Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.     

The Cold Box stem-loop proximal to the 5'-end of the Escherichia coli cspA gene stabilizes its mRNA at low temperature.

The 5'-end region of cspA mRNA contains a Cold Box sequence conserved among several cold-shock mRNAs. This region forms a stable stem-loop structure followed by an AU-rich sequence. Here we show that the Cold Box region is essential for the normal scale of cspA mRNA induction after cold shock because a deletion of the stem-loop significantly destabilizes the mRNA and reduces the cold shock-induced cspA mRNA amount by approximately 50%. The AU-rich track, however, slightly destabilizes the mRNA. The integrity of the stem is essential for the stabilizing function, whereas that of the loop sequence is less important. Overexpression of a mutant cspA mRNA devoid of both the AUG initiation codon and the coding sequence results in a severe growth inhibition at low temperature along with a derepression of the chromosomal cspA expression. Furthermore, the overexpressed RNA is stably associated with the 30 S and 70 S ribosomes. Our results demonstrate that the AUG initiation codon and the coding region containing the downstream box are not required for cspA mRNA to bind ribosomes and that the 5'-untranslated region by itself has a remarkable affinity to ribosomes at low temperature.     

Does boron play only a structural role in the growing tissues of higher plants?

In species in which boron (B) mobility is limited, B deficiency only occurs in growing plant organs. As a consequence of the highly localized patterns of plant growth and the general immobility of B it has been extremely difficult to determine the primary function of B in plants. In species in which B is phloem mobile, the removal of B from the growth medium results in the depletion of B present in mature leaves. Thus, it is possible to develop mature leaves with increasingly severe levels of B depletion, thereby overcoming the complications of experiments based on growing tissues. Utilizing this approach we demonstrate here that B depletion of mature plum (Prunus salicina) leaves did not result in any discernible change in leaf appearance, membrane integrity or photosynthetic capacity even though B concentrations were reduced to 6-8 µg/g dwt, which is less than 30% of the reported tissue B requirement. Boron depletion, however, results in a severe disruption of plant growth and metabolism in young growing tissues. This experimental evidence and theoretical considerations suggest that the primary and possibly sole function of B, is as a structural component of growing tissues.     

Using Echo Ultrasound from Schooling Fish to Detect and Classify Fish Types

Fish finders have already been widely available in the fishing market for a number of years.However,the sizes of these fishfinders are too big and their prices are expensive to suit for the research of robotic fish or mini-submarine.The goal of thisresearch is to propose a low-cost fish detector and classifier which suits for underwater robot or submarine as a proximity sensor.With some pre-condition in hardware and algorithms,the experimental results show that the proposed design has good per-formance,with a detection rate of 100 % and a classification rate of 94 %.Both the existing type of fish and the group behaviorcan be revealed by statistical interpretations such as hovering passion and sparse swimming mode.     

Evidence for the signaling role of methyl jasmonate, methyl salicylate and benzothiazole between poplar (Populus simonii × P. pyramidalis ‘Opera 8277’) cuttings

Interplant communication has been widely demonstrated in plants, especially in herbaceous plants. In this study, mechanical damage was shown to affect the levels of pyrochatechol, chlorogenic acid, gallic acid and p-hydroxyl benzoic acid in poplar (Populus simonii × P. pyramidalis ‘Opera 8277’) cuttings, indicating the activation of defense response. In neighboring intact cuttings, the levels of those phenolics also varied when compared to the control, suggesting the interplant communication between poplar cuttings. Three volatiles, methyl jasmonate, methyl salicylate and benzothiazole, were detected in volatiles emitted from mechanically damaged poplar cuttings. All of them can induce changes in the levels of four phenolics. Therefore, they could act as airborne signals between P. simonii × P. pyramidalis ‘Opera 8277’ cuttings. The different change patterns of phenolic contents induced by different volatiles imply that the defense response activated in neighboring plants may be regulated by multiple signaling pathways. The results also suggest that the entire defense response of plants involves a variety of airborne signals in wound-induced volatiles.     

Characterization of Viticultural Terroirs using a Simple Field Model Based on Soil Depth – II. Validation of the Grape Yield and Berry Quality in the Anjou Vineyard (France)

Soil and climate are major constituents of the French notion of Terroir. This concept implies that there is a strong relationship between the composition of the grape, the characteristics of the wine and the territory of production. To study this link, a new method of characterization of the Terroir, including geological and pedological factors, was investigated. It uses a field model based on depth and clay content of soil, together with the degree of weathering of the parent rock. Consequently, for every type of parent rock belonging to a given geologic stage, there are a series of soils that show different stages of pedological evolution. According to the model, three kinds of soils are distinguished with regards to the weathering intensity of the parent rock, that are named weakly weathered rock (WWR), moderately weathered rock (MWR) and strongly weathered rock (SWR). By hypothesis, each soil type is considered as a homogeneous unit for vine production from the viewpoint of ecophysiological factors. Each terroir unit defined by this method is called a Basic Terroir Unit (BTU). To validate this hypothesis, experimental plots planted with Chenin and Cabernet Franc vines were studied over three consecutive seasons (2000–2002), in the Anjou vineyard (Loire Valley – France). The major BTUs developed on the two most important geological systems of Anjou (Brioverian and Ordovician–Devonian), were studied. Results showed that the berries of vines cultivated in WWR were significantly smaller, richer in sugars and anthocyanins and had a Total Phenolic Index higher than those of the vines cultivated in SWR. They also had a lower titratable acidity. Cabernet Franc vines cultivated in MWR had berries with sugar and anthocyanin contents but also total phenolics very close to those of WWR. With Chenin vines there was a good relationship between the global pool of free aromas of berries and the BTU type. The study showed significant relationships between the quality of grapes and the measured values of several ecophysiological variables such as the water supply regime or the timing of budburst.