Bacteria associated with Zn-hyperaccumulators Arabidopsis halleri and Arabidopsis arenosa from Zn-Pb-Cd waste heaps in Poland as promising tools for bioremediation.

To identify metal adapted bacteria equipped with traits positively influencing the growth of two hyperaccumulator plant species Arabidopsis arenosa and Arabidopsis halleri, we isolated bacteria inhabiting rhizosphere and vegetative tissues (roots, basal and stem leaves) of plants growing on two old Zn-Pb-Cd waste heaps in Boleslaw and Bukowno (S. Poland), and characterized their potential plant growth promoting (PGP) traits as well as determined metal concentrations in rhizosphere and plant tissues. To determine taxonomic position of 144 bacterial isolates, 16S rDNA Sanger sequencing was used. A metabolic characterization of isolated strains was performed in vitro using PGP tests. A. arenosa and A. halleri accumulate high amounts of Zn in their tissues, especially in stem leaves. Among in total 22 identified bacterial taxa, the highest level of the taxonomical diversity (H' = 2.01) was revealed in A. halleri basal leaf endophytes originating from Bukowno waste heap area. The 96, 98, 99, and 98% of investigated strains showed tolerant to Cd, Zn, Pb and Cu, respectively. Generally, higher percentages of bacteria could synthesize auxins, siderophores, and acetoin as well as could solubilize phosphate. Nine of waste heap origin bacterial strains were tolerant to toxic metals, showed in vitro PGP traits and are potential candidates for bioremediation.

The Current State of Osteoarthritis Treatment Options Using Stem Cells for Regenerative Therapy: A Review.

Articular cartilage has very low metabolic activity. While minor injuries may be spontaneously repaired within the joint by chondrocytes, there is very little chance of a severely impaired joint regenerating itself when damaged. Therefore, any significant joint injury has little chance of spontaneously healing without some type of therapy. This article is a review that will examine the causes of osteoarthritis, both acute and chronic, and how it may be treated using traditional methods as well as with the latest stem cell technology. The latest regenerative therapy is discussed, including the use and potential risks of mesenchymal stem cells for tissue regeneration and implantation. Applications are then discussed for the treatment of OA in humans after using canine animal models. Since the most successful research models of OA were dogs, the first applications for treatment were veterinary. However, the treatment options have now advanced to the point where patients suffering from osteoarthritis may be treated with this technology. A survey of the literature was performed in order to determine the current state of stem cell technology being used in the treatment of osteoarthritis. Then, the stem cell technology was compared with traditional treatment options.

Community Profiling of Seed Endophytes from the Pb-Zn Hyperaccumulator Noccaea caerulescens and Their Plant Growth Promotion Potential.

Endophytes within plants are known to be crucial for plant fitness, and while their presence and functions in many compartments have been studied in depth, the research on seed endophytes is still limited. This work aimed to characterize the seed endophytic and rhizospheric bacterial community of two Noccaea caerulescens Pb-Zn hyperaccumulator populations, growing on two heavy-metal-polluted sites in Belgium. Cultured representatives were evaluated for their potential to enhance seed germination and root length of the model species Arabidopsis thaliana. The results indicated that the community structure within the seed is conserved between the two locations, comprising mainly of Proteobacteria (seeds), and Actinobacteria in the bulk soil. Root length of A. thaliana was significantly increased when inoculated with Sphingomonas vulcanisoli. The results of this paper offer insights into the importance of the selection of the core seed endophytic microbiome and highlight the precarious symbiotic relationship they have with the plant and seed.

An Alliance of Trifolium repens-Rhizobium leguminosarum bv. trifolii-Mycorrhizal Fungi From an Old Zn-Pb-Cd Rich Waste Heap as a Promising Tripartite System for Phytostabilization of Metal Polluted Soils.

The Boleslaw waste heap in South Poland, with total soil Zn concentrations higher than 50,000 mg kg-1, 5,000 mg Pb kg-1, and 500 mg Cd kg-1, is a unique habitat for metallicolous plants, such as Trifolium repens L. The purpose of this study was to characterize the association between T. repens and its microbial symbionts, i.e., Rhizobium leguminosarum bv. trifolii and mycorrhizal fungi and to evaluate its applicability for phytostabilization of metal-polluted soils. Rhizobia originating from the nutrient-poor waste heap area showed to be efficient in plant nodulation and nitrogen fixation. They demonstrated not only potential plant growth promotion traits in vitro, but they also improved the growth of T. repens plants to a similar extent as strains from a non-polluted reference area. Our results revealed that the adaptations of T. repens to high Zn-Pb-Cd concentrations are related to the storage of metals predominantly in the roots (excluder strategy) due to nodule apoplast modifications (i.e., thickening and suberization of cell walls, vacuolar storage), and symbiosis with arbuscular mycorrhizal fungi of a substantial genetic diversity. As a result, the rhizobia-mycorrhizal fungi-T. repens association appears to be a promising tool for phytostabilization of Zn-Pb-Cd-polluted soils.

Exopolysaccharide Carbohydrate Structure and Biofilm Formation by Rhizobium leguminosarum bv. trifolii Strains Inhabiting Nodules of Trifoliumrepens Growing on an Old Zn-Pb-Cd-Polluted Waste Heap Area.

Heavy metals polluting the 100-year-old waste heap in Boleslaw (Poland) are acting as a natural selection factor and may contribute to adaptations of organisms living in this area, including Trifolium repens and its root nodule microsymbionts-rhizobia. Exopolysaccharides (EPS), exuded extracellularly and associated with bacterial cell walls, possess variable structures depending on environmental conditions; they can bind metals and are involved in biofilm formation. In order to examine the effects of long-term exposure to metal pollution on EPS structure and biofilm formation of rhizobia, Rhizobium leguminosarum bv. trifolii strains originating from the waste heap area and a non-polluted reference site were investigated for the characteristics of the sugar fraction of their EPS using gas chromatography mass-spectrometry and also for biofilm formation and structural characteristics using confocal laser scanning microscopy under control conditions as well as when exposed to toxic concentrations of zinc, lead, and cadmium. Significant differences in EPS structure, biofilm thickness, and ratio of living/dead bacteria in the biofilm were found between strains originating from the waste heap and from the reference site, both without exposure to metals and under metal exposure. Received results indicate that studied rhizobia can be assumed as potentially useful in remediation processes.

Insight into probiotic properties of lactic acid bacterial endosymbionts of Apis mellifera L. derived from the Polish apiary.

Taking into account that fructophilic lactic acid bacteria (FLAB) can play an important role in the health of honey bees and can be used as probiotics, phenotypic properties of probiotic interest of Lactobacillus kunkeei (12 strains) and Fructobacillus fructossus bacteria (2 strains), isolated from Apis mellifera gastrointestinal tract, have been studied. We have evaluated survival of tested FLAB in honey bee gut, their susceptibility to antibiotics (ampicillin, erythromycin, tylosin), cell surface hydrophobicity, auto-aggregation ability, co-aggregation with model pathogenic bacteria, biofilm formation capacity, and effect of studied FLAB, added to sucrose syrup bee diet, on longevity of honey bees. The tested FLAB exhibited good gastrointestinal tract tolerance and high antibiotic susceptibility, which are important criteria in the screening of probiotic candidates. It was also found that all FLAB studied have high cell surface hydrophobicity and fulfil next selection criterion for their use as probiotics. Symbionts of A. mellifera showed also auto- and co-aggregation capacities regarded as valuable features for biofilm formation and inhibition of pathogens adhesion to the bee gut cells. Biofilm-development ability is a desired characteristic of probiotic lactic acid bacteria. As indicated by quantitative crystal violet-stained microplate assay and confocal laser scanning microscopy imaging, all studied A. mellifera gut isolates exhibit a biofilm positive phenotype. Moreover, it was also documented, on honey bees kept in cages, that supplementation of A. mellifera sucrose diet with FLAB decreases mortality and improves significantly longevity of honey bees. Presented research showed that A. mellifera FLAB symbionts are good candidates for application as probiotics.

Trifolium repens-Associated Bacteria as a Potential Tool to Facilitate Phytostabilization of Zinc and Lead Polluted Waste Heaps.

Heavy metals in soil, as selective agents, can change the structure of plant-associated bacterial communities and their metabolic properties, leading to the selection of the most-adapted strains, which might be useful in phytoremediation. Trifolium repens, a heavy metal excluder, naturally occurs on metal mine waste heaps in southern Poland characterized by high total metal concentrations. The purpose of the present study was to assess the effects of toxic metals on the diversity and metabolic properties of the microbial communities in rhizospheric soil and vegetative tissues of T. repens growing on three 70-100-years old Zn-Pb mine waste heaps in comparison to Trifolium-associated bacteria from a non-polluted reference site. In total, 113 cultivable strains were isolated and used for 16S rRNA gene Sanger sequencing in order to determine their genetic affiliation and for in vitro testing of their plant growth promotion traits. Taxa richness and phenotypic diversity in communities of metalliferous origin were significantly lower (p < 0.0001) compared to those from the reference site. Two strains, Bacillus megaterium BolR EW3_A03 and Stenotrophomonas maltophilia BolN EW3_B03, isolated from a Zn-Pb mine waste heap which tested positive for all examined plant growth promoting traits and which showed co-tolerance to Zn, Cu, Cd, and Pb can be considered as potential facilitators of phytostabilization.

Beneficial features of plant growth-promoting rhizobacteria for improving plant growth and health in challenging conditions: A methodical review.

New eco-friendly approaches are required to improve plant biomass production. Beneficial plant growth-promoting (PGP) bacteria may be exploited as excellent and efficient biotechnological tools to improve plant growth in various - including stressful - environments. We present an overview of bacterial mechanisms which contribute to plant health, growth, and development. Plant growth promoting rhizobacteria (PGPR) can interact with plants directly by increasing the availability of essential nutrients (e.g. nitrogen, phosphorus, iron), production and regulation of compounds involved in plant growth (e.g. phytohormones), and stress hormonal status (e.g. ethylene levels by ACC-deaminase). They can also indirectly affect plants by protecting them against diseases via competition with pathogens for highly limited nutrients, biocontrol of pathogens through production of aseptic-activity compounds, synthesis of fungal cell wall lysing enzymes, and induction of systemic responses in host plants. The potential of PGPR to facilitate plant growth is of fundamental importance, especially in case of abiotic stress, where bacteria can support plant fitness, stress tolerance, and/or even assist in remediation of pollutants. Providing additional evidence and better understanding of bacterial traits underlying plant growth-promotion can inspire and stir up the development of innovative solutions exploiting PGPR in times of highly variable environmental and climatological conditions.

Differentiation of Human Mesenchymal Stem Cells from Wharton's Jelly Towards Neural Stem Cells Using A Feasible and Repeatable Protocol.

The transplantation of neural stem cells (NSCs) capable of regenerating to the cells of the central nervous system (CNS) is a promising strategy in the treatment of CNS diseases and injury. As previous studies have highlighted mesenchymal stem cells (MSCs) as a source of NSCs, this study aimed to develop a feasible, efficient, and reproducible method for the neural induction of MSCs isolated from Wharton's jelly (hWJ-MSCs). We induced neural differentiation in a monolayer culture using epidermal growth factor, basic fibroblast growth factor, N2, and B27 supplements. This resulted in a homogenous population of proliferating cells that expressed certain neural markers at both the protein and mRNA levels. Flow cytometry and immunocytochemistry confirmed the expression of neural markers: nestin, sex-determining region Y (SRY) box 1 and 2 (SOX1 and SOX2), microtubule-associated protein 2 (MAP2), and glial fibrillary acidic protein (GFAP). The qRT-PCR analysis revealed significantly enhanced expression of nestin and MAP2 in differentiated cells. This study confirms that it is possible to generate NSCs-like cells from hWJ-MSCs in a 2D culture using a practical method. However, the therapeutic effectiveness of such differentiated cells should be extended to confirm the terminal differentiation ability and electrophysiological properties of neurons derived from them.

Genomic polymorphism of Trifolium repens root nodule symbionts from heavy metal-abundant 100-year-old waste heap in southern Poland.

In total, 77 rhizobial strains isolated from the root nodules of T. repens, inhabiting heavy metal-contaminated waste heap (36 isolates) and control grassland (41 ones) in southern Poland, were analyzed for genome polymorphism and strength of the heavy metals' (mainly Zn, Pb, Cd) selective pressure on bacterial genome polymorphism using two PCR-based techniques, ERIC- (enterobacterial repetitive intergenic consensus) and REP-PCR (repetitive extragenic palindromic) sequences. Both methods of different discriminatory power index (D) (ERIC-PCR D = 0.9737; REP-PCR D = 0.9826) allowed to distinguish 47 and 44 rhizobial strains, respectively. Combined analysis of ERIC-PCR and REP-PCR DNA amplicons differentiated all tested isolates. Both ERIC- and REP-PCR DNA fingerprinting techniques showed significant decline of the genome polymorphism (h) in rhizobial population from metalliferous waste heap (h = 0.89 ± 0.03; h = 0.90 ± 0.02, respectively) compared to rhizobia from control non-metalliferous area (h = 0.99 ± 0.01; h = 0.98 ± 0.02, respectively) as well as substantial differences in the genomic polymorphism between both these populations (FST = 0.162, p = 0.008; FST = 0.170, p = 0.000, respectively).

Alterations in porcine intrahepatic sympathetic nerves after bisphenol A administration.

INTRODUCTION: Bisphenol A (BPA) is used in the chemical industry for manufacturing plastics which are used as food packaging. Data indicate that BPA is released from such products and is widely present in the environment and the human body. So far, the EFSA and the US FDA have determined "safe" BPA oral exposure levels, and a large amount of data indicates that BPA is harmful even at low-doses. Our previously performed analyses concerning BPA exposure demonstrated the impact of this substance on parasympathetic and peptidergic nerve fibers present within the liver. Therefore, this study concerns BPA exposure and sympathetic intrahepatic in-nervation in reference to several neuropeptides which modulate neuronal responses: cocaine and amphetamine regulated transcript (CART), galanin (GAL), calcitonin gene-regulated peptide (CGRP) and substance P (SP). MATERIALS AND METHODS: Fifteen young swine at 8 weeks of age were used as experimental models of the juvenile human liver. The pigs were divided into 3 groups and received capsules orally with bisphenol at a dose of 0.05 mg/kg b.w./day; a dose of 0.5 mg/kg b.w./day and placebo capsules as a control. After 28 days of oral BPA intake, the animals were euthanized, perfused with 4% paraformaldehyde (PFA), and livers were collected and fixed in PFA. The cryostat sections were subjected to a routine double-labeling immunofluorescence technique. The primary antibodies were directed against dopamine beta hydroxylase (DbH), which is a marker for sympathetic nerves, and one of the investigated neuropeptides: CART, GAL, CGRP and SP, which co-localized the inves-tigated nerves. Immunoreactive nerves were counted in the liver and the percentage presence of each neuronal combination in particular samples of each experimental group were determined and analyzed statistically. RESULTS: The BPA oral intake at low and ten times higher dosage caused an increase of the number of sympa-thetic nerve fibers within the porcine liver by 48.6% and 63.7%, respectively. Moreover, BPA exposure caused an increased presence of sympathetic nerve fibers in these two experimental groups, which were co-localized with CART and GAL up to 65.9%/173.2% and 147.4%/126.3%, respectively. At the lower BPA doses of 50 µg/kg b.w./day, the percentages of SP+/DbH+ and CGRP+/DbH+ nerve fibers were similar to the control. However at a ten times higher dose, BPA caused an increased number of SP+/ DbH+ and CGRP+/ DbH+ nerve fibers in the liver, up to 46.4% and 73.5% respectively. CONCLUSIONS: BPA caused an increase in the number of sympathetic nerve fibers as well as sympathetic nerve fibers which co-localized with neuropeptides in the porcine liver. The increase in CART and GAL were excep-tionally high even at low BPA doses. BPA food contamination may dysregulate liver sympathetic innervation, and thereby may change the oxygenated blood supply, alter metabolism and disrupt the activity of hepatic pa-renchymal cells.

Application of MRI for the Diagnosis of Neoplasms.

AIM: The aim of the study was to determine the most commonly diagnosed neoplasms in the MRI scanned patient population and indicate correlations based on the descriptive variables. METHODS: The SPSS software was used to determine the incidence of neoplasms within the specific diagnoses based on the descriptive variables of the studied population. Over a five year period, 791 patients and 839 MRI scans were identified in neoplasm category (C00-D48 according to the International Statistical Classification of Diseases and Related Health Problems ICD-10). RESULTS: More women (56%) than men (44%) represented C00-D48. Three categories of neoplasms were recorded. Furthermore, benign neoplasms were the most numerous, diagnosed mainly in patients in the fifth decade of life, and included benign neoplasms of the brain and other parts of the central nervous system. CONCLUSIONS: Males ≤ 30 years of age with neoplasms had three times higher MRI scans rate than females of the same age group; even though females had much higher scans rate in every other category. The young males are more often selected for these scans if a neoplasm is suspected. Finally, the number of MRI-diagnosed neoplasms showed a linear annual increase.

Zinc Transporter 3 (Znt3) as an Active Substance in the Enteric Nervous System of the Porcine Esophagus.

Zinc transporter 3 (ZnT3), a member of the SLC 30 zinc transporter family, is involved in the transport of zinc ions from the cytoplasm into synaptic vesicles or intracellular organelles. The aim of the present study was to investigate for the first time the percentage of ZnT3-like immunoreactive (ZnT3-LI) neurons in the enteric nervous system (ENS) of the porcine esophagus and denotation of their neurochemical coding. Routine double- and triple-immunofluorescence labeling of cervical, thoracic, and abdominal fragments of esophagus for ZnT3 with protein gene product (PGP 9.5; used as pan-neuronal marker), nitric oxide synthase (NOS), somatostatin, vasoactive intestinal peptide (VIP), vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), and galanin (GAL) was performed. The percentage of ZnT3-LI neurons in myenteric ganglia amounted to 50.2 ± 4.7, 63.4 ± 8.3, and 77.1 ± 1.1 % of all PGP 9.5-like immunoreactive neuronal cells in cervical, thoracic, and abdominal esophagus, respectively. In submucous ganglia, these values in particular parts of esophagus amounted to 46.3 ± 6.3, 81.0 ± 8.1, and 74.4 ± 4.4 %. Znt3 co-localized mainly with VAChT, NPY, GAL, NOS, and VIP, but the degree of co-localization depended on the "kind" of enteric ganglia and part of esophagus studied. The obtained results suggest that both ZnT3 and zinc ions may play important and various roles in the neuronal regulation of esophageal functions.

Cadmium Accumulation and Pathological Alterations in the Midgut Gland of Terrestrial Snail Helix pomatia L. from a Zinc Smelter Area: Role of Soil pH.

The purpose of this study was to determine whether cadmium (Cd) accumulation and toxicity in the midgut gland of Helix pomatia snails living in a Cd-contaminated area were related to soil pH. Toxic responses in the midgut gland (i.e., increased vacuolization and lipid peroxidation) occurred in H. pomatia snails exhibiting the highest Cd levels in the gland (265-274 µg/g dry wt) and living on acidic soil (pH 5.3-5.5), while no toxicity was observed in snails accumulating less Cd (90 µg/g) and ranging on neutral soil (pH 7.0), despite the fact that total soil Cd was similar in the two cases. The accumulation of Cd in the gland was directly related to the water extractable Cd in soil, which in turn correlated inversely with soil pH, indicating that this factor had a significant effect on tissue Cd. It appeared further that the occurrence of Cd toxicity was associated with low levels of metallothionein in the gland of snails ranging on acidic soil.

Multilocus sequence analysis supports the taxonomic position of Astragalus glycyphyllos symbionts based on DNA-DNA hybridization.

In this study, the phylogenetic relationship and taxonomic status of six strains, representing different phenons and genomic groups of Astragalus glycyphyllos symbionts, originating from Poland, were established by comparative analysis of five concatenated housekeeping gene sequences (atpD, dnaK, glnA, recA and rpoB), DNA-DNA hybridization and total DNA G+C content. Maximum-likelihood phylogenetic analysis of combined atpD, dnaK, glnA, recA and rpoB sequence data placed the studied bacteria into the clade comprising the genus Mesorhizobium. In the core gene phylograms, four A. glycyphyllos nodule isolates (AG1, AG7, AG15 and AG27) formed a cluster common with Mesorhizobium ciceri, whereas the two other A. glycyphyllos symbionts (AG17 and AG22) were grouped together with Mesorhizobium amorphae and M. septentrionale. The species position of the studied bacteria was clarified by DNA-DNA hybridization. The DNA-DNA relatedness between isolates AG1, AG7, AG15 and AG27 and reference strain M. ciceri USDA 3383T was 76.4-84.2%, and all these A. glycyphyllos nodulators were defined as members of the genomospecies M. ciceri. DNA-DNA relatedness for isolates AG17 and AG22 and the reference strain M. amorphae ICMP 15022T was 77.5 and 80.1%, respectively. We propose that the nodule isolates AG17 and AG22 belong to the genomic species M. amorphae. Additionally, it was found that the total DNA G+C content of the six test A. glycyphyllos symbionts was 59.4-62.1 mol%, within the range for species of the genus Mesorhizobium.

Phylogeny of Symbiotic Genes and the Symbiotic Properties of Rhizobia Specific to Astragalus glycyphyllos L.

The phylogeny of symbiotic genes of Astragalus glycyphyllos L. (liquorice milkvetch) nodule isolates was studied by comparative sequence analysis of nodA, nodC, nodH and nifH loci. In all these genes phylograms, liquorice milkvetch rhizobia (closely related to bacteria of three species, i.e. Mesorhizobium amorphae, Mesorhizobium septentrionale and Mesorhizobium ciceri) formed one clearly separate cluster suggesting the horizontal transfer of symbiotic genes from a single ancestor to the bacteria being studied. The high sequence similarity of the symbiotic genes of A. glycyphyllos rhizobia (99-100% in the case of nodAC and nifH genes, and 98-99% in the case of nodH one) points to the relatively recent (in evolutionary scale) lateral transfer of these genes. In the nodACH and nifH phylograms, A. glycyphyllos nodule isolates were grouped together with the genus Mesorhizobium species in one monophyletic clade, close to M. ciceri, Mesorhizobium opportunistum and Mesorhizobium australicum symbiovar biserrulae bacteria, which correlates with the close relationship of these rhizobia host plants. Plant tests revealed the narrow host range of A. glycyphyllos rhizobia. They formed effective symbiotic interactions with their native host (A. glycyphyllos) and Amorpha fruticosa but not with 11 other fabacean species. The nodules induced on A. glycyphyllos roots were indeterminate with apical, persistent meristem, an age gradient of nodule tissues and cortical vascular bundles. To reflect the symbiosis-adaptive phenotype of rhizobia, specific for A. glycyphyllos, we propose for these bacteria the new symbiovar "glycyphyllae", based on nodA and nodC genes sequences.

Insight into the genomic diversity and relationship of Astragalus glycyphyllos symbionts by RAPD, ERIC-PCR, and AFLP fingerprinting.

We assessed the genomic diversity and genomic relationship of 28 Astragalus glycyphyllos symbionts by three methodologies based on PCR reaction, i.e., RAPD, ERIC-PCR, and AFLP. The AFLP method with one PstI restriction enzyme and selective PstI-GC primer pair had a comparable discriminatory power as ERIC-PCR one and these fingerprinting techniques distinguished among the studied 28 A. glycyphyllos symbionts 18 and 17 genomotypes, respectively. RAPD method was less discriminatory in the genomotyping of rhizobia analyzed and it efficiently resolved nine genomotypes. The cluster analysis of RAPD, ERIC-PCR, and AFLP profiles resulted in a generally similar grouping of the test strains on generated dendrograms supporting a great potential of these DNA fingerprinting techniques for study of genomic polymorphism and evolutionary relationship of A. glycyphyllos nodulators. The RAPD, ERIC-PCR, and AFLP pattern similarity coefficients between A. glycyphyllos symbionts studied was in the ranges 8-100, 18-100, and 23-100%, respectively.

Genetic differentiation of Trifolium repens microsymbionts deriving from Zn-Pb waste-heap and control area in Poland.

The aim of this work was to determine the genetic structure of Rhizobium leguminosarum bv. trifolii population isolated from root nodules of Trifolium repens growing in heavy metal contaminated Boleslaw waste-heap area and compare it with that of an unpolluted control Bolestraszyce population. The 684-bp long dinitrogenase reductase (nifH) gene fragments were amplified in a PCR reaction and then sequenced. An analysis of nifH gene amplicons of 21 rhizobial strains from each of the studied populations revealed substantially reduced genotype (h) and nucleotide (π) diversities in the metallicolous Boleslaw population in comparison to the non-metallicolous Bolestraszyce one, and showed a significant genetic differentiation between these populations (F(ST) = 0.159, p = 0.018). Among the strains under investigation, six genotypes (A-F) with 95-99% nifH gene sequence identities were distinguished. Studied T. repens nodule isolates indicated the highest nifH gene sequence similarities (95-100%) with R. leguminosarum bv. trifolii reference strains and on nifH phylogram all these strains formed monophyletic, highly supported clade (100%). The decreased genotype and nucleotide diversities of the waste-heap R. leguminosarum bv. trifolii population, compared to that from the control area and substantial genetic differentiation between populations of nifH gene, is arguably the consequence of the random genetic drift (Tajima's D = 2.042, p = 0.99).

Accumulation of cadmium in and its effect on the midgut gland of terrestrial snail Helix pomatia L. from urban areas in Poland.

The objectives of this study were (1) to determine cadmium (Cd) accumulation in the midgut gland of a land snail Helix pomatia L. inhabiting residential areas of the 14 largest cities in Poland, and (2) to examine whether the accumulated Cd exerted any toxic effects. The average accumulation of Cd in the midgut gland of snails, weighing 16-18 g, ranged from 7.00 to 87.3 µg/g dry weight (0.06-0.77 µmol/g) and differed significantly among animals from the various urban areas. This difference in Cd accumulation was not related to city population, but was associated with the topsoil Cd (R(2) = 0.868, p < 0.0001). The tissue Cd was not found to produce toxicity (histopathology, programmed cell death, lipofuscin formation or lipid peroxidation), probably due to the induction of sufficiently high quantities of metallothionein and glutathione, well-known protective molecules.

Differential susceptibility to cadmium-induced liver and kidney injury in wild and laboratory-bred bank voles Myodes glareolus.

The objective of the study was to compare the sensitivity of wild and laboratory-bred bank voles to cadmium (Cd)-induced histopathological changes in the liver and kidneys. For 4 weeks, the male bank voles-both wild and laboratory-bred-were provided with diet containing Cd in quantities <0.1 (control), 30, and 60 µg/g dry weight. At the end of exposure period, histopathology and analyses of Cd, metallothionein (MT), glutathione (GSH), zinc (Zn), copper (Cu), iron (Fe), and lipid peroxidation-all considered to be critical factors during the development of Cd toxicity in the liver and kidneys-were carried out. Histopathological changes (focal hepatocyte swelling, vacuolation and inflammation [leukocyte infiltration] in the liver, and focal proximal tubule degeneration [including epithelial cell swelling] in the kidneys) occurred only in the wild bank voles fed a diet containing 60 µg Cd/g. There were no differences in concentrations of Cd, MT, GSH, Zn, and Cu in liver and kidney between the respective groups of wild and laboratory-bred animals. However, a decrease of hepatic Fe and lipid peroxidation was observed in the wild voles exhibiting histopathological changes. These data indicate the following: (1) wild bank voles are more susceptible to Cd-induced liver and kidney injury than those bred and raised in the laboratory; (2) the difference in sensitivity may be associated with a distinct decrease of hepatic Fe in response to Cd exposure between the two groups of bank voles; and (3) dietary Cd may produce histopathological changes indirectly through decreasing the hepatic Fe and Fe-dependent oxidative processes. These results also suggest that histopathology in the liver and kidney of wild bank voles living in a contaminated environment may occur at relatively low levels of tissue Cd.