Molecular basis for pacemaker cells in epithelia

Intercellular signaling is highly coordinated in excitable tissues such as heart, but the organization of intercellular signaling in epithelia is less clear. We examined Ca(2+) signaling in hepatoma cells expressing the hepatocyte gap junction protein connexin32 (cx32) or the cardiac gap junction protein cx43, plus a fluorescently tagged V(1a) vasopressin receptor (V(1a)R). Release of inositol 1,4,5-trisphosphate (InsP(3)) in wild type cells increased Ca(2+) in the injected cell but not in neighboring cells, while the Ca(2+) signal spread to neighbors when gap junctions were expressed. Photorelease of caged Ca(2+) rather than InsP(3) resulted in a small increase in Ca(2+) that did not spread to neighbors with or without gap junctions. However, photorelease of Ca(2+) in cells stimulated with low concentrations of vasopressin resulted in a much larger increase in Ca(2+), which spread to neighbors via gap junctions. Cells expressing tagged V(1a)R similarly had increased sensitivity to vasopressin, and could signal to neighbors via gap junctions. Higher concentrations of vasopressin elicited Ca(2+) signals in all cells. In cx32 or cx43 but not in wild type cells, this signaling was synchronized and began in cells expressing the tagged V(1a)R. Thus, intercellular Ca(2+) signals in epithelia are organized by three factors: 1) InsP(3) must be generated in each cell to support a Ca(2+) signal in that cell; 2) gap junctions are necessary to synchronize Ca(2+) signals among cells; and 3) cells with relatively increased expression of hormone receptor will initiate Ca(2+) signals and thus serve as pacemakers for their neighbors. Together, these factors may allow epithelia to act in an integrated, organ-level fashion rather than as a collection of isolated cells.     

Molecular and immunological characterization of profilin from mugwort pollen

In late summer in Europe, pollen of mugwort is one of the major sources of atopic allergens. No information about the complete molecular structure of any mugwort allergen has been published so far. Here we report the isolation and characterization of mugwort pollen cDNA clones coding for two isoforms of the panallergen profilin. Thirty-six percent of the mugwort-allergic patients tested displayed IgE antibodies against natural and recombinant profilin, and no significant differences were observed in the IgE-binding properties of the isoforms. One profilin isoform was purified to homogeneity and detailed structural analysis indicated that the protein exists in solution as dimers and tetramers stabilized by sulfydryl and/or ionic interactions. Profilin monomers were detectable only after exposure of multimers to harsh denaturing conditions. Dimers and tetramers did not significantly differ in their ability to bind serum IgE from mugwort pollen-allergic patients. However, oligomeric forms might have a higher allergenic potential than monomers because larger molecules would have additional epitopes for IgE-mediated histamine release. Profilin isolated from mugwort pollen also formed multimers. Thus, oligomerization is not an artifact resulting from the recombinant production of the allergen. Inhibition experiments showed extensive IgE cross-reactivity of recombinant mugwort profilin and profilin from various pollen and food extracts.     

The curse of normalization

Despite its enormous promise to further our understanding of cellular processes involved in the regulation of gene expression, microarray technology generates data for which statistical pre-processing has become a necessity before any interpretation of data can begin. The process by which we distinguish (and remove) non-biological variation from biological variation is called normalization. With a multitude of experimental designs, techniques and technologies influencing the acquisition of data, numerous approaches to normalization have been proposed in the literature. The purpose of this short review is not to add to the many suggestions that have been made, but to discuss some of the difficulties we encounter when analysing microarray data.     

Xanosporic acid,an intermediate in bacterial degradation of the fungal phototoxin cercosporin

The red fungal perylenequinone phototoxin cercosporin is oxidized by Xanthomonas campestris pv zinniae to a non-toxic, unstable green metabolite xanosporic acid, identified via its lactone as 1,12-bis(2'R-hydroxypropyl)-4,9-dihydroxy-6,7-methylenedioxy-11-methoxy-3-oxaperylen-10H-10-one-2-carboxylic acid. Xanosporolactone was isolated in approximately 2:1 ratio of M:P atropisomers.     

Distribution of SERCA isoforms in human intrafusal fibers

The sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) is a membrane protein that plays a crucial role in muscle relaxation by transporting cytosolic Ca²⁺ into the lumen of the sarco/endoplasmic reticulum. In this study, the presence of SERCA1 and SERCA2 was investigated in human intrafusal fibers by immunocytochemistry. Nuclear bag₁ fibers contained both SERCA1 and SERCA2 isoforms, with predominant staining seen with SERCA2 in the A and B regions. Most nuclear bag₂ fibers also contained SERCA1 and SERCA2 isoforms and their coexistence frequently occurred in the A region. SERCA1 was present whereas SERCA2 was generally absent in the nuclear chain fibers. The staining intensity seen with the SERCA1 monoclonal antibody varied in the order of chain>bag₁>bag₂. The expression of SERCA1 isoform was found to correlate with the presence of fast myosin heavy chain (MyHC) isoform in nuclear chain fibers, whereas for nuclear bag fibers there was no such apparent correlation between patterns of expression of SERCA and MyHC isoforms. The phenotype revealed for the human bag fibers was very sophisticated and adapted to attain a very wide range of contraction and relaxation velocities.     

Modulation of granulomatous hypersensitivity against Schistosoma mansoni eggs in mice vaccinated with culture-derived macrophages loaded with PIII

Granuloma modulation induced by antigen is an attractive model for vaccination studies of experimental schistosomiasis to test the effect of anti-pathology vaccine. We describe here an immunization procedure with culture derived macrophages-pulsed PIII, a known anionic antigen purified from S. mansoni adult worm, involved in the inhibition of granulomatous response to eggs. For our studies, peritoneal or spleen macrophages cultured over 15 days were loaded with PIII. Both macrophage sub-populations were capable to efficiently take up and subsequently present PIII to lymphocytes as evidenced by immunofluorescence assay. The vaccination of mice with intravenous injection of PIII-loaded macrophages potently induced antigen-specific immune response to S. mansoni antigens as determined by cell proliferation assay. This immunization procedure of mice caused significant decrease in hepatic granuloma formation and in vitro granuloma reaction to S. mansoni antigens coupled to polyacrylamide beads (PB-SEA, PB-SWAP or PB-PIII). Assessment of in vitro granuloma supernatant of spleen cells from PIII-loaded macrophages vaccinated mice revealed significant amounts of Th1-cytokines IFN-gamma and IL-2 compared to control cells. Collectively, our results indicate that culture derived-macrophages provided a valuable research tool to investigate aspects of immune response that promote modulation of granulomatous hypersensitivity to S. mansoni eggs in mice.     

Effect of ethylene on enzymatic activities involved in the browning of Hevea brasiliensis callus

Browning, which is of varying intensity depending on species, develops in Hevea brasiliensis callus in vitro and can affect somatic embryogenesis. Endogenous ethylene appears to be involved since application of aminocyclopropane carboxylic acid (ACC) strongly enhances this browning. At the same time, peroxidases (EC 1.11.1.7), bonded polyphenol oxidase (EC 1.10.3.1) and NADH-quinone reductase (EC 1.6.99.5) are enhanced by ACC whereas superoxide dismutase (SOD: EC 1.15.1.1) and ascorbate peroxidase (EC 1.11.1.11) are decreased. In contrast, application of aminoacetic acid (AOA) causes spectacular decreases in bound and soluble polyphenol oxidase, peroxidase and NADH-quinone reductase activities whereas SOD and ascorbate peroxidase activities increase. Enzymatic activities in the control callus present a medium value, probably due to a considerable ethylene production in the culture medium. After 47 days culture. the initial catalase activity (EC 1.11.1.6) in the explants had disappeared completely. In contrast, after only 22 days, the silver nitrate and especially AOA treatments promoted considerable activities of catalase. The beneficial effect of the inhibitors of ethylene synthesis was reflected in the enhanced formation of embryogenic tissue, which varied from 1% (control or ACC) to 34% (AOA treatment).