Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology

The ability to rapidly identify small molecules that interact with RNA would have significant clinical and research applications. Low-molecular-weight molecules that bind to RNA have the potential to be used as drugs. Therefore, technologies facilitating the rapid and reliable identification of such activities become increasingly important. We have applied a fluorescence-based assay to screen for modulators of hammerhead ribozyme (HHR) catalysis from a small library of antibiotic compounds. Several unknown potent inhibitors of the hammerhead cleavage reaction were identified and further characterized. Tuberactinomycin A, for which positive cooperativity of inhibition in vitro was found, also reduced ribozyme cleavage in vivo. The assay is applicable to the screening of mixtures of compounds, as inhibitory activities were detected within a collection of 2,000 extracts from different actinomycete strains. This approach allows the rapid, reliable, and convenient identification and characterization of ribozyme modulators leading to insights difficult to obtain by classical methodology.     

Detection of insulin-releasing cells using in situ immunoblotting

Embryonic stem (ES) cells hold promise as a source for cell transplantation treatment of diseases such as type I diabetes. Further, cells releasing bioactive substances from ES cell progeny may be concentrated and purified for clinical applications. Although ES cell lines that express reporter genes have been established to isolate cells releasing bioactive substances, other difficulties must be overcome before these genetically modified cells can be used for gene therapy in human patients. Fluorescence- or magnetic-activated cell sorters are commonly used to isolate specific cells using antibodies against cell surface antigens. However, for some cells, such as insulin-producing beta cells, specific surface antigens have not yet been identified. In this study, we developed a simple and efficient method to identify and purify insulin- and alpha-fetoprotein-producing cells. A nitrocellulose membrane treated with anti-insulin or anti-alpha-fetoprotein antibodies was placed on a cell layer to trap insulin or alpha-fetoprotein released from the cells. The location of specific substance-producing cells was identified by immunostaining the membrane. The insulin-releasing cells were selectively collected from the culture dish using a cloning ring and transferred to another culture plate.     

Identification of new celiac disease autoantigens using proteomic analysis

Celiac disease is an autoimmune disorder in which gluten peptides presented by specific HLA-DQ2- and HLA-DQ8-positive antigen presenting cells elicit immune response in connective tissue of lamina propria. Immunoglobulin A (IgA) antiendomysial antibodies are specific for celiac disease and are used for screening, diagnosis and follow-up of this disease with an almost 100% sensitivity and specificity. The major target antigen of IgA antiendomysial antibodies was identified as tissue transglutaminase; nevertheless, the existence of the additional unique celiac disease-specific autoantigens is anticipated. In this study we have utilized a proteomic approach in order to search out new autoantigens recognized by serum antibodies of patients with active celiac disease. We report the detection of 11 proteins that were immunorecognized with various frequencies by sera of patients with celiac disease. Four autoantigens were identified by mass fingerprinting approach as actin, ATP synthase beta chain and two charge variants of enolase alpha. While production of IgA antibodies against actin molecules were described earlier, the existence of autoantibodies to ATP synthase beta chain and enolase alpha species in sera collected from patients with active celiac disease are described for the first time. These results are suggestive of the existence of additional celiac disease autoantigens with possible diagnostic utility.     

Corneal microsporidioses: characterization and identification.

Two ocular infectious disorders attributed to Microsporidia have been observed. They differ in that one infection involves the corneal stroma leading to corneal ulceration and suppurative keratitis whereas the other infection involves the conjunctival and corneal epithelium. The corneal stromal infection is caused by a binucleated oval spore that is Nosema-like in character. The conjunctival and corneal epithelial infection occurs in HIV-sero-positive individuals and is caused by a spore containing a single nucleus that is a member of the genus Encephalitozoon. Characteristics of these genera and the above-mentioned infections are presented.     

Human placental DNA polymerase delta: identification of a 170-kilodalton polypeptide by activity staining and immunoblotting

DNA polymerase delta was isolated from human placenta and identified as such on the basis of its association with a 3'- to 5'-exonuclease activity. The association of the polymerase and exonuclease activities was maintained throughout purification and attempted separations by physical or electrophoretic methods. Moreover, ratios of the two activities remained constant during the purification steps, and both activities were inhibited by aphidicolin, oxidized glutathione, and N-ethylmaleimide. The purified enzyme had an estimated molecular weight of 172,000, on the basis of a Stokes radius of 53.6 A and a sedimentation coefficient of 7.8 S. On sodium dodecyl sulfate (SDS) gel electrophoresis, polymerase delta preparations contained a band of ca. 170 kilodaltons (kDa) as well as several smaller polypeptides. The 170-kDa polypeptide was identified as the largest polypeptide component in the preparation possessing DNA polymerase activity by an activity staining procedure following gel electrophoresis in the presence of SDS. Western blotting of DNA polymerase delta with polyclonal antisera also revealed a single 170-kDa immunoreactive polypeptide. Monoclonal antibodies to KB cell polymerase alpha inhibited placental polymerase alpha but did not inhibit DNA polymerase delta, while the murine polyclonal antisera to polymerase delta inhibited delta but not alpha. These findings establish the existence of DNA polymerase delta in a human tissue and support the view that both its polymerase and its exonuclease activities may be associated with a single protein.     

Platelet plasma membrane: characterization of the serotonin transporter]

After treatment of human platelets by a sulfhydryl-dependent bacterial protein cytolysin, a glycoprotein was reproducibly purified by a one-step affinity chromatography using 6-fluorotryptamine as ligand and elution by serotonin (5-HT), cyanoimipramine, citalopram, or a Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa. The purified glycoprotein bound the 5-HT uptake blockers 3H-paroxetine, 3H-cyanoimipramine, and 3H-citalopram with Kds similar to the ones observed for intact human platelets. No binding was detected with 3H-hydroxytetrabenazine, 3H-ouabain, 3H-gamma aminobutyric acid or 3H-BTCP, the respective markers of the granular monoamine transporter, the plasma membrane Na+, K(+)-ATPase, the gamma aminobutyric acid and dopamine carriers. The purified 68-kDa glycoprotein is therefore likely to correspond at least to the paroxetine and imipramine binding domains of the 5-HT transporter located at the human platelet plasma membrane. Finally a 68-kDa protein was purified in the same conditions from the human megakaryocytic cell line Dami and to a lesser extent from the human megakaryoblastic cell line MEG-01 but not from the human erythroleukaemic cell line HEL.     

Liposome immunoblotting assay using a substrate-forming precipitate inside immunoliposomes

A new immunoblotting assay which uses antibody-coupled liposomes containing horseradish peroxidase is proposed. A substrate 4-chloro-1-naphthol permeated through the phospholipid membrane of the antibody-coupled liposomes and formed a colored product precipitating inside the liposomes. The precipitates accumulated in the liposomes and could be detected at the positions where the liposomes coupled with a target in blotted samples. Combination of liposomes with average diameter of 350 nm and a PVDF membrane with a pore size of 450 nm, 0.02 ng of IgM was detected, while the conventional immunoblotting using antibody-HRP conjugates detected 2 ng of IgM. The sensitivity increased about two orders of magnitude by the liposome immunoblotting assay. This liposome immunoblotting assay gives a simple detection method of proteins with a high sensitivity, as well as a high sensitivity Western blotting assay.     

Ecdysteroids in nemerteans: further characterization and identification

Ecdysteroids are a class of steroidal hormones thatare important in molting and reproduction inarthropods. These hormones have been recentlydetected in non-arthropodan groups, such assoft-bodied worms. To continue our efforts todetermine the presence of ecdysteroids in nemerteans,this study further documents the identification of20-hydroxyecdysone (20E), in Paranemertesperegrina, by gas chromatography/mass spectrometry(GC/MS). After C₁₈ Sep-Pak extraction,radioimmunoassay of the 40% and 80% methanolicfractions from an extract of 1000 worms indicated 98and 156 ng of ecdysteroids, respectively. Ecdysteroids of these two samples, as well as the 20Eand ecdysone standards, were N-trimethylsilyimidazole(TMSI)-derivatized before GC/MS analysis. Themethanolic samples contained a large number ofcompounds but only small, insignificant peaks in thearea where ecdysteroid standards eluted. However, thereconstruction ion current (RIC) chromatograms for m/z561 indicated the presence of 20E at the correctretention time of 12.48 min in the 40% methanolicfraction. Reanalysis of the samples under selectedion monitoring mode demonstrated the presence of 20Ein both the 40% and 80% fractions. These resultsdemonstrate conclusively the presence of an activeecdysteroid in the phylum Nemertea.     

Fish plasminogen activators: their identification and characterization

Immunoblots of proteins extracted from the skin of a small viviparous fish (Xiphophorus) showed that a monoclonal antibody against human urokinase recognizes multiple molecular weight species of antigens. The immunoaffinity-purified antigens had serine-protease activity for the hydrolysis of a chromogenic substrate and could convert human plasminogen to plasmin in a manner similar to that for human urokinase in vitro. Two antigens with apparent molecular weights of 55 and 50 kilodaltons that had been purified on a fibrin-Celite column were separable on SDS-polyacrylamide gels and were characterized as major plasminogen activators on fibrin-agar indicator plates. The 125I-tryptic peptide maps of both antigens were similar to that of human urokinase; therefore, the fish activators and human urokinase are structurally and functionally related.     

基于高通量测序的长穗偃麦草功能分子标记发掘和分析

长穗偃麦草是小麦重要的近源物种,含有丰富的抗逆基因,广泛地应用于小麦的遗传改良育种。本研究利用高通量测序,获得长穗偃麦草的转录组测序信息,利用比较基因组学方法研究其与小麦、水稻和玉米等作物的遗传关系,评估它们之间的亲缘关系。同时,将长穗偃麦草的高通量序列比对到小麦基因,利用软件Freebayes和SAMtools/Bcftools发掘功能基因的变异位点,并对这些含有变异位点的功能基因进行注释分析,揭示长穗偃麦草优异性状形成的分子机制,这将为长穗偃麦草优异基因资源的开发和应用奠定重要基础。     

The zebrafish erythropoietin: functional identification and biochemical characterization

In the present study, the zebrafish epo cDNA was cloned. The encoded protein displays 90%, 55% and 32% identity to the Epo from carp, fugu and human, respectively. Through RT-PCR, the expression of zepo mRNA was mainly in the heart and liver. In the COS-1 cell transfection experiments, the recombinant zEpo-HA protein was efficiently secreted into the culture medium as a glycoprotein and the carbohydrate moiety can be cleaved by the treatment of peptide-N-glycosidase F (PNGase F). Using the morpholino approach, we showed that zepo morphants displayed severe anemia leading to high mortality during development. Such an effect can be significantly rescued by zepo RNA. Furthermore, in the absence of functional zEpo, the expression of specific markers for adult globin genes, such as alphaA1- and betaA1-globin, but not the embryonic betae1-globin, was affected.     

MICE: multiple-peak identification, characterization, and estimation

MICE--multiple-peak identification, characterization, and estimation--is a procedure for estimating a lower bound of the number of frequency peaks and for estimating the frequency peak parameters. The leading application is protein structure determination using nuclear magnetic resonance (NMR) experiments. NMR frequency data are multiple-peak data, where each frequency peak corresponds to two connected atoms in the three-dimensional protein structure. We analyze the NMR frequency data through a series of steps: a preliminary step for separating the signal from the background followed by identification of local maxima up to a noise-level-dependent threshold, estimation of the frequency peak parameters using an iterative algorithm, and detection of mixtures of peaks using hypothesis testing.     

Platelet contractile proteins: separation and characterization of the actin and myosin-like components.

Solution of thrombosthenin, the contractile protein complex isolated from pig platelets, have been studied by analytical ultracentrifugation and zone sedimentation in sucrose density gradients. Freshly prepared thrombosthenin in 0.6 M KCl shows a prominent peak in the ultracentrifuge with S degrees 20w about 5.5 and higher molecular weight aggregates (greater than 100S) sedimenting quickly to the bottom of the cell. Short term storage of high ionic strength solutions of thrombosthenin induces actomyosin-like gel formation and these gels dissociate with ATP and Mg2+ ions into two components of S degrees 20w 8.0 and S degrees 20w50. The supernatant, after actomyosin gel removal, contains only the S degrees 20w5.5 protein. From results of Ca2+ ATPase activity measurements and SDS polyacrylamide gel electrophoretic mobilities of dissociated thrombosthenin separated into fractions in sucrose density gradients, it is concluded that the S degrees20w5.5 protein species is the myosin-like protein of thrombosthenin. The S degrees 20w8.0 protein is not fibrinogen but also has myosin-like properties and is believed to be myosin dimer. Species of higher S values seen in the presence of ATP and Mg2+ in the analytical ultracentrifuge and located in the higher density zones of the sucrose gradients all gave in SDS polyacrylamide gel electrophoresis a single band of molecular weight 46-47,000 daltons. These subunit proteins appear to be derived from a range of polymeric variants of the F-actin-like protein of the contractile complex. All these higher density F-actin-like proteins readily form superprecipitates and display syneresis when combined with rabbit skeletal muscle myosin or platelet myosin. They are also all capable of conferring upon these two myosins a Mg2+ activated ATPase activity. It is suggested that in thrombosthenin solutions a myosin monomer-dimer equilibrium state exists which can be directionally influenced by a number of factors. The coexistence in the solution of F-actin and Mg2+ ATP, for example, increases the propensity of the myosin-like protein to form the higher molecular weight aggregate. Such aggregation may be the initiating mechanism for the intracellular organization of the thick filaments of the actomyosin complex, preparatory to a contractile event.     

Schistosoma mansoni: identification and characterization of schistosomula polypeptides

Schistosomula proteins separated by a two-dimensional (NEPHGE) gel system identify 94 major silver-stained polypeptides. When compared to polypeptides similarly separated from cercariae and adult worms; cercariae share the same polypeptides as schistosomula, adult worms share ca. 60% of the polypeptides. A group of five schistosomula polypeptides 15-31 kDa (apparent pI 8.2-8.9) was not found in adult worm extracts. To identify which polypeptides were immunogens, Western blots of the NEPHGE gels were probed with sera either from humans with chronic schistosomiasis or from mice vaccinated with irradiated cercariae. For characterization studies, polyclonal antibodies were made against the five schistosomula-specific and selected immunogenic polypeptides by immunizing mice with silver-stained spots removed from NEPHGE gels. We show that the polyclonal serum against a polypeptide of 12.5 kDa and an apparent pI of 6.70 mediated complement and eosinophil-dependent killing of schistosomula in an in vitro assay. Epitopes recognized by antibody against the 12.5-kDa polypeptide show a diffuse distribution and are found on flame cells of the excretory system of the schistosomula.     

Platelet cryopreservation using a trehalose and phosphate formulation

Long-term storage of platelets is infeasible due to platelet activation at low temperatures. In an effort to address this problem, we evaluated the effectiveness of a formulation combining trehalose and phosphate in protecting platelet structure and function following cryopreservation. An annexin V binding assay was used to quantify the efficacy of the trehalose and phosphate formulation in suppressing platelet activation during cryopreservation. Of the platelets cryopreserved with the trehalose plus phosphate formulation, 23% +/- 1.2% were nonactivated, compared with 9.8% +/- 0.26% nonactivated following cryopreservation with only trehalose. The presence of both trehalose and phosphate in the cryopreservation medium is critical for cell survival and preincubation in trehalose plus phosphate solutions further enhances viability. The effectiveness of trehalose plus phosphate in preserving platelets in a nonactivated state is comparable to 6% dimethyl sulfoxide (Me(2)SO). Measurements of platelet metabolic activity using an alamarBlue assay also established that trehalose plus phosphate is superior to trehalose alone. Finally, platelets protected by the trehalose plus phosphate formulation exhibit similar aggregation response upon thrombin addition as fresh platelets, but an increase of cytosolic calcium concentration upon thrombin addition was not observed in the cryopreserved platelets. These results suggest that trehalose and phosphate protect several aspects of platelet structure and function during cryopreservation, including an intact plasma membrane, metabolic activity, and aggregation in response to thrombin, but not intracellular calcium release in response to thrombin.