Primary enzyme quantitation using substrates labeled with a second indicator enzyme. I. Elastase determination using peroxidase-labeled elastin

Quantitation of proteolytic enzyme concentration can be accomplished by measuring the release, due to primary enzyme catalysis, of a second enzyme bound to a particulate substrate. As the primary enzyme acts on the substrate, release of the indicator enzyme into the surrounding medium occurs, which in turn can be quantitated colorimetrically, and under suitable reaction conditions the amount of indicator enzyme released is directly proportional to the amount of primary enzyme present. A specific example of such an assay is that for elastolytic activity using powdered elastin labeled with horseradish peroxidase. The detection sensitivity of the system described is 1 ng/ml of pancreatic elastase, and the dynamic range of the assay is 2 orders of magnitude. The reaction time for optimal elastase detection sensitivity is 3 h. For the assay, horseradish peroxidase is coupled to insoluble elastin. Labeled elastin is incubated with varying amounts of pancreatic elastase. The elastase in the test sample solubilizes the elastin and the horseradish peroxidase bound to it. The amount of peroxidase released is then quantified using the colorimetic reaction produced by catalysis of 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonate)-H₂O₂. For a fixed, nonsaturating concentration of elastase, the amount of peroxidase released is proportional to the elastase concentration.     

Mycorrhizal response of <Emphasis Type="Italic">Clusia pusilla</Emphasis> growing in two different soils in the field

Arbuscular mycorrhizas (AM) are important for promoting the mineral nutrition, growth and survival of plants used to rehabilitate degraded areas. Clusia pusilla is an evergreen shrub which is tolerant of high irradiance, germinates readily and can be easily reproduced by cuttings. All these characteristics make this species useful in the recovery of deforested areas. The aim of this work was to explore the response of C. pusilla to AM in the field, in two types of soil: the shrubland soil in which the species naturally grows and in a soil of a riparian forest. Eight treatments were performed in each type of soil. The treatments consisted of a non-mycorrhizal control and mycorrhizal plants colonized by one of the three AM inocula tested in the presence or absence of triple superphosphate (150 kg ha^-1). After 11 months of growth in the shrubland soil, C. pusilla seedlings showed an increase in height and dry weight in response to the fertilizer but not to mycorrhizas. In contrast, in the forest soil the arbuscular mycorrhizal fungi (AMF) effect was equivalent to the fertilizer effect, and the two effects interacted positively. The lack of response to AM in shrubland soil was caused by its high sand content, which hinders the retention of the inocula. Due to a higher clay content, the forest soil binds inocula more tightly than shrubland soil. In conclusion, C. pusilla appears to benefit greatly from the addition of AMF in forest soil, though it requires an additional P source for such benefits in shrubland soil. This P source must be organic so that phosphorus is not lost by leaching. Although the growth rate of this species is very low, its survival can be guaranteed with the application of AMF inocula together with P-fertilizer applied at a low rate.     

Association between Fibrillin1 Polymorphisms (rs2118181, rs10519177) and Transforming Growth Factor β1 Concentration in Human Plasma

Transforming growth factor (TGF)-β1 is a cytokine that participates in a broad range of cellular regulatory processes and is associated with various diseases including aortic aneurysm. Increased TGF-β1 levels are linked to Marfan syndrome (MFS) caused by fibrillin1 (FBN1) mutations and subsequent defects in signaling system. FBN1 single nucleotide polymorphisms (SNPs) rs2118181 and rs1059177 do not cause MFS but are associated with dilative pathology of aortic aneurysms (DPAAs). TGF-β1 and FBN1 SNPs rs2118181 and rs1059177 are potential biomarkers for early diagnosis of DPAA. We investigated the relationship between TGF-β1 levels in human blood plasma and FBN1 rs2118181 and rs1059177 in 269 individuals. The results showed a quantitative dependence of SNP genotype and TGF-β1 concentration. Presence of a single rs2118181 minor allele (G) increased the amount of TGF-β1 by roughly 1 ng/mL. Two copies of FBN1 rs1059177 minor allele (G) were required to have an additive effect on TGF-β1 levels. We found higher TGF-β1 concentrations in men compared with women (p = 0.001). A strong correlation between TGF-β1 levels and FBN1 SNPs suggests that a single nucleotide substitution in FBN1 sequence might reduce bioavailability or binding properties of fibrillin-1 and have an effect on TGF-β1 activation and cytokine concentration in blood plasma. By establishing the relationship between TGF-β1 and FBN1 SNPs rs2118181 and rs1059177, we provide evidence that their combination might be used as molecular biomarkers to identify patients at risk for sporadic ascending aortic aneurysm and aortic dissection.     

Documenti palinologici del paesaggio forestale al Monte Amiata nei sedimenti del Bacino lacustre delle lame (Abbadia S. Salvatore)

Abstract

PALYNOLOGICAL PAPERS OF M. AMIATA FORESTAL LANDSCAPE IN THE SEDIMENTS OF LE LAME LACUSTRINE BASIN (ABBADIA S. SALVATORE). — This paper deals with Le Lame lacustrine deposits (Abbadia S. Salvatore, M. Amiata area). This lake basin, like others in the same area, was located along the border of trachitic- ignimbritic terranes, variously degradated.

The examined sediments have shown a large predominance of chestnut-tree. The pollinic curve of this plant undergoes some fluctuations, due to climatic pulsations emphasized by Pinus, Abies, Fagus, Betula, Carpinus, Quercetum mixtum. These genera are present in the lower part of the diagram, related to a peat bed and are more evident in the upper one.

Pteridophyta spores are common and Graminaceae and Cyperaceae grains are very frequent in the lower part of the diagram.

Regarding the age problem, the chestnut-tree predominance gives evidence of an age not far from Recent, although in postglacial diagrams such a high per cent is not usually found. On the other hand we can tentatively anribuie these deposits to infraglacial age, because other basins in the same area are of Riss, Riss-Würm and Würm age.

The abundance of chestnut-tree can be ascribed to particularly good substratum conditions.

The age problems and the chestnut-tree predominance meaning will be developed in following researches.     

Association of salivary steroid hormones and their ratios with time-domain heart rate variability indices in healthy individuals

BackgroundStress system consists of the hypothalamicpituitary-adrenal (HPA) axis and the locus caeruleus/norepinephrine-autonomic nervous system (ANS). Traditionally, HPA axis activity is evaluated by measuring its end-product cortisol, while the activity of ANS is assessed using heart rate variability (HRV) indices. Alterations in cortisol levels and HRV measures during laboratory-based stress tasks were extensively studied in previous research. However, scarce data exist on the associations of HRV measures with the levels of other adrenal steroid hormones under baseline conditions. Thus, we aimed to evaluate the activity of the HPA axis by measuring salivary cortisol, cortisone, dehydroepiandrosterone (DHEA) levels, and their ratios and to examine its association with HRV measures in a sample of healthy young and middle-aged adults.MethodsFor each participant (n=40), three data collection sessions taking place at the same time of the day were scheduled within five working days. Participants completed a self-reported questionnaire on sociodemographic and lifestyle characteristics, filled out t h e Perceived Stress Scale and State-Trait Anxiety Inventory. Also, saliva samples were collected, and physiological measures, including resting HR and HRV, were recorded during three data collection sessions.ResultsStatistically significant associations between diminished parasympathetic vagal tone evaluated by time domain HRV measures and higher salivary cortisol, lower DHEA levels, as well as decreased DHEA to cortisol ratio, were found. Also, physiological stress indicators (i.e., HRV) showed greater intraindividual stability compared with biochemical biomarkers (i.e., salivary steroid hormones) within five days.ConclusionsOur findings suggest that both cortisol and DHEA mediate the link between two stress-sensitive homeostatic systems.