Michael P. Popp

A global view of the selectivity of zinc deprivation and excess on genes expressed in human THP-1 mononuclear cells

Among the micronutrients required by humans, zinc has particularly divergent modes of action. cDNA microarray and quantitative PCR technologies were used to investigate the zinc responsiveness of known genes that influence zinc homeostasis and to identify, through global screening, genes that may relate to phenotypic outcomes of altered dietary zinc intake. Human monocytic/macrophage THP-1 cells were either acutely zinc depleted, using a cell-permeable zinc-specific chelator, or were supplemented with zinc to alter intracellular zinc concentrations. Initially, genes associated with zinc homeostasis were evaluated by quantitative PCR to establish ranges for fold changes in transcript abundance that might be expected with global screening. Zinc transporter-1 and zinc transporter-7 expression increased when cellular zinc increased, whereas Zip-2 expression, the most zinc-responsive gene examined, was markedly increased by zinc depletion. Microarrays composed of ≈22,000 elements were used to identify those genes responsive to either zinc depletion, zinc supplementation, or both conditions. Hierarchal clustering and ANOVA revealed that ≈5% or 1,045 genes were zinc responsive. Further sorting based on this pattern of the zinc responsiveness of these genes into seven groups revealed that 104 genes were linearly zinc responsive in a positive mode (i.e., increased expression as cellular zinc increases) and 86 genes that were linearly zinc responsive in a negative mode (i.e., decreased expression as cellular zinc increases). Expression of some genes was responsive to only zinc depletion or supplementation. Categorization by function revealed numerous genes needed for host defense were among those identified as zinc responsive, including cytokine receptors and genes associated with amplification of the Th1 immune response.

Hypobaric biology: Arabidopsis gene expression at low atmospheric pressure.

As a step in developing an understanding of plant adaptation to low atmospheric pressures, we have identified genes central to the initial response of Arabidopsis to hypobaria. Exposure of plants to an atmosphere of 10 kPa compared with the sea-level pressure of 101 kPa resulted in the significant differential expression of more than 200 genes between the two treatments. Less than one-half of the genes induced by hypobaria are similarly affected by hypoxia, suggesting that response to hypobaria is unique and is more complex than an adaptation to the reduced partial pressure of oxygen inherent to hypobaric environments. In addition, the suites of genes induced by hypobaria confirm that water movement is a paramount issue at low atmospheric pressures, because many of gene products intersect abscisic acid-related, drought-induced pathways. A motivational constituent of these experiments is the need to address the National Aeronautics and Space Administrations plans to include plants as integral components of advanced life support systems. The design of bioregenerative life support systems seeks to maximize productivity within structures engineered to minimize mass and resource consumption. Currently, there are severe limitations to producing Earth-orbital, lunar, or Martian plant growth facilities that contain Earth-normal atmospheric pressures within light, transparent structures. However, some engineering limitations can be offset by growing plants in reduced atmospheric pressures. Characterization of the hypobaric response can therefore provide data to guide systems engineering development for bioregenerative life support, as well as lead to fundamental insights into aspects of desiccation metabolism and the means by which plants monitor water relations.

Gene Expression Profiling in Squamous Cell Carcinoma of the Oral Cavity Shows Abnormalities in Several Signaling Pathways

Objectives/Hypothesis: To examine gene expression profiles in squamous cell carcinoma of the oral cavity (oral SCC) compared with histologically matched normal tissue.

Molecular cloning, structure and expression of an elicitor-inducible chitinase gene from pine trees

We have cloned, sequenced, and examined the expression of genes from pine trees that appear to encode extracellular class II chitinase. Nucleotide sequence analysis indicates a coding sequence composed of three exons interrupted by two introns at locations identical to those found in other chitinase genes that possess introns. One of the genes, Pschi4, potentially encodes a protein that shares 62% amino acid sequence identity through the catalytic domain with class II chitinase from tobacco. In contrast, Pschi1 contains a stop codon in the first exon and may be a pseudogene. Pschi4 genes are conserved in several species of pine, and appear to comprise a small multigene family. Treatment of pine cell suspension cultures with the general elicitor chitosan induced Pschi4 expression. The regulatory sequences associated with the Pschi4 gene were sufficient to direct chitosan-inducible expression of Pschi4 in transgenic tobacco plants, which indicates that Pschi4 is an actively expressed member of the multigene family. The observation that the Pschi4 gene from pine (a gymnosperm) was appropriately regulated by chitosan in tobacco (an angiosperm) suggests that the signaling pathways that mediate chitosan-induced transcription are highly conserved in the plant kingdom.

Defense responses elicited in pine cell suspension cultures

To understand mechanisms of disease resistance in pine trees, we took advantage of the fact that suspension cultured cells exhibit many of the defense responses that are characteristic of intact tissues. In this study, we measured constitutive and elicitor-induced levels of ethylene production, chitinase activity and glucanase activity in cells of loblolly pine (Pinus taeda L). Increased ethylene production was induced similarly by a live fungus (Ophiostoma minus Hedgc. H.P. Sydow) and chitosan, a general elicitor. Culture age, relative to the most recent transfer, affected the constitutive level of all defense responses. Culture age also had a pronounced effect on the ability of the cells to produce ethylene and cellular chitinase, but not on secreted chitinase, cellular glucanase, secreted glucanase, or lignification. In older cultures, elicitation induced a 4- to 10-fold increase in ethylene production and a 2-fold increase in cellular chitinase, secreted chitinase and cellular glucanase. Chitosan elicitation did not affect secreted glucanase. The overall regulation of the defense response in pine cells appears complex, but individual components of the response can be differentially induced in cell cultures under appropriate experimental conditions.

Ant opsins: Sequences from the Saharan silver ant and the carpenter ant

AbstractcDNA clones encoding opsins from compound eyes of carpenter ant,Camponotus abdominalis, and Saharan silver ant,Cataglyphis bombycina, were isolated from cDNA libraries. The opsin cDNAs from each species code for deduced proteins with 378 amino acids which are 92% identical. Of the 30 amino acid differences between the two proteins, 13 are non-conservative. Eight of these non-conservative substitutions are within the membrane spanning domain. The presence of a potential Schiff-base counterion in helix III in both species suggests that these opsins are the protein moiety of the visible range pigments. When compared to all known opsins, these opsins are most similar to the opsin from preying mantis (76% identity at the amino acid level). Phyletic comparisons group the two ant opsins with the other arthropod long wavelength opsins.

Expression of CYP2L1 in the yeast Pichia pastoris, and determination of catalytic activity with progesterone and testosterone

Abstract Cytochrome P450 (P450) enzymes are widely distributed throughout the plant and animal kingdoms. In previous work, two forms of P450 partially purified from spiny lobster, Panulirus argus, hepatopancreas microsomes were shown to metabolize several substrates, including testosterone and progesterone at the 16α, 6β and 21 positions f James (1990) Archives of Biochemistry and Biophysics 282, 8–17]. We have recently cloned and sequenced a major form of P450, CYP2L1, from hepatopancreas cDNA [James et al. (1996) Archives of Biochemistry and Biophysics 329, 31–38]. CYP2L1 is one of at least four P450s detected in a cDNA library constructed from spiny lobster hepatopancreas. The spiny lobster CYP2L1 cDNA was introduced into the methylotrophic yeast Pichia pastoris and the crustacean CYP2L1 was expressed in microsomes of the methanol-induced yeast. On the basis of immunoblot analysis and ability to catalyse the 16α-hydroxylation of testosterone and progesterone, the catalytically active, heterologously expressed spiny lobster CYP2L1 appears to be very similar to the major spiny lobster hepatopancreas P450 previously purified in this laboratory. This is the first report of heterologous expression of a crustacean P450.

Short wavelength-sensitive opsins from the Saharan silver and carpenter ants.

We have previously cloned the opsins coding for the long-wavelength visual pigments from the Saharan silver ant and carpenter ant. Here we report two new cDNA clones isolated from cDNA libraries which also code for opsin proteins. These cDNAs code for deduced proteins with 369 amino acids which are 91% identical to each other, but only 38% identical to the previously cloned opsins. Phyletic comparisons suggest that these opsins are likely the ultraviolet sensitive visual pigments, a conclusion that is supported by the presence of a phenylalanine at the counterion position in the third transmembrane segment.

Flow Regime Analysis of Forced Flow Boiling Hydrogen Subjected to High Heat Flux

This paper presents an analysis of forced flow two-phase hydrogen heat transfer data for the purpose of interpreting the various flow regimes of inverted annular flow. Hydrogen heat transfer data available from an early 1960s NASA experimental investigation were used for the analysis. The data was evaluated in light of a heat transfer characteristics map and related flow pattern map which were established by more recent work on inverted flow film boiling of freon R113. It was shown that the hydrogen data exhibit the same three flow patterns as found in the R113 data. This may allow the use of film boiling heat transfer models developed for such fluids to be used for hydrogen film boiling heat transfer predictions.Copyright