Location of determinants of stability to neomycin and kanamycin as well as DNA segments, responsible for amplification in Streptomyces plasmids pSU3 and pSU10

The S. rimosus amplifying sequence AUD-Sr1 encodes kanamycin and neomycin resistance, defined in the case of neomycin by aminoglycoside phosphotransferase. Its cloning on plasmid SLP1.2 makes possible the co-amplification of the obtained hybrid plasmids in S. lividans. In our study the regions responsible for resistance to aminoglycoside antibiotics and the capacity for amplification the two hybrid plasmids pSU10 and pSU3 were determined. Experiments on subcloning of the AUD-Sr1 sequence fragments on vector pIJ702 revealed localization of kanamycin and neomycin resistance determinants between PvuII(6) and BglII(7) on the AUD-Sr1 sequence fragments of 2.0 kb length. Two regions responsible for amplification of the hybrid plasmids were detected with deletion and insertion mapping. The first region is localized in the region of the plasmid SLP1.2 BamHI site and the second region is localized on the PstI(4)-PvuII(6) of the AUD-Sr1 sequence fragment of 1.1 kb length.     

Characterization of multiple changes of antibiotic resistance characters in Streptomyces coelicolor A3(2)

Among mutants of Streptomyces coelicolor A3(2) studied which were sensitive to chloramphenicol (Cmls), strains sensitive to a number of antibiotics (ristomycin, tetracycline, polymyxin, lincomycin) amount of 46%. Antibiotic-sensitive mutants are capable to form different classes of resistant revertants with frequency varying from 10(-2) to 10(-6) in independent strains. Ristomycin-sensitive clones (Rims) have been found to occur with high frequency in Cmls strains and Cmlr revertants. Mutations mediating the Rims phenotype are mapped in a locus linked to the gene for resistance to chloramphenicol. The results obtained are discussed, in accordance with the notion about possible role of cml mutation in induction of secondary mutational changes in the genome of S. coelicolor A3(2).     

The capacity of bottle-nosed dolphins for generalization according to a relative sign

In bottle-nosed dolphins by a new modification of previously suggested method, a conditioned reflex was elaborated and consolidated to a relative spatial sign--choice of always left object from two similar ones in conditions of a permanent change of place of this pair of stimuli against the background of one of the sides of an open-air cage. Specialization of the reflex was checked by presentation of a pair of stimuli under different changes of surroundings, including discordance of external orientors and also the way of stimuli presentation. Then the initial objects were substituted by others (12 pairs of stimuli) sharply differing from the initial ones by the form, size, material etc. Retention of adequate choice also at the change of the medium of stimuli presentation, testified to the ability of dolphins of abstraction and generalization by relative sign, i.e. at the choice from two objects of the type "to the right--to the left in general".     

Determination of proteomic and metabolic composition of exhaled breath condensate of newborns

Here, the possibility of proteomic and metabolomic analysis of the composition of exhaled breath condensate of neonates with respiratory support. The developed method allows non-invasive collecting sufficient amount of the material for identification of disease-specific biomarkers. Samples were collected by using a condensing device that was incorporated into the ventilation system. The collected condensate was analyzed by liquid chromatography coupled with high resolution mass spectrometry and tandem mass spectrometry. The isolated substances were identified with a use of databases for proteins and metabolites. As a result, a number of compounds that compose the exhaled breath condensate was determined and can be considered as possible biomarkers of newborn diseases or stage of development.     

Preconcentration of organic solutes in urine by bubble bursting

Introduction

The chemical sensitivity of urine metabolomics analysis is greatly compromised due to the large amounts of inorganic salts in urine (NaCl, KCl), which are detrimental to analytical instrumentation, e.g. chromatographic columns or mass spectrometers. Traditional desalting approaches applied to urine pretreatment suffer from the chemical losses, which reduce the information depth of analysis.

Objectives

We aimed to test a simple approach for the simultaneous preconcentration and desalting of organic solutes in urine based on the collection of induced bursting bubble aerosols above the surface of urine samples.

Method

Bursting bubbles were generated at ambient conditions by feeding gas through an air diffuser at the bottom of diluted (200 times in ultrapure water) urine solution (50–500 mL). Collected aerosols were analyzed by the direct-infusion electrospray ionization mass spectrometry (ESI–MS).

Results

The simultaneous preconcentration (ca. 6–12 fold) and desalting (ca. six–tenfold) of organic solutes in urine was achieved by the bursting bubble sample pretreatment, which allowed ca. three-times higher number of identified urine metabolites by high-resolution MS analysis. No chemical losses due to bubbling were observed. The increased degree of MS data clustering was demonstrated on the principal component analysis of data sets from the urine of healthy people and from the urine people with renal insufficiency. At least ten times higher sensitivity of trace drug detection in urine was demonstrated for clenbuterol and salbutamol.

Conclusion

Our results indicate the high versatility of bubble bursting as a simple pretreatment approach to enhance the chemical depth and sensitivity of urine analysis. The approach could be attractive for personalized medicine as well as for the diagnostics of renal disorders of different etiology (diabetic nephropathy, chronic renal failure, transplant-associated complications, oncological disorders).

Graphical Abstract

Urine desalting and preconcentration in bursting bubbles.

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Systemic Neurochemical Alterations in Schizophrenic Brain: Glutamate Metabolism in Focus

We have used a systemic approach to establish a relationship between enzyme measures of glial glutamate and energy metabolism (glutamine synthetase and glutamine synthetase-like protein, glutamate dehydrogenase isoenzymes, brain isoform creatine phosphokinase) and two major glial proteins (glial fibrillary acidic protein and myelin basic protein) in autopsied brain samples taken from patients with schizophrenia (SCH) and mentally healthy subjects (23 and 22 cases, respectively). These biochemical parameters were measured in tissue extracts in three brain areas (prefrontal cortex, caudate nucleus, and cerebellum). Significant differences in the level of at least one of the glutamate metabolizing enzymes were observed between two studied groups in all studied brain areas. Different patterns of correlative links between the biochemical parameters were found in healthy and schizophrenic brains. These findings give a new perspective to our understanding of the impaired regulation of enzyme levels in the brain in SCH.     

Methyl Jasmonate Increases the Tropane Alkaloid Scopolamine and Reduces Natural Herbivory in Brugmansia suaveolens: Is Scopolamine Responsible for Plant Resistance?

The tropane alkaloid (TA) scopolamine is suggested to protect Brugmansia suaveolens (Solanaceae) against herbivorous insects. To test this prediction in a natural environment, scopolamine was induced by methyl jasmonate (MJ) in potted plants which were left 10?days in the field. MJ-treated plants increased their scopolamine concentration in leaves and herbivory decreased. These findings suggest a cause?Ceffect relationship. However, experiments in laboratory showed that scopolamine affect differently the performance of the specialist larvae of the ithomiine butterfly Placidina euryanassa (C. Felder & R. Felder) and the generalist fall armyworm Spodoptera frugiperda (J. E. Smith): the specialist that sequester this TA from B. suaveolens leaves was not negatively affected, but the generalist was. Therefore, scopolamine probably acts only against insects that are not adapted to TAs. Other compounds that are MJ elicited may also play a role in plant resistance against herbivory by generalist and specialist insects, and deserve future investigations.     

Cyanotoxins in small artificial dams in Kenya utilised for cage fish farming – a threat to local people?

Nine small artificial dams located in different climatic regions of Kenya were studied. The local communities use the stored water for various purposes, such as irrigation, domestic use, watering of livestock and cage fish farming. Such intense use is commonly accompanied by eutrophication, including fast growth of cyanobacteria, which at times produce cyanotoxins threatening human and animal life. We studied the pelagic community, analysed abiotic variables and identified microcystins by means of high performance liquid chromatography and ELISA kits at monthly intervals over a period of one year. Mass spectrometry (MALDI-TOF MS) was used to identify structural variants of microcystins by their protonated masses (M + H). Three dams contained microcystins, with the highly toxic Microcystin-LR being identified as the most prominent substance. Cell content of the toxin varied from 7.2 to 686.7 fg cell^?1. Basic limnological variables that indicate the probability of toxin presence were also recorded. Non-parametric Mann–Whitney U-test revealed significant differences in soluble reactive phosphorous, nitrate-N, water depth, total hardness and post-Nauplii stages sampled between toxin-producing and non-toxin-producing dams. Although most of the samples did not contain high amounts of cyanobacteria, the cyanotoxin-problem was evident, suggesting the need for regular cyanotoxin monitoring programs.     

Peroxynitrite regulates exocytosis of neutrophil granules

Peroxynitrite is formed in the organism by activated neutrophils as a result of the enhanced production of nitrogen monoxide and superoxide anion radical in the inflammation foci. Since peroxynitrite modifies the structure of macromolecules, including the elements of actin cytoskeleton, it can influence signal transduction pathways that regulate intracellular granule exocytosis. In this paper we explore a dual effect of peroxynitrite on the processes of neutrophil degranulation by the methods of flow cytometry, light microscopy, and atomic force microscopy. We showed that peroxynitrite at concentrations less than 300 μM activated graded exocytosis of neutrophil intracellular granules, which resulted in the enhancement of neutrophil adhesion to the substrate, cell spreading on the substrate, and activation of neutrophil ability to kill microorganisms. Peroxynitrite at higher concentrations inhibited exocytosis of neutrophil granules and hindered cell adhesion to the substrate. The character of influence of the specific agents, such as colchicine and cytochalasin that selectively disrupt cytoskeletal structures, on peroxynitrite-induced changes in neutrophil morphology indicates an important role of actin cytoskeleton in the regulation of intracellular granule exocytosis induced by peroxynitrite. Our results support the hypothesis suggesting that peroxynitrite is a natural regulator of neutrophil effector functions.