Structural, spectroscopic and catalytic activity studies on glutathione reductase reconstituted with FAD analogues

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66378/1/j.1432-1033.1991.tb16100.x.pd

Impact of changes in environmental parameters (pH and elevated CO2) on soil microbial communities involved in N-cycling

Microorganisms involved in the nitrogen (N)-cycle in soils are the major drivers of N-transformation changes and the main source of the potent greenhouse gas nitrous oxide (N2O) from soil, which has a global warming potential of 298 times that of carbon dioxide (CO2). Accordingly, it is of great interest to explore shifts in the rates, balances and reactions of the N-cycle impacted by climate changes, in order to offer more accurate predictions. Particularly, since increases in CO2 concentrations or changes in the pH of agricultural fields due to anthropogenic influences often lead to changes in the N-transformation rates, along with an increase of N2O emissions. However, the N-cycle and its corresponding pathways are very complex and the response to different environmental changes is difficult to predict. Many of the interactions between microorganisms and their contribution to N-transformation rates as well as N2O emission are not well understood, controversially discussed and plenty of important interactions remain puzzling. Therefore, the main objective of this thesis was to shed light on the interaction of the overall and active microbial communities involved in the N-cycle in response to pH shifts or elevated atmospheric CO2 concentrations in soils, two variables known to influence N2O fluxes from soils. In the first part we studied the influence of an acidic pH on a denitrifier community from an initial neutral pH. We followed the abundance and composition of an overall and active denitrifier community extracted from soil (pH = 7.1) when exposed to pH 5.4 and drifting back to pH 6.6. When exposed to pH 5.4, the denitrifier community was able to actively grow, but only reduced N2O to N2 after a near neutral pH was reestablished by the alkalizing metabolic activity of an acid-tolerant part of the community. The genotypes proliferating under these conditions differed from those dominant at neutral pH. Denitrifiers of the nirS-type appeared to be severely suppressed by low pH whereas nirK-type and nosZ-containing denitrifiers showed strongly reduced transcriptional activity and growth, even after restoration of neutral pH. Our study suggests that low pH episodes alter transcriptionally active populations which shape denitrifier communities and determine their gas kinetics. The second part of this thesis analyses the effect of elevated CO2 (eCO2) on the N-cycle to reveal the underlying microbial mechanisms and process inside the N-cycle causing the enhanced emission of N2O. To gain a better understanding of the impact of eCO2 on soil microbial communities, we applied a molecular approach targeting several microbial groups involved in soil N-cycling (N-fixers, denitrifiers, archaeal and bacterial ammonia oxidizers, and dissimilatory nitrate reducers to ammonia) at the Gießen Free Air Carbon dioxide Enrichment (GiFACE) site. Remarkably, soil parameters, overall microbial community abundance and composition in the top soil under eCO2 differed only slightly from soil under ambient CO2. We concluded that +20% eCO2 had little to no effect on the overall microbial community involved in N-cycling. Based on these findings, in a third part we conducted a comprehensive study monitoring N-transformation rates, nutrient fluxes, and gaseous emission, while analyzing the dynamics in microbial communities involved in N-cycling under eCO2 accompanied with simultaneous addition of N-fertilizer. We could show that long-term fumigation with eCO2 influences the response of the soil microbial communities to N inputs via fertilization. Compared to aCO2 distinct parts of the community were transcriptionally activated. Here, nirS-type denitrifiers showed the greatest positive feedback to eCO2, which correlated with increasing N2O emissions. This stimulation may be an effect of higher labile C input in the rhizosphere by increased photosynthesis. However, the input of N by fertilization rather seems to exert short term effects on the expression of functional marker genes with consequences for N-transformations which do not translate into the development of distinct communities under eCO2 in the long-term. In conclusion this thesis provides evidence that already small changes in abundance and composition of the microbial community involved in N-cycling are sufficient to strongly influence emission of N2O from soil under changing environmental parameters such as pH and elevated CO2

Inhibition of prostate cancer cell growth by human secreted PDZ domain-containing protein 2, a potential autocrine prostate tumor suppressor

A possible role of the PDZ domain-containing protein 2 (PDZD2) in prostate tumorigenesis has been suggested. Besides, PDZD2 is posttranslationally cleaved by a caspase-dependent mechanism to form a secreted PDZ domain-containing protein 2 (sPDZD2) with unknown functions in humans. In this study, we demonstrate the endogenous expression of PDZD2 and secretion of sPDZD2 in cancerous DU145, PC-3, 22Rv1, LNCaP, and immortalized RWPE-1 prostate epithelial cells. Inhibition of endogenous sPDZD2 production and secretion by DU145, PC-3, 22Rv1, and RWPE-1 cells via the caspase-3 inhibitor Z-DEVD-FMK resulted in increased cell proliferation, which was abrogated by treatment with exogenous recombinant sPDZD2. Whereas sPDZD2-induced antiproliferation in DU145, PC-3, and 22Rv1 cells, it induced apoptosis in LNCaP cells. The data suggest that endogenous sPDZD2, produced by caspase-3-mediated cleavage from PDZD2, may function as a novel autocrine growth suppressor for human prostate cancer cells. The antiproliferative effect of sPDZD2 was apparently mediated through slowing the entry of DU145, PC-3, and 22Rv1 cells into the S phase of the cell cycle. In DU145 cells, this can be attributed to stimulated p53 and p21 CIP1/WAF1 expression by sPDZD2. On the other hand, the apoptotic effect of sPDZD2 on LNCaP cells was apparently mediated via p53-independent Bad stimulation. Together our results indicate the presence of p53-dependent and p53-independent PDZD2/sPDZD2 autocrine growth suppressive signaling pathways in human prostate cancer cells and suggest a novel therapeutic approach of harnessing the latent tumor-suppressive potential of an endogenous autocrine signaling protein like sPDZD2 to inhibit prostate cancer growth. Copyright © 2006 by The Endocrine Society.postprin

Diverse hypolithic refuge communities in the McMurdo Dry Valleys

Hyper-arid deserts present extreme challenges to life. The environmental buffering provided by quartz and other translucent rocks allows hypolithic microbial communities to develop on sub-soil surfaces of such rocks. These refuge communities have been reported, for many locations worldwide, to be predominantly cyanobacterial in nature. Here we report the discovery in Antarctica’s hyper-arid McMurdo Dry Valleys of three clearly distinguishable types of hypolithic community. Based on gross colonization morphology and identification of dominant taxa, we have classified hypolithic communities as Type I (cyanobacterial dominated), Type II (fungal dominated) and Type III (moss dominated). This discovery supports a growing awareness of the high biocomplexity in Antarctic deserts, emphasizes the possible importance of cryptic microbial communities in nutrient cycling and provides evidence for possible successional community processes within a cold arid landscape

A new intrinsic thermal parameter for enzymes reveals true temperature optima

Two established thermal properties of enzymes are the Arrhenius activation energy and thermal stability. Arising from anomalies found in the variation of enzyme activity with temperature, a comparison has been made of experimental data for the activity and stability properties of five different enzymes with theoretical models. The results provide evidence for a new and fundamental third thermal parameter of enzymes, Teq, arising from a subsecond timescale-reversible temperature-dependent equilibrium between the active enzyme and an inactive (or less active) form. Thus, at temperatures above its optimum, the decrease in enzyme activity arising from the temperature-dependent shift in this equilibrium is up to two orders of magnitude greater than what occurs through thermal denaturation. This parameter has important implications for our understanding of the connection between catalytic activity and thermostability and of the effect of temperature on enzyme reactions within the cell. Unlike the Arrhenius activation energy, which is unaffected by the source (“evolved”) temperature of the enzyme, and enzyme stability, which is not necessarily related to activity, Teq is central to the physiological adaptation of an enzyme to its environmental temperature and links the molecular, physiological, and environmental aspects of the adaptation of life to temperature in a way that has not been described previously. We may therefore expect the effect of evolution on Teq with respect to enzyme temperature/activity effects to be more important than on thermal stability. Teq is also an important parameter to consider when engineering enzymes to modify their thermal properties by both rational design and by directed enzyme evolution

Dietary Level of the Omega-3 Fatty Acids EPA and DHA Influence the Flesh Pigmentation in Atlantic Salmon

publishedVersio

Changes in sulfhydryl groups of honeybee glyceraldehyde phosphate dehydrogenase associated with generation of the intermediate plateau in its saturation kinetics

Honeybee and rabbit muscle GPDH were studied to obtain information at the chemical level regarding anomolous saturation kinetics of the honeybee enzyme. Results demonstrate that the enzyme's sulfhydryl groups are implicated in the process. Measured by DTNB titration, native honeybee GPDH has one less active SH than the native rabbit muscle enzyme and displays changes in overall sulfhydryl reactivity after preincubation with G-3-P or G-3-P plus NAD+. The total DTNB reactive sulfhydryls of rabbit muscle GPDH are not changed by preincubation with NAD+ or G-3-P; honeybee GPDH, under certain conductions of preincubation with these ligands, shows a decrease of two total DTNB reactive SH groups. This difference has been confirmed by an independent experiment in which the two enzymes were carboxymethylated with C-14 bromoacetic acid

Algal responses to abiotic and biotic environmental changes

L’eterogeneità delle risposte fisiologiche delle microalghe ai cambiamenti ambientali rappresenta uno dei fattori più importanti nel determinare le interazioni tra le specie in ambiente. La mia ricerca ha rivelato che specie differenti sono diversamente inclini a modificare la propria composizione cellulare in risposta ai cambiamenti ambientali. La scelta tra acclimatazione e omeostasi dipende anche dal tipo e dalla durata della perturbazione in esame. La maggior parte delle alghe considerate nel mio studio, per esempio, ha mostrato una risposta omeostatica ai cambiamenti nelle concentrazioni ambientali di CO2 e nella forma di azoto disponibile. Non è stato ritrovato nessun legame tra la strategia di risposta e la tassonomia delle alghe. Particolare attenzione è stata rivolta a Chromera velia, parente prossima dei parassiti Apicomplexa e probabile simbionte di coralli dell’ordine Scleractinia. C. velia si è dimostrata perfettamente in grado di vivere ad alta CO2. Questa condizione ha stimolato la produzione di C organico da parte di C. velia, incrementato la sua efficienza di utilizzo dei nutrienti e ha determinato cambiamenti nei rapporti stechiometrici tra gli elementi. Si può ipotizzare, dunque, che l’elevata concentrazione di CO2 rinvenuta all’interno dei tessuti del corallo che circondano il simbionte possa facilitare la vita di quest’alga in simbiosi. Infine, ho potuto dimostrare che le interazioni tra alghe e ambiente possono avere conseguenze nei rapporti tra alghe e loro predatori. I miei esperimenti hanno mostrato che i copepodi (ma non i rotiferi) possono discriminare tra alghe che sono identiche in ogni aspetto tranne che nella composizione cellulare. La storia nutrizionale delle alghe, dunque, essendo uno dei principali determinanti della loro composizione cellulare, risulta un elemento di grande importanza nelle relazioni tra alghe e predatori.Algae exhibit a large variety of physiological responses to the environmental changes. Such heterogeneity of responses, which is a major determinant of species interaction in natural algal assemblages, was the target of my research. My results show that different species are differently prone to change their cell composition in response to environmental changes, depending on the type and duration of the perturbation. When algae are exposed to changes in the N source and in the CO2 availability, for instance, homeostasis appears as a much more common strategy than usually believed. No link between the response modes and the taxonomy of the examined species was found. I paid special attention to Chromera velia, a photosynthetic relative of apicomplexan parasites that is likely involved in symbiotic associations with scleractinian corals. This alga seems perfectly capable of copying with very high CO2. Life at high CO2 stimulates the overall organic C production of C. velia, increases its nutrient use efficiency and changes the stoichiometric relationships among elements within the cell. The high CO2 concentrations that has been reported in the animal tissue surrounding the photosynthetic cells may therefore facilitate C. velia life in symbiosis. Finally, I have demonstrated that the interactions between algae and environment can affect the relationships between algae and their grazers. My experiments show that the copepods are able to discriminate among algae identical in all aspects but in cell composition, while the rotifers are not. Therefore, the nutritional history of algae, which has the potential to affect algal cell composition, appears as a major determinant of the relationships between algae and grazers