A continuous kinetic assay for RNA-cleaving deoxyribozymes,exploiting ethidium bromide as an extrinsic fluorescent probe

We describe a rapid and inexpensive method to monitor the kinetics of small RNA-cleaving deoxyribozymes, based on the exogenous fluorophore ethidium bromide. Ethidium binds preferentially to double-stranded nucleic acids, and its fluorescence emission increases dramatically upon intercalation. Thus, ethidium can be used in single-turnover experiments to measure both annealing of the deoxyribozyme to its substrate and release of the products. Under conditions in which dissociation of the product is fast compared with cleavage, the apparent rate of product release reflects the cleavage step. The method was developed for characterizing the so-called 8-17 catalytic DNA, but its general applicability in the deoxyribozyme field was verified using the 10-23 RNA-cleaving construct. Catalysis by both deoxyribozymes was not inhibited in the presence of substoichiometric amounts of ethidium, and the rates obtained through the ethidium assay were virtually identical to the rates determined using radiolabeled substrates. In contrast, the assay cannot be applied to the large, structured ribozymes, and its use to study the kinetics of the small hammerhead ribozyme was hampered by the presence on the catalyst of at least one high-affinity ethidium binding site.     

Cytology and mendelism: early connection between Michael F. Guyer's contribution

This paper examines the contribution of the PhD dissertation of the American cytologist Michael F. Guyer (1874-1959) to the early establishment (in 1902-1903) of the parallel relationship between cytological chromosome behaviour in meiosis and Mendel's laws. Guyer's suggestions were among the first, which attempted to relate the variation observed in the offspring in hybridisation studies by a coherent cytological chromosome mechanism to meiosis before the rediscovery of Mendel's principles. This suggested for the first time that the chromosome mechanism involved a conjugation of maternal and paternal chromosomes during the synapsis followed by a segregation of parental chromosomes in the final germ cells and a random union of the final germ cells in the fertilization. It shows that this early suggestion was similar to William Austin Cannon's later chromosome proposal attempting to explain Mendel's principles and had some influence on Walter Sutton's cytological suggestion explaining correctly the behaviour of Mendel's particle by 1903.     

One-step purification of Kunitz soybean trypsin inhibitor

A fast and simple method for the extraction and purification of Kunitz trypsin inhibitor from soybean seeds is described. The first step consisted in the heat treatment of whole soybean seeds in water at 60 degrees C for 90 min. It was found that 8.4% of total trypsin inhibitory activity of the seeds was secreted during heat treatment. The aqueous medium was loaded onto an affinity chromatography column with immobilized trypsin. The retained fraction, eluted with 0.01 N HCl, contained the purified Kunitz trypsin inhibitor, which was subsequently stabilized by freeze-drying without loss of activity. From 1g soybean seeds, 0.7 mg inhibitor with a specific trypsin inhibitory (TI) activity of 11,430 TIU/mg was obtained. The yield was greater than that obtained with established procedures. Due to the ease of the procedure proposed, the method is readily scalable to pilot plant or industrial preparations.     

Mechanism of constitutive export from the golgi: bulk flow via the formation,protrusion, and en bloc cleavage of large trans-golgi network tubular domains

Transport of constitutive cargo proteins from the Golgi complex to the plasma membrane (PM) is known to be mediated by large tubular-saccular carriers moving along microtubules. However, the process by which these large structures emerge from the trans-Golgi network (TGN) remains unclear. Here, we address the question of the formation of Golgi-to-PM carriers (GPCs) by using a suitable cluster of morphological techniques, providing an integrated view of their dynamics and three-dimensional structure. Our results indicate that exit from the TGN of a constitutive traffic marker, the VSVG protein, occurs by bulk flow and is a three-step process. First, the formation of a tubular-reticular TGN domain (GPC precursor) that includes PM-directed proteins and excludes other cargo and Golgi-resident proteins. Notably, this step does not require membrane fusion. Second, the docking of this preformed domain on microtubules and its kinesin-mediated extrusion. Finally, the detachment of the extruded domain by membrane fission. The formation of GPCs does not involve cargo concentration and is not associated with the presence of known coat proteins on GPC precursors. In summary, export from the Golgi occurs via the formation, protrusion and en bloc cleavage of specialized TGN tubular-saccular domains.     

In vivo and in vitro effects of cadmium on H+ ATPase activity of plasma membrane vesicles from oat (Avena sativa L.) roots

The effect of an in vivo and in vitro treatment with cadmium on transport activities of root plasma membrane enriched vesicles was studied in oat (Avena sativa L. cv. Argentina) plants. Addition of 100 mumol/L CdSO4 to nutrient solution decreases both proton transport activity and ATPase activity to the same level. In vitro experiments show that cadmium seems to have a differential inhibiting effect on proton transport activity and ATPase activity, the most pronounced one on ATP-dependent H(+)-accumulation, suggesting that cadmium would interfere with membrane permeability properties. This is indeed the case. The results demonstrate that cadmium decreases passive permeability to protons.     

Double muscling in Marchigiana beef breed is caused by a stop codon in the third exon of myostatin gene

Double muscling is a partially recessive trait present in some beef breeds. It shows a high frequency in some breeds, while in others the frequency is low, and double-muscled individuals are rare. The double muscling is caused by an allelic series of mutations that cause a loss of function of the myostatin gene ( GDF8). We describe here a new mutation in the myostatin gene in Marchigiana breed, a typical beef breed of Central Italy, in which rare double-muscling individuals have been described. A PCR product of the third exon was sequenced in subjects phenotypically showing double muscling, and a G > T transversion was discovered that introduces a premature stop codon. The variant found adds to the large series of mutations present in cattle, and particularly to the only two causative of double muscling in the third exon. A PCR-RFLP test is described for the rapid and effective identification of both heterozygous and homozygous subjects. It was applied to a larger survey carried on the same and also in two other beef breeds, Chianina and Romagnola. Further individuals carrying the new variant were found in Marchigiana, but none in the other breeds. The results may be important for a better comprehension of the role of myostatin in muscular development, for commercial use and for the inference of phylogeny of this gene.