Polyadenylated mRNA for the light-harvesting chlorophyll a/b protein

In thylakoid membranes isolated from green plants of parsley, pea, and barley, the light-harvesting chlorophyll a/b protein complex (LHCP, mol. weight: 25,000), is a major constituent. Poly(A)RNA isolated from these species was translated in a wheat germ, cell-free system. The in vitro translation products were treated with antibodies raised against the LHCP. This treatment resulted in the precipitation of a precursor protein (mol. weight: 29,000). Poly(A)RNA was also prepared from a cell culture ofPetroselinum that does not develop chloroplasts upon illumination. This poly(A)RNA is capable of stimulating amino acid incorporation in the in vitro translation system, however, it does not direct the synthesis of LHCP.     

Regulation of levels of nuclear transcripts for C4 photosynthesis in bundle sheath and mesophyll cells of maize leaves

In vitro translation of polyA⁺ mRNAs isolated from purified maize bundle sheath and mesophyll cells results in the production of distinctive, cell-specific polypeptides. Immunoprecipitation experiments show that translatable polyA⁺ mRNAs for phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK) and NADP-malate dehydrogenase (MDH) are prominent in mesophyll but not bundle sheath cells. On the contrary, those for sedoheptulose-1,7-bisphosphatase (SBP), fructose-1,6-bisphosphatase (FBP), NADP-malic enzyme (ME) and the small subunit of ribulose-1,5-bisphosphate carboxylase (RuBPC SS) are present only in bundle sheath cells. Moreover, polyA⁺ mRNAs encoding the 33 kD, 23 kD and 16 kD polypeptides of the oxygen-evolving complex (OE33, OE23 and OE16) and the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCP II) are much more abundant in mesophyll than in bundle sheath cells. Northern blot analyses with cDNA clones of PEPC, PPDK, ME, RuBPC SS, OE33, OE23, OE16 and LHCP II are consistent with the conclusion that the cell-specific expression of these genes is regulated at the RNA level. The RNA level differences are especially dramatic in dark-grown maize seedlings after illumination for 24 h.     

A new evaluation of chlorophyll absorption in photosynthetic membranes

The three major chlorophyll-proteins of spinach chloroplasts were solubilized with digitonin and isolated by electrophoresis with deoxycholate. The gel bands were identified from their absorption and fluorescence spectra measured at 77 K. The slowest moving band was a Photosystem I complex (CPI); the second, a Photosystem II complex (Cpa); and the third, a chlorophyll a-b, antenna complex (LHCP). When absorption spectra (630–730 nm) of the bands were added in the proportions found in the gel, the sum closely matched the absorption of the chloroplasts both before and after solubilization. Thus these spectra represent the native absorption of the major antenna chlorophyll-proteins of green plants. Each of these spectra was resolved with a computer assisted, curve-fitting program into 8 mixed Gaussian-Lorentzian shaped components. The major, Chl a components in the 3 fractions were different both in peak positions and bandwidths. This result suggests that each chlorophyll-protein has its own unique set of chlorophyll a spectral forms or components.     

Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.     

Adenovirus-Mediated NDRG2 Inhibits the Proliferation of Human Renal Cell Carcinoma Cell Line OS-RC-2 in Vitro

The objective of the study is to investigate the inhibitory effects of adenovirus-mediated N-Myc downstream-regulated gene 2 (NDRG2) on the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro. NDRG2 was harvested by RT-PCR, confirmed by DNA sequencing, and then cloned into the eukaryotic expression vector pIRES2-EGFP, which encodes green fluorescent protein (GFP), to construct pIRES2-EGFP-NDRG2 plasmid. OS-RC-2 cells with NDRG2 negative expression were transfected with pIRES2-EGFP-NDRG2 plasmid. The growth of transfected OS-RC-2 cells was observed under the light and fluorescence microscopes. After colony-forming cell assays, cell proliferation detection, and MTT assays, the growth curves of cells in each group were plotted to investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of OS-RC-2 cells. Cell cycle was determined by flow cytometry. Confocal laser scanning microscopy was applied to determine the specific location of NDRG2 protein in subcellular level. A eukaryotic expression vector pIRES2-EGFP-NDRG2 was successfully constructed. After NDRG2 transfection, the growth of OS-RC-2 cells was inhibited. Flow cytometry showed that cells were arrested in S phase but the peak of cell apoptosis was not present, and confocal laser scanning microscopy showed that NDRG2 protein was located in mitochondrion. In conclusion, NDRG2 can significantly inhibit the proliferation of OS-RC-2 cells in vitro and its protein is specifically expressed in the mitochondrion.     

Substitution of selenocysteine for cysteine in a reticulocyte lysate protein synthesis system

Selenocysteine occurs in the peptide backbone of several selenoenzymes. The mechanism, of selenocysteine incorporation has not been well characterized. The incorporation of selenocysteine into protein in a rabbit reticulocyte lysate (RRL) was studied at high levels of selenocysteine. [⁷⁵Se]Selenocysteine incorporation was inhibited by cycloheximide and by nuclease treatment. Random RNA copolymers were tested for protein synthesis activity in the messenger RNA-dependent RRL system. Of the active polymers, poly CIU and GU most strongly stimulated the incorporation of selenocysteine. In a series of four polymers with different ratios of U to G, incorporation of selenocysteine and cysteine increased with increasing percentages of U, suggesting that selenocysteine and cysteine responded to the same codon, presumably UGU. Of the 20 protein amino acids, only cysteine and cystine competed with selenocysteine incorporation. Selenocysteine was charged to cysteine-accepting tRNA in RRL. These results show that at supraphysiological concentrations selenocysteine can substitute for cysteine in RRL protein synthesis. Misincorporation of selenocysteine could be important when animal tissues contain high levels of selenium.     

Molecular cloning,expression and characterisation of Afp4, an antifreeze protein from Glaciozyma antarctica

Antifreeze proteins (AFPs) are proteins with affinity towards ice and contribute to the survival of psychrophiles in subzero environment. Limited studies have been conducted on how AFPs from psychrophilic yeasts interact with ice. In this study, we describe the functional properties of an antifreeze protein from a psychrophilic Antarctic yeast, Glaciozyma antarctica. A cDNA encoding the antifreeze protein, AFP4, from G. antarctica PI12 was amplified from the mRNA extracted from cells grown at 4 °C. Sequence characterisation of Afp4 showed high similarity to fungal AFPs from Leucosporidium sp. AY30, LeIBP (93 %). The 786-bp cDNA encodes a 261-amino-acid protein with a theoretical pI of 4.4. Attempts to produce the recombinant Afp4 in Escherichia coli resulted in the formation of inclusion bodies (IB). The IB were subsequently denatured and refolded by dilution. Gel filtration confirmed that the refolded recombinant Afp4 is monomeric with molecular mass of ~25 kDa. Thermal hysteresis (TH) and recrystallisation inhibition assays confirmed the function of Afp4 as an antifreeze protein. In the presence of Afp4, ice crystals were modified into hexagonal shapes with TH values of 0.08 °C and smaller ice grains were observed compared with solutions without AFP. Structural analyses via homology modelling showed that Afp4 folds into β-helices with three distinct faces: a, b and c. Superimposition analyses predicted the b-face as the ice-binding surface of Afp4, whereby the mechanism of interaction is driven by hydrophobic interactions and the flatness of surface. This study may contribute towards an understanding of AFPs from psychrophilic yeasts.     

The Use of Amphipols for NMR Structural Characterization of 7-TM Proteins

While amphipols have been proven useful for refolding of seven transmembrane helical (7-TM) proteins including G-protein-coupled receptors (GPCRs) and it could be shown that an amphipol environment is in principle suitable for NMR structural studies of the embedded protein, high-resolution NMR insights into amphipol refolded and isotopically labeled GPCRs are still very limited. Here we report on the recent progress toward NMR structural studies of the melanocortin-2 and -4 receptors, two class A GPCRs which so far have not been reported to be incorporated into an amphipol environment. Making use of the established 7-TM protein bacteriorhodopsin (BR) we initially tested and optimized amphipol refolding conditions. Most promising conditions were transferred to the refolding of the two melanocortin receptors. Analytical-scale refolding experiments on the melanocortin-2 receptor show very similar behavior to the results obtained on BR. Using cell-free protein expression we could generate sufficient amounts of isotopically labeled bacteriorhodopsin as well as melanocortin-2 and -4 receptors for an initial NMR analysis. Upscaling of the amphipol refolding protocol to protein amounts needed for NMR structural studies was, however, not straightforward and impeded detailed NMR insights for the two GPCRs. While well-resolved and dispersed NMR spectra could only be obtained for bacteriorhodopsin, a comparison of NMR data recorded on the melanocortin-4 receptor in SDS and in an amphipol environment indicates that amphipol refolding induces larger structural modifications in the receptor.     

Rutin modulates ASC expression in NLRP3 inflammasome: a study in alcohol and cerulein-induced rat model of pancreatitis

Inflammasomes are protein complexes formed in response to tissue injury and inflammation to regulate the formation of proinflammatory cytokines. Nod-like receptor pyrin domain containing 3 (NLRP3) is one such inflammasome involved in pancreatic inflammation. Caspase activation recruitment domain (CARD) is an interaction motif found in all the major components of NLRP3 inflammasome such as apoptosis associated speck-like CARD containing protein (ASC) and procaspase-1. NLRP3 activates procaspase-1 with the concerted action of CARD domain of ASC. In the present study, the effect of rutin, a natural flavonoid on the expression of ASC of NLRP3, was investigated in rats treated with ethanol (EtOH) and cerulein (Cer). Male albino Wistar rats were divided into four groups. Groups 1 and 2 rats were fed normal diet, whereas groups 3 and 4 rats were fed EtOH (36 % of total calories) containing diet for a total period of 5 weeks and also administered Cer (20 µg/kg body weight i.p.) thrice weekly for the last 3 weeks. In addition, groups 2 and 4 rats received daily 100 mg/kg body weight of rutin from third week. Rutin co-administration significantly decreased the level of pancreatic marker enzymes, oxidative stress markers, inflammatory markers, mRNA expression of caspase-1, cytokines, ASC–NLRP3, and protein expression of caspase-1 and ASC in rats received EtOH–Cer. The results of the study revealed that rutin can reduce inflammation in pancreas probably by influencing the down regulation of ASC–NLRP3 which might result in the reduced activation of caspase-1 and controlled cytokine production.     

Age related changes in blood-to-brain amino acid transport and incorporation into brain protein

Blood-to-brain amino acid transport consists of at least two components: 1. a fast rate or early process, commonly measured by the intra-carotid bolus injection method and attributed to transport across the capillary endothelium and entry into the astrocytes, and, 2. a slow rate or later component measured over 2 to 15 minutes probably associated with exit from the astrocytes and entry into the neurons. Incorporation into brain protein is temporally related to the second process. In the present study the slow and fast rate transport components and the incorporation into brain protein of tyrosine (Tyr) and Valine (Val) was measured in young adult and aged male C57BL/6 mice. The results indicate that the fast rate transport component is unaffected by age while the rates of the slow process and protein turnover show an exponential decline most marked between 3 and 8 months of age. Changes in the relative incorporation of Tyr and Val suggest that brain protein metabolism is altered qualitatively as well as quantitatively in aging, in these animals.     

Multiple correlations of mRNA expression and protein abundance in human cytokine profile

With the development of genomic study, researchers found that it is insufficient to predict protein expression from quantitative mRNA data in large scale, which is contrary to the traditional opinion that mRNA expression correlates with protein abundance at the single gene level. To try to solve the apparent conflicting views, here we set up a series of research models and chose soluble cytokines as targets. First, human peripheral blood mononuclear cell (PBMC) from one health donor was treated with 16 continuously changing conditions, the protein and mRNA profile were analyzed by multiplex Luminex and genomic microarray, respectively. Among the tested genes, around half mRNA correlated well with their corresponding proteins (ρ > 0.8), however if we put all the genes together, the correlation coefficient for the 16 conditions varied from 0.29 to 0.71. Second, PBMC from 14 healthy donors were stimulated with the same condition and it was found that the correlation coefficient went down (ρ < 0.6). Third, 28 rheumatoid arthritis (RA) patients were tested for their response to the same external stimuli and it turned out different individual displayed different protein expression pattern as expect. Lastly, autoimmune disease cohorts (8 diseases including RA, 103 patients in total) were assayed on the whole view. It was observed that there was still some similarity in the protein profile among patients from the single disease type although completely different patterns were displayed across different disease categories. This study built a good bridge between single gene analysis and the whole genome study and may give a reasonable explanation for the two conflicting views in current biological science.     

Modulation of inducible nitric oxide synthase (iNOS) expression and cardiovascular responses during static exercise following iNOS antagonism within the ventrolateral medulla

Previous reports indicate that inducible nitric oxide synthase (iNOS) blockade within the rostral ventrolateral medulla (RVLM) and caudal ventrolateral medulla (CVLM) differentially modulated cardiovascular responses, medullary glutamate, and GABA concentrations during static skeletal muscle contraction. In the current study, we determined the role of iNOS antagonism within the RVLM and CVLM on cardiovascular responses and iNOS protein expression during the exercise pressor reflex in anesthetized rats. Following 120 min of bilateral microdialysis of a selective iNOS antagonist, aminoguanidine (AGN; 10 µM), into the RVLM, the pressor responses were attenuated by 72 % and changes in heart rate were reduced by 38 % during a static muscle contraction. Furthermore, western blot analysis of iNOS protein abundance within the RVLM revealed a significant attenuation when compared to control animals. In contrast, bilateral administration of AGN (10 µM) into the CVLM augmented the increases in mean arterial pressure by 60 % and potentiated changes in heart rate by 61 % during muscle contractions, but did not alter expression of the iNOS protein within the CVLM. These results demonstrate that iNOS protein expression within the ventrolateral medulla is differentially regulated by iNOS blockade that may, in part, contribute to the modulation of cardiovascular responses during static exercise.     

Microporation is an efficient method for siRNA-induced knockdown of PEX5 in HepG2 cells: evaluation of the transfection efficiency,the PEX5 mRNA and protein levels and induction of peroxisomal deficiency

The pathomechanism of peroxisomal biogenesis disorders (PBDs), a group of inherited autosomal recessive diseases with mutations of peroxin (PEX) genes, is not yet fully understood. Therefore, several knockout models, e.g., the PEX5 knockout mouse, have been generated exhibiting a complete loss of peroxisomal function. In this study, we wanted to knockdown PEX5 using the siRNA technology (1) to mimic milder forms of PBDs in which the mutated peroxin has some residual function and (2) to analyze the cellular consequences of a reduction of the PEX5 protein without adaption during the development as it is the case in a knockout animal. First, we tried to optimize the transfection of the hepatoma cell line HepG2 with PEX5 siRNA using different commercially available liposomal and non-liposomal transfection reagents (Lipofectamine^® 2000, FuGENE 6, HiPerFect^®, INTERFERin?, RiboJuice?) as well as microporation using the Neon? Transfection system. Microporation was found to be superior to the transfection reagents with respect to the transfection efficiency (100 vs. 0–70 %), to the reduction of PEX5 mRNA (by 90 vs. 0–50 %) and PEX5 protein levels (by 70 vs. 0–50 %). Interestingly, we detected that a part of the cleaved PEX5 mRNA still existed as 3′ fragment (15 %) 24 h after microporation. Using microporation, we further analyzed whether the reduced PEX5 protein level impaired peroxisomal function. We indeed detected a reduced targeting of SKL-tagged proteins into peroxisomes as well as an increased oxidative stress as found in PBD patients and respective knockout mouse models. Knockdown of the PEX5 protein and functional consequences were at a maximum 48 h after microporation. Thereafter, the PEX5 protein was resynthesized, which may allow the temporal analysis of the loss as well as the reconstitution of peroxisomes in the future. In conclusion, we propose microporation as an efficient and reproducible method to transfect HepG2 cells with PEX5 siRNA. We succeeded to transiently knockdown PEX5 mRNA and its protein level leading to functional consequences similar as observed in peroxisome deficiencies.     

Increased Oxidative Stress and Altered Levels of Nitric Oxide and Peroxynitrite in Tunisian Patients with Chronic Obstructive Pulmonary Disease: Correlation with Disease Severity and Airflow Obstruction

This study was aimed to evaluate the oxidant–antioxidant imbalance in the pathogenesis of chronic obstructive pulmonary disease (COPD) in Tunisians. We assessed 16 parameters related to the oxidative status that include malondialdehyde (MDA), total protein carbonyls (PCs), and advanced oxidation protein products (AOPP). We also examined the activity of glutathione peroxydase (GSH-Px), catalase, and superoxide dismutase (SOD) in the plasma and erythrocytes. Levels of total thiols, reduced glutathione (GSH), total antioxidant status (TAS), hydrogen peroxide, ascorbic acid, iron, and protein sulfhydryls were determined using spectrophotometry. We also evaluated the level of nitric oxide (NO) and peroxynitrite in plasma from COPD patients and healthy controls. Estimation of DNA damage was determined using the comet assay. Pulmonary functional tests were performed by body plethysmography. Levels of MDA, PC, DNA damage, and AOPP were significantly increased while total thiols, GSH, and TAS were decreased in COPD patients. GSH-Px activity was higher in COPD patients while no difference was found for catalase and SOD. We also observed a lower level of NO and peroxynitrite in COPD patients. Decreased levels of peroxynitrite were found to correlate with disease progression, as well as with forced expiratory volume in 1 s/forced vital capacity among COPD patients. Multivariate analysis revealed that NO is associated with pathological pathways that help to predict patient outcome independently of the degree of airflow obstruction. These results indicate the presence of a systemic oxidative stress and highlight the importance of NO and peroxynitrite as major effectors in COPD development and airflow obstruction.     

Arrest of yellowing in senescing leaf discs of maize by growth retardants,coumarin and inhibitors of RNA and protein synthesis

Kinetin, coumarin and four growth retardants including Phosfon D (2,4-dichlorobenzyltributylphosphonium chloride), CCC [(2-chloroethyl) trimethylammonium chloride], B-Nine (N-dimethylaminosuccinamic acid) and AMO-1618 (2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride) arrested chlorophyll, protein and RNA degradation in the leaf discs of maize kept in darkness; GA₃ was without effect. Coumarin and Phosfon D markedly lowered the level of TCA soluble nitrogen compounds in the tissue; other compounds were inactive in this respect. Puromycin, 4-fluorophenylalanine, actinomycin D, 5-diazouracil and 2-thiouracil also retarded the loss of chlorophyll from the leaf discs of maize; chloramphenicol was without effect on yellowing. The inhibitors of RNA and protein synthesis slightly decreased, increased or had no influence on the chlorophyll preserving effect of coumarin and growth retarding chemicals. 2-Thiouracil markedly decreased the loss of protein content from the discs. Coumarin and growth retardants inhibited the synthesis of chlorophyll in the leaf tissue of etiolated seedlings of maize. It is suggested that synthesis of some specific protein(s) is required for senescence in the detached leaf tissue. Synthesis of this protein(s) was possibly arrested by the compounds which were under examination.