Volume 71, Issue 5

The genetic modification of lactic acid bacteria being used in medicine and food industries has been limited due to the scarcity of food-grade cloning vectors for the bacteria. The 4.46-kb food-grade cloning vector pUBU constructed in this study consisted of 3 major components from food-approved organisms, the theta-type replicon from pUCL287 of Tetragenococcus halophilus , the lactococcal cadmium resistance (Cd r ) determinant from pND918 and the promoter of L-lactate dehydrogenase (ldh L ) gene from Lactobacillus plantarum . The Cd r determinant was used as a dominant selectable marker and the ldh L promoter, a strong constitutive promoter, was used to drive the expression of inserted genes. The newly constructed vector was able to transform several genera of lactic acid bacteria and stable in the bacteria under non-selective pressure for at least 100 generations. In addition, it allowed inserted genes to express in lactic acid bacteria under the control of ldh L promoter. The host range of pUBU extended to Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Tetragenococcus . These results suggest that pUBU is a potential food-grade cloning vector for genetic modification of a wide range of lactic acid bacteria.

The antimicrobial activity of anthraquinone emodin isolated from Aspergillus awamori WAIR120 (LC032125) culture was investigated against some clinical, phytopathogenic and foodborne pathogenic microorganisms using an agar diffusion method. Among bacterial and fungal strains tested, the highest activity was obtained against Enterococcus faecalis AHR7 as well as Aspergillus niger OC20 with minimal inhibitory emodin concentration of 125 and 85 μg/mL, respectively. Emodin was found to induce morphogenic effects including swelling and elongation of bacterial cell and conidiation decrease, pigmentation loss, and cytoplasmic retraction of fungal cell, as was shown by light microscopy. Additionally, cellular effects were also resulted, in which emodin caused considerable changes in the nature of cell membrane and submicroscopic structure of bacterial and fungal cell, as was shown by transmission electron microscopy. Furthermore, there was an evidence of a disruption of lipid metabolism of fungal cell. These findings thus indicate the future possibility of exploiting emodin as an effective inhibitor of clinical, phytopathogenic and foodborne pathogenic microorganisms.

Saccharomyces cerevisiae was able to degrade a highly toxic textile dye malachite green (MG) at 100 mg/L concentration. Although 99% decolourization was observed, a tremendous metabolic and oxidative stress was exerted on the cells. Ethanolic extracts of Terminalia chebula, Clitoria ternatea and Boerhaavia diffusa at a concentration of 1 mg/mL were independently supplied to S. cerevisiae cells to counter the stress. T. chebula, C. ternatea and B. diffusa extracts reduced the activities of glutathione peroxidase (67, 8 and 71%), superoxide dismutase (2, 7 and 16%) and catalase (16, 52 and 57%), respectively. Inductions in the activities of laccase (66, 82 and 50%), lignin peroxidase (35, 75 and 10%), NADH-DCIP reductase (43, 52 and 91%) and MG reductase (66, 126 and 117%) were observed respectively. Presence of dye (MG) extended the lag phase of the growth cycle of S. cerevisiae up to 36 h, which was observed to be restored to normal (4 h) after phytoextract supplementation. Scanning electron microscope imaging revealed the restored cell morphology upon exposure to plant extracts. The accumulation of reactive oxygen species (ROS) was observed to be 355% greater in cells exposed to MG, which was significantly reduced after phytoextracts augmentation when compared to control cells. Phytoextracts proved to be beneficial in increasing the viability of S. cerevisiae cells and reduced the intracellular ROS and nuclear damage. Inclusion of plant extracts during decolourization proved to be beneficial and protected the cells so that 20 treatment cycles could be run achieving significant removal of MG.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium responsible for several infections in humans. The infections caused by this bacterial strain are difficult to treat due to the resistance of MRSA to clinically used antibiotics. Several medicinal plants extracts and their phytoconstituents have been reported to possess modulation and efflux pump inhibitory (EPI) activity against MRSA strains. Alpinia calcarata rhizomes have been reported to be used in Ayurveda for several ailments including fungal infections. Based on this information and in continuation with our efforts to discover EPIs from Indian medicinal plants, we describe EPI activity of flavonoids isolated from A. calcarata . Galangin and kaempferol showed ≥ 32-fold modulation in minimum inhibitory concentration (MIC) of ethidium bromide (EtBr) as well as norfloxacin in NorA-overexpressed S. aureus (SA-1199B) strain. Pinocembrin showed 32-fold modulation of EtBr MIC in SA-1199 strain, but not in SA-1199B and K1758 strains. A significant diference was not observed in the modulation of norfloxacin MIC by galangin in SA-1199 and SA-1199B strains, which may be due to non-specific nature of galangin as modulator or EPI. However, kaempferol modulated the MIC of EtBr as well as norfloxacin 64-fold and 32-fold, respectively. Also, the best modulatory effect of kaempferol was observed only in SA-1199B strain compared to two other strains. The EPI activity of kaempferol and galangin were found to be competitive with respect to verapamil. In dose-response assay, kaempferol at 31.25 μg/mL concentration was found to be better EPI by inhibiting NorA pump in SA-1199B strain and also demonstrated further confocal microscopy.

This study illustrates the sporadic distribution of metals in fluvial systems flowing from catchments to urban settlements. This is a detailed study prognosticating the deteriorating quality of rivers at specific locations due to metal pollution. Heavy metals like cadmium, lead, nickel and mercury are prominent in industrial sector. Contour plots derived using spatial and temporal data could determine the focal point of metal pollution and its gradation. Metal values recorded were cadmium 157 mg/L, lead 47 mg/L, nickel 61 mg/L and mercury 0.56 mg/L. Prokaryote diversity was less in polluted water and it harboured metal tolerant bacteria, which were isolated from these polluted sites. Actinomycetes like Streptomyces and several other bacteria like Stenotrophomonas and Pseudomonas isolated from the polluted river sites exhibited changes in morphology in presence of heavy metals. This stress response offered remedial measures as Streptomyces were effective in biosorption of cadmium, nickel and lead and Stenotrophomonas and Pseudomonas were effective in the bioaccumulation of lead and cadmium. Eighty-nine mg of lead and 106 mg of nickel could be adsorbed on one gram of Streptomyces biomass-based biosorbent. Such biological remedies can be further explored to remove metals from polluted sites and from metal contaminated industrial or waste waters.

Indoor air pollution occurs due to hazardous pollutants, such as tobacco smoke, pesticides and carbon oxides, sulphur oxides and nitrogen oxides arising from combustion of biomass fuels. Exposure to these pollutants results in respiratory conditions like asthma, chronic obstructive pulmonary disease, lung cancer, pneumonia and other lower respiratory infections. Several of these infections are a result of inflammation and oxidative stress. Here we demonstrate the ability of N , N ’-diacetylchitobiose in preventing oxidative DNA damage in peripheral blood mononuclear cells exposed to biomass smoke extracts and cigarette smoke extract. The cytotoxic effect of these pollutants was determined by trypan blue exclusion assay in peripheral blood mononuclear cells, where cytotoxicity in decreasing order was cigarette > wood > sawdust > cowdung. Cytotoxicity could be due to single- and double-strand breaks in the DNA as a result of oxidative stress. Comet assay measures the extent of DNA damage in the cells exposed to toxic agents. When mononuclear cells were treated with N , N ′-diacetylchitobiose and later exposed to smoke extracts, the extent of DNA damage decreased by 44.5% and 57.5% as compared to untreated cells. The protection offered by N , N ′-diacetylchitobiose towards oxidative DNA damage was at par with quercetin, a popular herbal medicine. Glutathione-S-transferase activity was determined in mononuclear cells exposed to smoke extracts, where oxidative stress in cells exposed to cigarette smoke extract was maximum. The present study demonstrates for the first time the ability of N , N ′-diacetylchitobiose to alleviate the harmful effects of indoor air pollutants.

Our study provides new knowledge about macrophyte assemblages and environmental variables of Slovak drainage and irrigation canals. The canal system, comprising ca. 15% of all flows in Slovakia, is one of the oldest in Central Europe. This information represents a first step toward exploitation of macrophytes as an obligatory biological quality element for the assessment of ecological potential according to the requirements of the Water Framework Directive. Among 61 canals studied, a total of 85 aquatic plant species (35 hydrophytes, 16 amphiphytes and 34 helophytes) were recorded during the years 2012–2014, including three neophytes and 18 threatened species. Physico-chemical variables explained the highest proportion of species variability, whilst hydromorphological variables and landscape variables were much less significant. Chemical oxygen demand, ammonium nitrogen, electrical conductivity, water pH, flow velocity and water depth were identified as most important drivers of species composition of aquatic macrophytes and explained 21.1% of the variability. The CCA results revealed differences in species composition of canals reflecting the geographical origin and environmental conditions as well, relating to South-Eastern and South-Western Slovakia. In conclusion, macrophytes in drainage and irrigation canals are able to reflect several environmental variables and might provide valuable information for bioindication in assessing the ecological potential.

This study is aimed at the responses of grapevine adventitious root explants to zinc (Zn 2+ ) excess. Within 24 h Zn 2+ induced oxidative burst in concentration-dependent manner. The time course analysis revealed biphasic response in superoxide (O 2–· ) production. Hydrogen peroxide (H2O2) accumulation rose gradually within 24 h. Enhanced Zn 2+ concentrations did not induce progression of cell death. Immediately upon Zn 2+ addition to the perfusion solution, root epidermal cells exhibited hyperpolarization of their electrical membrane potential (E M ), that was transient and independent of Zn 2+ concentration. The subsequent, transient depolarization of EM was concentration-dependent and its magnitude increased with increasing Zn 2+ concentration. After 24 h the EM in treated roots recovered and its values were identical with those of control roots. Membrane permeability of root cells increased in the roots treated with 5 mM Zn 2+ within 24 h while the lower concentrations did not show any impact on membrane permeability. Differences in protein expression pattern identified by proteomic approach involving antibody microarray expression profiling revealed Zn 2+ -induced upregulation of apoptosis-related protein dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit (DAD1), extracellular signal-regulated kinase 1/2 (ERK1/2), some antioxidant enzymes and structural proteins in the roots. Moreover, the proteins involved in plant defense mechanisms endochitinase I (CHIT 1) and phenylalanine ammonia-lyase (PAL) were down-regulated indicating a cross-talk between defense and heavy metal signaling pathways. Taken together, these results showed that the grape cultivar Limberger is highly Zn 2+ -responsive and could be used as a model plant for studying physiological and molecular responses to heavy metal excess.

Tumourous stem mustard, a variant of Brassica juncea (Cruciferae), is an economically important crop. In a preliminary study, the expression of a fragment of a MYB (v-myb avian myeloblastosis viral oncogene homolog) gene was screened out during stem swelling. The four complete cDNA sequences of novel MYB genes were cloned by rapid amplification of cDNA ends based on the MYB sequence fragment. These sequences, which represent distinct alternatively spliced transcripts of a BjMYB1 ( Brassica juncea var. tumida Tsen et Lee MYB gene 1), possessed a similar alternative splicing pattern to several reported MYB genes of other species. RT-PCR analysis revealed different expression patterns for these four spliced transcripts in various tissues. However, stem expression of BjMYB1 gene was detected in the non-swelling growth phase, and different expression patterns were detected at other stages during stem swelling. The expression levels of the transcripts differed after treatment with various phytohormones (e.g., ABA) and NaCl. In addition, prokaryotic expression and Western blotting showed that BjMYB1 -2 encodes a 5.3 kDa protein while BjMYB1-3 encodes a 21.6 kDa protein. The overexpression of BjMYB1-3 and BjMYB1-4 in Arabidopsis thaliana showed earlier germination, promoting growth and having tolerance to abiotic stress such as osmotic stress or high salt. The research of this article set the foundation for further study of the function of MYB genes in tumourous stem mustard and other Brassica plants.

During epizootological monitoring of local pet-shops and aquarium fish breeders, 45 juvenile fish of Astronotus ocellatus Agassiz, 1831 were examined in March 2013 and October 2014. Fish were imported from a Singapore farm. We found massive infection by Gussevia asota Kritsky, Thatcher & Boeger, 1989 with 100% prevalence and high mean intensities 246 and 265 parasites. Fish displayed visible asphyxia, neurological symptoms, uncoordinated locomotion and sudden death. High 98% mortality of imported juveniles suggests reduced capacity to tolerate hypoxia among oscar juveniles. This is the first report of infection by G. asota in A. ocellatus juveniles in commercial shop from Slovak Republic, in Europe.

Environmental stressors are known to play an important role in determining the distribution and abundance of intertidal species. Marine molluscs are particularly susceptible to changes in water temperature and salinity in inter-tidal zones. Ruditapes decussatus and Ruditapes philippinarum are marine intertidal clams, constantly exposed to salinity variations in their intertidal habitat. The goal of this study was to investigate whether these species would handle salinity increase as well as salinity decrease, given the general paucity of data on R. decussatus and R. philippinarum exposed to salinity changes. In this context, leucine aminopeptidase (LAP) activity changes following exposure to salinity change were investigated in clams from Tunisia and from Brittany. Samples of R. decussatus from Tunisia were maintained in salinity of l0% 0 , 20% 0 , 30% 0 , 45% 0 and 55% 0 for four weeks. Effects of salinity on LAP activity are also investigated in R. decussatus and in R. philippinarum from Brittany in salinities of 5% 0 , l0% 0 , 15% 0 , 17% 0 , 25% 0 , 30% 0 , 40% 0 , 45 % 0 and 55% 0 . Three sets of experiments were conducted: Short (20 h), medium (96 h) and long-term (four weeks). During salinity trials, LAP activity of R. decussatus ranged from 0.07 ± 0.04 to 0.2 ± 0.05 μmol of paranitroanilide mg protein –1 min −1 and from 0.15 ± 0.015 to 0.56 ± 0.41 mg protein –1 min –1 for clams from Tunisia and from Brittany, respectively. In the R. philippinarum species, results showed the occurrence of higher activity levels, values ranged from 0.26 ± 0.11 to 1.04 ± 0.38 μmol of paranitroanilide mg protein –1 min –1 . None of the salinity regimes gave significant differences in LAP ctivity in R. decussatus from Tunisia. Conclusion has also been deduced from experiments carried out on R. decussatus and R. philippinarum from Brittany. Results reported here suggest that LAP ctivity seems to be not affected by salinity changes in populations investigated. The study provided some useful insights into response of the bivalves R. decussatus and R. philippinarum to salinity stress and offered a number of candidate hypothesis as potential explanations for moderate changes in LAP activity.

The qualitative and quantitative structure of rotifer and crustacean zooplankton inhabiting Stratiotes beds and pelagial zone have been studied in different types of lakes. In 12 lakes we recorded 151 rotifer and 52 crustacean species, with more than 90% of them being present in the Stratiotes beds. There were twice more zooplankton species in the Stratiotes stands than in open water zone of lakes. The dense beds of a simple-built water soldier supported high zooplankton diversity. The obtained results suggest that the degree of leaf dissection is not the only factor responsible for microhabitat heterogeneity in macrophyte beds. The dense Stratiotes stands can provide a diverse array of surfaces for colonization, feeding (detritus) and shelter against predators for many plant-associated and benthic species, as well as they can act as a daytime refuge for some pelagic crustaceans (Diaphanosoma brachyurum, Mesocyclops leuckarti and Ceriodaphnia spp.). Composition of rotifer and crustacean assemblages in the Stratiotes stands were very similar in all studied lakes and clearly differed from open water zones. Our study suggested that similar zooplankton communities could develop in the same macrophyte habitat.

The effect of longitudinal zonation patterns and macroinvertebrate responses to changes in habitat characteristics have been given a lot of attention. But studies of changes in macroinvertebrate assemblages along small upland undisturbed watercourses are still lacking. The aim of the study is to analyse variability in macroinvertebrate communities between two different habitats/morphological sequences – shallow (riffle/run/step/) and deeper (pool) channel-bed morphological units on the background of the environmental parameters (local relief, slope, channel confinement ratio, channel-valley walls connectivity, floodplain continuity and channel abut, channel sinuosity and predominant land cover of riparian zone) of seven valley segments (functional process zones) in two seasons of the year (spring and autumn). The longitudinal-downstream gradient research was conducted on the semi-natural upland headwater brook in the Little Carpathians (9,330 m long, average gradient 2.8%) at 15 morphological sequences (30 sampling points). Each sampling point in spring as well as in autumn was characterised by mean flow velocity, discharge, water depth, channel width, channel bottom particle size, and flow types. Selected physico-chemical variables: pH, dissolved oxygen content (DO), oxygen saturation (DO %), temperature (t) conductivity and total dissolved solids (TDS) were measured directly in the field using the multisonde measuring device. Organisms were identified into the family level. The results showed that differences in zonation of benthic invertebrates between seasons are more apparent than seasonal variability between pools and riffles. Spring samples followed the increasing gradient of total dissolved solids downstream with characteristic families for upper and lower stretch. The disruption of macroinvertebrates zonation was more evident in autumn samples with greatest effects in pools. Based on RDA, the distribution of families was driven by three significant variables in riffles and four in pools. Alike environmental factors of pool sequences in the middle and upper reaches were responsible for similarity in macroinvertebrate structure. In conclusion, the distribution pattern of benthic invertebrates of the natural part of the small headwater stream in terms of abundance of macroinvertebrate families followed the longitudinal zonation in spring, but in autumn were the local habitat conditions more important.

A study was carried out in different type of habitats in an urban ecosystem of Bydgoszcz. The aim of the study was to determine aphid species spectrum and to compare dominance and species richness of the two aphid communities connected with the two different plant community structures in an urban ecosystem. There were two types of habitats: so-called southern slope, it was a part of the city on the hill, and so-called downtown – middle-northern part of the city. The differences were evaluated taking into consideration plant community structures. Observations of the occurrence of aphids were conducted every seven-ten days by monitoring of aphid host plants from the beginning of the appearance of aphids, that is from the end of April to the beginning of August, when aphids disappear. Aphids were counted in three replications: on the same host plant species, each time on different plant individuals bearing aphids in a given habitat. Based on the collected material, 32 aphid species were found on 31 plant species on southern slope areas, and 24 species of aphids on 23 plant species in downtown environments. In both years of the study of aphid species feeding on plants, Aphis fabae Scopoli, 1763 , Aphis sambuci L., 1758, Brachycaudus prunicola (Kaltenbach, 1843) and Hyalopterus pruni (Geoffroy, 1762) were most numerous. The city on the hill was characterized by a larger number of aphids colonizing host plants and a higher species diversity in the communities than in the downtown.

Leaf beetles were studied in the Kovada Lake National Park (KL) and Kızıldağ National Park (KD) in Isparta province (Turkey) during April–October in 2009 and 2010. A total of 131 leaf beetle species were collected. In total, 88 species were detected from KL and 95 species from KD. Adult activity periods of the collected leaf beetle species were determined and their frequency and dominancy values were provided. The results showed slight differences in terms of the seasonal activity of the species in the two national parks. According to Shannon-Wiener index, the highest leaf beetle diversity among the studied months was found in early June (H’: 4.23) and late May (H’: 4.17). Sørensen similarity index of the species according to different seasonal periods showed three distinct seasonal periods: a) early April, b) late April to early July, c) late July to late October. Almost all leaf beetles showed a seasonal distribution, with the highest dominance during spring and early summer. The main reason for these different seasonal activities is that leaf beetle species are related to the quality and availability of host-plants.

The ependyma, lining of the brain ventricular system, is a heterogeneous structure. The brain ventricles, including the lateral-, third-, fourth-, and mesencephalic ones and cerebral aqueduct, are covered by a single uninterrupted layer, composed of squamous to columnar ependymocytes, possessing cilia, microvilli or cytoplasmic protrusions. Small ependymal areas are considered to be functionally different and therefore, accurate and systematic classification of ependymal areas might be helpful to perform mutual comparisons of the same types of ependymal areas under different experimental conditions. Thus, the aim of the present study was to create an anatomical guide that will be able to offer an easy and repeatable technique for the selection of anatomically precisely identified small ependymal areas. For this purpose, the periventricular structures, as a stable part of brain, localized most closely to the brain ventricle walls, were chosen as reference points. Classification of the ependymal areas is presented in a form of tables, i.e. ependymal tables, which prevent of an interchange of different types of ependymal areas, avoiding of their misinterpretation. Each table brings all the information needed to localize the selected ependymal sector in 5 columns, indicating: (1) the number of the frontal sections; (2) the labelling of the ventricle walls; (3) Latin names of the periventricular structures; (4) abbreviations of the periventricular structures; and (5) the final designation of the selected ependymal areas. The final designation of a small ependymal area is composed of several letters (e.g., "LvE-ca", where Lv = lateral ventricle, E = ependyma, and ca = caudate nucleus). The proposed anatomical classification of the ependymal lining represents an original approach with more unambiguous classification of ependymal areas than is only a structural naming of ependyma. This anatomical guide will be helpful in performing of an accurate mutual comparison of the same types of ependymal areas under different experimental conditions as well as a possibility to compare mutually the data from different morphological/biochemical studies.