Effect of Inorganic and Organic Tin Compounds on ACh- and Voltage-Activated Na Currents

1. Inorganic tin and organotin compounds, occurring in aquatic ecosystems, are toxic and can cause behavioral abnormalities in living organisms. To determine the possible neuronal basis of these actions, the effects of both forms of Sn were studied on identified neurones of the mollusk, Lymnaea stagnalis L.2. SnCl₂ caused a dose-dependent decrease in the acetylcholine (Ach)-induced inward current. The effective threshold concentration, measured by a two microelectrode voltage clamp technique, was 0.1 M, and the maximal effect occurred at 5 M SnCl₂. The depression of the inward current was greater after a 10 min preapplication (20%) than after 3 min treatment (7%).3. The next series of experiments compared the actions of inorganic or organic tin compounds. In whole cell clamp experiments both (CH₃)₂SnCl₂ and (CH₃)₃SnCl, like inorganic Sn, decreased the amplitude of Ach-induced current. Increasing the duration of the preapplication time resulted in an increase in the effect, but the action was not reversible. SnCl₂ treatment caused a concentration-dependent alteration (initial potentiation followed by depression) of the amplitude of I _Na(V) over the whole voltage range and slightly shifted the I–V curves to the left. In contrast, trimethyl tin decreased the amplitude of I _Na(V) only at high concentration (100 M). The activation time course of I _Na was increased ( = 0.43 ms in control and 0.55 ms in Sn), but Sn did not alter the inactivation parameters ( = 3.43 and 3.41 ms).4. These results support earlier findings that agonist- and voltage-activated channels are direct targets of toxic metals. We conclude that tin in both inorganic and organic forms acts at neuronal membranes to modulate synaptic transmission through direct actions on agonist-activated ion channels, and suggest that these actions may be the basis of the altered behavior of animals in tin-polluted environments.     

Expression of the Oligodendrocyte-Myelin Glycoprotein by Neurons in the Mouse Central Nervous System

Abstract: The oligodendrocyte-myelin glycoprotein (OMgp) is a 110-kDa glycosylphosphatidylinositol-linked protein that was initially identified as a myelin-specific protein but whose precise function remains unknown. In this study, immunohistochemistry, western blots, in situ hybridization, and northern blots were used to determine the distribution of OMgp in the mouse brain. OMgp is present in a concentration detectable on western blots in the brains of newborn mice, and its concentration gradually increases until day 24 of life. OMgp mRNA is also present in amounts detectable on northern blots in the brains of newborn mice, and its concentration gradually increases until day 21 of life, after which the concentration diminishes a little. Most of the OMgp in the mouse brain appears to be expressed in diverse groups of neurons, but it is particularly prominent in large projection neurons such as the pyramidal cells of the hippocampus, the Purkinje cells of the cerebellum, motoneurons in the brainstem, and anterior horn cells of the spinal cord. However, OMgp is not confined to these cells and is expressed in cells in the white matter as well. The OMgp gene is placed within an intron of the neurofibromatosis type I gene and on the opposite strand. This organization raises the possibility that there may be a relationship between the functions of the products of the two genes. In support of this possibility, we show that within the mouse CNS OMgp and neurofibromin are expressed in the same cell types.     

A simple and sensitive ribonucleotide reductase assay

Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA synthesis pathway and is the target of the cancer chemotherapeutic agent hydroxyurea. The study of RR is significantly hindered by the tedious and labor-intensive nature of enzymatic assay. In this report, we present a novel RR assay in which detection of the deoxyribonucleotides produced by RR occurs via coupling to the DNA polymerase reaction, and is enhanced by using RNase to degrade endogenous RNA. Cell extracts from various cell lines were treated with RNase and then reacted with ATP and radioactive ribonucleotide diphosphate as the substrate. Incorporation of the radioactive substrate [¹⁴C]CDP into DNA was linear over 30 min and was linear with the amount of extract, which provided RR activity. The reaction was inhibited by hydroxyurea and required Mg²⁺ and ATP, suggesting that the assay is specific to RR activity. While RR activities determined by our method and by a conventional method were comparable, this novel method proved to be simpler, faster, more sensitive and less expensive. In addition, assay of the RR activity for multiple samples can easily be performed simultaneously. It is superior to other RR assays in all aspects.     

Bird predation by tawny owls (Strix aluco L.) and its effect on the reproductive performance of tits

The density of great tit Parus major L. and blue tit Parus caeruleus L. was artificially increased by placing nest-box colonies for these species in the vicinity of the nests of breeding tawny owls during 1993–1997. Bird prey composition in the owl nests, the proportion of parents disappearing from the breeding tit populations and the reproductive performance of the widowed parents were analysed. The frequency of predation on tits by tawny owls was greater in areas where tit density had been artificially increased. Owls preyed more on tits during the feeding period of owlets than during the incubation period and more in years when snow covered the ground during the incubation period than when it did not. Mortality due to predation was male biased and more females lost their mates in populations breeding near tawny owl nests. Reproductive performance of the widowed parents was lower and their body weights were lighter at the end of the nestling period than those found in birds rearing youngs with their mates. Predation by owls increased the between-year turnover in the breeding tit population: widowed parents did not return to the nesting site for the next breeding season.     

Urinary Tract Infections in Kidney Transplant Patients Due to Escherichia coli and Klebsiella pneumoniae-Producing Extended-Spectrum β-Lactamases: Risk Factors and Molecular Epidemiology

Urinary tract infection (UTI) is a common complication after kidney transplantation, often associated to graft loss and increased healthcare costs. Kidney transplant patients (KTPs) are particularly susceptible to infection by Enterobacteriaceae-producing extended-spectrum β-lactamases (ESBLs). A retrospective case-control study was conducted to identify independent risk factors for ESBL-producing Escherichia coli and Klebsiella pneumoniae in non-hospitalized KTPs with UTI. Forty-nine patients suffering from UTI by ESBL-producing bacteria (ESBL-P) as case group and the same number of patients with UTI by ESBL negative (ESBL-N) as control-group were compared. Clinical data, renal function parameters during UTI episodes, UTI recurrence and relapsing rate, as well as risk factors for recurrence, molecular characterization of isolates and the respective antimicrobial susceptibility profile were evaluated. Diabetes mellitus (p <0.007), previous antibiotic prophylaxis (p=0.017) or therapy (p<0.001), previous UTI (p=0.01), relapsing infection (p=0.019) and patients with delayed graft function after transplant (p=0.001) represented risk factors for infection by ESBL positive Enterobacteriaceae in KTPs. Interestingly, the period of time between data of transplantation and data of UTI was shorter in case of ESBL-P case-group (28.8 months) compared with ESBL-N control-group (50.9 months). ESBL-producing bacteria exhibited higher resistance to fluoroquinolones (p=0.002), trimethoprim-sulfamethoxazole (p<0.001) and gentamicin (p<0.001). Molecular analysis showed that bla _CTX-M was the most common ESBL encoding gene (65.3%), although in 55.1% of the cases more than one ESBL gene was found. In 29.4% of K. pneumoniae isolates, three bla-genes (bla _CTX-M-bla _TEM-bla _SHV) were simultaneously detected. Low estimated glomerular filtration rate (p=0.009) was found to be risk factor for UTI recurrence. Over 60% of recurrent UTI episodes were caused by genetically similar strains. UTI by ESBL-producing Enterobacteriaceae in KTPs represent an important clinical challenge regarding not only hospitalized patients but also concerning outpatients.     

In vitro assessment of the antimicrobial activity of new N-acyl-thiourea derivatives

The qualitative screening of the susceptibility spectra of different microbial strains to the newly synthesized substances complexes was performed by adapted disk diffusion techniques, while the quantitative assay of the minimal inhibitory concentration (M.I.C., microg/cm3) value was based on liquid medium serial microdilutions. The compounds were solubilized in dimethylsulfoxide (DMSO). The in vitro biological screening effects were tested against a microbial inoculum of approximately 1.5 x 10(8) UFC/cm3, corresponding to 0.5 McFarland standard density, obtained from Gram positive (Staphylococcus aureus, Bacillus subtilis), Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) and fungal strains (Candida albicans). In order to investigate the influence of the subinhibitory concentration of the tested substances on the expression of different virulence features, the strains were incubated overnight in the presence of the newly synthesized thiourea derivatives (vol:vol) and different virulence features were investigated, i.e: adherence capacity to the cellular substrate represented by HeLa cells and to inert substrata quantified by slime test and soluble enzymatic virulence factors (haemolysins and other pore-forming toxins, proteases activity, DN-ase and siderophores production). The cytotoxicity was assessed microscopically, by observing the effect of the tested compounds on the cellular substratum integrity.     

Antimicrobial activity of copper and silver nanofilms on nosocomial bacterial species

Contaminated surfaces are possible vehicles in infection transmission. It is known that both Copper (Cu) and Silver (Ag) efficiently inactivate microbes by direct contact. Aiming at using these metals for benefitting from their antimicrobial effect, but to avoid subsequent toxic effects, we evaluated the antimicrobial activity of nanometric thin Silver and Copper films covering less expensive materials. Using a modified version of the Japan Industrial Standard JIS Z 2801:2000, we demonstrated the antimicrobial activity of the surfaces covered with metal ions nanofilms on microorganisms possibly involved in nosocomial infections and on Bacillus anthracis, bacteria with possible implication in bioterrorist attacks. Copper covered surfaces proved to have better antimicrobial activity than Silver surfaces. Silver covered surfaces showed better activity on Gram negative bacteria than on Gram positive cocci. Going deeper with studies on antimicrobial effects using new methods with better direct and/or functional discriminatory capacity is needed in order to provide additional information on the mechanisms of Silver and Copper nanofilms antimicrobial activity.     

Cytokine profiles of HeLa and human diploid cells induced by different fractions of Vibrio parahaemolyticus cultures exposed to stress conditions

Vibrio (V.) parahaemolyticus is an aquatic halophilic bacteria which produces gastroenteritis and in rare cases septicaemia after the consumption of raw or under-cooked contaminated seafood.The severity of diarrheal illness caused by this bacterium is closely related to the presence of two types of hemolysins (the thermostable direct hemolysin-TDH and TDH related hemolysin-TRH) and also of type III secretion system (TTSS) proteins. The TTSS type 1 induces a wide array of effects on infected HeLa cells such as autophagy, oncosis, cell rounding and lysis. Previous studies have shown that heat shock proteins have the ability to stimulate the production of interleukins in different cellular cultures. In our studies we have stimulated two cellular lines (HeLa and human diploid cells) with different V. parahaemolyticus culture fractions in order to observe the effect on cytokines production. Thus, the purpose of this study was to analyze the expression of IL-1, IL-2, IL-4, IL-6, IL-10 and TNF-alpha induced by the cell treatment with total cellular lysate, periplasmic fractions and culture supernatants extracted from V. parahaemolyticus exposed to normal and also to stress conditions. The ELISA assay of the cytokine profile of the HeLa and HDC cell lines stimulated with different bacterial fractions revealed that in the V. parahemolyticus cultures submitted to osmotic and heat shock stress are accumulating factors (probably heat shock proteins) which are exhibiting immunomodulatory activity, responsible for the induction of a pro-inflammatory response associated with increased levels of IL-6 and TNF-alpha expression, however balanced by the stimulation of the anti-inflammatory cytokine IL-4 synthesis.