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Robust-LongSAGE (RL-SAGE): a substantially improved LongSAGE method for gene discovery and transcriptome analysis.
Serial analysis of gene expression (SAGE) is a widely used technique for large-scale transcriptome analysis in mammalian systems. Recently, a modified version called LongSAGE (S. Saha, A.B. Sparks, C. Rago, V. Akmaev, C.J. Wang, B. Vogelstein, K.W. Kinzler [2002] Nat Biotechnol 20: 508-512) was reported by increasing tag length up to 21 bp. Although the procedures for these two methods are similar, a detailed protocol for LongSAGE library construction has not been reported yet, and several technical difficulties associated with concatemer cloning and purification have not been solved. In this study, we report a substantially improved LongSAGE method called Robust-LongSAGE, which has four major improvements when compared with the previously reported protocols. First, a small amount of mRNA (50 ng) was enough for a library construction. Second, enhancement of cDNA adapter and ditag formation was achieved through an extended ligation period (overnight). Third, only 20 ditag polymerase chain reactions were needed to obtain a complete library (up to 90% reduction compared with the original protocols). Fourth, concatemers were partially digested with NlaIII before cloning into vector (pZEro-1), greatly improving cloning efficiency. The significant contribution of Robust-LongSAGE is that it solved the major technical difficulties, such as low cloning efficiency and small insert sizes associated with existing SAGE and LongSAGE protocols. Using this protocol, one can generate two to three libraries, each containing over 4.5 million tags, within a month. We recently have constructed five libraries from rice (Oryza sativa), one from maize (Zea mays), and one from the rice blast fungus (Magnaporthe grisea).
Nutritional study of two Brazilian soybean (Glycine max) cultivars differing in the contents of antinutritional and toxic proteins.
The research was conducted with two different recently released Brazilian soybean cultivars (Rio Balsas and Bays) to evaluate whether there is any correlation between the different levels of antinutritional and/or toxic proteins in the cultivars and their nutritive value as sources of protein for monogastric animals (rats). Furthermore, it is discussed, for the first time, the role of the dietary soyatoxin on the performance of rats fed on diets containing soyatoxin-rich (cv. Bays) and soyatoxin-free (cv. Rio Balsas) soybean cultivars. Feeding rats with diets containing raw soybean cultivars showed a lower growth rate, net protein utilization and digestibility, a much higher dry matter and nitrogen excretion and macroscopic alterations in internal organs when compared to rats fed on egg-white protein. The nutritional parameters measured for the diet based on raw Bays cultivar were poorer than those of the diet prepared with Rio Balsas. In the raw soybeans, trypsin inhibitor and lectin, and urease to a lesser extent, significantly affected at different fashion the soybean protein utilization. Heating treatment of the Bays seeds increased the growth rate, NPU, in vivo protein digestibility and practically eliminated or attenuated all the organ alterations observed. This study might be helpful in the choice of safe and nutritious soybean cultivars.
A model for short alpha-neurotoxin bound to nicotinic acetylcholine receptor from Torpedo californica: comparison with long-chain alpha-neurotoxins and alpha-conotoxins.
Short-chain alpha-neurotoxins from snakes are highly selective antagonists of the muscle-type nicotinic acetylcholine receptors (nAChR). Although their spatial structures are known and abundant information on topology of binding to nAChR is obtained by labeling and mutagenesis studies, the accurate structure of the complex is not yet known. Here, we present a model for a short alpha-neurotoxin, neurotoxin II from Naja oxiana (NTII), bound to Torpedo californica nAChR. It was built by comparative modeling, docking and molecular dynamics using 1H NMR structure of NTII, cross-linking and mutagenesis data, cryoelectron microscopy structure of Torpedo marmorata nAChR [Unwin, N., 2005. Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J. Mol. Biol. 346, 967-989] and X-ray structures of acetylcholine-binding protein (AChBP) with agonists [Celie, P.H., van Rossum-Fikkert, S.E., van Dijk, W.J., Brejc, K., Smit, A.B., Sixma, T.K., 2004. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41 (6), 907-914] and antagonists: alpha-cobratoxin, a long-chain alpha-neurotoxin [Bourne, Y., Talley, T.T., Hansen, S.B., Taylor, P., Marchot, P., 2005. Crystal structure of Cbtx-AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors. EMBO J. 24 (8), 1512-1522] and alpha-conotoxin [Celie, P.H., Kasheverov, I.E., Mordvintsev, D.Y., Hogg, R.C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S.E., Zhmak, M.N., Bertrand, D., Tsetlin, V., Sixma, T.K., Smit, A.B., 2005. Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat. Struct. Mol. Biol. 12 (7), 582-588]. In complex with the receptor, NTII was located at about 30 A from the membrane surface, the tip of its loop II plunges into the ligand-binding pocket between the alpha/gamma or alpha/delta nAChR subunits, while the loops I and III contact nAChR by their tips only in a 'surface-touch' manner. The toxin structure undergoes some changes during the final complex formation (for 1.45 rmsd in 15-25 ps according to AMBER'99 molecular dynamics simulation), which correlates with NMR data. The data on the mobility and accessibility of spin- and fluorescence labels in free and bound NTII were used in MD simulations. The binding process is dependent on spontaneous outward movement of the C-loop earlier found in the AChBP complexes with alpha-cobratoxin and alpha-conotoxin. Among common features in binding of short- and long alpha-neurotoxins is the rearrangement of aromatic residues in the binding pocket not observed for alpha-conotoxin binding. Being in general very similar, the binding modes of short- and long alpha-neurotoxins differ in the ways of loop II entry into nAChR.
The compliance of contracting skeletal muscle.
1. The method of controlled releases was used to obtain tension-extension curves in toad (Bufo bufo) sartorii under a variety of conditions at 0 degrees C.2. The curves obtained were approximately linear over a considerable range of force (0.4P(0) to P(0)) if the releases were given from the plateau of tetanic tension. The slope of this linear region was little affected by changes of release velocity in the range 10-120 mm/sec.3. Such changes as did occur with alterations in release velocity could be quantitatively accounted for in terms of the internal shortening predicted by A. V. Hill's two-component model.4. As the muscles were stretched above l(0), we found that the maximum stiffness of the tetanized muscles fell in much the same way as the maximum developed force, P(0).5. In another series of experiments we found a rapid change in the overall shape of the tension-extension curve during the early phase of force development in an isometric tetanus. The stiffness of the muscle increased with increasing developed force during this period.6. The force-velocity curve in these muscles was measured by two methods, both giving a similar result. Surprisingly, toad muscle appears to have about the same intrinsic speed as frog muscle at 0 degrees C. The a.b product from our experiments is considerably greater than the reported values for the maintenance heat rate at 0 degrees C in these muscles.7. The probable site of the variable compliance in active muscle is discussed. It seems most likely that this is within the A-band, perhaps in the cross-bridges themselves.
The energetics of tortoise muscle.
1. A study has been made of the mechanical behaviour and the heat production of tortoise skeletal muscle during tetanic contractions.2. The relation between force (P) and velocity (v) is more curved than that of frog muscle. It can be fitted by Hill's equation (v/v(max) = (1 - P/P(0))/(1 + P/a)) using a value of P(0)/a considerably less than for frog muscle.3. The value of the shortening heat constant is less than in frog muscle. It is about a half of a. No evidence was found that the value depended on the load.4. The maintenance heat rate is much less than in frog muscle and, as in frog muscle, is roughly equal to a.b (where b = v(max).a/P(0)). The maintenance heat rate does not diminish during a long tetanus as it does in frog muscle.5. The ratio of work/(work + initial heat) is greater in tortoise muscle than in frog muscle. As in frog muscle, the recovery heat is about equal to (work + initial heat). It follows that tortoise muscle is more efficient than frog muscle at converting free energy into work.6. Possible theoretical bases for connexions between the shape of the force-velocity curve of a muscle and its efficiency are discussed.
Overexpression of polygalacturonase in transgenic apple trees leads to a range of novel phenotypes involving changes in cell adhesion.
Polygalacturonases (PGs) cleave runs of unesterified GalUA that form homogalacturonan regions along the backbone of pectin. Homogalacturonan-rich pectin is commonly found in the middle lamella region of the wall where two adjacent cells abut and its integrity is important for cell adhesion. Transgenic apple (Malus domestica Borkh. cv Royal Gala) trees were produced that contained additional copies of a fruit-specific apple PG gene under a constitutive promoter. In contrast to previous studies in transgenic tobacco (Nicotiana tabacum) where PG overexpression had no effect on the plant (K.W. Osteryoung, K. Toenjes, B. Hall, V. Winkler, A.B. Bennett [1990] Plant Cell 2: 1239-1248), PG overexpression in transgenic apple led to a range of novel phenotypes. These phenotypes included silvery colored leaves and premature leaf shedding due to reduced cell adhesion in leaf abscission zones. Mature leaves had malformed and malfunctioning stomata that perturbed water relations and contributed to a brittle leaf phenotype. Chemical and ultrastructural analyses were used to relate the phenotypic changes to pectin changes in the leaf cell walls. The modification of apple trees by a single PG gene has offered a new and unexpected perspective on the role of pectin and cell wall adhesion in leaf morphology and stomatal development.
Role of B-1a cells in autoimmunity.
B-1a cells are distinguished from conventional B cells (B2) by their developmental origin, their surface marker expression and their functions. They were originally identified as a B cell subset of fetal origin that expresses the pan-T cell surface glycoprotein, CD5. B-1a cells also differ from B2 by the expression levels of several surface markers, including IgM, IgD, CD43 and B220 [R. Berland, H.H. Wortis, Origins and functions of B-1 cells with notes on the role of CD5. Ann Rev Immunol, 20 (2002) 253-300.]. The majority of B-1a cells are located in peritoneal and pleural cavities. Compared to B2 cells, B-1a are long-lived, non-circulating, with reduced BCR diversity and affinity [A.B. Kantor, C.E. Merrill, L.A. Herzenberg, J.L. Hillson, An unbiased analysis of V-H-D-J(H) sequences from B-1a, B-1b, and conventional B cells. J Immunol, 158 (1997) 1175-1186.]. B-1a cells are largely responsible for the production of circulating IgM referred to as natural antibodies. These low affinity antibodies are polyreactive and constitute as such a first line of defense against bacterial pathogens [M.C. Carroll, A.P. Prodeus, Linkages of innate and adaptive immunity. Curr Opin Immunol, 10 (1998) 36-40.]. This polyreactivity also results into the recognition of autoantigens, which serves in the clearance of apoptosis products. The weak autoreactivity of the B-1a cells has been postulated to play a role in autoimmune pathogenesis. In addition, other characteristics, such as the production of high level of IL-10 [A. O'Garra, R. Chang, N. Go, R. Hastings, G. Haughton, M. Howard, et al. Ly-1 B (B-1) cells are the main source of B cell-derived interleukin 10. Eur J Immunol, 22 (1992) 711-717.] and enhanced antigen presentation capacities [C. Mohan, L. Morel, P. Yang, E.K. Wakeland, Accumulation of splenic B1a cells with potent antigen-presenting capability in NZM2410 lupus-prone mice. Arthritis and Rheumatism, 41 (1998) 1652-1662.], have implicated B-1a cells in autoimmunity. This review will discuss the current understandings of their role in autoimmune diseases with focus on lupus.
Inactivation of the SNF5 transcription factor gene abolishes the lethal phenotype induced by the expression of HIV-1 integrase in yeast.
The ubiquitous human transcription factor Ini1 has been shown to interact with HIV-1 integrase (IN) and to stimulate in vitro the reactions catalyzed by this enzyme. We have previously used a yeast model to study the effect of HIV-1 IN expression (Caumont, A.B., Jamieson, G.A., Pichuantes, S., Nguyen, A.T., Litvak, S., Dupont, C. -H., 1996. Expression of functional HIV-1 integrase in the yeast Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening. Curr. Genet. 29, 503-510). Here, we describe the effect of the inactivation of the gene encoding for SNF5, a yeast transcription factor homologous to Ini1, on the lethality induced by the expression of HIV-1 IN in yeast. We observed that the retroviral IN was unable to perform its lethal activity in cells where the SNF5 gene has been disrupted, suggesting that SNF5 may play a role in the lethal effect induced by IN in yeast. SNF5 inactivation affects neither yeast viability nor expression of HIV-1 IN. Given the homology between SNF5 and its human counterpart Ini1, our results suggest that this factor may be important for IN activity in infected cells. Moreover, given the important role proposed for this transcription factor in the integration step and the fact that it is dispensable for cell viability, the interaction between Ini1/ySNF5 and HIV-1 IN should become a potential target in the search for new antiretroviral agents.
Differential expression within a three-gene subfamily encoding a plasma membrane H(+)-ATPase in Nicotiana plumbaginifolia.
Genomic and cDNA clones for the three members of a gene subfamily (pma) encoding a plasma membrane H(+)-translocating ATPase in Nicotiana plumbaginifolia were isolated and sequenced. They are between 95 and 96% identical at the deduced amino acid sequence level. Sequence comparisons with the corresponding tomato genes (Ewing, N.N., Wimmers, L.E., Meyer, D.J., Chetelat, R.T., and Bennett, A.B. (1990) Plant Physiol. 94, 1874-1881) indicate that divergence among the three N. plumbaginifolia pma genes occurred before the development of the Solanaceae family. Here, determination of pma1 transcription initiation sites reveals several 5' boundaries located 266 to 120 nucleotides upstream from the plasma membrane H(+)-ATPase translation initiation codon. The 5'-untranslated region contains a small open reading frame, 9 residues long. pma3 has a single, 264-nucleotide long 5' leader containing a 5-residue open reading frame. The latter is completely conserved in a corresponding tomato gene. These features suggest the possibility of translational regulation of plant pma genes. S1 nuclease protection assays on total cellular RNA isolated from different organs reveals that all three genes are expressed in leaf, stem, flower, and root tissues, albeit at different levels according to the organ and gene. The different genes for the plant H(+)-translocating ATPase are thus subject to differential regulation of transcription, possibly related to specific aspects of enzyme function.
Radiogenic argon distribution within a mineral grain: implications for dating of hydrothermal mineral-forming event in Sludyanka complex, Siberia, Russia.
Usually, in age-determination procedures, the experimenter is interested in the mean concentrations of parent and daughter substances. However, the structure of a solution of the Fick's diffusion equation enables one to specify a procedure, allowing, through macroscopic experiments, one to determine the distribution of radiogenic argon (as well as any of other isotopes) within a mineral grain. Argon, influenced in the geological past by a metamorphic event, has a half-sinusoidal distribution, whereas argon preserved in geological time has a rectangular distribution. The ratio of the quantity of argon of the first type to the total argon is called an alpha-factor. It is believed that this coefficient can be of great interest for geochronological studies, being a symptom of virtual Ar losses and the time instant, at which they happened. A laboratory procedure and a mathematical algorithm to determine the alpha-factor are developed. As an example, a determination of the alpha-factor on a vein-phlogopite from the well-studied Sludyanka deposit, Southern Baikal, Siberia, Russia, is described. Rb-Sr isochron age on phlogopite-calcite-apatite paragenetic assemblage yielded 460+/-7 Ma and is constrained by U-Pb zircon ages of 471+/-1 and 447+/-2 Ma, respectively, on an emplacement of early syenites and monzonites and later 'post-phlogopitic' pegmatites [E.B. Salnikova, S.A. Sergeev, A.B. Kotov, S.Z. Yakovleva, R.H. Steiger, L.Z. Reznitskiy, E.P. Vasil'ev. U-Pb zircon dating of granulite metamorphism in the Sludyanskiy complex, Eastern Siberia. Gondwana Res., 1, 195-205 (1998). L.Z. Reznitskii, G.P. Sandimirova, Y.A. Pakhol'chenko, S.V. Kuznetsova. The Rb-Sr age of phlogopite deposits in Sludyanka, southern Baikal region. Dokl. Earth Sci., 367, 711-713 (1999). L.Z. Reznitskii, A.B. Kotov, E.B. Salnikova, E.P. Vasil'ev, S.Z. Yakovleva, V.P. Kovach, A.M. Fedoseenko. The age and time span of the origin of phlogopite and lazurite deposits in the Southwestern Baikal area: U-Pb geochronology. Petrology, 8, 66-76 (2000).]. In this study, the phlogopite gave an apparent K-Ar age of 365.6+/-10.4 Ma which has no geological meaning. The alpha-factor of 0.27 was derived from a laboratory kinetic experiment. The age determined by the rectangularly distributed argon corresponded to alphax0.129 nmm3/g (total radiogenic argon in the sample)=94.17 nmm3/g, and an age of approximately 274 Ma (e.g. a period without any argon losses). Here, at the first sight, we deal with common argon losses not conjugated with any geological event, because after 447 Ma only lower temperature (in a range of 50-300 degrees C) hydrothermal processes were recorded. A paragenetic phlogopite-bearing vein mineral, hyalophane, having a disturbed Rb-Sr isotopic system, however, yielded 40Ar/39Ar subplateau age of 271+/-2 Ma [V.V. Ivanenko, M.I. Karpenko, M.A. Litsarev. Age of the Sludyanka phlogopite deposits (in Russian; data of the 39Ar-40Ar method). In Geological Series 5, pp. 92-98, Izvestiya Akademii Nauk SSSR, Moskva (1990).], marking similar timing of the K-Ar isotopic system closure. This indicates that K-Ar isotopic system in the phlogopite was not closed (or was partially reset) long after its crystallisation and closing of the Rb-Sr system, in spite of the fact that the crystal was not affected by processes having temperatures above the accepted closure temperatures for micas. There seems to have acted an exchange mechanism of OH-groups between surrounding fluid and hydrocrystal structure. Thus the argon loss did not occur, spontaneously, but argon particles seem to have been captured and carried to the mineral surface by vacancies formed during displacements of OH-groups.