Controlled expression of recombinant proteins in Physcomitrella patens by a conditional heat-shock promoter: a tool for plant research and biotechnology.

The ability to express tightly controlled amounts of endogenous and recombinant proteins in plant cells is an essential tool for research and biotechnology. Here, the inducibility of the soybean heat-shock Gmhsp17.3B promoter was addressed in the moss Physcomitrella patens, using beta-glucuronidase (GUS) and an F-actin marker (GFP-talin) as reporter proteins. In stably transformed moss lines, Gmhsp17.3B-driven GUS expression was extremely low at 25 degrees C. In contrast, a short non-damaging heat-treatment at 38 degrees C rapidly induced reporter expression over three orders of magnitude, enabling GUS accumulation and the labelling of F-actin cytoskeleton in all cell types and tissues. Induction levels were tightly proportional to the temperature and duration of the heat treatment, allowing fine-tuning of protein expression. Repeated heating/cooling cycles led to the massive GUS accumulation, up to 2.3% of the total soluble proteins. The anti-inflammatory drug acetyl salicylic acid (ASA) and the membrane-fluidiser benzyl alcohol (BA) also induced GUS expression at 25 degrees C, allowing the production of recombinant proteins without heat-treatment. The Gmhsp17.3B promoter thus provides a reliable versatile conditional promoter for the controlled expression of recombinant proteins in the moss P. patens.

Phosphorylation of neurofilament subunit NF-M is regulated by activation of NMDA receptors and modulates cytoskeleton stability and neuronal shape.

The cytoskeleton is essential for the structural organization of neurons and is influenced during development by excitatory stimuli such as activation of glutamate receptors. In particular, NMDA receptors are known to modulate the function of several cytoskeletal proteins and to influence cell morphology, but the underlying molecular and cellular mechanisms remain unclear. Here, we characterized the neurofilament subunit NF-M in cultures of developing mouse cortical neurons chronically exposed to NMDA receptor antagonists. Western blots analysis showed that treatment of cortical neurons with MK801 or AP5 shifted the size of NF-M towards higher molecular weights. Dephosphorylation assay revealed that this increased size of NF-M observed after chronic exposure to NMDA receptor antagonists was due to phosphorylation. Neurons treated with cyclosporin, an inhibitor of the Ca(2+)-dependent phosphatase calcineurin, also showed increased levels of phosphorylated NF-M. Moreover, analysis of neurofilament stability revealed that the phosphorylation of NF-M, resulting from NMDA receptor inhibition, enhanced the solubility of NF-M. Finally, cortical neurons cultured in the presence of the NMDA receptor antagonists MK801 and AP5 grew longer neurites. Together, these data indicate that a blockade of NMDA receptors during development of cortical neurons increases the phosphorylation state and the solubility of NF-M, thereby favoring neurite outgrowth. This also underlines that dynamics of the neurofilament and microtubule cytoskeleton is fundamental for growth processes.

Bone metabolism and risk of secondary hyperparathyroidism 12 months after gastric banding in obese pre-menopausal women.

OBJECTIVE: To evaluate, during the first postoperative year in obese pre-menopausal women, the effects of laparoscopic gastric banding on calcium and vitamin D metabolism, the potential modifications of bone mineral content and bone mineral density, and the risk of development of secondary hyperparathyroidism. SUBJECTS: Thirty-one obese pre-menopausal women aged between 25 and 52 y with a mean body mass index (BMI) of 43.6 kg/m(2), scheduled for gastric banding were included. Patients with renal, hepatic, metabolic and bone disease were excluded. METHODS: Body composition and bone mineral density (BMD) were measured at baseline, 6 and 12 months after gastric banding using dual-energy X-ray absorptiometry. Serum calcium, phosphate, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, bilirubin, urea, creatinine, uric acid, proteins, parathormone, vitamin D(3), IGF-1, IGF-BP3 and telopeptide, as well as urinary telopeptide, were measured at baseline and 1, 3, 6, 9 and 12 months after surgery. RESULTS: After 1 y vitamin D3 remained stable and PTH decreased by 12%, but the difference was not significant. Serum telopeptide C increased significantly by 100% (P<0.001). There was an initial drop of the IGF-BP3 during the first 6 months (P<0.05), but the reduction was no longer significant after 1 y. The BMD of cortical bone (femoral neck) decreased significantly and showed a trend of a positive correlation with the increase of telopeptides (P<0.06). The BMD of trabecular bone, at the lumbar spine, increased proportionally to the reduction of hip circumference and of body fat. CONCLUSION: There is no evidence of secondary hyperparathyroidism 1 y after gastric banding. Nevertheless biochemical bone markers show a negative remodelling balance, characterized by an increase of bone resorption. The serum telopeptide seems to be a reliable parameter, not affected by weight loss, to follow up bone turnover after gastroplasty.

Characterization of sustained BOLD activation in the rat barrel cortex and neurochemical consequences.

To date, only a couple of functional MR spectroscopy (fMRS) studies were conducted in rats. Due to the low temporal resolution of (1)H MRS techniques, prolonged stimulation paradigms are necessary for investigating the metabolic outcome in the rat brain during functional challenge. However, sustained activation of cortical areas is usually difficult to obtain due to neural adaptation. Anesthesia, habituation, high variability of the basal state metabolite concentrations as well as low concentrations of the metabolites of interest such as lactate (Lac), glucose (Glc) or γ-aminobutyric acid (GABA) and small expected changes of metabolite concentrations need to be addressed. In the present study, the rat barrel cortex was reliably and reproducibly activated through sustained trigeminal nerve (TGN) stimulation. In addition, TGN stimulation induced significant positive changes in lactate (+1.01μmol/g, p<0.008) and glutamate (+0.92μmol/g, p<0.02) and significant negative aspartate changes (-0.63μmol/g, p<0.004) using functional (1)H MRS at 9.4T in agreement with previous changes observed in human fMRS studies. Finally, for the first time, the dynamics of lactate, glucose, aspartate and glutamate concentrations during sustained somatosensory activation in rats using fMRS were assessed. These results allow demonstrating the feasibility of fMRS measurements during prolonged barrel cortex activation in rats.

Review on polyhydroxyalkanoate formation in the model plant Arabidopsis thaliana

In order to explore potential alternatives to the production of polyhydroxyalkanoates (PHAs) in bacteria, the enzymes of Alcaligenes eutrophus involved in the synthesis of polyhydroxybutyrate (PHB) have been expressed in the model plant Arabidopsis thaliana. Following the successful production of low amounts of high molecular weight PHB in plants expressing the acetoacetyl-CoA reductase and the PHB synthase in the cytoplasm of Arabidopsis cell, expression of the PHB pathway in the pastids was achieved by modifying the PHB enzymes with plastid targeting signals. This strategy resulted in a significant increase in the formation of PHB in Arabidopsis, with a maximum of 14% of the leaf dry weight . The increase in PHB production is most likely due to the higher flux in the plastids of acetyl-CoA, the precursor for PHB synthesis. A detailed study of metabolic fluxes in Arabidopsis plants producing high levels of PHB could help to determine the potential problems and limitations of PHB synthesis in Arabidopsis and could be useful for optimising strategies for the production of PHB in crop plants. The knowledge on PHB production could also be used for the production of PHAs other than PHB. Apart from PHB, no other PHAs have been produced in an eukaryotic system. Arabidopsis will therefore be used as a model system for the production in eukaryotes of more complex PHAs, such as poly(hydroxybutyrate-co-hydroxyvylerate) or medium-chain-lenght-PHAs.

Chemical Functionalization of Bioceramics To Enhance Endothelial Cells Adhesion for Tissue Engineering.

To control the selective adhesion of human endothelial cells and human serum proteins to bioceramics of different compositions, a multifunctional ligand containing a cyclic arginine-glycine-aspartate (RGD) peptide, a tetraethylene glycol spacer, and a gallate moiety was designed, synthesized, and characterized. The binding of this ligand to alumina-based, hydroxyapatite-based, and calcium phosphate-based bioceramics was demonstrated. The conjugation of this ligand to the bioceramics induced a decrease in the nonselective and integrin-selective binding of human serum proteins, whereas the binding and adhesion of human endothelial cells was enhanced, dependent on the particular bioceramics.

Idarubicin-loaded beads for chemoembolisation of hepatocellular carcinoma: results of the IDASPHERE phase I trial.

BACKGROUND: A phase I dose-escalation trial of transarterial chemoembolisation (TACE) with idarubicin-loaded beads was performed in cirrhotic patients with hepatocellular carcinoma (HCC). AIM: To estimate the maximum-tolerated dose (MTD) and to assess safety, efficacy, pharmacokinetics and quality of life. METHODS: Patients received a single TACE session with injection of 2 mL drug-eluting beads (DEBs; DC Bead 300-500 μm) loaded with idarubicin. The idarubicin dose was escalated according to a modified continuous reassessment method. MTD was defined as the dose level closest to that causing dose-limiting toxicity (DLT) in 20% of patients. RESULTS: Twenty-one patients were enrolled, including nine patients at 5 mg, six patients at 10 mg, and six patients at 15 mg. One patient at each dose level experienced DLT (acute myocardial infarction, hyperbilirubinaemia and elevated aspartate aminotransferase (AST) at 5-, 10- and 15-mg, respectively). The calculated MTD of idarubicin was 10 mg. The most frequent grade ≥3 adverse events were pain, elevated AST, elevated γ-glutamyltranspeptidase and thrombocytopenia. At 2 months, the objective response rate was 52% (complete response, 28%, and partial response, 24%) by modified Response Evaluation Criteria in Solid Tumours. The median time to progression was 12.1 months (95% CI 7.4 months - not reached); the median overall survival was 24.5 months (95% CI 14.7 months - not reached). Pharmacokinetic analysis demonstrated the ability of DEBs to release idarubicin slowly. CONCLUSIONS: Using drug-eluting beads, the maximum-tolerated dose of idarubicin was 10 mg per TACE session. Encouraging responses and median time to progression were observed. Further clinical investigations are warranted (NCT01040559).

Role of the c-Jun N-terminal kinase pathway in retinal excitotoxicity, and neuroprotection by its inhibition.

J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06705.x Abstract Retinal excitotoxicity is associated with retinal ischemia, and with glaucomatous and traumatic optic neuropathy. The present study investigates the role of c-Jun N-terminal kinase (JNK) activation in NMDA-mediated retinal excitotoxicity and determines whether neuroprotection can be obtained with the JNK pathway inhibitor, d-form of JNK-inhibitor 1 (d-JNKI-1). Young adult rats received intravitreal injections of 20 nmol NMDA, which caused extensive neuronal death in the inner nuclear and ganglion cell layers. This excitotoxicity was associated with strong activation of calpain, as revealed by fodrin cleavage, and of JNK. The cell-permeable peptide d-JNKI-1 was used to inhibit JNK. Within 40 min of its intravitreal injection, FITC-labeled d-JNKI-1 spread through the retinal ganglion cell layer into the inner nuclear layer and interfered with the NMDA-induced phosphorylation of JNK. Injections of unlabeled d-JNKI-1 gave unprecedentedly strong neuroprotection against cell death in both layers, lasting for at least 10 days. The NMDA-induced calpain-specific fodrin cleavage was likewise strongly inhibited by d-JNKI-1. Moreover the electroretinogram was partially preserved by d-JNKI-1. Thus, the JNK pathway is involved in NMDA-mediated retinal excitotoxicity and JNK inhibition by d-JNKI-1 provides strong neuroprotection as shown morphologically, biochemically and physiologically.

Short-term oleoyl-estrone treatment affects capacity to manage lipids in rat adipose tissue

Background: Short-term OE (oleoyl-estrone) treatment causes significant decreases in rat weight mainly due to adipose tissue loss. The aim of this work was to determine if OE treatment affects the expression of genes that regulate lipid metabolism in white adipose tissue. Results: Gene expression in adipose tissue from female treated rats (48 hours) was analysed by hybridization to cDNA arrays and levels of specific mRNAs were determined by real-time PCR. Treatment with OE decreased the expression of 232 genes and up-regulated 75 other genes in mesenteric white adipose tissue. The use of real-time PCR validate that, in mesenteric white adipose tissue, mRNA levels for Lipoprotein Lipase (LPL) were decreased by 52%, those of Fatty Acid Synthase (FAS) by 95%, those of Hormone Sensible Lipase (HSL) by 32%, those of Acetyl CoA Carboxylase (ACC) by 92%, those of Carnitine Palmitoyltransferase 1b (CPT1b) by 45%, and those of Fatty Acid Transport Protein 1 (FATP1) and Adipocyte Fatty Acid Binding Protein (FABP4) by 52% and 49%, respectively. Conversely, Tumour Necrosis Factor (TNF¿) values showed overexpression (198%). Conclusion: Short-term treatment with OE affects adipose tissue capacity to extract fatty acids from lipoproteins and to deal with fatty acid transport and metabolism.

Semiquantitative RT-PCR measurement of gene expression in rat tissues including a correction for varying cell size and number

Background: Current methodology of gene expression analysis limits the possibilities of comparison between cells/tissues of organs in which cell size and/or number changes as a consequence of the study (e.g. starvation). A method relating the abundance of specific mRNA copies per cell may allow direct comparison or different organs and/or changing physiological conditions. Methods: With a number of selected genes, we analysed the relationship of the number of bases and the fluorescence recorded at a present level using cDNA standards. A lineal relationship was found between the final number of bases and the length of the transcript. The constants of this equation and those of the relationship between fluorescence and number of bases in cDNA were determined and a general equation linking the length of the transcript and the initial number of copies of mRNA was deduced for a given pre-established fluorescence setting. This allowed the calculation of the concentration of the corresponding mRNAs per g of tissue. The inclusion of tissue RNA and the DNA content per cell, allowed the calculation of the mRNA copies per cell. Results: The application of this procedure to six genes: Arbp, cyclophilin, ChREBP, T4 deiodinase 2, acetyl-CoA carboxylase 1 and IRS-1, in liver and retroperitoneal adipose tissue of food-restricted rats allowed precise measures of their changes irrespective of the shrinking of the tissue, the loss of cells or changes in cell size, factors that deeply complicate the comparison between changing tissue conditions. The percentage results obtained with the present methods were essentially the same obtained with the delta-delta procedure and with individual cDNA standard curve quantitative RT-PCR estimation. Conclusion: The method presented allows the comparison (i.e. as copies of mRNA per cell) between different genes and tissues, establishing the degree of abundance of the different molecular species tested.

Activities of amino acid metabolizing enzymes in the stomach and small intestitine of developing rats

The activities of aspartate and alanine transaminase, serine dehydratase, arginase, glutamate dehydrogenase, adenylate deaminase and glutamine synthetase were determined in the stomach and small intestine of developing rats. Despite the common embryonic origin of the intestine and stomach, their enzymes showed quite different activity levels and patterns of development, depending on their roles. Most enzyme activities were low during late intrauterine life and after birth, attaining adult levels with the change of diet at weaning. No arginase activity was found in the stomach and no changes were detected in adenylate deaminase in the stomach or intestine throughout the period studied. Alanine transaminase, serine dehydratase and, to some extent, glutamine synthetase levels, significantly higher in late intrauterine life, decreased after birth, suggesting that the foetal stomach has a transient ability to handle amino acids.

Glutamatergic and GABAergic energy metabolism measured in the rat brain by (13) C NMR spectroscopy at 14.1 T.

Energy metabolism supports both inhibitory and excitatory neurotransmission processes. This study investigated the specific contribution of astrocytic metabolism to γ-aminobutyric acid (GABA) synthesis and inhibitory GABAergic neurotransmission that remained to be ilucidated in vivo. Therefore, we measured (13) C incorporation into brain metabolites by dynamic (13) C nuclear magnetic resonance spectroscopy at 14.1 T in rats under α-chloralose anaesthesia during infusion of [1,6-(13) C]glucose. The enhanced sensitivity at 14.1 T allowed to quantify incorporation of (13) C into the three aliphatic carbons of GABA non-invasively. Metabolic fluxes were determined with a mathematical model of brain metabolism comprising glial, glutamatergic and GABAergic compartments. GABA synthesis rate was 0.11 ± 0.01 μmol/g/min. GABA-glutamine cycle was 0.053 ± 0.003 μmol/g/min and accounted for 22 ± 1% of total neurotransmitter cycling between neurons and glia. Cerebral glucose oxidation was 0.47 ± 0.02 μmol/g/min, of which 35 ± 1% and 7 ± 1% was diverted to the glutamatergic and GABAergic tricarboxylic acid cycles, respectively. The remaining fraction of glucose oxidation was in glia, where 12 ± 1% of the TCA cycle flux was dedicated to oxidation of GABA. 16 ± 2% of glutamine synthesis was provided to GABAergic neurons. We conclude that substantial metabolic activity occurs in GABAergic neurons and that glial metabolism supports both glutamatergic and GABAergic neurons in the living rat brain. We performed (13) C NMR spectroscopy in vivo at high magnetic field (14.1 T) upon administration of [1,6-(13) C]glucose. This allowed to measure (13) C incorporation into the three aliphatic carbons of GABA in the rat brain, in addition to those of glutamate, glutamine and aspartate. These data were then modelled to determine fluxes of energy metabolism in GABAergic and glutamatergic neurons and glial cells.

Severe transient neonatal lactic acidosis during prophylactic zidovudine treatment.

Zidovudine (ZDV) treatment during pregnancy, delivery and the postnatal period is effective in reducing the maternal-infant transmission of the human immunodeficiency virus. Reported adverse effects in the neonate during this longterm treatment are bone marrow suppression and elevation in aspartate aminotransferase activity. We report a case of severe ZDV-associated lactic acidosis in a neonate, which resolved rapidly following discontinuation of ZDV. The mechanisms leading to this side effect are poorly understood.

Hypoxia-induced inhibition of epithelial Na(+) channels in the lung. Role of Nedd4-2 and the ubiquitin-proteasome pathway.

Transepithelial sodium transport via alveolar epithelial Na(+) channels (ENaC) and Na(+),K(+)-ATPase constitutes the driving force for removal of alveolar edema fluid. Alveolar hypoxia associated with pulmonary edema may impair ENaC activity and alveolar Na(+) absorption through a decrease of ENaC subunit expression at the apical membrane of alveolar epithelial cells (AECs). Here, we investigated the mechanism(s) involved in this process in vivo in the β-Liddle mouse strain mice carrying a truncation of β-ENaC C-terminus abolishing the interaction between β-ENaC and the ubiquitin protein-ligase Nedd4-2 that targets the channel for endocytosis and degradation and in vitro in rat AECs. Hypoxia (8% O2 for 24 h) reduced amiloride-sensitive alveolar fluid clearance by 69% in wild-type mice but had no effect in homozygous mutated β-Liddle littermates. In vitro, acute exposure of AECs to hypoxia (0.5-3% O2 for 1-6 h) rapidly decreased transepithelial Na(+) transport as assessed by equivalent short-circuit current Ieq and the amiloride-sensitive component of Na(+) current across the apical membrane, reflecting ENaC activity. Hypoxia induced a decrease of ENaC subunit expression in the apical membrane of AECs with no change in intracellular expression and induced a 2-fold increase in α-ENaC polyubiquitination. Hypoxic inhibition of amiloride-sensitive Ieq was fully prevented by preincubation with the proteasome inhibitors MG132 and lactacystin or with the antioxidant N-acetyl-cysteine. Our data strongly suggest that Nedd4-2-mediated ubiquitination of ENaC leading to endocytosis and degradation of apical Na(+) channels is a key feature of hypoxia-induced inhibition of transepithelial alveolar Na(+) transport.

Liver, gastrointestinal and cardiac toxicity in intermediate hepatocellular carcinoma treated with PRECISION TACE with drug-eluting beads: Results from the PRECISION V randomized trial : B-240

Purpose: To evaluate the toxicity focussing on hepatic, gastrointestinal and cardiac parameters following PRECISION TACE with DC Bead? versus conventional transarterial chemoembolization (cTACE) in the treatment of intermediate-stage hepatocellular carcinoma (HCC). Methods and Materials: This prospective, randomized, multicentre study was conducted under best practice trial management and authorized by local institutional review boards. Informed consent was obtained. 212 patients (185 men/27 women; mean: 67 years) were randomized to be treated with DC Beads? or cTACE. The majority of both groups presented in a more advanced stage. Safety was measured by rate of adverse events (South West Oncology Group criteria) and changes in laboratory parameters. Cardiotoxicity was assessed by means of left ventricular ejection fraction (LVEF) in MRI or echocardiography. The results of the two groups were compared using the chi-square test and Student`s t-test. Results: Mean maximum alanine transaminase increase in the DC Bead group was 50% in the cTACE group (p < 0.001) and 59% for aspartate transaminase (p < 0.001). For bilirubin, mean increase was 5.30±15.13 vs. 13.53±73.89 µmol/L. Concerning gastrointestinal disorders, 120 adverse events (AEs) occurred in 57/93 (61.3%) patients in the DC Bead group vs. 114 in 49/108 (45.4%) in cTACE. Concerning hepatobiliary disorders, serious AEs occurred in 8/93 (8.6%) vs. 11/108 (10.2%) patients. LVEF showed an increase in the DC Bead group by +2.7±10.1 percentage points and a small decrease by -1.5±7.6 in the cTACE group, p=0.018. Conclusion: PRECISION TACE is safe, even in more advanced HCC patients. Serious liver and cardiac toxicity were significantly lower in the DC Bead group.