Co-suppression of AtMIPS demonstrates cooperation of MIPS1, MIPS2 and MIPS3 in maintaining myo-inositol synthesis.

KEY MESSAGE: Co-suppressed MIPS2 transgenic lines allow bypass of the embryo lethal phenotype of the previously published triple knock-out and demonstrate the effects of MIPS on later stages of development. Regulation of inositol production is of interest broadly for its effects on plant growth and development. The enzyme L-myo-inositol 1-phosphate synthase (MIPS, also known as IPS) isomerizes D-glucose-6-P to D-inositol 3-P, and this is the rate-limiting step in inositol production. In Arabidopsis thaliana, the MIPS enzyme is encoded by three different genes, (AtMIPS1, AtMIPS2 and AtMIPS3), each of which has been shown to produce proteins with biochemically similar properties but differential expression patterns. Here, we report phenotypic and biochemical effects of MIPS co-suppression. We show that some plants engineered to overexpress MIPS2 in fact show reduced expression of AtMIPS1, AtMIPS2 and AtMIPS3, and show altered vegetative phenotype, reduced size and root length, and delayed flowering. Additionally, these plants show reduced inositol, increased glucose levels, and alteration of other metabolites. Our results suggest that the three AtMIPS genes work together to impact the overall synthesis of myo-inositol and overall inositol homeostasis.

Fluorescent acid-fast microscopy for measuring phagocytosis of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and their intracellular growth.

Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in the ciliated phagocytic protozoan Tetrahymena pyriformis. There was a linear relationship between FAM and colony counts of M. avium cells both from cultures and within protozoa. The Ziehl-Neelsen acid-fast stain could not be used to enumerate intracellular mycobacteria because uninfected protozoa contained acid-fast, bacterium-like particles. Starved, 7-day-old cultures of T. pyriformis transferred into fresh medium readily phagocytized M. avium, M. intracellulare, and M. scrofulaceum. Phagocytosis was rapid and reached a maximum in 30 min. M. avium, M. intracellulare, and M. scrofulaceum grew within T. pyriformis, increasing by factors of 4- to 40-fold after 5 days at 30 degrees C. Intracellular M. avium numbers remained constant over a 25-day period of growth (by transfer) of T. pyriformis. Intracellular M. avium cells also survived protozoan encystment and germination. The growth and viability of T. pyriformis were not affected by mycobacterial infection. The results suggest that free-living phagocytic protozoa may be natural hosts and reservoirs for M. avium, M. intracellulare, and M. scrofulaceum.

Molecular characterization of At5PTase1, an inositol phosphatase capable of terminating inositol trisphosphate signaling.

The inositol triphosphate (IP(3))-signaling pathway has been associated with several developmental and physiological processes in plants, but we currently know little about the regulation of this pathway. Inositol 5' phosphatases (5PTases) are enzymes that remove a 5' phosphate from several potential second messengers, including IP(3). In catalyzing the removal of a 5' phosphate from second messenger substrates, 5PTases can act to terminate signal transduction events. We describe the molecular analysis of At5PTase1, a 5PTase gene from Arabidopsis. When expressed transiently in Arabidopsis leaf tissue or ectopically in transgenic plants, At5PTase1 allowed for the increased hydrolysis of I(1,4,5)P(3) and I(1,3,4,5)P(4) substrates. At5PTase1 did not hydrolyze I(1)P, I(1,4)P(2), or PI(4,5)P(2) substrates. This substrate specificity was similar to that of the human Type I 5PTase. We identified 14 other potential At5PTase genes and constructed an unrooted phylogenetic tree containing putative Arabidopsis, human, and yeast 5PTase proteins. This analysis indicated that the Arabidopsis 5PTases were grouped in two separate branches of the tree. The multiplicity of At5PTases indicates that these enzymes may have different substrate specificities and play different roles in signal termination in Arabidopsis.

Plant inositol monophosphatase is a lithium-sensitive enzyme encoded by a multigene family.

myo-Inositol monophosphatase (IMP) is a soluble, Li(+)-sensitive protein that catalyzes the removal of a phosphate from myo-inositol phosphate substrates. IMP is required for de novo inositol synthesis from glucose 6-phosphate and for breakdown of inositol trisphosphate, a second messenger generated by the phosphatidylinositol signaling pathway. We cloned the IMP gene from tomato (LeIMP) and show that the plant enzyme is encoded by a small gene family. Three different LeIMP cDNAs encode distinct but highly conserved IMP enzymes that are catalytically active in vitro. Similar to the single IMP from animals, the activities of all three LeIMPs are inhibited by low concentrations of LiCl. LeIMP mRNA levels are developmentally regulated in seedlings and fruit and in response to light. Immunoblot analysis detected three proteins of distinct molecular masses (30, 29, and 28 kD) in tomato; these correspond to the predicted molecular masses of the LeIMPs encoded by the genes. Immunoreactive proteins in the same size range are also present in several other plants. Immunolocalization studies indicated that many cell types within seedlings accumulate LeIMP proteins. In particular, cells associated with the vasculature express high levels of LeIMP protein; this may indicate a coordinate regulation between phloem transport and synthesis of inositol. The presence of three distinct enzymes in tomato most likely reflects the complexity of inositol utilization in higher plants.

Antigenic and differentiative heterogeneity among human glioblastomas.

Increasing evidence suggests that in mammals, astrocytes are a heterogenous family of cells all of which share certain properties, but differ in lineage, biochemical and functional aspects. It seems likely that glioblastomas, arising from glial precursors, may also represent a family of related but distinct cell types. We have examined the antigenic characteristics and differentiative potential of 7 different human glioblastoma cell lines in vitro. All the cell lines were labeled with a monoclonal antibody 7B11 which labels all classes of astrocytes and their precursors in the rat CNS. U138MG and Tm3 cells expressed antigens on their surfaces recognized by the monoclonal antibodies A2B5 and HNK-1. When grown in serum-free medium in the presence of cAMP and theophylline, U138MG cells assumed a process-bearing morphology and some cells expressed the Gal-C antigen specific for oligodendrocytes. Under identical conditions, Tm3 cells converted to process-bearing cells, some of which expressed glial fibrillary acidic protein (GFAP) specific for astrocytes. Other cell lines with similar antigenic characteristics did not respond similarly to cAMP and theophylline. Finally, A2781 cells were GFAP immunoreactive and unlabeled by either A2B5 or HNK-1 antibodies. These observations suggest that individual glioblastoma cell lines may be derived from distinct glial precursor cells in the vertebrate CNS.

Transcriptional patterns of growth factors and proto-oncogenes in human glioblastomas and normal glial cells.

To document over-expression of proto-oncogenes in tumors, it is necessary to determine the level of expression in the progenitor normal tissue. These studies compare the levels of nuclear transcription of a series of growth-factor related genes and proto-oncogenes in human glioblastoma cell lines with those in three normal glial cell populations. The unusual finding was that levels in the three normal glial cell populations varied considerably for several genes and thus overexpression of a specific gene in a tumor cell when compared to just one normal glial cell population would not necessarily represent overexpression. In this study, we compared the level of 17 genes in 7 tumors to the highest level of each gene found in any of three normal glial cell populations. Over-expression of PDGF-B in 4/7 glioblastoma cell lines, EGFR in 1/7, neu in 1/7 IGF-2 in 1/7 and ros in 2/7 was observed. The variation observed in the normal glial cell populations emphasizes the possibility that the normal glial cell populations represent different glial cell lineages and/or stages of differentiation and that the tumors could have arisen from different normal glial cells. Matching lineages of normal and tumor cells, probably by monoclonal antibody reactions, may be required to accurately define over-expression.