The Effects of N Helix Deletion and Mutant F29W on the Ca2+ Binding and Functional Properties of Chicken Skeletal Muscle Troponin C

To assess the structural and functional significance of the N helix (residues 3-13) of avian recombinant troponin C (rTnC), we have constructed NHdel, in which residues 1-11 have been deleted, both in rTnC and in the spectral probe mutant F29W (Pearlstone, J. R., Borgford, T., Chandra, M., Oikawa, K., Kay, C. M., Herzberg, O., Moult, J., Herklotz, A., Reinach, F. C., and Smillie, L.B. (1992) Biochemistry 31, 6545-6553). Comparison of the far- and near-UV CD spectra (±Ca2+) of F29W and F29W/ NHdel and titration of the Ca2+-induced ellipticity and fluorescence changes indicates that the deletion has little effect on the global fold of the molecule but reduces the Ca2+ affinity of the N domain, but not the C domain, by 1.6-1.8-fold. Comparisons of the mutants NHdel, F29W, and F29W/NHdel with rTnC have been made using several functional assays. In reconstituted troponin-tropomyosin actomyosin subfragment 1 and myofibrillar ATPase systems, both F29W and NHdel have significantly reduced Ca2+-activated enzymic activities. These effects are cumulative in the double mutant F29W/ NHdel. On the other hand, maximal isometric tension development in Ca2+-activated reconstituted skinned fibers is not affected with F29W and NHdel, although the Ca2+ sensitivity of NHdel in this system is markedly reduced. We conclude that both mutations, NHdel and F29W, are functionally deleterious, possibly affecting interactions of the N domain with troponin I and/or T.

Dwarf gynes in Nannotrigona testaceicornis (Apidae, Meliponinae, Trigonini). Behaviour, exocrine gland morphology and reproductive status

The behaviour and morphology of dwarf gynes produced in worker-sized cells of normal colonies in Nannotrigona testaceicornis (Meliponinae, Trigonini) were studied. The behaviour of these dwarf virgin queens was the same as observed for normal Trigonine gynes. The glandular equipment is also the same: Dufour glands, fat bodies and spermathecae are present. Despite these similarities, their ovaries are different. The functional significance of dwarf gynes is unknown, but may be a basis for an alternative reproductive strategy.

Cellulolytic activity of wild type and mutant Trichoderma pseudokoningii

Strains of Trichoderma pseudokoningii are promising objects for genetic studies and cellulase production. Auxotrophic mutants with deficiencies in the biosynthesis of aminoacids, nucleotides and vitamins (up to five markers) in addition to morphological aspects like conidial colour were obtained from two strains of double auxotrophic mutants using UV radiation. In order to compare the cellulolytic capabilities of the T. pseudokoningii (wild type strain), some of its mutants and T. reesei QM9414 we performed semiquantitative cellulase assays and quantitative determination of the enzymes exoglucanase and endoglucanase. The semiquantitative test showed that the strains with minimal mycelial growth rate were better producers. Both tests revealed that two of the studied mutants, TG3 and TG4 presented a yield higher than the wild type, reaching 30% more exoglucanase and 70% more endoglucanase. These results indicate that the wild type was improved for cellulase production. Highly significant values of correlation were found for exoglucanase and endoglucanase activities, suggesting that these enzymes may be co-regulated in T. pseudokoningii.

Chromosome damage induced by 5-azacytidine under the influence of caffeine or cytosine arabinoside in CHO-K1 (wild-type) and XRS-5 (mutant) cell lines

5-azacytidine (5-azaC) treatment combined with cytosine arabinoside (ara-C) or caffeine were performed in vitro in Chinese hamster cells, CHO-K1 (wild-type) and xrs-5 (mutant) cell lines, in order to compare the cell response to the induction of chromosomal aberrations. Exponentially growing cells were treated with 5-azaC (4-16 uM) for 1 h, the cells were washed and incubated for 7 h, and 500 uM caffeine or 5 uM ara-C were added to the cultures for the last 2 h. In both cell lines, 5-azaC induced a significantly increase (P<0.01) in the frequencies of aberrations; in the combined treatments (5-azaC + Ara-C), a significant reduction (P<0.05) was observed for the aberrations which were randomly distributed. Caffeine had no influence at the same conditions. 5-azaC induced-DNA lesions were probably processed at S/G2 phase in a common pathway in both cell lines, but alternatively, 5-azaC may cause xrs-5 cells to revert to the wild-type.

Bacillus Thuringiensis mutant increase activity against Apodoptera Frugiperda larvae

Bacillus thuringiensis is a Gram-positive bacterium which main characteristic is the production of Cry proteins, that is toxic to some insects. These proteins, when ingested by susceptible insects, become active causing their death. In nature, it is possible to found B. thuringiensis strains which produce these proteins, but they differ in productivity (some of these isolates are more productive then others), and as to the toxicity levels of the produced proteins. Two B. thuringiensis strains that were highly effective against Spodoptera frugiperda larvae were chosen to verifying genetic mutation implication on Cry proteins productivity. One strain with a prolific spores production, while the other one only produced small amounts of spores. A genomic mutant library of these two isolates was, separately, constructed by genome Tn-5 transposon random insertion. Data analysis showed that mutation had a direct effect on the spores production, inducing an increase as well as a decrease in the production, according to the different strain observed. These results indicate, for the first time, that it is possible to use the described technique with B. thuringiensis, as well as the possibility to genetically breeding this bacteria. Another possibility introduced here is the possibility to do functional genetic studies mediated by mutagenesis in this bacterium.

Action of dwarf mucuna, pigeon pea and stylos anthes on weed under field and laboratory conditions

The quality and allelopathy properties of dwarf mucuna, dwarf pigeon pea and stylosanthes as cover crops on corn and weed species were evaluated. Seeds were sown in October 2007, with a control treatment, in 20 plots of 4×5m, with five replicates. Weed population was determined 30 and 60 days after sowing. At 90 days, plants were mowed and the residues left to remain on the plot. Fresh and dry mass of the cover crops were determined and the allelopathic potential of aqueous extract of their aerial part was tested. The extract was chemically characterized and applied on seeds of weeds and corn. The experimental design was completely randomized and averages compared by the Scott-Knott test at 5% significance. The cover crops showed to be effective in the control of weeds. The highest values in fresh and dry mass were obtained for dwarf pigeon pea, followed by dwarf mucuna; fresh mass increased 72 and 34%, respectively, compared to the control. The extract with dwarf mucuna affected arrowleaf sida germination. The use of green manure in the summer or between harvests ensures that crop rotation is carried out properly and warrants its benefits.

Fontes e doses de giberelina no desempenho de arroz anão em biotestes

Mutant of dwarf rice (Oryza sativa L.) were treated with three different concentrations of gibberellic acid originated from two sources (Gibberellin and Pro-Gibb) and submitted to the determination of bigger height, in relation to the soil leveI. Mensurations were realized in each seven days at greenhouse conditions under mean temperature of 25°C. lt was concluded that concentrations of 10 mg L-1 of gibberellin originated from Pro-Gibb or Gibberellin was enough to promote a differentiated performance of the test plant. For the study of leaf anatomy Pro-Gibb presented, under all tested concentration, increase of mesophyll thickness in relation to Gibberellin. In this analysis, source and interaction between source and concentration applied promoted significant variations between the mean data.

The Deoxyhypusine Synthase Mutant dys1-1 Reveals the Association of eIF5A and Asc1 with Cell Wall Integrity

The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1) catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1) and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1Δ and overexpression of TIF51A (eIF5A) or DYS1 is toxic for an asc1Δ strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1Δ mutants in comparison with the pkc1Δ mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of mRNAs associated with cell integrity. © 2013 Galvão et al.

Search for methylation-sensitive amplification polymorphisms in mutant figs

Fig (Ficus carica) breeding programs that use conventional approaches to develop new cultivars are rare, owing to limited genetic variability and the difficulty in obtaining plants via gamete fusion. Cytosine methylation in plants leads to gene repression, thereby affecting transcription without changing the DNA sequence. Previous studies using random amplification of polymorphic DNA and amplified fragment length polymorphism markers revealed no polymorphisms among select fig mutants that originated from gamma-irradiated buds. Therefore, we conducted methylation-sensitive amplified polymorphism analysis to verify the existence of variability due to epigenetic DNA methylation among these mutant selections compared to the main cultivar 'Roxo-de-Valinhos'. Samples of genomic DNA were double-digested with either HpaII (methylation sensitive) or MspI (methylation insensitive) and with EcoRI. Fourteen primer combinations were tested, and on an average, non-methylated CCGG, symmetrically methylated CmCGG, and hemimethylated hmCCGG sites accounted for 87.9, 10.1, and 2.0%, respectively. MSAP analysis was effective in detecting differentially methylated sites in the genomic DNA of fig mutants, and methylation may be responsible for the phenotypic variation between treatments. Further analyses such as polymorphic DNA sequencing are necessary to validate these differences, standardize the regions of methylation, and analyze reads using bioinformatic tools. © FUNPEC-RP.

Experimental infection of commercial layers with wild or attenuated Salmonella Gallinarum mutant strains: Anatomic pathology, total blood cell count and serum protein levels

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Rhamnolipids Production by a Pseudomonas eruginosa LBI Mutant: Solutions and Homologs Characterization

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation

Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development.

Wood and leaf anatomy of Copaifera langsdorffii dwarf trees

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cetacean Notes. I. Sei and Rorqual Whales on the Mississippi Coast, a Correction. II. A Dwarf Sperm Whale in Mississippi Sound and Its Helminth Parasites

I. Gunter and Christmas (1973) described the events leading to the stranding of a baleen whale on Ship Island, Mississippi, in 1968, giving the species as Balaenopteru physalus, the Rorqual. Unfortunately the identification was in error, but fortunately good photographs were shown. The underside of the tail was a splotched white, but there was no black margin. The specimen also had fewer throat and belly grooves than the Rorqual, as a comparison with True’s (1904) photograph shows. Dr. James Mead (in litt.) pointed out that the animal was a Sei Whale, Balaenoptera borealis. This remains a new Mississippi record and according to Lowery’s (1974) count, it is the fifth specimen reported from the Gulf of Mexico. The stranding of a sixth Sei Whale on Anclote Keys in the Gulf, west of Tarpon Springs, Florida on 30 May 1974, was reported in the newspapers and by the Smithsonian Institution (1974). II. Gunter, Hubbs and Beal (1955) gave measurements on a Pygmy Sperm Whale, Kogia breviceps, which stranded on Mustang Island on the Texas coast and commented upon the recorded variations of proportional measurements in this species. Then according to Raun, Hoese and Moseley (1970) these questions were resolved by Handley (1966), who showed that a second species, Kogia simus, the Dwarf Sperm Whale, is also present in the western North Atlantic. Handley’s argument is based on skull comparisons and it seems to be rather indubitable. According to Raun et al. (op. cit.), the stranding of a species of Kogia on Galveston Island recorded by Caldwell, Ingles and Siebenaler (1960) was K. simus. They also say that Caldwell (in litt.) had previously come to the same conclusion. Caldwell et al. also recorded another specimen from Destin, Florida, which is now considered to have been a specimen of simus. The known status of these two little sperm whales in the Gulf is summarized by Lowery (op. cit.).

Comparison of Two Roadside Survey Procedures for Dwarf Mistletoes on the Sawtooth National Forest, Idaho

Two roadside surveys were conducted for dwarf mistletoes parasitizing lodgepole pine and Douglas-fir on the Sawtooth National Forest, Idaho. One survey used variable-radius plots located less than 150 m from roads. The 2nd survey used variable-radius plots established at 200-m intervals along 1600-m transects run perpendicular to the same roads. Estimates of the incidence (percentage of trees infected and percentage of plots infested) and severity (average dwarf mistletoe rating) for both lodgepole pine and Douglas-fir dwarf mistletoes were not significantly different for the 2 survey methods. These findings are further evidence that roadside-plot surveys and transect-plot surveys conducted away from roads provide similar estimates of the incidence of dwarf mistletoes for large forested areas.