RNA editing of wheat mitochondrial ATP synthase subunit 9: direct protein and cDNA sequencing.

RNA editing of subunit 9 of the wheat mitochondrial ATP synthase has been studied by cDNA and protein sequence analysis. Most of the cDNA clones sequenced (95%) showed that editing by C-to-U transitions occurred at eight positions in the coding region. Consequently, 5 amino acids were changed in the protein when compared with the sequence predicted from the gene. Two edited codons gave no changes (silent editing). One of the C-to-U transitions generated a stop codon by modifying the arginine codon CGA to UGA. Thus, the protein produced is 6 amino acids shorter than that deduced from the genomic sequence. Minor forms of cDNA with partial or overedited sequences were also found. Protein sequence and amino acid composition analyses confirmed the results obtained by cDNA sequencing and showed that the major form of edited atp9 mRNA is translated.     

Diving behaviour and prey of the Humboldt Penguin (Spheniscus humboldti)

Summary Humboldt Penguins diving in clear water in Chile had two distinct behaviours; short dives where the birds remained within a metre of the surface and bounce dives, where the penguins descended the water column to the sea bed where they immediately returned to the surface. Here, dive time was correlated with water depth. Stomach pumped penguins had eaten predominantly pelagic school fish,Scomberesox, Engraulis andSardinops. Most prey (94 %) had been seized from below. We suggest that short, shallow dives are typical of travelling penguins. Bounce dives, however, would enable foraging penguins, which have an enhanced binocular field above the line of the bill, to perceive surface-swimming prey more easily because silvery clupeid fish appear as silhouettes.

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Zusammenfassung In klaren Gewässern in Chile tauchende Humboldt-Pinguine zeigten zwei deutlich verschiedene Verhaltensweisen. Während kurzer Tauchgänge blieben die Tiere im Bereich von 1 m unter der Oberfläche. Tauchgänge durch unmittelbares Abtauchen in einem Winkel von 45° eingeleitet, führten direkt zum Meeresboden; die Vögel kehrten sofort wieder an die Oberfläche zurück. In diesem Fall war die Tauchzeit von der Wassertiefe abhängig. Pinguine, deren Magen durch Auspumpen untersucht wurde, hatten vorwiegend pelagische Schwarmfische gefressen:Scomberesox, Engraulis undSardinops. Die Mehrzahl der Fische (94 %) wurde von unten kommend erfaßt. Nach unserer Auffassung sind die kurzen und flachen Tauchgänge typisch für nicht jagende Pinguine, die längere horizontale Strecken zurücklegen. Das tiefe Abtauchen dagegen ermöglicht jagenden Pinguinen, die ein besonders gutes Fernsichtvermögen oberhalb des Schnabels besitzen, nahe der Oberfläche schwimmende Beute leichter zu erfassen. Die sonst silbrigen clupeiden Fische sind als dunkle Silhouetten gegen die Wasseroberfläche besser zu erkennen.

     

Essential role of non-canonical Wnt signalling in neural crest migration

Migration of neural crest cells is an elaborate process that requires the delamination of cells from an epithelium and cell movement into an extracellular matrix. In this work, it is shown for the first time that the non-canonical Wnt signalling [planar cell polarity (PCP) or Wnt-Ca2+] pathway controls migration of neural crest cells. By using specific Dsh mutants, we show that the canonical Wnt signalling pathway is needed for neural crest induction, while the non-canonical Wnt pathway is required for neural crest migration. Grafts of neural crest tissue expressing non-canonical Dsh mutants, as well as neural crest cultured in vitro, indicate that the PCP pathway works in a cell-autonomous manner to control neural crest migration. Expression analysis of non-canonical Wnt ligands and their putative receptors show that Wnt11 is expressed in tissue adjacent to neural crest cells expressing the Wnt receptor Frizzled7 (Fz7). Furthermore, loss- and gain-of-function experiments reveal that Wnt11 plays an essential role in neural crest migration. Inhibition of neural crest migration by blocking Wnt11 activity can be rescued by intracellular activation of the non-canonical Wnt pathway. When Wnt11 is expressed opposite its normal site of expression, neural crest migration is blocked. Finally, time-lapse analysis of cell movement and cell protrusion in neural crest cultured in vitro shows that the PCP or Wnt-Ca2+ pathway directs the formation of lamellipodia and filopodia in the neural crest cells that are required for their delamination and/or migration.     

Development of highly stable galectins: truncation of the linker peptide confers protease-resistance on tandem-repeat type galectins

Galectin-9 and galectin-8, members of beta-galactoside-binding animal lectin family, are promising agents for the treatment of immune-related and neoplastic diseases. The proteins consist of two carbohydrate recognition domains joined by a linker peptide, which is highly susceptible to proteolysis. To increase protease resistance, we prepared mutant proteins by serial truncation of the linker peptide. As a result, mutant forms lacking the entire linker peptide were found to be highly stable against proteolysis and retained their biological activities. These mutant proteins might be useful tools for analyzing the biological functions and evaluating the therapeutic potential of galectin-9 and galectin-8.     

Loss of RPA1 induces Chk2 phosphorylation through a caffeine-sensitive pathway

RPA is an important component of DNA replication, repair and recombination, but its involvement in the signaling of cell-cycle checkpoints is not well understood. In this study, we show that knockdown of RPA1 by siRNA duplexes induces ATM (Ser1981) and Chk2 (Thr68), but not Chk1 (Ser345) phosphorylation and results in p21 upregulation in HeLa cells. However, the induction of Chk2 (Thr68) phosphorylation and p21 expression by RPA1 siRNA transfection can be completely blocked by the ATM inhibitor caffeine. Moreover, transfection of siRNAs targeting ATM dramatically reduces Chk2 (Thr68) phosphorylation in RPA1 knockdown cells. Taken together, these results suggest that loss of RPA1 activates the Chk2 signaling pathway in an ATM-dependent manner.     

Isolation and identification of a plant growth promotive substance from mixture of essential plant oils

The PCS (commercial products by Field Science Co, Japan, used for air fresheners) was analyzed for the presence of bioactive constituents and their role as root growth promoters. Chromatographic separation of the methanolic solution of PCS resulted in the isolation of an promoting active substance, which was identified using GC-mass spectrometry and NMR spectroscopy as 1,2-propanediol (CH₃CH(OH)CH₂OH). Lettuce seedling growth bioassay as test plant revealed that 1,2-propanediol can act as potent root growth promoter; enhancing the growth of lettuce seedling radicle at a concentration 0.01 ppm. The concentration of 1,2-propanediol in PCS mixture was estimated as 4 g/l. These studies suggest that 1,2-propanediol might play an important role in the plant growth promoting activity of PCS.     

L-3-(3,4-Dihydroxyphenyl)alanine (<Emphasis Type="SmallCaps">L</Emphasis>-DOPA), an allelochemical exuded from velvetbean (<Emphasis Type="Italic">Mucuna pruriens</Emphasis>) roots

We investigated whether or not lettuce growth was inhibited by diffused L-3-(3,4-dihydroxyphenyl)alanine (L-DOPA), an allelochemical exuded from the roots of velvetbean (Mucuna pruriens (L.) DC. var. utilis) cultivars using a modified plant-box bioassay. For all the cultivars and one accession examined L-DOPA diffused from the roots and caused radicle and hypocotyl growth inhibition. A high correlation co-efficient (r = 0.838 to 0.982) was observed between L-DOPA concentration and lettuce seed sowing distance. L-DOPA diffused equally in all directions from roots at 0 mm position (close to root surface) in the plant-box, while the inhibition (%) of lettuce radicle growth gradually decreased with distance from the roots. For all cultivars the concentration of L-DOPA was significantly different at 0 mm position: being highest in cv. preta (167 g/ml) and lowest in cv. jaspeada and cv. ana (13 g/ml). The correlation between lettuce radicle growth inhibition and concentration of diffused L-DOPA was high (r = 0.856 to 0.966) in all cultivars and accession examined. However, the concentration of diffused L-DOPA did not correlate with the fresh weight concentration of L-DOPA measured in roots. The lettuce radicle growth inhibition from mucuna diffused L-DOPA was very similar that induced by synthetic L-DOPA, suggesting that diffused L-DOPA was the allelochemical responsible for growth inhibition.     

Possibility of long-term preservation of freeze-dried mouse spermatozoa

Freeze-dried mouse spermatozoa are capable of participating in normal embryonic development after injection into oocytes. When the freeze-dried spermatozoa are used as a method for storage of genetic materials, however, it is essential to assure the relevance of long-term preservation over several decades or centuries. Thus, we applied the theory of accelerated degradation kinetics to freeze-dried mouse spermatozoa. Thermal denaturation kinetics were determined based on Arrhenius plots derived from transition-state theory analysis at three elevated temperatures: 30, 40, and 50 degrees C. Accelerated degradation kinetics were calculated by extrapolation of Arrhenius plots. This theory also is being applied to the long-term stability of drugs. The estimated rate of development to the blastocyst stage at 3 and 6 mo and at 1, 10, and 100 yr of sperm storage at 4 degrees C were 21.60%, 7.91%, 1.00%, 0%, and 0%, respectively. At -80 degrees C, estimated development rates to the blastocyst stage that would be expected after 100 yr of storage did not decline significantly. In addition, after 3 or 6 mo of storage at 4 or -80 degrees C, preimplantation development of the embryos derived from intracytoplasmic sperm injection (ICSI) was examined. The actual developmental rates to the blastocyst stage from ICSI by freeze-dried sperm stored for 3 mo at 4 and -80 degrees C were 21% and 62%, respectively, and the rates for such sperm stored for 6 mo were 13% and 59%, respectively. These results indicate that the determination of accelerated degradation kinetics can be applied to the preservation of freeze-dried mouse spermatozoa. Furthermore, for long-term preservation, freeze-dried mouse spermatozoa appear to require being kept at lower than -80 degrees C.     

Germination growth response of different plant species to the allelochemical L-3,4-dihydroxyphenylalanine (L-DOPA)

The aim of this study was to investigate the seed germination response of different plant families to L-3,4-dihydroxyphenylalanine (L-DOPA), one of the strongest allelochemicals in nature. Three types of responses in terms of colouration changes on filter paper were obtained; black and gray (Gramineae and Compositae), no change (Leguminosae, Brassicaceae, and Cucurbitaceae) and an obstructed-circle around the seeds with black colouration on the outer side of the circle (Hydrophyllaceae) when L-DOPA solution was applied during seed germination. Radicle growth in the Gramineae and Leguminosae families was inhibited less by a single treatment of L-DOPA solution (250 g/ml) than in the other families. However, continuous treatment with L-DOPA demonstrated that the Gramineae family was less affected in terms of the inhibition of radicle growth than the Leguminosae family. When more seeds were added to the L-DOPA solution less inhibition of radicle growth was observed in all plants tested. The EC₅₀ of L-DOPA for bluebell (Hydrophyllaceae), white clover (Leguminosae), and lettuce (Compositae) was approximately 200, 100, and 50 g/ml, respectively. However, in perennial ryegrass (Gramineae) no EC₅₀ was observed even at 250 g/ml L-DOPA. In the Gramineae family, addition of more seeds into the L-DOPA solution increased the colouration on the filter paper. These results demonstrated that each seed functions to oxidize or dissolve L-DOPA. In the Gramineae, Leguminosae, Compositae, and Hydrophyllaceae, increasing the number of seeds imbibed in the L-DOPA solution increased the rate of L-DOPA disappearance from the petri-dish. Of the Grammaceous plants tested, only perennial ryegrass, which showed fairly weak allelopathic activity, metabolised L-DOPA to dopamine. Although the relationships between the changes in colouration of the filter paper and the inhibition of radicle growth in these experiments are still unknown, there appears to be a strong response in each species to protect the cell from L-DOPA damage.