Free amino acid levels and the regulation of nitrate uptake in maize cell suspension cultures

The ability of individual amino acids to regulate nitrate uptakeand induction was studied in a Zea mays embryo cell line grownin suspension culture. The maize cells exhibited a marked preferencefor absorbing amino acids over nitrate when both were presentin culture medium. The addition of an individual amino acid(2 mM glutamine, glycine, aspartic acid, or arginine) to theculture medium with 1 mM nitrate completely inhibited nitrateuptake and resulted in a cycle of low levels of nitrate influxfollowed by efflux to the growth medium. Glutamine was readilyabsorbed by the cells and was particularly effective in supportingoptimum cell growth in the absence of an inorganic nitrogensource as compared to the three other amino acids evaluated.However, neither glutamine nor any of the remaining 19 proteinaceousamino acids appeared to be solely responsible for regulationof nitrate uptake and induction. The ability of amino acidsto regulate nitrate uptake and assimilation appears to be morerelated to their overall levels in the cell rather than to anaccumulation of a specific amino acid. Key words: Amino acids, nitrate uptake, maize, regulation, cell suspension culture     

Protein Tyrosine Kinase-Mediated Potentiation of Currents from Cloned NMDA Receptors

Abstract: Although serine/threonine phosphorylation has been more commonly recognized as a mechanism to modulate the function of ion channels and receptors, tyrosine phosphorylation is under increasing scrutiny. An important subtype of glutamate receptor, the NMDA receptor, is shown to be regulated by insulin via protein tyrosine kinase (PTK). NMDA currents through cloned receptors are potentiated by insulin in a subunit-specific manner. The insulin-mediated potentiation of NMDA current is diminished by inhibitors of PTKs. At least one exogenous cytosolic PTK, pp60^{c- src} , is also able to potentiate NMDA current. Because later application of PTK inhibitors can reverse the seemingly stable insulin-mediated potentiation of NMDA current, it appears that tyrosine residues responsible for potentiation are continually rephosphorylated by some long-term PTK activity that was induced via insulin treatment.     

Interactions between the regulatory regions of twoAdh alleles

A region (NS1) that acts like an enhancer is located approximately 300 bp upstream of the larval cap site in theAdh gene ofD. melanogaster. When this sequence is deleted (NS1), the gene fails to express ADH protein. Gene expression can be restored by placing a secondAdh gene with an intact enhancer elsewhere on the same plasmid. In these circumstances, both genes are expressed equally regardless of their orientation on the plasmid. In this report we further characterize the interactions that occur when a single enhancer activates expression from a proximal and distant promoter. We have made the following observations: (1) While the two genes are expressed equivalently, their expression relative to a plasmid carrying two intact genes is reduced by a factor of 2 to 6 depending on the orientation of the two genes. (2) The single enhancer drives expression of both genes on any given plasmid molecule. (3) The enhancer does not interact with theAdh gene from which the NS7 region (which spans the larval TATA box) is removed. (4) Expression of the NS1 gene can be restored by an intact gene when both are inserted together into theDrosophila genome via P element-mediated transformation. (5) Increasing the separation between the two genes on a plasmid by up to 15 kbp does not prevent the restoration of expression of the NS1 gene. We propose a model that explains how a single enhancer can stimulate equal expression from two genes.     

High-conductance calcium-activated potassium channels; Structure,pharmacology, and function

High-conductance calcium-activated potassium (maxi-K) channels comprise a specialized family of K⁺ channels. They are unique in their dual requirement for depolarization and Ca²⁺ binding for transition to the open, or conducting, state. Ion conduction through maxi-K channels is blocked by a family of venom-derived peptides, such as charybdotoxin and iberiotoxin. These peptides have been used to study function and structure of maxi-K channels, to identify novel channel modulators, and to follow the purification of functional maxi-K channels from smooth muscle. The channel consists of two dissimilar subunits, and . The subunit is a member of theslo Ca²⁺-activated K⁺ channel gene family and forms the ion conduction pore. The subunit is a structurally unique, membrane-spanning protein that contributes to channel gating and pharmacology. Potent, selective maxi-K channel effectors (both agonists and blockers) of low molecular weight have been identified from natural product sources. These agents, together with peptidyl inhibitors and site-directed antibodies raised against and subunit sequences, can be used to anatomically map maxi-K channel expression, and to study the physiologic role of maxi-K channels in various tissues. One goal of such investigations is to determine whether maxi-K channels represent novel therapeutic targets.     

An IL-2 response element in the human IL-2 receptor alpha chain promoter is a composite element that binds Stat5, Elf-1, HMG-I(Y) and a GATA family protein.

Expression of the human interleukin-2 (IL-2) receptor alpha chain gene is potently upregulated by its own ligand, IL-2. In this study, we characterize an essential upstream IL-2 response element that contains both consensus and non-consensus GAS motifs, two putative Ets binding sites (EBS), one of which overlaps the consensus GAS motif, and a GATA motif, which overlaps the non-consensus GAS motif. We demonstrate that although the individual components of this element do not respond to IL-2, together they form a composite element capable of conferring IL-2 responsiveness to a heterologous promoter. Multiple factors including Stat5, Elf-1, HMG-I(Y) and GATA family proteins bind to the IL-2 response element and mutation of any one of these binding sites diminishes the activity of this element. An unidentified Ets family protein binds to the EBS overlapping the consensus GAS motif and appears to negatively regulate the human IL-2R alpha promoter. Thus, IL-2-induced IL-2R alpha promoter activity requires a complex upstream element, which appears to contain binding sites for both positive and negative regulatory factors.     

Differences between observed and predicted energy costs at rest and during exercise in three subsistence-level populations

Estimates of daily energy expenditure are important for many areas of research in human ecology and adaptability. The most common technique for estimating human energy expenditure under field conditions, the factorial method, generally relies on activity-specific energy costs derived from published sources, based largely on North American and European subjects. There is concern that such data may not be appropriate for non-Western populations because of differences in metabolic costs. The present study addresses this concern by comparing measured vs. predicted energy costs at rest and during sub-maximal exercise in 83 subjects (52 males, 31 females) from three subsistence-level populations (Siberian herders and high-land and coastal Ecuadorian farmers). Energy costs at rest (i.e., lying, sitting and standing) and while performing a standard stepping exercise did not significantly differ among the three groups. However, resting energy costs were significantly elevated over predicted levels (+16% in men, +11% in women), whereas exercising costs were comparable to predicted values (?6% in men, +3% in women). Elevations in resting energy needs appear to reflect responses to thermal stress. These results indicate that temperature adjustments of resting energy costs are critical for accurately predicting daily energy needs among traditionally living populations. o 1996 Wiley-Liss, Inc.     

Efficient lipid-mediated transfection of DNA into primary rat hepatocytes

Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids, however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic (EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio. Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions for transfecting 5 μg of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration of 15 μg DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical, easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes.     

Structure of corneal scar tissue: an X-ray diffraction study.

Full-thickness corneal wounds (2 mm diameter) were produced in rabbits at the Schepens Eye Research Institute, Boston. These wounds were allowed to heal for periods ranging from 3 weeks to 21 months. The scar tissue was examined using low- and wide-angle x-ray diffraction from which average values were calculated for 1) the center-to-center collagen fibril spacing, 2) the fibril diameter, 3) the collagen axial periodicity D, and 4) the intermolecular spacing within the collagen fibrils. Selected samples were processed for transmission electron microscopy. The results showed that the average spacing between collagen fibrils within the healing tissue remained slightly elevated after 21 months and there was a small increase in the fibril diameter. The collagen D-periodicity was unchanged. There was a significant drop in the intermolecular spacing in the scar tissues up to 6 weeks, but thereafter the spacing returned to normal. The first-order equatorial reflection in the low-angle pattern was visible after 3 weeks and became sharper and more intense with time, suggesting that, as healing progressed, the number of nearest neighbor fibrils increased and the distribution of nearest neighbor spacings reduced. This corresponded to the fibrils becoming more ordered although, even after 21 months, normal packing was not achieved. Ultrastructural changes in collagen fibril density measured from electron micrographs were consistent with the increased order of fibril packing measured by x-ray diffraction. The results suggest that collagen molecules have a normal axial and lateral arrangement within the fibrils of scar tissue. The gradual reduction in the spread of interfibrillar spacings may be related to the progressive decrease in the light scattered from the tissue as the wound heals.