolonies of six Legionella species growing in Vero cells showed distinctly varied morphologies. The varieties were observed by light microscopy of Gimenez-stained, Legionella-infected Vero cells and by electron microscopy (EM). Legionella pneumophila Philadelphia-1 formed needle-shaped crystal-like microcolonies. Legionella bozemanii WIGA formed microcolonies like wool balls containing filamentous cells. In EM, these organisms proliferated in endosomes, which were adjacent to swollen rough endoplasmic reticula. Legionella oakridgensis OR-10 showed serpentine chains. Many mitochondria were observed around the microcolonies. Legionella jordanis BL-540 formed spherical moss-like microcolonies which were or were not surrounded by endoplasmic membranes. Legionella feeleii WO-44C spread throughout the cytoplasm without making clusters. Legionella dumoffii Tex-KL made big clusters that spread in the cytoplasm, a portion of which was outside the endosome membranes. These different morphologies imply diversity in modes of intracellular multiplication of Legionella spp.     

Critical requirement for the membrane-proximal cytosolic tyrosine residue for CD28-mediated costimulation in vivo

The YMNM motif that exists in the CD28 cytoplasmic domain is known as a binding site for phosphatidylinositol 3-kinase and Grb-2 and is considered to be important for CD28-mediated costimulation. To address the role of the YMNM motif in CD28 cosignaling in primary T cells, we generated transgenic mice on a CD28 null background that express a CD28 mutant lacking binding ability to phosphatidylinositol 3-kinase and Grb-2. After anti-CD3 and anti-CD28 Ab stimulation in vitro, the initial proliferative response and IL-2 secretion in CD28 Y189F transgenic T cells were severely compromised, while later responses were intact. In contrast to anti-CD3 and anti-CD28 Ab stimulation, PMA and anti-CD28 Ab stimulation failed to induce IL-2 production from CD28 Y189F transgenic T cells at any time point. Using the graft-vs-host reaction system, we assessed the role of the YMNM motif for CD28-mediated costimulation in vivo and found that CD28 Y189F transgenic spleen cells failed to engraft and could not induce acute graft-vs-host reaction. Together, these results suggest that the membrane-proximal tyrosine of CD28 is required for costimulation in vivo. Furthermore, these results indicate that the results from in vitro assays of CD28-mediated costimulation may not always correlate with T cell activation in vivo.     

Conjugating oligosaccharides to proteins by squaric acid diester chemistry: rapid monitoring of the progress of conjugation, and recovery of the unused ligand

Samples that are periodically withdrawn from the mixture of a conjugation reaction can be analyzed on a picomolar scale without any work-up or pre-purification using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) in combination with the ProteinChip^® System. The technique provides rapid information about the increasing molecular mass of the glycoconjugate formed, thereby allowing termination of the process when the desired incorporation of the ligand onto the carrier protein is achieved. The excess oligosaccharide used at the onset of conjugation can be recovered and used in preparation of a similar neoglycoconjugate. The overall economy of conjugations, which often involve labor-intensive linker-equipped oligosaccharides, can be markedly increased in this way.     

'Working' cardiomyocytes exhibiting plateau action potentials from human placenta-derived extraembryonic mesodermal cells

The clinical application of cell transplantation for severe heart failure is a promising strategy to improve impaired cardiac function. Recently, an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and embryonic stem cells, have become important candidates for cell sources for cardiac repair. In the present study, we focused on the placenta as a cell source. Cells from the chorionic plate in the fetal portion of the human placenta were obtained after delivery by the primary culture method, and the cells generated in this study had the Y sex chromosome, indicating that the cells were derived from the fetus. The cells potentially expressed 'working' cardiomyocyte-specific genes such as cardiac myosin heavy chain 7beta, atrial myosin light chain, cardiac alpha-actin by gene chip analysis, and Csx/Nkx2.5, GATA4 by RT-PCR, cardiac troponin-I and connexin 43 by immunohistochemistry. These cells were able to differentiate into cardiomyocytes. Cardiac troponin-I and connexin 43 displayed a discontinuous pattern of localization at intercellular contact sites after cardiomyogenic differentiation, suggesting that the chorionic mesoderm contained a large number of cells with cardiomyogenic potential. The cells began spontaneously beating 3 days after co-cultivation with murine fetal cardiomyocytes and the frequency of beating cells reached a maximum on day 10. The contraction of the cardiomyocytes was rhythmical and synchronous, suggesting the presence of electrical communication between the cells. Placenta-derived human fetal cells may be useful for patients who cannot supply bone marrow cells but want to receive stem cell-based cardiac therapy.     

Expression level of c-FLIP versus Fas determines susceptibility to Fas ligand-induced cell death in murine thymoma EL-4 cells

The caspase-8 inhibitor c-FLIP blocks death receptor-mediated cell death and plays an essential role in the regulation of lymphocyte homeostasis and the immune escape of tumors. The murine thymoma cell line EL-4 was resistant to Fas ligand (FasL)-induced apoptosis by constitutive expression of FLIP (L). Cycloheximide downregulated the expression of FLIP (L) and markedly sensitized EL-4 cells to FasL-induced apoptosis. In contrast, DNA-damaging agents sensitized EL-4 cells to FasL-induced cell death via an increase of cell-surface Fas without any influence on FLIP (L) expression. Enforced expression of transfected Fas rendered EL-4 cells highly susceptible to FasL-induced cell death. These findings demonstrate that susceptibility to FasL-induced cell death mainly depends on the expression level of c-FLIP versus cell-surface Fas.