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Abstract: Recent studies have revealed that at lower cultivation temperatures (25 °C) much higher percentage of correctly folded recombinant hG-CSF protein can be extracted from inclusion bodies. Hence, the goal of our research was to investigate mechanisms determining characteristics of non-classical inclusion bodies production using gene expression profiling, focusing on proteases and chaperones gene expression. Statistical analysis of microarray data showed prominent changes in energy metabolism, in metabolism of amino acids and nucleotides, as well as in biosynthesis of cofactors and secondary metabolites if the culture was grown below its optimal temperature. Moreover, 24 differentially expressed up to now known genes classified among proteases, chaperones and other heat or stress related genes. Among chaperones UspE and among proteases YaeL and YeaZ might play an important role in accumulation of correctly folded recombinant proteins. Membrane localized protease yaeL gene was found to have higher activity at 25 °C and is thus potentially functionally related to the more efficient recombinant protein production at lower temperatures. The results of this study represent advance in the understanding of recombinant protein production in E. coli. Genes potentially influencing production of recombinant protein at lower growth temperature represent basis for further research towards improvement of E. coli production strains as well as fermentation process.
Keywords: recombinant protein production, non-classical inclusion bodies, expression microarrays, YaeL protease, GroEL chaperone
Published in DiRROS: 05.08.2024; Views: 401; Downloads: 330
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Abstract: Od konca februarja 2007 do sredine februarja 2008 je bilo na Cerkniškem polju (južna Slovenija) v desetdnevnih obdobjih (dekadah) opravljenih 73 sistematičnih popisov vodnih ptic in ujed. Cilji raziskave so bili: ugotoviti številčnost in časovno dinamiko pojavljanja vrst, opredeliti njihov status, napraviti primerjavo s preteklim obdobjem sistematičnega zbiranja podatkov v letih 1991–1992, podati ocene gnezdečih, selitvenih in prezimujočih populacij ter na njihovi osnovi naravovarstveno ovrednotiti to območje. Zbrani so bili tudi drugi ornitološki podatki, pridobljeni do konca leta 2010. Med začetkom maja in začetkom septembra leta 2007 je voda na območju raziskave presahnila, tako da se je zadržala le v strugah potokov, vodna površina pa je bila deloma ali v celoti zaledenela od sredine novembra 2007 do začetka februarja 2008. Do konca leta 2010 je bilo na Cerkniškem polju skupaj zabeleženih 129 vrst vodnih ptic in ujed, v obdobju 2007/2008 pa 83. Dinamika pojavljanja vrst z več kot devetimi opazovanji je podrobneje predstavljena v pregledu vrst. Status gnezdilke ima 27 vrst, še nadaljnjih sedem pa je prehranskih gostov, ki gnezdijo v okolici območja raziskave. Največ vrst (118) ima status preletnega gosta, medtem ko je vrst s statusom poletnega (34) oziroma zimskega gosta (40) manj. Med zabeleženimi vrstami je 34 izjemnih in 21 redkih gostov. Prezimovalcev je 16, letovalcev sedem, vse leto pa se je tukaj zadrževalo šest vrst. V vseh dekadah obdobja raziskave so bile opažene štiri vrste: mlakarica Anas plathyrhynchos, siva čaplja Ardea cinerea, kanja Buteo buteo in postovka Falco tinnunculus. V več kot 90 % dekad je bila zabeležena še velika bela čaplja Ardea alba. Dve vrsti sta bili v obdobju 2007/2008 evdominantni (mlakarica 27,2 %, kanja 10,1 %), še dve pa dominantni (reglja Anas querquedula 7,4 %, liska Fulica atra 6,4 %). Največ osebkov je bilo zabeleženih konec marca in v začetku aprila (do 1978 os.), največ vrst (48) pa sredi aprila. Število vrst in osebkov je bilo med majem in avgustom zaradi presahlega jezera majhno. Najmanjše območje pojavljanja so imeli kormoran Phalacrocorax carbo ter skupina rac iz rodov Aythya, Bucephala in Mergellus. Galebi, čaplje, pobrežniki, lunji Circus sp. in rdečenoga postovka Falco vespertinus so se pojavljali na večjem delu območja raziskave. 10 gnezdilk izpolnjuje kriterije za oznako varstveno najpomembnejše vrste, od katerih sta dve (kostanjevka Aythya nyroca, veliki škurh Numenius arquata) vrsti globalne varstvene pozornosti, sedem pa vrste evropske varstvene pozornosti. Med negnezdilkami se 14 vrst vodnih ptic in ujed pojavlja v pomembnem številu (> 0,1 % biogeografske populacije), od tega se jih pet vsaj občasno pojavlja z več kot 1 % biogeografske populacije. Dve vrsti (rjavovrati ponirek Podiceps grisegena, kozica Gallinago gallinago) v Sloveniji gnezdita samo na Cerkniškem polju, še šest vrst pa ima tukaj vsaj 40 % slovenske gnezdeče populacije.
Keywords: vodne ptice, ujede, številčnost (biologija), dinamika pojavljanja (biologija), naravovarstveno vrednotenje
Published in DiRROS: 02.08.2024; Views: 320; Downloads: 200
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Abstract: OBJECTIVE Increased levels of vascular endothelial growth factor (VEGF) in human plasma samples have suggested that circulating VEGF is a cause of endothelial dysfunction in diabetes mellitus. However, artificial release of VEGF from platelets as a source of VEGF in plasma samples, as also occurs in serum samples, has not been ruled out in these studies. RESEARCH DESIGN AND METHODS We determined VEGF levels in plasma collected in both citrate and PECT, a medium that inactivates platelets, in a cross-sectional cohort of 21 healthy subjects and 64 patients with type 1 diabetes. In addition, we evaluated whether VEGF levels in both types of plasma correlated with the presence of diabetes, glycemic control, markers of in vivo or ex vivo platelet activation, and degree of diabetic retinopathy and nephropathy. RESULTS VEGF levels were invariably low in PECT plasma of both nondiabetic and diabetic subjects and were unrelated to any other diabetes-related variable studied. In contrast, VEGF levels in citrate plasma were 150% higher in diabetic patients than in control subjects and correlated with diabetes-related variables. Multiple linear regression analysis showed that levels of platelet factor 4, a marker for ex vivo platelet activation, and HbA1c were the independent predictors of VEGF levels in citrate plasma. Platelet activation, in vivo and ex vivo, was similar in diabetic persons and control subjects. CONCLUSIONS Like serum, citrate plasma is not suitable for reliable measurements of circulating VEGF. The low levels of VEGF in vivo, as represented by measurements in PECT plasma in our study, do not support a role of circulating VEGF in endothelial dysfunction in type 1 diabetes. Higher levels of VEGF in citrate plasma samples of diabetic persons do not represent the in vivo situation, but mainly originate from higher artificial ex vivo release from platelets correlating with the degree of glycemic control.
Keywords: vascular endothelial growth factor, VEGF, diabetes mellitus
Published in DiRROS: 02.08.2024; Views: 280; Downloads: 146
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Abstract: Despite the increasing urban and industrial development in coastal areas our knowledge on direct consequences of coastal modifications on benthic communities is still limited. The aim of the present study was to assess the response of rocky macrozoobenthos mediolittoral communities to human-induced hydromorphological pressures. Sampling was carried out by SCUBA diving and snorkeling in June 2008. Ten sites were selected along a gradient of hydromorphological alterations in the southern part of the Gulf of Trieste. Variables used to describe the stressor gradient were: water retention (from normal hydrology in unprotected coast to closed areas with only one opening), substrate composition, texture and rugosity. Despite natural differences between upper and lower mediolittoral subbelts, the present work showed that human-induced alterations of the coastal zone impact biological assemblages. There was a marked difference in biodiversity among sites with pristine conditions and stressed zones, mainly due to evenness of species distribution. Structural complexity of the substrate resulted to be the main factor influencing benthic diversity in the upper mediolittoral subbelt, while in the lower subbelt also the human-induced water retention seemed to play a key role. Anyhow, this response was complex, and the major human-induced alterations considered had different level of pressure within the two subbelts. The current study has a good potential to contribute to existing coastal assessment methods, since the impact of hydromorphological pressures on mediolittoral communities was almost neglected in the past. However, further work is needed to fully explain the impact of main human-induced threats on benthic communities.
Keywords: sea, Zoobenthos, artificial substrata, intertidal environment, hydromorphological modifications, ecological distribution, coastal waters, biodiversity, bio-indicators, macrozoobenthos, rocky bottom, biocoenoses, ecological aspects, Gulf of Trieste
Published in DiRROS: 02.08.2024; Views: 294; Downloads: 192
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Abstract: Soil salinity and drought are among the most serious agricultural and environmental problems of today. Therefore, investigations of plant resistance to abiotic stress have received a lot of attention in recent years. In this study, we identified the complete coding sequence of a 3′-phosphoadenosine-5′-phosphatase protein, ApHal2, from the halotolerant yeast Aureobasidium pullulans. Expression of the ApHAL2 gene in a Saccharomyces cerevisiae hal2 mutant complemented the mutant auxotrophy for methionine, and rescued the growth of the hal2 mutant in media with high NaCl concentrations. A 21-amino-acids-long region of the ApHal2 enzyme was inserted into the Arabidopsis thaliana homologue of Hal2, the SAL1 phosphatase. The inserted sequence included the META motif, which has previously been implicated in increased sodium tolerance of the Hal2 homologue from a related fungal species. Transgenic Arabidopsis plants overexpressing this modified SAL1 (mSAL1) showed improved halotolerance and drought tolerance. In a medium with an elevated salt concentration, mSAL1-expressing plants were twice as likely to have roots in a higher length category in comparison with the wild-type Arabidopsis and with plants overexpressing the native SAL1, and had 5% to 10% larger leaf surface area under moderate and severe salt stress, respectively. Similarly, after moderate drought exposure, the mSAL1-expressing plants showed 14% increased dry weight after revitalisation, with no increase in dry weight of the wild-type plants. With severe drought, plants overexpressing native SAL1 had the worst rehydration success, consistent with the recently proposed role of SAL1 in severe drought. This was not observed for plants expressing mSAL1. Therefore, the presence of this fungal META motif sequence is beneficial under conditions of increased salinity and moderate drought, and shows no drawbacks for plant survival under severe drought. This demonstrates that adaptations of extremotolerant fungi should be considered as a valuable resource for improving stress-tolerance in plant breeding in the future.
Keywords: soil salinity and drought, plant resistance, abiotic stress
Published in DiRROS: 02.08.2024; Views: 288; Downloads: 270
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