Thiols as biomarkers of heavy metal tolerance in the aquatic macrophytes of Middle Urals,Russia

Aquatic macrophytes, viz. Sagittaria sagittifolia L., Lemna gibba L., Elodea canadensis Michx., Batrachium trichophyllum (Chaix.) Bosch., Ceratophyllum demersum L. and Potamogeton sp. (P. perfoliatus L., P. alpinus Balb., P. crispus L., P. berchtoldii Fieber, P. friesii Rupr., P. pectinatus L.) were collected from 11 sites for determining their metal accumulation and thiols content. Cu²⁺, Ni²⁺, Mn²⁺, Zn²⁺, and Fe³⁺ exceeded maximum permissible concentrations in chosen sites. Significant transfer of metals from water to leaves is observed in the order of Ni²⁺ < Cu²⁺ < Zn²⁺ < Fe³⁺ < Mn²⁺. The maximum variation of bioconcentration factor was noticed for manganese. The accumulation of heavy metals in leaves was correlated with non-protein and protein thiols, confirming their important role in metal tolerance. The largest contribution was provided by Cu²⁺ (on the average r = 0.88, p < 0.05), which obviously can be explained as an important role of these ions in thiols synthesis. Increased synthesis of thiols in the leaves allows the usage of SH-containing compounds as biomarkers of metal tolerance. Considering accumulation of metals and tolerance, B. trichophyllum, C. demersum and L. gibba are the most suitable species for phytoremediation of highly multimetal contamination, while E. canadensis and some species of Potamageton are suitable for moderately metal-polluted sites.     

The case of an infertile male with an uncommon reciprocal X-autosomal translocation: how does this affect male fertility?

Infertility is defined as the inability to conceive after one year of regular unprotected intercourse. Constitutional numerical and/or structural chromosomal aberrations like sex-chromosome aberrations are one of the possible factors involved in fertility problems. Reciprocal translocations between an X-chromosome and an autosome are rarely seen in men. Male carriers of an X-autosome translocation are invariably sterile, regardless of the position of the breakpoint in the X-chromosome. Breakpoints in autosomal chromosomes could also be involved in male infertility. In this paper, we describe a 31-year-old male with azoospermia. GTG banding with high resolution multicolor-banding (MCB) techniques revealed a karyotype 46,Y,t(X;1)(p22.3;q25), and we discuss how the breakpoint of this translocation could affect male infertility. As a conclusion, cytogenetic evaluation of infertile subjects with azoospermia should be considered in the first place before in vitro fertilisation procedures are planned.     

Up-regulation of ectonucleotidase activity after cortical stab injury in rats

The objective of this study was to examine the changes in the activity and expression of ectonucleotidase enzymes in the model of unilateral cortical stab injury (CSI) in rat. The activities of ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1) and ecto 5'-nucleotidase were assessed by measuring the levels of ATP, ADP and AMP hydrolysis in the crude membrane preparations obtained from injured left cortex, right cortex, left and right caudate nucleus, whole hippocampus and cerebellum. Significant increase in NTPDase and ecto 5'-nucleotidase activities was observed in the injured cortex following CSI, whereas in other brain areas only an increase in ecto 5'-nucleotidase activity was seen. Immunohistochemical analysis performed using antibodies specific to NTPDase 1 and ecto 5'-nucleotidase demonstrated that CSI induced significant changes in enzyme expression around the injury site. Immunoreactivity patterns obtained for NTPDase 1 and ecto 5'-nucleotidase were compared with those obtained for glial fibrillary acidic protein, as a marker of astrocytes and complement receptor type 3 (OX42), as a marker of microglia. Results suggest that up-regulation of ectonucleotidase after CSI is catalyzed by cells that activate in response to injury, i.e. cells immunopositive for NTPDase 1 were predominantly microglial cells, whereas cells immunopositive for ecto 5'-nucleotidase were predominantly astrocytes.     

Polysaccharides, tightly bound to cellulose in cell wall of flax bast fibre: Isolation and identification

Part of matrix polymers of flax bast fibre cell wall is tightly bound to cellulose and can not be extracted by conventional methods. To analyze these polymers, the residue, remaining after cell wall treatment with chelators and alkali, was dissolved in solution of lithium chloride in N,N-dimethylacetamide. Cellulose was precipitated by water and completely degraded by cellulase, giving the possibility to separate matrix polysaccharides, which remained in polymeric form. The obtained polymers were fractionated by gel permeation chromatography and characterized by monosaccharide analysis, staining with LM5 antibody and Yariv reagent, ¹H and ¹³C NMR. The total yield of the polysaccharides that are tightly bound to cellulose in flax fibre, was 4.6%. The major fractions (molecular mass 100–400 kDa) were composed of galactose, accompanied by two other significant monomers, GalA and Rha, with the ratio 1.1–1.4. Composition and structure of the cellulose bound galactan permit to consider it as fragment of the high-molecular mass (2000 kDa) galactan, synthesized by the developing fibres, while forming the secondary cell wall of gelatinous type.     

Medicinal plant extracts can variously modify biofilm formation in Escherichia coli

Low concentrations of black tea and water extracts from medicinal plants Arctostaphylos uva-ursi, Vaccinium vitis-idaea, Tilia cordata, Betula pendula and Zea mays stimulated biofilm formation in Escherichia coli BW25113 up to three times. Similar effect was observed for tannic acid and low concentrations of quercetin. In contrast, the extract from Urtica dioica reduced biofilm production. Pretreatment with plant extracts variously modified antibiotic effects on specific biofilm formation (SBF). Extract from V. vitis-idaea increased SBF, while the extracts from Achillea millefolium, Laminaria japonica and U. dioica considerably decreased SBF in the presence of ciprofloxacin, streptomycin and cefotaxime. Stimulatory effect of the extracts and pure polyphenols on biofilm formation was probably related to their prooxidant properties. The rpoS deletion did not affect SBF significantly, but stimulation of biofilm formation by the compounds tested was accompanied by inhibition of rpoS expression, suggesting that a RpoS-independent signal transduction pathway was apparently used.     

Functional Analysis of Drosophila HSP70 Promoter with Different HSE Numbers in Human Cells

The activation of genetic constructs including the Drosophila hsp70 promoter with four and eight HSE sequences in the regulatory region has been described in human cells. The promoter was shown to be induced at lower temperatures compared to the human hsp70 promoter. The promoter activity increased after a 60-min heat shock already at 38°C in human cells. The promoter activation was observed 24 h after heat shock for the constructs with eight HSEs, while those with four HSEs required 48 h. After transplantation of in vitro heat-shocked transfected cells, the promoter activity could be maintained for 3 days with a gradual decline. The promoter activation was confirmed in vivo without preliminary heat shock in mouse ischemic brain foci. Controlled expression of the Gdnf gene under a Drosophila hsp70 promoter was demonstrated. This promoter with four and eight HSE sequences in the regulatory region can be proposed as a regulated promoter in genetic therapeutic systems.     

Using the dual isotope method to assess cecal amino acid absorption of goat whey protein in rats,a pilot study

Measurement of ileal amino acids (AA) bioavailability is recommended to evaluate protein quality. A dual isotope tracer method, based on plasma isotopic enrichment ratios, has been proposed to determine true digestibility in humans. In a pilot study, we aimed to evaluate whether this method could be implemented in rats to determine AA bioavailability based on isotopic enrichment ratios measured in cecal digesta or plasma samples. Goat milk proteins were intrinsically labeled with ¹⁵N and ²H. Wistar rats were fed a meal containing the doubly labeled goat whey proteins and a tracer dose of ¹³C-spirulina. Blood samples were collected 0, 1 h and 3 h after meal ingestion from the tail vein. The rats were euthanized 4 h (n?=?6) or 6 h (n?=?6) after meal to collect plasma and intestinal contents. True orocecal protein digestibility and AA bioavailability were assessed by means of ¹⁵N and ²H enrichment in cecum content and compared with absorption indexes determined at the plasma or cecum level using isotopic ratios. Plasma kinetics of isotopic enrichment could not be completed due to the limited quantity of plasma obtained with sequential blood collection. However, the absorption indexes determined from cecal ¹⁵N or ²H/¹³C ratios gave coherent values with true orocecal AA bioavailability. This dual isotope approach with measurements of isotopic ratios in digestive content could be an interesting strategy to determine true AA bioavailability in ileal digesta of rats.

     

Array painting using microdissected chromosomes to map chromosomal breakpoints

Molecular characterization of breakpoints of chromosomal rearrangements is a successful strategy for the identification of candidate disease genes. Mapping translocation breakpoints and rearranged chromosomal boundaries is labor intensive and/or time consuming. Here, we present a novel and rapid procedure to map such chromosomal breakpoints by hybridizing amplified microdissection derived DNA of aberrant chromosomes to arrays containing genomic clones. We illustrate the potential of the technique by molecularly delineating the breakpoints in five small supernumerary marker chromosomes (sSMC) and mapping the breakpoints of five different chromosomal translocations.     

Thoracic 9 Spinal Transection-Induced Model of Muscle Spasticity in the Rat: A Systematic Electrophysiological and Histopathological Characterization

The development of spinal hyper-reflexia as part of the spasticity syndrome represents one of the major complications associated with chronic spinal traumatic injury (SCI). The primary mechanism leading to progressive appearance of muscle spasticity is multimodal and may include loss of descending inhibitory tone, alteration of segmental interneuron-mediated inhibition and/or increased reflex activity to sensory input. Here, we characterized a chronic thoracic (Th 9) complete transection model of muscle spasticity in Sprague-Dawley (SD) rats. Isoflurane-anesthetized rats received a Th9 laminectomy and the spinal cord was transected using a scalpel blade. After the transection the presence of muscle spasticity quantified as stretch and cutaneous hyper-reflexia was identified and quantified as time-dependent changes in: i) ankle-rotation-evoked peripheral muscle resistance (PMR) and corresponding electromyography (EMG) activity, ii) Hoffmann reflex, and iii) EMG responses in gastrocnemius muscle after paw tactile stimulation for up to 8 months after injury. To validate the clinical relevance of this model, the treatment potency after systemic treatment with the clinically established anti-spastic agents baclofen (GABA_B receptor agonist), tizanidine (α₂-adrenergic agonist) and NGX424 (AMPA receptor antagonist) was also tested. During the first 3 months post spinal transection, a progressive increase in ankle rotation-evoked muscle resistance, Hoffmann reflex amplitude and increased EMG responses to peripherally applied tactile stimuli were consistently measured. These changes, indicative of the spasticity syndrome, then remained relatively stable for up to 8 months post injury. Systemic treatment with baclofen, tizanidine and NGX424 led to a significant but transient suppression of spinal hyper-reflexia. These data demonstrate that a chronic Th9 spinal transection model in adult SD rat represents a reliable experimental platform to be used in studying the pathophysiology of chronic spinal injury-induced spasticity. In addition a consistent anti-spastic effect measured after treatment with clinically effective anti-spastic agents indicate that this model can effectively be used in screening new anti-spasticity compounds or procedures aimed at modulating chronic spinal trauma-associated muscle spasticity.