Sucrose Gradient Method Research Articles

Sequencing and Annotation of the Chloroplast Genome of <i>Triticum militinae</i> – a “Natural Mutant” of Tetraploid Wheat <i>Triticum timopheevii</i> Zhuk.

Triticum militinae Zhuk. et Migusch. – tetraploid wheat with the GAA genome, considered a natural naked mutant of T. timopheevii Zhuk. Previously, experiments were conducted for this wheat to study the karyotype and crossing characteristics. To clarify its origin and relationship with other representatives of the wheat family, analysis of the chloroplast genome of T. militinae, which has previously remained unstudied, is of great interest. The sucrose gradient method was used to isolate chloroplast DNA from leaves. Sequencing was performed using the FASTASeq 300 Sequencing Kit V1.0 100 M reads/flow cell on a Genolab M sequencer (GeneMind, China). For the first time, we sequenced and annotated the complete chloroplast genome of T. militinae with a size of 135898 bp. In the structure of the plastid genome there are a pair of inverted repeats with a size of 21552 bp each, the region of a small single copy (SSC) – 12791 bp and the region of a large single copy (LSC) – 80003 bp. As part of the chloroplast genome of T. militinae 132 structural genes were found, of which 85 protein-coding genes, 31 tRNAs and 4 rRNA genes.

Detection and Characterization of Nosema bombycis Using TEM and SEM Techniques.

Pebrine disease is the most important and dangerous disease of silkworm caused by Nosema bombycis as an obligate intracellular parasitic fungus. It has caused tremendous economic losses in the silk industry in recent years. Given the fact that light microscopy method (with low accuracy) is the only method for diagnosing pebrine disease in the country, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) methods were adopted in this study for accurate morphological identification of the spores causing pebrine disease. Infected larvae and mother moth samples were collected from several farms (Parand, Parnian, Shaft, and Iran Silk Research Center in Gilan province, Iran). The spores were then purified using the sucrose gradient method. From each region, 20 and 10 samples were prepared for SEM and TEM analysis, respectively. In addition, an experiment was performed to evaluate the symptoms of pebrine disease by treating fourth instars with the spores purified for the present study, along with a control group. The results of SEM analysis showed that the mean±SD length and width of spores were 1.99±0.25 to 2.81±0.32 μm, respectively. Based on the obtained results, the size of spores was smaller than the Nosema bombycis (N. bombycis) as the classic species that cause pebrine disease. In addition, transmission electron microscopy (TEM) pictures showed that the grooves of the adult spores were deeper than those of other Nosema species, Vairomorpha, and Pleistophora, and resembled N. bombycis in other studies. Examination of pathogenicity of the studied spores indicated that the disease symptoms in controlled conditions were similar to those in the sampled farms. The most important symptom in fourth and fifth instrars were the small size and no growth in the treatment group compared with the control group. Findings of SEM and TEM analysis showed better morphological and structural details of parasite compared with light microscopy, and demonstrated that the studied species were a native strain of N. bombycis specific to Iran, whose size and other characteristics were unique and introduced for the first time in this study.

Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus.

Oncolytic viruses (OVs), such astheoncolytic herpes simplex virus (oHSV), are a rapidly growing treatment strategy in the field of cancer immunotherapy. OVs, including oHSV, selectively replicate in and kill cancer cells (sparing healthy/normal cells) while inducing anti-tumor immunity. Because of these unique properties, oHSV-based treatment strategies are being increasingly used for the treatment of cancer, preclinically and clinically, including FDA-approved talimogene laherparevec (T-Vec). Growth, purification, and titration are three essential laboratory techniques for any OVs, including oHSVs, before they can be utilized for experimental studies. This paper describes a simple step-by-step method to amplify oHSV in Vero cells. As oHSVs multiply, they produce a cytopathic effect (CPE) in Vero cells. Once 90-100% of the infected cells show a CPE, they are gently harvested, treated with benzonase and magnesium chloride (MgCl2), filtered, and subjected to purification using the sucrose-gradient method. Following purification, the number of infectious oHSV (designated as plaque-forming units or PFUs) is determined by a "plaque assay" in Vero cells. The protocol described herein can be used to prepare high-titer oHSV stock for in vitro studies in cell culture and in vivo animal experiments.

Purification of Recombinant Wild Type and Mutant Ryanodine Receptors Expressed in HEK293 Cells.

High quantities of purified ryanodine receptor (RyR), a large (2.26 MDa) intracellular homotetrameric membrane protein, can be obtained from heterologous expression in HEK293 cells and used for structure determination by cryo-EM. The advantage of using recombinant protein is that the variability due to post-translational modifications can be minimized, to which the high resolution of up to 2.4 Å achieved for RyR2 can be attributed ( Iyer et al., 2020 ). In addition, recombinant protein expression enables the study of mutations that are deleterious when expressed homozygously in animals. Protein purification was achieved using two strategies, sucrose density gradient and affinity chromatography, which have previously been used for purification of RyR from tissue. The sucrose gradient method was developed from ( Lee et al., 1994 ) and later adapted for cryo-EM ( Samsó et al., 2005 ). The affinity chromatography method takes advantage of the high affinity of RyR for its ligand FKBP12/12.6, by using a construct between FKBP and streptavidin binding protein (SBP) ( Cabra et al., 2016 ). While the sucrose gradient method can yield a higher protein concentration (≥ 2 mg/ml), the affinity purification method is faster. Both methods are suitable and applicable to the purification of recombinant proteins and were successfully used in the first 3D near-atomic reconstructions of RyRs purified from cells expressing disease mutants ( Iyer et al., 2020 ). This purification protocol is also suitable for functional studies, such as single-channel analysis, that require pure RyR protein.

Investigating the Response of Human Neutrophils to Hydrophilic and Hydrophobic Micro-Rough Titanium Surfaces.

Various treatments have been used to change both the topography and chemistry of titanium surfaces, aiming to enhance tissue response and reduce healing times of endosseous implants. Most studies to date focused on bone healing around dental implants occurring later during the healing cascade. However, the impact of the initial inflammatory response in the surgical wound site on the success and healing time of dental implants is crucial for implant integration and success, yet it is still poorly understood. The purpose of this study was to investigate the effect of titanium surface hydrophilicity on the response of human neutrophils by monitoring oxygen radical production, which was measured as chemiluminescence activity. Materials and Methods: Neutrophils were isolated from human donors’ blood buffy coats using the double sucrose gradient method. Neutrophils were exposed to both hydrophilic and hydrophobic titanium surfaces with identical topographies in the presence and absence of human serum. This resulted in six experimental groups including two different implant surfaces, with and without exposure to human serum, and two control groups including an active control with cells alone and a passive control with no cells. Two samples from each group were fixed and analyzed by SEM. Comparisons between surface treatments for differences in chemiluminescence values were performed using analysis of variance ANOVA. Results and Conclusion: In the absence of exposure to serum, there was no significant difference noted between the reaction of neutrophils to hydrophilic and hydrophobic surfaces. However, there was a significant reduction in the mean and active chemiluminescence activity of neutrophils to serum-coated hydrophilic titanium surfaces than to serum-coated hydrophobic titanium surfaces. This suggests that surface hydrophilicity promotes enhanced adsorption of serum proteins, which leads to decreased provocation of initial immune cells and reduction of local oxygen radical production during wound healing. This can help explain the faster osseointegration demonstrated by hydrophilic titanium implants.

Investigation of Blastocystis Subspecies in Water Samples Collected from Ordu Province

The aim of the study was to determine the prevalence of Blastocystis subspecies in water samples collected from Ordu province. Seventy-five surface water samples and 25 drinking water samples were collected from Ordu and its boroughs. The samples were flocculated by aluminum sulphate and concentrated by sucrose gradient method. Small subunit ribosomal RNA (SSU rRNA) gene was amplified by polymerase chain reaction (PCR). Sequence analysis was performed on the positive PCR products and the base sequences were aligned using Bioedit. Phylogeny trees were drawn and Blastocystis subspecies were identified. Four out of the 100 water samples were positive for Blastocystis spp. No positivity was found in the drinking water. Blastocystis ST-1 subspecies was detected in Bülbül River, Kacalı River and Bolaman Stream. Blastocystis ST-3 subspecies were found in Karabalçık River. The lowest genetic distance was found between ST-1 subspecies and the samples from Bülbül River, Kacalı River and Bolaman Stream. The nucleotide similarities between them were 98.8%, 76.6 and 98.8%, respectively. The lowest genetic distance was found between Karabalçık River and ST-3 subspecies and the nucleotide similarity between them was 99.1%. This is the first study on the presence of Blastocystis spp. in the surface water and drinking water samples in Ordu province of the Black Sea area in Turkey.

Investigation of Cryptosporidium spp. Positivity in Formol-Fixed Stool Samples by Polymerase Chain Reaction Method

Objective: It was aimed to determine Cryptosporidium spp., which is thought to cause digestive system complaints in primary school children, by Modified Acid-Fast (MAF) staining method and Polymerase Chain Reaction (PCR). Method: One hundred sixty four stool samples taken from children were examined under light microscopy with MAF. Positive samples were fixed in 10% formalin and stored at + 4 °C until analysis. These samples were then purified using the Sucrose Gradient method and, DNA isolation was performed using the modified QIAamp DNA Mini Kit (Qiagen) protocol. In order to increase sensitivity of the study, SSU rRNA gene region was amplified by PCR method. Results: Microscopic examination showed that 89 of the 164 stool samples (54,26%) were found to be positive for Cryptosporidium spp.oocysts. Eighty nine samples which were found to be positive by microscopic examination and fixed in %10 formalin were studied by PCR and 74 (45,12%) of them were positive. Conclusions: Cryptosporidiosis, which is known to cause severe infections, especially in immunocompromised individuals and children, is transmitted by fecal-oral route from infected humans and animals, and contaminated water and nutrients. Cryptosporidiosis, which is the result of fecal pollution, causes a great risk for human health even with a low number of oocysts due to the fact that oocysts are resistant to chlorine and they can survive for a long time in the outer environment. It was observed that, primary school children living in area of the study were under risk for cryptosporidiosis.

Neonatal Rat; A Suitable Animal Model for Experimental Cryptosporidiosis

Background: Cryptosporidiosis is a major public health problem for neonatal livestock worldwide. Cryptosporidium parvum infects intestinal epithelial cells via contaminated food or drinking water and leads to cryptosporidiosis. Most of the animal model studies on infectivity of C. parvumare conducted on the neonatal mice. Objectives:: The current study aimed at evaluating the infectivity of C. parvum in neonatal rat as an animal model. Methods: A dose of 100,000 to 120,000 C. parvum oocysts (Iowa strain, BTF Company, Sydney, Australia) was orally inoculated in a group of 30 neonatal Wistar rats aged 2 days old. Eight days postinfection, jejunum, ileum, cecum, colon, and rectum were removed and contents were homogenized and purified using sucrose gradient method. Results: Our results indicated that 6 to 12 million C. parvum was found per rat Conclusions: Analysis of the study results revealed that the neonatal rat could be used as an alternative animal model to investigate C. parvum.

Análisis comparativo de diferentes métodos de extracción de DNA y su eficiencia de genotipificación en población mexicana

Los estudios de variabilidad genetica han reportado inconsistencias de resultados entre poblaciones, en gran medida, debido a que los metodos de extraccion de acido desoxirribonucleico (DNA, por sus siglas en ingles) y las tecnicas de genotipificacion son altamente variables entre ellas, lo que conduce a la asignacion erronea de genotipos. El objetivo de este estudio es comparar distintas tecnicas de extraccion de DNA y de genotipificacion de polimorfismos. Para llevar a cabo el estudio, se analizo el DNA de 10 muestras sanguineas correspondientes a individuos mestizos mexicanos, y se purifico por los metodos de fenol-cloroformo-alcohol isoamilico (FCI), gradiente de sales (GS), gradiente de sacarosa (GSC), DNAzol® y DNeasy Blood & Tissue Kit ®. Se genotipificaron los polimorfismos GSTT1 *0 y GSTM1*0 por reaccion en cadena de la polimerasa (PCR, por sus siglas en ingles) multiple, CYP1A1*2C por RFLP’s y AhR Arg554Lys por PCR tiempo real. Los resultados mostraron diferencias significativas (p < 0.001) en cantidad, pureza e integridad entre los distintos metodos. En los analisis moleculares se observo que el metodo de FCI presenta inhibidores de la reaccion de PCR, ya que no fue posible amplificar los fragmentos por PCR multiple y PCR punto final, aunque si hubo amplificacion en el PCR tiempo real. Los metodos GS y GSC amplificaron todas las muestras en las tres modalidades de PCR y mostraron resultados concordantes, mientras que solo el 80% de las muestras extraidas mediante DNAzol ® y DNeasy ® amplificaron, y los resultados no fueron concordantes para DNAzol ® (50%) y DNeasy ® (80%) en el analisis de PCR - RFLP’s. Los metodos de extraccion GS y GSC mostraron mayor recuperacion de DNA, con parametros de calidad e integridad optimos para los analisis moleculares por PCR

Investigation on Toxoplasma gondii by polymerase chain reaction and loop-mediated isothermal amplification in water samples from Giresun, Turkey

Toxoplasmosis is generally asymptomatic in immunocompetent subjects, however serious manifestations of the disease may develop in immunocompromised patients and in pregnant women. The mean Toxoplasma gondii seroprevalence which is approximately 40% in Turkish population, indicates the high risk for the development of acute toxoplasmosis in those cases. One of the transmission ways of T.gondii is the consumption of contaminated water. The aim of this study was to detect the presence of T.gondii in the environmental and drinking water samples by using standard polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) methods. A total of 96 water samples, of them 76 were environmental water samples collected from creeks in Giresun province center and its various districts (Piraziz, Bulancak, Keşap, Espiye) and 20 from drinking water samples, were included in the study. The samples were precipitated by the aluminium sulfate method and DNAs were isolated from the pellets formed with the sucrose gradient method. PCR and LAMP were applied to the isolated DNAs, by the use of primers F3 and B3 specific to B1 gene of the parasite. In our study, T.gondii was detected in none of the drinking water samples by PCR and LAMP, however T.gondii DNAs were positive in 13.2% (10/76) of the environmental water samples. Both of the methods yielded the same results in those samples. The stations that were positive for T.gondii were Aksu creek in Giresun province center; Gelivera creek in Espiye; Yolağzı, Keşap, Keşap Login Bridge creeks in Keşap; Bulancak, Karadere and İncivez creeks in Bulancak; Piraziz and Çayırağzı creeks in Piraziz counties. Resistance of T.gondii to chlorination and the inadequacy of the filtration processes, create a serious threat among people in contact with rivers, sea and drinking waters particularly in areas with high humidity. As far as the national literature was considered, no report about the water-borne T.gondii infections were detected in Giresun province of Black Sea region, Turkey. Thus this study will aid to the literature related to water-borne T.gondii infections. The results of this study emphasizes the essential need for appropriate disinfection procedures and purification processes before the discharge of waste water to prevent the cases of water-borne toxoplasmosis.

English

In order to obtain different actinomycetes isolates from the East Black Sea Region plateau soil, an isolation study was carried out by using sucrose gradient method and different growth media. A molecular taxonomic method, 16S rDNA gene sequencing, was applied to obtain different actinomycetes isolates from East Black Sea region plateau soil. Phylogenetic dendrograms based on 16S rDNA nucleotide sequences were constructed by using neighbour-joining algorithm. In the light of phylogenetic analyses, it was determined that out of 15 organisms, two belong to Actinomadura, three Kribbella, three Nocardia, six Micromonospora and an organism of Microbacterium. Soil isolates obtained from this study contribute to taxonomy of actinomycetes and also many species will emerge. &nbsp; Key words: Actinomadura, Kribbella, Microbacterium, Micromonospora, Nocardia, 16S rDNA gene sequencing.

Fetal iron deficiency alters the proteome of adult rat hippocampal synaptosomes

Fetal and neonatal iron deficiency results in cognitive impairments in adulthood despite prompt postnatal iron replenishment. To systematically determine whether abnormal expression and localization of proteins that regulate adult synaptic efficacy are involved, we used a quantitative proteomic approach (isobaric tags for relative and absolute quantitation, iTRAQ) and pathway analysis to identify dysregulated proteins in hippocampal synapses of fetal iron deficiency model. Rat pups were made iron deficient (ID) from gestational day 2 through postnatal day (P) 7 by providing pregnant and nursing dams an ID diet (4 ppm Fe) after which they were rescued with an iron-sufficient diet (200 ppm Fe). This paradigm resulted in a 40% loss of brain iron at P15 with complete recovery by P56. Synaptosomes were prepared from hippocampi of the formerly iron-deficient (FID) and always iron-sufficient controls rats at P65 using a sucrose gradient method. Six replicates per group that underwent iTRAQ labeling and LC-MS/MS analysis for protein identification and comparison elucidated 331 differentially expressed proteins. Western analysis was used to confirm findings for selected proteins in the glutamate receptor signaling pathway, which regulates hippocampal synaptic plasticity, a cellular process critical for learning and memory. Bioinformatics were performed using knowledge-based Interactive Pathway Analysis. FID synaptosomes show altered expression of synaptic proteins-mediated cellular signalings, supporting persistent impacts of fetal iron deficiency on synaptic efficacy, which likely cause the cognitive dysfunction and neurobehavioral abnormalities. Importantly, the findings uncover previously unsuspected pathways, including neuronal nitric oxide synthase signaling, identifying additional mechanisms that may contribute to the long-term biobehavioral deficits.

Activation of Transcription Factor MEF2D by Bis(3)-cognitin Protects Dopaminergic Neurons and Ameliorates Parkinsonian Motor Defects

Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP(+)-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3β (GSK3β) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP(+)-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP(+)-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo.

Direct magnetic resonance detection of myelin and prospects for quantitative imaging of myelin density.

Magnetic resonance imaging has previously demonstrated its potential for indirectly mapping myelin density, either by relaxometric detection of myelin water or magnetization transfer. Here, we investigated whether myelin can be detected and possibly quantified directly. We identified the spectrum of myelin in the spinal cord in situ as well as in myelin lipids extracted via a sucrose gradient method, and investigated its spectral properties. High-resolution solution NMR spectroscopy showed the extract composition to be in agreement with myelin's known chemical make-up. The 400-MHz (1)H spectrum of the myelin extract, at 20 °C (room temperature) and 37 °C, consists of a narrow water resonance superimposed on a broad envelope shifted ∼3.5 ppm upfield, suggestive of long-chain methylene protons. Superimposed on this signal are narrow components resulting from functional groups matching the chemical shifts of the constituents making up myelin lipids. The spectrum could be modeled as a sum of super-Lorentzians with a T(2)* distribution covering a wide range of values (0.008-26 ms). Overall, there was a high degree of similarity between the spectral properties of extracted myelin lipids and those found in neural tissue. The normalized difference spectrum had the hallmarks of membrane proteins, not present in the myelin extract. Using 3D radially ramp-sampled proton MRI, with a combination of adiabatic inversion and echo subtraction, the feasibility of direct myelin imaging in situ is demonstrated. Last, the integrated signal from myelin suspensions is shown, both spectroscopically and by imaging, to scale with concentration, suggesting the potential for quantitative determination of myelin density.

Use of the Sucrose Gradient Method for Bacterial Cell Cycle Synchronization

Although many undergraduate and graduate Cell and Molecular Biology courses study the bacterial cell cycle and the mechanisms that regulate prokaryotic cell division, few laboratory projects exist for the enhanced study of cell cycle characteristics in a standard teaching laboratory. One notable reason for this lack of engaging laboratory projects is, although bacterial cells can be grown fairly easily, these cultured cells are in a variety of cell cycle states. As such, to study and understand the factors that regulate bacterial cell division in morphological, physiological, and even molecular respects, it is necessary to have bacterial cells in the same stage of its cell cycle. This matching can be performed by a procedure called cell cycle synchronization.

Lipid raft‐dependent NADPH oxidase regulates polychlorinated biphenyls‐induced expression of cell adhesion molecules in human brain endothelial cells

Exposure to persistent organic pollutants, such as polychlorinated biphenyls (PCBs), can lead to expression of cell adhesion molecules (CAMs) of the brain endothelium leading to chronic neuroinflammation. In the present study, we have explored whether NADPH oxidase (NOX) modulates ortho‐substituted PCBs‐induced upregulation of CAMs in brain endothelial cells. Superoxide production, as measured by dihydroethidium (DHE), was increased in the cells exposed to PCB153. Exposure to PCB153 induced phosphorylation of cytosolic components including p47 and Rac and translocation into the lipid rafts domain for NOX activation. Upregulation of ICAM‐1 and VCAM‐1 was impeded by inhibitors to NAPDH oxidase, Rac1, and nitric oxide synthase. Moreover, isolation of lipid raft fraction using non‐detergent sucrose gradient method confirmed predominant distribution of ICAM‐1 in lipid raft domain. Results of the present study indicate that accumulation of ROS through lipid raft‐dependent NADPH oxidase regulates PCB153‐mediated induction of CAMs in brain endothelial cells. Due to its role in leukocyte infiltration, induction of CAMs may contribute to PCBs‐induced cerebrovascular disorders and neurotoxic effects in the CNS. Supported in part by NIH grants (P42 ES 07380, MH63022, MH072567, and NS39254) and the American Heart Association (09SDG2300037).

Aqueous biphasic system to extract arbuscular mycorrhizal spores from soils

The aqueous biphasic system (ABS) method using PEG and ammonium sulphate improved the recovery of spores from tropical soils as compared with the sucrose gradient method. The ABS was not affected by low temperatures. The optimal recovery of spores was achieved when 100% PEG and 50% ammonium sulphate concentration and PEG 8000 instead of PEG 4000 were used. Under these conditions an optimal interface formation which facilitates the localization and recovery of spores was obtained. Both phases of the ABS are aqueous and did not affect either the viability of spores or the level of root colonization reached by these spores. Soil composition did not affect the number of spores obtained by the ABS method either.

Generation of Nicotinic Acid Adenine Dinucleotide Phosphate and Cyclic ADP-Ribose by Glucagon-Like Peptide-1 Evokes Ca2+ Signal That Is Essential for Insulin Secretion in Mouse Pancreatic Islets

Glucagon-like peptide-1 (GLP-1) increases intracellular Ca(2+) concentrations ([Ca(2+)](i)), resulting in insulin secretion from pancreatic beta-cells. The molecular mechanism(s) of the GLP-1-mediated regulation of [Ca(2+)](i) was investigated. GLP-1-induced changes in [Ca(2+)](i) were measured in beta-cells isolated from Cd38(+/+) and Cd38(-/-) mice. Calcium-mobilizing second messengers were identified by measuring levels of nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (ADPR), using a cyclic enzymatic assay. To locate NAADP- and cyclic ADPR-producing enzyme(s), cellular organelles were separated using the sucrose gradient method. A GLP-1-induced [Ca(2+)](i) increase showed a cooperative Ca(2+) signal, i.e., an initial [Ca(2+)](i) rise mediated by the action of NAADP that was produced in acidic organelles and a subsequent long-lasting increase of [Ca(2+)](i) by the action of cyclic ADPR that was produced in plasma membranes and secretory granules. GLP-1 sequentially stimulated production of NAADP and cyclic ADPR in the organelles through protein kinase A and cAMP-regulated guanine nucleotide exchange factor II. Furthermore, the results showed that NAADP production from acidic organelles governed overall Ca(2+) signals, including insulin secretion by GLP-1, and that in addition to CD38, enzymes capable of synthesizing NAADP and/or cyclic ADPR were present in beta-cells. These observations were supported by the study with Cd38(-/-) beta-cells, demonstrating production of NAADP, cyclic ADPR, and Ca(2+) signal with normal insulin secretion stimulated by GLP-1. Our findings demonstrate that the GLP-1-mediated Ca(2+) signal for insulin secretion in pancreatic beta-cells is a cooperative action of NAADP and cyclic ADPR spatiotemporally formed by multiple enzymes.

A mass spectrometric approach to identify arbuscular mycorrhiza-related proteins in root plasma membrane fractions

One of the most important morphological changes occurring in arbuscular mycorrhizal (AM) roots takes place when the plant plasma membrane (PM) invaginates around the fungal arbuscular structures resulting in the periarbuscular membrane formation. To investigate whether AM symbiosis-specific proteins accumulate at this stage, two complementary MS approaches targeting the root PM from the model legume Medicago truncatula were designed. Membrane extracts were first enriched in PM using a discontinuous sucrose gradient method. The resulting PM fractions were further analysed with (i) an automated 2-D LC-MS/MS using a strong cation exchange and RP chromatography, and (ii) SDS-PAGE combined with a systematic LC-MS/MS analysis. Seventy-eight proteins, including hydrophobic ones, were reproducibly identified in the PM fraction from non-inoculated roots, representing the first survey of the M. truncatula root PM proteome. Comparison between non-inoculated and Glomus intraradices-inoculated roots revealed two proteins that differed in the mycorrhizal root PM fraction. They corresponded to an H(+)-ATPase (Mtha1) and a predicted glycosylphosphatidylinositol-anchored blue copper-binding protein (MtBcp1), both potentially located on the periarbuscular membrane. The exact role of MtBcp1 in AM symbiosis remains to be investigated.

Regulation of GTP hydrolysis prior to ribosomal AUG selection during eukaryotic translation initiation

Genetic studies in yeast have shown that the translation initiation factor eIF5 plays an important role in the selection of the AUG start codon. In order to ensure translation fidelity, the hydrolysis of GTP bound to the 40S preinitiation complex (40S.Met-tRNA(i).eIF2.GTP), promoted by eIF5, must occur only when the complex has selected the AUG start codon. However, the mechanism that prevents the eIF5-promoted GTP hydrolysis, prior to AUG selection by the ribosomal machinery, is not known. In this work, we show that the presence of initiation factors eIF1, eIF1A and eIF3 in the 40S preinitiation complex (40S.eIF1.eIF1A.eIF3.Met-tRNA(i).eIF2.GTP) and the subsequent binding of the preinitiation complex to eIF4F bound at the 5'-cap structure of mRNA are necessary for preventing eIF5-promoted hydrolysis of GTP in the 40S preinitiation complex. This block in GTP hydrolysis is released upon AUG selection by the 40S preinitiation complex. These results, taken together, demonstrate the biochemical requirements for regulation of GTP hydrolysis and its coupling to the AUG selection process during translation initiation.