Highest Steady-state mRNA Levels Research Articles

Increased light damage susceptibility at night does not correlate with RPE65 levels and rhodopsin regeneration in rats

The susceptibility of rats to light-induced retinal degeneration is increased at night. In mice, an important determinant of light damage susceptibility is the efficacy of rhodopsin regeneration after bleaching. The rate of rhodopsin regeneration is at least partly controlled by RPE65, a protein expressed in the retinal pigment epithelium. We therefore tested a potential involvement of RPE65 and rhodopsin regeneration in the increased light damage susceptibility of rats at night. For this purpose, rats were exposed to visible light at noon or at midnight and extent of light damage was determined by retinal morphology and TUNEL staining. Rpe65 gene expression was analyzed by semiquantitative RT-PCR and levels of RPE65 protein were determined by Western blotting. Rhodopsin regeneration kinetics was determined by measuring rhodopsin content immediately after a strong bleach and after different times of recovery in darkness. Rats were more susceptible to light damage at night as described by Organisciak and collegues [Invest. Ophthalmol. Vis. Sci. 41 (2000) 3694]. Rpe65 gene expression followed a day–night rhythm with highest steady-state mRNA levels at the beginning and lowest levels at the end of the day period. However, RPE65 protein levels remained constant. Rhodopsin regeneration kinetics did not differ during day and night. We conclude that levels of RPE65 protein and rhodopsin regeneration kinetics do not correlate with the increased light damage susceptibility observed in rats at night. Additional genetic or physiologic modifiers may exist in rats that regulate the retinal responsiveness to acute light exposure.

Estrogen receptor-alpha gene deficiency enhances androgen biosynthesis in the mouse Leydig cell.

Leydig cells, which produce the primary male steroid hormone testosterone (T), express the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, and have the capacity to convert testosterone to the natural estrogen 17beta-estradiol. Thus, Leydig cells are subject to estrogen action. The development of transgenic mice that are homozygous for targeted deletion of genes encoding the ER subtypes provides an opportunity to examine the role of estrogen in Leydig cell function. In this study androgen biosynthesis was analyzed in Leydig cells from mice that were homozygous for targeted deletion of the ERalpha gene (alphaERKO). T production by alphaERKO Leydig cells was 2-fold higher than that in wild-type (WT) cells. Serum T levels were accordingly higher in alphaERKO compared with WT mice (5.1 +/- 1.1 vs. 2.2 +/- 0.4 ng/ml; P </= 0.01) as were serum LH levels (1.31 +/- 0.3 vs. 0.45 +/- 0.08 ng/ml; P </= 0.01). Mice that were treated with the pure antiestrogen ICI 182,780 at 100 micro g/kg.d for 7 d, effectively abrogating ER-mediated activity, also had 2-fold elevations in the serum levels of LH (1.15 +/- 0.3 vs. 0.45 +/- 0.2 ng/ml) and T (4.3 +/- 1.1 vs. 2.2 +/- 0.2 ng/ml; P </= 0.01). Increased androgen biosynthesis by alphaERKO Leydig cells was associated with higher steroidogenic enzyme activity, especially of cytochrome P450 17alpha-hydroxylase/17-20 lyase (P450(17alpha)) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD), as measured by conversion of radiolabeled steroid substrates to T or its precursors. The largest increases in enzymatic activity were observed for P450(17alpha) (423 +/- 45 pmol/min.10(6) cells in alphaERKO Leydig cells vs. 295 +/- 27 pmol/min.10(6) cells in WT cells; P < 0.01). Consistent with steroidogenic enzyme activity, the testis of alphaERKO mice expressed higher steady state mRNA levels for steroidogenic acute regulatory protein and two enzymes involved in androgen biosynthesis, P450(17alpha) and 17beta-HSD type III, as determined by semiquantitative RT-PCR. Compared with the controls, higher steady state mRNA levels for steroidogenic acute regulatory protein and P450(17alpha) were also measured in the testis of ICI 182,780-treated mice. In a second set of experiments estrogen administration reduced serum LH and T levels in WT controls, whereas alphaERKO mice were unaffected. Although exposure of WT and alphaERKO Leydig cells to estrogen in vitro did not affect androgen biosynthesis, incubation with ICI 182,780 reduced T production by WT, but not alphaERKO, Leydig cells. These observations indicate that abrogation of the ERalpha gene by targeted deletion or treatment with an antiestrogen increases Leydig cell steroidogenesis in association with elevations in the serum levels of LH, which presumably is the result of estrogen insensitivity at the level of the hypothalamus and/or pituitary gonadotropes. Furthermore, the decrease in T production by WT Leydig cells and not alphaERKO Leydig cells occasioned by incubation with ICI 182,780 suggests that of the ER subtypes, ERalpha has a regulatory role in Leydig cell steroidogenic function.

The four subunits of mitochondrial respiratory complex II are encoded by multiple nuclear genes and targeted to mitochondria in Arabidopsis thaliana.

Mitochondrial respiratory complex II contains four subunits: a flavoprotein (SDHI), an iron-sulphur subunit (SDH2) and two membrane anchor subunits (SDH3 and SDH4). We have found that in Arabidopsis thaliana SDH I and SDH3 are encoded by two, and SDH4 by one nuclear genes, respectively. All these encoded polypeptides are found to be imported into isolated plant mitochondria. While both SDHI proteins are highly conserved when compared to their counterparts in other organisms, SDH3 and SDH4 share little similarity with non-plant homologues. Expression of SDH1-1, SDH3 and SDH4 genes was detected in all tissues analysed, with the highest steady-state mRNA levels found in flowers and inflorescences. In contrast, the second SDH1 gene (SDH1-2) is expressed at a low level.

Three different genes encode the iron-sulfur subunit of succinate dehydrogenase in Arabidopsis thaliana.

The iron-sulfur protein is an essential component of mitochondrial complex II (succinate dehydrogenase, SDH), which is a functional enzyme of both the citric acid cycle and the respiratory electron transport chain. This protein is encoded by a single-copy nuclear gene in mammals and fungi and by a mitochondrial gene in Rhodophyta and the protist Reclinomonas americana. In Arabidopsis thaliana, the homologous protein is now found to be encoded by three nuclear genes. Two genes (sdh2-1 and sdh2-2) likely arose from a relatively recent duplication event since they have similar structures, encode nearly identical proteins and show similar expression patterns. Both genes are interrupted by a single intron located at a conserved position. Expression was detected in all tissues analysed, with the highest steady-state mRNA levels found in flowers and inflorescences. In contrast, the third gene (sdh2-3) is interrupted by 4 introns, is expressed at a low level, and encodes a SDH2-3 protein which is only 67% similar to SDH2-1 and SDH2-2 and has a different N-terminal presequence. Interestingly, the proteins encoded by these three genes are probably functional because they are highly conserved compared with their homologues in other organisms. These proteins contain the cysteine motifs involved in binding the three iron-sulfur clusters essential for electron transport. Furthermore, the three polypeptides are found to be imported into isolated plant mitochondria.

Adenosine kinase of Arabidopsis. Kinetic properties and gene expression.

To assess the functional significance of adenosine salvage in plants, the cDNAs and genes encoding two isoforms of adenosine kinase (ADK) were isolated from Arabidopsis. The ADK1- and ADK2-coding sequences are very similar, sharing 92% and 89% amino acid and nucleotide identity, respectively. Each cDNA was overexpressed in Escherichia coli, and the catalytic activity of each isoform was determined. Both ADKs had similar catalytic properties with a K(m) and V(max)/K(m) for adenosine of 0.3 to 0.5 microM and 5.4 to 22 L min(-1) mg(-1) protein, respectively. The K(m) and V(max)/K(m) for the cytokinin riboside N(6)(isopentenyl) adenosine are 3 to 5 microM and 0.021 to 0.14 L min(-1) mg(-1) protein, respectively, suggesting that adenosine is the preferred substrate for both ADK isoforms. In Arabidopsis plants, both ADK genes are expressed constitutively, with the highest steady-state mRNA levels being found in stem and root. ADK1 transcript levels were generally higher than those of ADK2. ADK enzyme activity reflected relative ADK protein levels seen in immunoblots for leaves, flowers, and stems but only poorly so for roots, siliques, and dry seeds. The catalytic properties, tissue accumulation, and expression levels of these ADKs suggest that they play a key metabolic role in the salvage synthesis of adenylates and methyl recycling in Arabidopsis. They may also contribute to cytokinin interconversion.

Studies on the production of fungal peroxidases in Aspergillus niger.

To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been studied. For this purpose, a protease-deficient A. niger strain and different expression cassettes have been used. Northern blotting experiments indicated high steady-state mRNA levels for the recombinant genes. Manganese peroxidase was secreted into the culture medium as an active protein. The recombinant protein showed specific activity and a spectrum profile similar to those of the native enzyme, was correctly processed at its N terminus, and had a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant MnP production could be increased up to 100 mg/liter upon hemoglobin supplementation of the culture medium. Lignin peroxidase was also secreted into the extracellular medium, although the protein was not active, presumably due to incorrect processing of the secreted enzyme. Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase gene did not result in improved production yields.

Increased steady state mRNA levels of the STM and KNAT1 homeobox genes in cytokinin overproducing Arabidopsis thaliana indicate a role for cytokinins in the shoot apical meristem.

This study investigates the consequences of endogenously enhanced biosynthesis of the plant hormone cytokinin in Arabidopsis thaliana (L.) Heynh. Transcriptional control of the bacterial ipt gene by the Drosophila melanogaster hsp70 promoter enabled temperature-dependent increased cytokinin production in transgenic plants. Heat-treated plants accumulated higher levels of unbound and bound zeatin-type cyto-kinins, the latter being preferentially N-conjugated glucosides. Cytokinin overproduction significantly increased the biomass of seedlings. Ipt transgenics had higher steady state mRNA levels of the shoot meristem specifying homeobox genes KNAT1 and STM, similar to the cytokinin-overproducing shoot meristem mutant amp1 (hpt, cop2, pt) This finding, together with previously described phenotypic similarities between transgenic cytokinin-overproducing plants and plants overexpressing the KNAT1 or KN1 genes, suggests that these factors act on the same pathway. We hypothesize that cytokinins act upstream of KNAT1 and STM. The influence of cytokinins on homeobox genes provides a link between the hormone and the developmental genes and indicates a role for cytokinins in the shoot apical meristem.

The LOXL2 Gene Encodes a New Lysyl Oxidase-like Protein and Is Expressed at High Levels in Reproductive Tissues

We have reported in this paper the complete cDNA sequence, gene structure, and tissue-specific expression of LOXL2, a new amine oxidase and a member of an emerging family of human lysyl oxidases. The predicted amino acid sequence, from several overlapping cDNA clones isolated from placenta and spleen cDNA libraries, shared extensive sequence homology with the conserved copper-binding and catalytic domains of both lysyl oxidase (LOX) and the lysyl oxidase-like (LOXL) protein. These conserved domains are encoded by five consecutive exons within the LOX, LOXL, and LOXL2 genes that also maintained exon-intron structure conservation. In contrast, six exons encoding the amino-terminal domains diverged both in sequence and structure. Exon 1 of the LOXL2 gene does not encode a signal sequence that is present in LOX and LOXL, suggesting a different processing and intracellular localization for this new protein. Expression of the LOXL2 gene was detected in almost all tissues with the highest steady state mRNA levels in the reproductive tissues, placenta, uterus and prostate. In situ hybridization identified placental syncytial and cytotrophoblasts responsible for the synthesis of LOXL2 mRNA and demonstrated a spatial and temporal expression pattern unique to the LOXL2 gene.

Nucleotide sequence of psbQ gene for 16-kDa protein of oxygen-evolving complex from Arabidopsis thaliana and regulation of its expression.

The psbQ gene encoding a 16-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana and characterized. The gene consists of a 28 nucleotide long leader sequence, two introns and three exons encoding a 223-amino-acid precursor polypeptide. The first 75 amino acids act as a transit peptide for the translocation of the polypeptide into the thylakoid lumen. Expression studies show that the gene is light-inducible and expresses only in green tissues with high steady-state mRNA levels in leaves. Using this gene as a probe, restriction fragment length polymorphism between two ecotypes, Columbia and Estland, has also been detected.

Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains.

A study was carried out to obtain more insight into the parameters that determine the secretion of heterologous proteins from filamentous fungi. A strategy was chosen in which the mRNA levels and protein levels of a number of heterologous genes of different origins were compared. All genes were under control of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) expression signals and were integrated in a single copy at the A. awamori pyrG locus. A Northern (RNA) analysis showed that large differences occurred in the steady-state mRNA levels obtained with the various genes; those levels varied from high values for genes of fungal origin (A. awamori 1,4-beta-endoxylanase A, Aspergillus niger glucoamylase, and Thermomyces lanuginosa lipase) to low values for genes of nonfungal origin (human interleukin 6 and Cyamopsis tetragonoloba [guar] alpha-galactosidase). With the C. tetragonoloba alpha-galactosidase wild-type gene full-length mRNA was even undetectable. Surprisingly, small amounts of full-length mRNA could be detected when a C. tetragonoloba alpha-galactosidase gene with an optimized Saccharomyces cerevisiae codon preference was expressed. In all cases except human interleukin 6, the protein levels corresponded to the amounts expected on basis of the mRNA levels. For human interleukin 6, very low protein levels were observed, whereas relatively high steady-state mRNA levels were obtained. Our data suggest that intracellular protein degradation is the most likely explanation for the low levels of secreted human interleukin 6.

Isolation and expression of an Arabidopsis 14-3-3-like protein gene

We have isolated an Arabidopsis gene, AFT1, which encodes a 14-3-3-like protein (AFT1). The wide distribution of the 14-3-3 protein family in eukaryotes and the high steady-state mRNA levels of the AFT1 gene in most tissues throughout Arabidopsis growth and development suggest that AFT1 plays an important role(s) in Arabidopsis. DNA blot analysis indicates that AFT1 is a single copy gene, therefore AFT1 should be a good target for studies involving antisense or sense RNA technologies as a means to determine AFT1's function in vivo.

Plasma lipopolysaccharide (LPS)-binding protein. A key component in macrophage recognition of gram-negative LPS.

LPS-binding protein (LBP) binds with high affinity (Kd approximately equal to 10(-9) M) to lipid A of LPS isolated from rough (R)- or smooth (S)-form Gram-negative bacteria as well as to lipid A partial structures such as precursor IVA. To define the role of LBP in regulating responses to LPS we have examined TNF release in rabbit peritoneal exudate macrophages (M phi) stimulated with LPS or with complete or partial lipid A preparations in the presence or absence of LBP. In the presence of LBP, M phi showed increased sensitivity to S- and R-form LPS as well as synthetic lipid A. Compared with LPS or lipid A, up to 1000-fold greater concentrations of partial lipid A structures were required to induce TNF production. However, consistent with our previous observations that these structures bind to LBP, TNF production was increased in the presence of LBP. In contrast, LBP did not enhance or inhibit TNF production produced by heat-killed Staphylococcus aureus, peptidoglycan isolated from S. aureus cell walls, or PMA. Potentiated M phi responsiveness to LPS was observed with as little as 1 ng LBP/ml. Heat-denatured LBP (which no longer binds LPS), BPI (an homologous LPS-binding protein isolated from neutrophils), or other serum proteins were without effect. LBP-treated M phi also showed a more rapid induction of cytokine mRNA (TNF and IL-1 beta), higher steady-state mRNA levels and increased TNF mRNA stability. These data provide additional evidence that LBP is part of a highly specific recognition system controlling M phi responses to LPS. The effects of LBP are lipid A dependent and importantly, extend to LPS preparations isolated from bacteria of R- and S-form phenotype.

Expression of antifreeze proteins in transgenic plants

The quality of frozen fruits and vegetables can be compromised by the damaging effects of ice crystal growth within the frozen tissue. Antifreeze proteins in the blood of some polar fishes have been shown to inhibit ice recrystallization at low concentrations. In order to determine whether expression of genes of this type confers improved freezing properties to plant tissue, we have produced transgenic tobacco and tomato plants which express genes encoding antifreeze proteins. The afa3 antifreeze gene was expressed at high steady-state mRNA levels in leaves from transformed plants, but we did not detect inhibition of ice recrystallization in tissue extracts. However, both mRNA and fusion proteins were detectable in transgenic tomato tissue containing a chimeric gene encoding a fusion protein truncated staphylococcal protein A and antifreeze protein. Furthermore, ice recrystallization inhibition was detected in this transgenic tissue.