Nandrolone decanoate does not enhance training effects but increases IGF-I mRNA in rat diaphragm.

To examine whether concomitant anabolic steroid treatment combined with training might enhance previously observed training effects (A. Bisschop, G. Gayan-Ramirez, H. Rollier, R. Gosselink, R. Dom, V. de Bock, and M. Decramer. Am. J. Respir. Crit. Care Med. 155: 1583-1589, 1997) and whether insulin-like growth factor I (IGF-I) was involved in these changes, male and female rats were submitted to inspiratory muscle training (IMT) for 8 wk (30 min/day, 5 times/wk) and were compared with untrained controls. During the last 5 wk of training, trained rats were divided to receive weekly either low-dose (LD; 1.5 mg/kg) or high-dose (HD; 7.5 mg/kg) nandrolone decanoate or saline for the IMT and control rats. In both sexes, diaphragm muscle mass and contractile properties were unchanged with treatment. In males, HD resulted in decreased diaphragm type I cross-sectional area (-15%; P < 0.05, HD vs. IMT), whereas no changes were observed in females. Finally, an increase in IGF-I mRNA levels was present in HD male (+73%; P < 0.05, HD vs. IMT) and female treated rats [LD (+58%) and HD (+96%) vs. IMT; P < 0.001]. We conclude that administration of nandrolone decanoate did not enhance the previously observed training effects in rat diaphragm, although it increased the IGF-I mRNA expression levels.     

The amino acid sequence of the light chain of human high-molecular-mass kininogen

The complete amino acid sequence of the light chain of human high-molecular-mass kininogen has been determined. The peptide chain contains 255 amino acid residues. The half-cystine, which forms the disulfide bridge to the heavy chain, was identified in position 225. Nine carbohydrate attachment sites were found. All carbohydrate side chains are O-glycosidically linked. Alignment of the present sequence with the bovine kininogen light chain sequence shows a high degree of homology, except for an extension of 22 amino acids within the histidine-rich part of the sequence. The histidine-rich region may have arisen by gene multiplication during evolution.     

Nuclear protein import, but not mRNA export, is defective in all Saccharomyces cerevisiae mutants that produce temperature-sensitive forms of the Ran GTPase homologue Gsp1p

A series of ts mutations in the GSP1 gene of Saccharomyces cerevisiae was isolated by error-prone PCR. A total of 25 ts gsp1 strains was obtained. Each of these mutants showed between one and seven different amino acid alterations. In several of these ts gsp1 strains, the same amino acid residues in Gsp1p were repeatedly mutated, indicating that our screen for ts gsp1 mutations was saturating. All of the ts gsp1 strains isolated had a defect in nuclear protein import, but only 16 of the 25 ts gsp1 strains had a defect in mRNA export. Thus, Gsp1p is suggested to be directly involved in nuclear protein import, but not in mRNA export. Following release from α-factor arrest, 11 of the ts gsp1 mutants arrested in G1; the remainder did not show any specific cell-cycle arrest, at 37°?C, the nonpermissive temperature. While the mutants that are defective in both mRNA export and protein import have a tendency to arrest in G1, there was no clear correlation between the cell cycle phenotype and the defects in mRNA export and nuclear protein import. Based on this, we assume that Ran/Gsp1p GTPase regulates the cell cycle and the nucleus/cytosol exchange of macromolecules through interactions with effectors that were independent of each other, and are differentially affected by mutation.     

The liver fluke Opisthorchis viverrini expresses nitric oxide synthase but not gelatinases

Host-parasite interaction during infection with the liver fluke Opisthorchis viverrini plays an important role in opisthorchiasis-associated cholangiocarcinoma via nitric oxide (NO) production. Host cells induce nitric oxide synthase (NOS)-dependent DNA damage and secrete Ras-related C3 botulinum toxin substrate (Rac)1, heme oxygenase (HO)-1, and gelatinases (matrix metalloproteinase (MMP)2 and MMP9). We evaluated whether these enzymes are expressed in O. viverrini. Colocalization of NOS and Rac1 was most prominently detected on day 30 post-infection (p.i.) in the gut, reproductive organ, eggs, acetabular and tegument. Expression of HO-1, an antioxidative enzyme, increased in a similar pattern to NOS, but was not present in the tegument. The levels of nitrate/nitrite, end products of NO, and ferric reducing antioxidant capacity, an indicator of antioxidant enzyme capacity, in parasite homogenates were highest on day 30 p.i. and then decreased on day 90 p.i. In contrast, zymography revealed that MMP2 and MMP9 were not present in parasite homogenates at all time points. In conclusion, O. viverrini induces NOS expression and NO production, but does not express gelatinases. The study may provide basic information and an insight into drug design for prevention and/or intervention approaches against O. viverrini infection.     

Clonal rat parathyroid cell line expresses a parathyroid hormone-related peptide but not parathyroid hormone itself

A novel parathyroid hormone-related peptide has been identified in tumors associated with the syndrome of humoral hypercalcemia of malignancy. Subsequently, mRNAs encoding this peptide have been found to be expressed in a number of normal tissues, including the parathyroids. Using Northern blotting, RNase protection, and immunochemical techniques, we examined a clonal rat parathyroid cell line originally developed as a model system for studying parathyroid cell physiology. We found that this line expresses the parathyroid hormone-related peptide but not parathyroid hormone itself. Secretion of the parathyroid hormone-related peptide varied inversely with extracellular calcium concentration, but neither calcium nor 1,25-dihydroxyvitamin D3 appeared to influence steady-state parathyroid hormone-related peptide mRNA levels. This clonal line may prove to be an interesting system for studying the factors responsible for tissue-specific parathyroid hormone and parathyroid hormone-related peptide gene expression.     

Vagotomy and accompanying pyloroplasty down-regulates ghrelin mRNA but does not affect ghrelin secretion

In this study, we have examined how the lack of vagus activity affects the long-term secretion of total and active ghrelin. We subjected mice to sham-operation, pyloroplasty or vagotomy and pyloroplasty. The study lasted for 2 weeks, during which body weight development and daily food intake was monitored. At the end of the study, the mice were sacrificed, and serum and fundus were collected. Measurements of total and active serum ghrelin revealed no difference between the surgical groups and sham-operated mice, despite the fact that fundic ghrelin mRNA was down-regulated. The results presented here suggest that the vagus activity is not required for the long-term secretion of neither total nor active ghrelin in mice. They also suggest that fundic ghrelin mRNA expression is affected by pyloroplasty and vagotomy but that this effect does not translate into changes in ghrelin levels in the circulation.     

Nuclear protein import, but not mRNA export, is defective in all Saccharomyces cerevisiae mutants that produce temperature-sensitive forms of the Ran GTPase homologue Gsp1p

A series of ts mutations in the GSP1 gene of Saccharomyces cerevisiae was isolated by error-prone PCR. A total of 25 ts gsp1 strains was obtained. Each of these mutants showed between one and seven different amino acid alterations. In several of these ts gsp1 strains, the same amino acid residues in Gsp1p were repeatedly mutated, indicating that our screen for ts gsp1 mutations was saturating. All of the ts gsp1 strains isolated had a defect in nuclear protein import, but only 16 of the 25 ts gsp1 strains had a defect in mRNA export. Thus, Gsp1p is suggested to be directly involved in nuclear protein import, but not in mRNA export. Following release from α-factor arrest, 11 of the ts gsp1 mutants arrested in G1; the remainder did not show any specific cell-cycle arrest, at 37° C, the nonpermissive temperature. While the mutants that are defective in both mRNA export and protein import have a tendency to arrest in G1, there was no clear correlation between the cell cycle phenotype and the defects in mRNA export and nuclear protein import. Based on this, we assume that Ran/Gsp1p GTPase regulates the cell cycle and the nucleus/cytosol exchange of macromolecules through interactions with effectors that were independent of each other, and are differentially affected by mutation. Received: 30 June 1997 / Accepted: 23 October 1997     

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins

When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper, we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein are present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon’s neuronal cell bodies, but despite the presence of NF mRNA in the giant axon no labeled NF protein is detected in the giant axon. This lends support to the glia–axon protein transfer hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.     

The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation

The untranslated leader RNA is the most conserved part of the human immunodeficiency virus type I (HIV-1) genome. It contains many regulatory motifs that mediate a variety of steps in the viral life cycle. Previous work showed that the full-length leader RNA can adopt two alternative structures: a long distance interaction (LDI) and a branched multiple-hairpin (BMH) structure. The BMH structure exposes the dimer initiation site (DIS) hairpin, whereas this motif is occluded in the LDI structure. Consequently, these structures differ in their capacity to form RNA dimers in vitro. The BMH structure is dimerization-competent, due to DIS hairpin formation, but also presents the splice donor (SD) and RNA packaging (Psi) hairpins. In the LDI structure, an extended RNA packaging (Psi(E)) hairpin is folded, which includes the splice donor site and gag coding sequences. The gag initiation codon is engaged in a long distance base pairing interaction with sequences in the upstream U5 region in the BMH structure, thus forming the evolutionarily conserved U5-AUG duplex. Therefore, the LDI-BMH equilibrium may affect not only the process of RNA dimer formation but also translation initiation. In this study, we designed mutations in the 3'-terminal region of the leader RNA that alter the equilibrium of the LDI-BMH structures. The mutant leader RNAs are affected in RNA dimer formation, but not in their translation efficiency. These results indicate that the LDI-BMH status does not regulate HIV-1 RNA translation, despite the differential presentation of the gag initiation codon in both leader RNA structures.     

Nigrostriatal denervation does not affect glutamate transporter mRNA expression but subsequent levodopa treatment selectively increases GLT1 mRNA and protein expression in the rat striatum

There is growing evidence that the loss of the nigrostriatal dopaminergic neurones induces an overactivity of the corticostriatal glutamatergic pathway which seems to be central to the physiopathology of parkinsonism. Moreover, glutamatergic mechanisms involving NMDA receptors have been shown to interfere with the therapeutical action of levodopa. Given the key role played by uptake processes in glutamate neurotransmission, this study examined the effects of nigrostriatal deafferentation and of levodopa treatment on the striatal expression of the glutamate transporters GLT1, GLAST and EAAC1 in the rat. No significant changes in striatal mRNA levels of these transporters were detected after either levodopa treatment (100 mg/kg; i.p., twice a day for 21 days) or unilateral lesion of the nigrostriatal pathway by intranigral 6-hydroxydopamine injection. In contrast, animals with the lesion subsequently treated with levodopa showed a selective increase (36%) in GLT1 mRNA levels in the denervated striatum versus controls. These animals also showed increased GLT1 protein expression, as assessed by immunostaining and western blotting. These data provide the first evidence that levodopa therapy may interfere with striatal glutamate transmission through change in expression of the primarily glial glutamate transporter GLT1. We further suggest that levodopa-induced GLT1 overexpression may represent a compensatory mechanism preventing neurotoxic accumulation of endogenous glutamate.     

Trigger factor depletion or overproduction causes defective cell division but does not block protein export.

Trigger factor is an abundant cytosolic protein of Escherichia coli which can stabilize proOmpA for in vitro translocation across inner membrane vesicles. The gene encoding E. coli trigger factor was isolated and sequenced, allowing construction of strains in which the expression of trigger factor is readily regulated. We found no defect in the in vivo rate of synthesis or secretion of proOmpA in trigger factor-depleted cells. The primary physiological defect in trigger factor-depleted or -overproducing cells is an enrichment of filamented cells. Filamentation of the trigger factor-overproducing strain is suppressed by a multicopy plasmid expressing the essential division gene ftsZ, suggesting that trigger factor has an important role in cell division.     

A diet high in cholesterol and deficient in vitamin E induces lipid peroxidation but does not enhance antioxidant enzyme expression in rat liver

Expression of antioxidant enzymes (AOE), an important mechanism in the protection against oxidative stress, could be modified by the redox status of the cells. The aim of this project was to evaluate the role of vitamin E deficiency in association with a high-cholesterol diet in the hepatic lipid peroxidation and the expression of AOE. Two groups of 6 male rats were fed with a high-cholesterol or a high-cholesterol vitamin E-deficient diet. All animals were sacrificed at 72 days of treatment. Liver lipid peroxidation index (Malondialdehyde; MDA) and hepatic AOE were evaluated. Total liver RNA was extracted, and the steady state messenger RNA (mRNA) levels of glutathion peroxydase, manganese superoxide dismutase, Cu/Zn superoxide dismutase and catalase were examined by northern blot. After 72 days on the diet, a significant increase in the lipid peroxidation index was observed in the vitamin E deficient group (MDA : 4.45 +/- 0.29 nmol/mg protein versus 3.65 +/- 0.1 nmol/mg protein in vitamin E normal group). Despite this oxidative stress, the activities and mRNA levels of liver AOE were not significantly different in the 2 groups. These preliminary results show that chronic vitamin E deficiency associated with high cholesterol diet is able to increase lipid peroxidation without modulation of AOE expression and activity in the liver. This suggests that beneficial effects of dietary vitamin E are due to a plasma antioxidant effect or a cell mediated action, rather than to a specific modulation of cellular enzymes.     

Age does not alter protein kinase C isozymes mRNA expression in rat brain

Calcium and phospholipid dependent Protein kinase C (PKC) may play a role in memory function and pathogenesis of many neurodegenerative disorders such as Alzheimer's disease (AD). Abnormal phosphorylation by PKC as well as reduced levels of PKC has been implicated in the neurodegeneration associated with AD and aging. Recently, many subtypes of PKC isozymes have been identified by molecular biology techniques which are expressed differentially in various regions of the brain. The reduction and alterations in the activities and distribution of these subtypes of PKC isozymes may be accountable for the decline of selective neurons during aging. In order to investigate the role of PKC isozymes during aging, we examined the distribution of PKC-, , and mRNA, expressions between young (4 months) and old (25 months) rat brains using in situ hybridization histochemistry. Our studies showed that signals of three isoforms of PKC mRNA vary in cortical and hippocampal regions. However, no change was detected in any of the PKC isoforms mRNA expressions in aged animals.     

Diurnal Lhc gene expression is present in many but not all species of the plant kingdom

The diurnal and circadian expression of light-harvesting genes (Lhc) is well documented for many plant species of the Angiospermae division. Here we present the diurnal mRNA levels of species of the Gymnospermae, Pteridophyta, Bryophyta and Phycophyta divisions. Except for four Coniferophytina species, diurnal Lhc mRNA accumulation is detected in fern, moss and algae, supporting the idea that the concept of circadian clock-controlled gene expression is an ancient process. Possible reasons why plants need the circadian clock control mechanism are discussed.Dedicated to Dr H. W. Heldt on the occasion of his 60th birthday.     

Nuclear export of mRNA by TAP/NXF1 requires two nucleoporin-binding sites but not p15

Metazoan NXF1/p15 heterodimers promote export of bulk mRNA through nuclear pore complexes (NPC). NXF1 interacts with the NPC via two distinct structural domains, the UBA-like domain and the NTF2-like scaffold, which results from the heterodimerization of the NTF2-like domain of NXF1 with p15. Both domains feature a single nucleoporin-binding site, and they act synergistically to promote NPC translocation. Whether the NTF2-like scaffold (and thereby p15) contributes only to NXF1/NPC association or is also required for other functions, e.g., to impart directionality to the export process by regulating NXF1/NPC or NXF1/cargo interactions, remains unresolved. Here we show that a minimum of two nucleoporin-binding sites is required for NXF1-mediated export of cellular mRNA. These binding sites can be provided by an NTF2-like scaffold followed by a UBA-like domain (as in the wild-type protein) or by two NTF2-like scaffolds or two UBA-like domains in tandem. In the latter case, the export activity of NXF1 is independent of p15. Thus, as for the UBA-like domain, the function of the NTF2-like scaffold is confined to nucleoporin binding. More importantly, two copies of either of these domains are sufficient to promote directional transport of mRNA cargoes across the NPC.     

The NES-Crm1p export pathway is not a major mRNA export route in Saccharomyces cerevisiae.

Nuclear export signal (NES)-containing proteins are recognized by the NES receptor CRM1/Crm1p (also called exportin 1/Xpo1p). In vertebrates and Schizosaccharomyces pombe, the toxin leptomycin B (LMB) inhibits CRM1-mediated export by interacting directly with CRM1 and disrupting the trimeric Ran-GTP-CRM1-NES export complex. In Saccharomyces cerevisiae, LMB is not toxic and is apparently unable to interact with Crm1p. A second difference between the systems is that LMB has no effect on mRNA export in vertebrate systems, whereas there is evidence that S.cerevisiae Crm1p plays a role in mRNA export. Here we show that a single amino acid change converts S. cerevisiae Crm1p from being LMB insensitive to fully LMB sensitive, indicating that Crm1p is the only relevant LMB target. This new strain has no phenotype, but LMB has a rapid and potent inhibitory effect on NES-mediated export. In situ hybridization assays show that LMB also causes nuclear accumulation of poly(A)+ RNA but with a significant delay compared with the effect on NES-mediated export. Biochemical assays indicate little or no LMB effect on cytoplasmic protein synthesis, indicating that the NES-Crm1p pathway is not a major mRNA export route in S.cerevisiae. We conclude that Crm1p structure and function is conserved from S.cerevisiae to man.     

Primase p49 mRNA expression is serum stimulated but does not vary with the cell cycle.

Expression of the small-subunit p49 mRNA of primase, the enzyme that synthesizes oligoribonucleotides for initiation of DNA replication, was examined in mouse cells stimulated to proliferate by serum and in growing cells. The level of p49 mRNA increased approximately 10-fold after serum stimulation and preceded synthesis of DNA and histone H3 mRNA by several hours. Expression of p49 mRNA was not sensitive to inhibition by low concentrations of cycloheximide, which suggested that the increase in mRNA occurred before the restriction point control for cell cycle progression described for mammalian cells and was not under its control. p49 mRNA levels were not coupled to DNA synthesis, as observed for the replication-dependent histone genes, since hydroxyurea or aphidicolin had no effect on p49 mRNA levels when added before or during S phase. These inhibitors did have an effect, however, on the stability of p49 mRNA and increased the half-life from 3.5 h to about 20 h, which suggested an interdependence of p49 mRNA degradation and DNA synthesis. When growing cells were examined after separation by centrifugal elutriation, little difference was detected for p49 mRNA levels in different phases of the cell cycle. This was also observed when elutriated G1 cells were allowed to continue growth and then were blocked in M phase with colcemid. Only a small decrease in p49 mRNA occurred, whereas H3 mRNA rapidly decreased, when cells entered G2/M. These results indicate that the level of primase p49 mRNA is not cell cycle regulated but is present constitutively in proliferating cells.     

Up-regulation of IL-18BP,but not IL-18 mRNA in rat liver by LPS

Interleukin (IL)-18 is a pro-inflammatory cytokine that plays a critical role in inflammation leading to liver damage, through promotion of Fas-mediated apoptosis. Inhibition of IL-18 activity protects against LPS-induced lethality in mice and against liver damage induced by LPS after sensitisation of mice with Proprionibacterium acnes. A specific, potent, endogenous inhibitor of IL-18 (IL-18BP) has been identified in mice and humans, and IL-18BP mRNA is expressed constitutively in liver. The objectives of this study were to compare changes in IL-1beta and IL-18 mRNA expression in the liver of rats in response to peripheral injection of LPS, using real-time PCR, and also to investigate whether IL-18BP mRNA expression is affected by this treatment. LPS rapidly up-regulated IL-1beta mRNA expression, but IL-18 mRNA expression was unaffected by LPS treatment. Unlike IL-18, IL-18BP mRNA was up-regulated dramatically by approximately 12-fold above nai;ve levels, peaking 3 h after LPS injection. This ability of LPS to up-regulate expression of the endogenous IL-18 inhibitor may indicate a mechanism by which the inflammatory response to LPS is regulated.