New matrices for the purification of pectinases by affinity chromatography

Polygalacturonic acid was used as a ligand in the affinity technique for pectinases purification from the filtrate of Aspergillus niger 71 culture. For this purpose four matrices were examined, namely, alkylamine controlled porous glass (CPG), alkylamine silica gel as well as keratin or polyamide coated silica gel. Good results of pectinase purification was obtained on silanized CPG or keratin coated silica gel supports.     

Successful immunotherapy with matrix metalloproteinase-derived peptides in adjuvant arthritis depends on the timing of peptide administration

We have recently found that matrix metalloproteinases (MMPs) are targets for T-cell and B-cell reactivity in experimental arthritis. In the present article, we investigate whether modulation of MMP-specific T-cell responses could influence the course of adjuvant arthritis (AA). Lewis rats were treated nasally with MMP peptides prior to or after AA induction. Administration of the MMP-10 or the MMP-16 peptide prior to AA induction reduced the arthritic symptoms. In contrast, administration of the MMP-10 peptide after AA induction aggravated the arthritic symptoms. The present study shows the possible usefulness of MMP peptides for immunotherapy. However, a clear understanding of proper timing of peptide administration is crucial for the development of such therapies.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Separation of dissociated thyroid follicular and parfollicular cells: association of serotonin binding protein with parafollicular cells

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.     

Effects of medium viscosity on kinetics of the enzymatic reaction catalyzed by bacterial RNase

Effects of medium viscosity on kinetic parameters of poly(U) hydrolysis catalyzed by RNase from Bac. intermedius 7P (binase) were studied in solutions of sucrose (4-50 wt. %) and glycerol (35-62 wt. %) in Tris--sodium acetate buffer (pH 7.5) at 25 degreesC. The rate constant of reaction kcat was practically unchanged over a wide range of viscosities (1-15 cP for sucrose and 2.5-3 cP for glycerol). In glycerol solutions, kcat slightly increased with viscosity increase from 4 to 10 cP. Addition of NaCl to the buffer medium resulted in an inhibitory effect of Na+ on kcat, prevented by 50% sucrose or 60% glycerol. It is concluded that binase-catalyzed poly(U) cleavage occurs through a "tense"-substrate mechanism, similarly to reactions catalyzed by alpha-chymotrypsin, trypsin, and laccase.     

Tropical wetlands: seasonal hydrologic pulsing,carbon sequestration,and methane emissions

This paper summarizes the importance of climate on tropical wetlands. Regional hydrology and carbon dynamics in many of these wetlands could shift with dramatic changes in these major carbon storages if the inter-tropical convergence zone (ITCZ) were to change in its annual patterns. The importance of seasonal pulsing hydrology on many tropical wetlands, which can be caused by watershed activities, orographic features, or monsoonal pulses from the ITCZ, is illustrated by both annual and 30-year patterns of hydrology in the Okavango Delta in southern Africa. Current studies on carbon biogeochemistry in Central America are attempting to determine the rates of carbon sequestration in tropical wetlands compared to temperate wetlands and the effects of hydrologic conditions on methane generation in these wetlands. Using the same field and lab techniques, we estimated that a humid tropical wetland in Costa Rica accumulated 255 g C m⁻² year⁻¹ in the past 42 years, 80% more than a similar temperate wetland in Ohio that accumulated 142 g C m⁻² year⁻¹ over the same period. Methane emissions averaged 1,080 mg-C m⁻² day⁻¹ in a seasonally pulsed wetland in western Costa Rica, a rate higher than methane emission rates measured over the same period from humid tropic wetlands in eastern Costa Rica (120–278 mg-C m⁻² day⁻¹). Tropical wetlands are often tuned to seasonal pulses of water caused by the seasonal movement of the ITCZ and are the most likely to be have higher fire frequency and changed methane emissions and carbon oxidation if the ITCZ were to change even slightly.     

A comparative study of hourly and daily relationships between selected meteorological parameters and airborne fungal spore composition

Air sampling was conducted in Szczecin (Poland) throughout April–September 2013. The final data set included 177 daily and 4248 hourly samples. The total of 21 types of spores, which occurred in a number >10 in the season, were taken into account. The following meteorological parameters were analyzed: air temperature, relative humidity, precipitation and wind speed. Effects of individual weather parameters on hourly and daily concentrations of different fungal spore types were examined using Spearman’s rank association test, whereas effects of complex of meteorological factors on hourly and daily compositions of spore were assessed using detrended correspondence analysis (DCA) and redundancy analysis (RDA). Airborne fungal spore distribution patterns in relation to meteorological variables were determined by RDA, after DCA results detected a linear structure of the spore data. The RDA results obtained indicated that all the applied variables accounted for 20 and 22% of the total variance in the hourly and daily spore data, respectively. The results of stepwise forward selection of variables revealed all included hourly and daily meteorological variables were statistically significant. The largest amount of the total variance in the spore composition was explained by the air temperature in both cases (16%). Multivariate ordination did not show large differences between the hourly and daily relationships (with exception of wind speed impact), while the differences between simple hourly and daily correlations were more clear. Correlations between daily values of variables were in most cases higher than between hourly values of variables.     

Characterization and <Emphasis Type="Italic">in vitro</Emphasis> expression patterns of extracellular degradative enzymes from non-pathogenic binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis> AG-G

Many filamentous fungi produce an array of extracellular enzymes that acting in cell walls release elicitors of the plant defense response These enzymes may therefore be important in biocontrol applications. The aim of this study was to characterize extracellular degradative enzymes produced by a non-pathogenic binucleate isolate of Rhizoctonia AG-G. The fungus was grown in liquid culture supplemented with pectin, polygalacturonic acid or glucose as a carbon sources and filtrates of the culture media were analyzed for the detection of pectinolytic and glucan hydrolytic enzymes. Using only pectin as a carbon source, secretion of polygalacturonases and methylesterases was found. When the liquid medium was supplemented with polygalacturonic acid, only polygalacturonase activity was detected. However, when glucose was used as carbon source -1,3 and -1,6 glucanases activities were detected, using laminarin and pustulan as substrates, but none of the pectinolytic activities were found. These enzymes were partially purified and characterized. The -(1,3)(1,6) glucanase and polygalacturonase enzymes showed to be active against cell wall polysaccharides from potato sprouts. These enzymes may have an important role in fungus-plant cell wall interaction. This is the first study about the production of extracellular enzymes by non-pathogenic binucleate Rhizoctonia AG-G.