Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1

Muscle tissue is the major site for insulin-stimulated glucose uptake in vivo, due primarily to the recruitment of the insulin-sensitive glucose transporter (GLUT4) to the plasma membrane. Surprisingly, virtually all cultured muscle cells express little or no GLUT4. We show here that adenovirus-mediated expression of the transcriptional coactivator PGC-1, which is expressed in muscle in vivo but is also deficient in cultured muscle cells, causes the total restoration of GLUT4 mRNA levels to those observed in vivo. This increased GLUT4 expression correlates with a 3-fold increase in glucose transport, although much of this protein is transported to the plasma membrane even in the absence of insulin. PGC-1 mediates this increased GLUT4 expression, in large part, by binding to and coactivating the muscle-selective transcription factor MEF2C. These data indicate that PGC-1 is a coactivator of MEF2C and can control the level of endogenous GLUT4 gene expression in muscle.

ParAlign: a parallel sequence alignment algorithm for rapid and sensitive database searches

There is a need for faster and more sensitive algorithms for sequence similarity searching in view of the rapidly increasing amounts of genomic sequence data available. Parallel processing capabilities in the form of the single instruction, multiple data (SIMD) technology are now available in common microprocessors and enable a single microprocessor to perform many operations in parallel. The ParAlign algorithm has been specifically designed to take advantage of this technology. The new algorithm initially exploits parallelism to perform a very rapid computation of the exact optimal ungapped alignment score for all diagonals in the alignment matrix. Then, a novel heuristic is employed to compute an approximate score of a gapped alignment by combining the scores of several diagonals. This approximate score is used to select the most interesting database sequences for a subsequent Smith–Waterman alignment, which is also parallelised. The resulting method represents a substantial improvement compared to existing heuristics. The sensitivity and specificity of ParAlign was found to be as good as Smith–Waterman implementations when the same method for computing the statistical significance of the matches was used. In terms of speed, only the significantly less sensitive NCBI BLAST 2 program was found to outperform the new approach. Online searches are available at http://dna.uio.no/search/

An Arabidopsis GSK3/shaggy-Like Gene That Complements Yeast Salt Stress-Sensitive Mutants Is Induced by NaCl and Abscisic Acid

GSK3/shaggy-like genes encode kinases that are involved in a variety of biological processes. By functional complementation of the yeast calcineurin mutant strain DHT22-1a with a NaCl stress-sensitive phenotype, we isolated the Arabidopsis cDNA AtGSK1, which encodes a GSK3/shaggy-like protein kinase. AtGSK1 rescued the yeast calcineurin mutant cells from the effects of high NaCl. Also, the AtGSK1 gene turned on the transcription of the NaCl stress-inducible PMR2A gene in the calcineurin mutant cells under NaCl stress. To further define the role of AtGSK1 in the yeast cells we introduced a deletion mutation at the MCK1 gene, a yeast homolog of GSK3, and examined the phenotype of the mutant. The mck1 mutant exhibited a NaCl stress-sensitive phenotype that was rescued by AtGSK1. Also, constitutive expression of MCK1 complemented the NaCl-sensitive phenotype of the calcineurin mutants. Therefore, these results suggest that Mck1p is involved in the NaCl stress signaling in yeast and that AtGSK1 may functionally replace Mck1p in the NaCl stress response in the calcineurin mutant. To investigate the biological function of AtGSK1 in Arabidopsis we examined the expression of AtGSK1. Northern-blot analysis revealed that the expression is differentially regulated in various tissues with a high level expression in flower tissues. In addition, the AtGSK1 expression was induced by NaCl and exogenously applied ABA but not by KCl. Taken together, these results suggest that AtGSK1 is involved in the osmotic stress response in Arabidopsis.

Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension

Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality. Here we show that disruption of the Cyp 4a14 gene causes hypertension, which is, like most human hypertension, more severe in males. Male Cyp 4a14 (−/−) mice show increases in plasma androgens, kidney Cyp 4a12 expression, and the formation of prohypertensive 20-hydroxyarachidonate. Castration normalizes the blood pressure of Cyp 4a14 (−/−) mice and minimizes Cyp 4a12 expression and arachidonate ω-hydroxylation. Androgen replacement restores hypertensive phenotype, Cyp 4a12 expression, and 20-hydroxy-arachidonate formation. We conclude that the androgen-mediated regulation of Cyp 4a arachidonate monooxygenases is an important component of the renal mechanisms that control systemic blood pressures. These results provide direct evidence for a role of Cyp 4a isoforms in cardiovascular physiology, establish Cyp 4a14 (−/−) mice as a monogenic model for the study of cause/effect relationships between blood pressure, sex hormones, and P450 ω-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.

Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels

Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K+ (KATP) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with KATP channel-dependent membrane excitability remains elusive. Here, we identify that the response of KATP channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the KATP channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and KATP channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with KATP channels and associated functions.

Differential Regulation of Sugar-Sensitive Sucrose Synthases by Hypoxia and Anoxia Indicate Complementary Transcriptional and Posttranscriptional Responses1

The goal of this research was to resolve the hypoxic and anoxic responses of maize (Zea mays) sucrose (Suc) synthases known to differ in their sugar regulation. The two maize Suc synthase genes, Sus1 and Sh1, both respond to sugar and O2, and recent work suggests commonalities between these signaling systems. Maize seedlings (NK508 hybrid, W22 inbred, and an isogenic sh1-null mutant) were exposed to anoxic, hypoxic, and aerobic conditions (0, 3, and 21% O2, respectively), when primary roots had reached approximately 5 cm. One-centimeter tips were excised for analysis during the 48-h treatments. At the mRNA level, Sus1 was rapidly up-regulated by hypoxia (approximately 5-fold in 6 h), whereas anoxia had less effect. In contrast, Sh1 mRNA abundance increased strongly under anoxia (approximately 5-fold in 24 h) and was much less affected by hypoxia. At the enzyme level, total Suc synthase activity rose rapidly under hypoxia but showed little significant change during anoxia. The contributions of SUS1 and SH1 activities to these responses were dissected over time by comparing the sh1-null mutant with the isogenic wild type (Sus+, Sh1+). Sh1-dependent activity contributed most markedly to a rapid protein-level response consistently observed in the first 3 h, and, subsequently, to a long-term change mediated at the level of mRNA accumulation at 48 h. A complementary midterm rise in SUS1 activity varied in duration with genetic background. These data highlight the involvement of distinctly different genes and probable signal mechanisms under hypoxia and anoxia, and together with earlier work, show parallel induction of “feast and famine” Suc synthase genes by hypoxia and anoxia, respectively. In addition, complementary modes of transcriptional and posttranscriptional regulation are implicated by these data, and provide a mechanism for sequential contributions from the Sus1 and Sh1 genes during progressive onset of naturally occurring low-O2 events.

Identification of a Role for an Azide-Sensitive Factor in the Thylakoid Transport of the 17-Kilodalton Subunit of the Photosynthetic Oxygen-Evolving Complex1

We have examined the transport of the precursor of the 17-kD subunit of the photosynthetic O2-evolving complex (OE17) in intact chloroplasts in the presence of inhibitors that block two protein-translocation pathways in the thylakoid membrane. This precursor uses the transmembrane pH gradient-dependent pathway into the thylakoid lumen, and its transport across the thylakoid membrane is thought to be independent of ATP and the chloroplast SecA homolog, cpSecA. We unexpectedly found that azide, widely considered to be an inhibitor of cpSecA, had a profound effect on the targeting of the photosynthetic OE17 to the thylakoid lumen. By itself, azide caused a significant fraction of mature OE17 to accumulate in the stroma of intact chloroplasts. When added in conjunction with the protonophore nigericin, azide caused the maturation of a fraction of the stromal intermediate form of OE17, and this mature protein was found only in the stroma. Our data suggest that OE17 may use the sec-dependent pathway, especially when the transmembrane pH gradient-dependent pathway is inhibited. Under certain conditions, OE17 may be inserted across the thylakoid membrane far enough to allow removal of the transit peptide, but then may slip back out of the translocation machinery into the stromal compartment.

The Distal Part of the Transition Zone Is the Most Aluminum-Sensitive Apical Root Zone of Maize1

For a better understanding of Al inhibition of root elongation, knowledge of the morphological and functional organization of the root apex is a prerequisite. We developed a polyvinyl chloride-block technique to supply Al (90 μm monomeric Al) in a medium containing agarose to individual 1-mm root zones of intact seedlings of maize (Zea mays L. cv Lixis). Root elongation was measured during a period of 5 h. After Al treatment, callose (5 h) and Al (1 h) contents of individual 1-mm apical root segments were determined. For comparison, callose and Al levels were also measured in root segments after uniform Al supply in agarose blocks to the 10-mm root apex. Only applying Al to the three apical 1-mm root zones inhibited root elongation after 1 h. The order of sensitivity was 1 to 2 > 0 to 1 > 2 to 3 mm. In the 1- to 2-mm root zone high levels of Al-induced callose formation and accumulation of Al was found, independently of whether Al was applied to individual apical root zones or uniformly to the whole-root apex. We conclude from these results that the distal part of the transition zone of the root apex, where the cells are undergoing a preparatory phase for rapid elongation (F. Baluška, D. Volkmann, P.W. Barlow [1996] Plant Physiol 112: 3–4), is the primary target of Al in this Al-sensitive maize cultivar.

Molecular and Physiological Responses to Water Deficit in Drought-Tolerant and Drought-Sensitive Lines of Sunflower1 : Accumulation of Dehydrin Transcripts Correlates with Tolerance

To investigate correlations between phenotypic adaptation to water limitation and drought-induced gene expression, we have studied a model system consisting of a drought-tolerant line (R1) and a drought-sensitive line (S1) of sunflowers (Helianthus annuus L.) subjected to progressive drought. R1 tolerance is characterized by the maintenance of shoot cellular turgor. Drought-induced genes (HaElip1, HaDhn1, and HaDhn2) were previously identified in the tolerant line. The accumulation of the corresponding transcripts was compared as a function of soil and leaf water status in R1 and S1 plants during progressive drought. In leaves of R1 plants the accumulation of HaDhn1 and HaDhn2 transcripts, but not HaElip1 transcripts, was correlated with the drought-adaptive response. Drought-induced abscisic acid (ABA) concentration was not associated with the varietal difference in drought tolerance. Stomata of both lines displayed similar sensitivity to ABA. ABA-induced accumulation of HaDhn2 transcripts was higher in the tolerant than in the sensitive genotype. HaDhn1 transcripts were similarly accumulated in the tolerant and in the sensitive plants in response to ABA, suggesting that additional factors involved in drought regulation of HaDhn1 expression might exist in tolerant plants.

Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells.

Decreased nitric oxide (NO) activity, the formation of reactive oxygen species, and increased endothelial expression of the redox-sensitive vascular cell adhesion molecule 1 (VCAM-1) gene in the vessel wall are early and characteristic features of atherosclerosis. To explore whether these phenomena are functionally interrelated, we tested the hypothesis that redox-sensitive VCAM-1 gene expression is regulated by a NO-sensitive mechanism. In early passaged human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the NO donor diethylamine-NO (DETA-NO, 100 microM) reduced VCAM-1 gene expression induced by the cytokine tumor necrosis factor alpha (TNF-alpha, 100 units/ml) at the cell surface level by 65% and intracellular adhesion molecule 1 (ICAM-1) gene expression by 35%. E-selectin gene expression was not affected. No effect on expression of cell adhesion molecules was observed with DETA alone. Moreover, DETA-NO suppressed TNF-alpha-induced mRNA accumulation of VCAM-1 and TNF-alpha-mediated transcriptional activation of the human VCAM-1 promoter. Conversely, treatment with NG-monomethyl-L-arginine (L-NMMA, 1 mM), an inhibitor of NO synthesis, augmented cytokine induction of VCAM-1 and ICAM-1 mRNA accumulation. By gel mobility shift analysis, DETA-NO inhibited TNF-alpha activation of DNA binding protein activity to the VCAM-1 NF-kappa B like binding sites. Peroxy-fatty acids such as 13-hydroperoxydodecanoeic acid (linoleyl hydroperoxide) may serve as an intracellular signal for NF-kappa B activation. Using thin layer chromatography, DETA-NO (100 microM) suppressed formation of this metabolite, suggesting that DETA-NO modifies the reactivity of oxygen intermediates in the vascular endothelium. Through this mechanism, NO may function as an immunomodulator of the vessel wall and thus mediate inflammatory events involved in the pathogenesis of atherosclerosis.

Haemonchus contortus GA1 antigens: related, phospholipase C-sensitive, apical gut membrane proteins encoded as a polyprotein and released from the nematode during infection.

It was previously shown that the Haemonchus contortus apical gut surface proteins p46, p52, and p100 induced protective immunity to challenge infections in goats. Here, it is shown that the three proteins are all encoded by a single gene (GA1) and initially expressed in adult parasites as a polyprotein (p100GA1). p46GA1 and p52GA1 are related proteins with 47% sequence identity, including a cysteine-containing region, which appears to confer secondary structure to these proteins, and a region with sequence similarity to bacterial Tolb proteins. GA1 protein expression is regulated during the life cycle at the level of transcript abundance. Only p52GA1 has characteristics of a glycosylinositolphospholipid membrane-anchored protein. However, both p46GA1 and p52GA1 were released from the gut membrane by phosphatidylinositol specific-phospholipase C, suggesting that p46GA1 membrane association depends on interactions with a glycosylinositolphospholipid gut membrane protein. Finally, GA1 proteins occur in abomasal mucus of infected lambs, demonstrating possible presentation to the host immune system during H. contortus infection. The results identify multiple characteristics of the GA1 proteins that should be considered for design of recombinant antigens for vaccine trials and that implicate a series of cellular processes leading to modification and expression of GA1 proteins at the nematode apical gut surface.

Imaging ROMK1 inwardly rectifying ATP-sensitive K+ channel protein using atomic force microscopy.

The inwardly rectifying K+ channel ROMK1 has been implicated as being significant in K+ secretion in the distal nephron. ROMK1 has been shown by immunocytochemistry to be expressed in relevant nephron segments. The development of the atomic force microscope has made possible the production of high resolution images of small particles, including a variety of biological macromolecules. Recently, a fusion protein of glutathione S-transferase (GST) and ROMK1 (ROMK1-GST) has been used to produce a polyclonal antibody for immunolocalization of ROMK1. We have used atomic force microscopy to examine ROMK1-GST and the native ROMK1 polypeptide cleaved from GST. Imaging was conducted with the proteins in physiological solutions attached to mica. ROMK1-GST appears in images as a particle composed of two units of similar size. Analyses of images indicate that the two units have volumes of approximately 118 nm3, which is close to the theoretical volume of a globular protein of approximately 65 kDa (the molecular mass of ROMK1-GST). Native GST exists as a dimer, and the images obtained here are consistent with the ROMK1-GST fusion protein's existence as a heterodimer. In experiments on ROMK1 in aqueous solution, single molecules appear to aggregate, but contact to the mica was maintained. Addition of ATP to the solution produced a change in height of the aggregates. This change (which was reversible) suggests that ATP induces a structural change in the ROMK1 protein. The data show that atomic force microscopy is a useful tool for examination of purified protein molecules under near-physiological conditions, and furthermore, that structural alterations in the proteins may be continuously investigated.

Sensitive detection of bacterial transcription initiation sites and differentiation from RNA processing sites in the pheromone-induced plasmid transfer system of Enterococcus faecalis.

A method was developed to detect 5' ends of bacterial RNAs expressed at low levels and to differentiate newly initiated transcripts from processed transcripts produced in vivo. The procedure involves use of RNA ligase to link a specific oligoribonucleotide to the 5' ends of cellular RNAs, followed by production of cDNA and amplification of the gene of interest by PCR. The method was used to identify the precise sites of transcription initiation within a 10-kb region of the pheromone-inducible conjugative plasmid pCF10 of Enterococcus faecalis. Results confirmed the 5' end of a very abundant, constitutively produced transcript (from prgQ) that had been mapped previously by primer extension and defined the initiation point of a less abundant, divergently transcribed message (from prgX). The method also showed that the 5' end of a pheromone-inducible transcript (prgB) that had been mapped by primer extension was generated by processing rather than new initiation. In addition, the results provided evidence for two promoters, 3 and 5 kb upstream of prgB, and indicated that only the transcripts originating 5 kb upstream may be capable of extending to prgB.