Acinetobacter sp. as the key player in diesel oil degrading community exposed to PAHs and heavy metals.

The aim of this study was to verify the hypothesis that a hydrocarbon degrading community isolated from a site heavily polluted with polycyclic aromatic hydrocarbons (PAHs) and heavy metals should exhibit a high activity and biodegradation efficiency, despite decreased biodiversity resulting from the presence of such contaminants. Microbial community isolated from soil collected at an abandoned creosote railway wood-sleepers impregnation plant using diesel oil was used during the studies. Four parallel systems spiked with diesel oil, diesel oil + PAHs, diesel oil + heavy metals and diesel oil + PAHs + heavy metals were analysed in terms of relative abundance and biodiversity of the microbial community (Illumina), biodegradation efficiency (GCMS) and cellular metabolic activity (flow cytometry). Principal Component Analysis and biodiversity parameters indicated that the mixture of PAHs and heavy metals was the dominant factor which resulted in the enrichment of the Gammaproteobacteria class. This was associated with higher degradation of additional PAHs in the presence of heavy metals and an increase of metabolically active sub-populations during flow cytometry analysis. The increased abundance of the Acinetobacter genus in systems with both PAHs and heavy metals implies that it may play a crucial role in soil populations exposed to mixed contaminations.

Production of ZFN-mediated GGTA1 knock-out pigs by microinjection of gene constructs into pronuclei of zygotes.

Animals as a source of organs and tissues for xenotransplantation could become a backup solution for the growing shortage of human donors. The presence of human xenoreactive anti- bodies directed against Galα1,3Gal antigens on the cell surface of a pig donor triggers the activa- tion of the complement leading to a hyperacute reaction. The development of genetic engineer- ing techniques has enabled the modification of genomes by knocking in and/or knocking out genes. In this paper, we report the generation of modified pigs with ZFN mediated disruption of the GGTA1 gene encoding the enzyme responsible for synthesis of Galα1,3Gal antigens. ZFN plasmids designed to target the exon 9 region of the pig GGTA1 gene encoding the catalytic domain were injected into the pronuclei of fertilized egg cells. Among 107 piglets of the F0 gene- ration analyzed, one female with 9-nt deletion in exon 9 of the GGTA1 gene was found. 13 of 33 piglets of the F1 generation represented the +/- GGTA1 genotype and 2 of 13 F2 piglets repre- sented the -/- GGTA1 genotype. No changes in the animals' behavior, phenotype or karyotype were observed. Analysis confirmed heredity of the trait in all animals. A complex functional analysis of the modified animals, including flow cytometry, human serum cytotoxicity test and immunohistochemical detection, was performed to estimate the phenotype effect of genetic modification and this indicated an efficient GGTA1 knock-out in modified pigs.

Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

Transgenic pigs designed to express human α-galactosidase to avoid humoral xenograft rejection.

The use of animals as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human organ donors. However, the presence of xenoreactive antibodies in humans directed against swine Gal antigen present on the surface of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a consequence of hyperacute reaction. To prevent hyperacute rejection, it is possible to change the swine genome by a human gene modifying the set of donor's cell surface proteins. The gene construct pGal-GFPBsd containing the human gene encoding α-galactosidase enzyme under the promoter of EF-1α elongation factor ensuring systemic expression was introduced by microinjection into a male pronucleus of the fertilised porcine oocyte. As a result, the founder male pig was obtained with the transgene mapping to chromosome 11p12. The polymerase chain reaction (PCR) analysis revealed and the Southern analysis confirmed transgene integration estimating the approximate number of transgene copies as 16. Flow cytometry analysis revealed a reduction in the level of epitope Gal on the cell surface of cells isolated from F0 and F1 transgenic animals. The complement-mediated cytotoxicity assay showed increased viability of the transgenic cells in comparison with the wild-type, which confirmed the protective influence of α-galactosidase expression.

Potent bradykinin antagonists containing N-benzylglycine or N-benzyl-l-alanine in position 8.

Two new analogues of a previously designed bradykinin (BK) antagonist, d-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-d-Phe-Thi-Arg, substituted in position 8 by N-benzylglycine and N-benzyl-l-alanine were designed, synthesized and bioassayed. The results show an impressive enhancement of B2 antagonistic potencies of both peptides in comparison with the model. In two further analogues these modifications were combined with acylation of the N-terminus with 1-adamantanacarboxylic acid. Acylated analogues exhibited higher antagonistic potency in comparison with the parent compounds, however, the range of effect was not as high as in previously described cases. The activity of analogues was assessed by their ability to inhibit vasodepressor response to exogenous BK (rat blood pressure test). Our results may be of value in the design of more potent BK antagonists.

Controversial aspect of using GFP as a marker for the production of transgenic cattle.

The objective of this study was to examine the feasibility of identification and selection of cattle embryos based on green fluorescence (GFP-positive) in order to obtain calves carrying an integrated transgene. The construct used (pbLGTNF-EGFP) contained the human tumor necrosis factor alpha (hTNFalpha) gene fused to the bovine beta-lactoglobulin promoter (bLG) in plasmid vector pCX-EGFP. In four experiments, 76 zygotes were injected; eight of them developed to the morulae/blastocysts stage of which only five were GFP positive (one of them 100%, one-50%, three- 25%). All of the GFP positive embryos were transferred to recipients. Two calves were born: one after transfer of the 100% GFP positive embryo and the other after transfer of one of the 25% GFP positive embryos. Both animals were healthy with normal weight when compared to two control calves. The integration of pbLGTNF-EGFP in the host genome could not be detected in either of the calves, suggesting that GFP is an unreliable marker for preimplantation screening of embryos.

Theoretical foundations for noninvasive measurement of variations in the width of the subarachnoid space.

Numerical modeling was used for the theoretical analysis of the propagation of optical radiation in the tissues of the human head, generated by a single source placed on the surface of the scalp. Of special interest and importance is the propagation of radiation within the layer of cerebrospinal fluid contained in the subarachnoid space (SAS), which is the only low absorption/high transmittance medium whose width can vary rapidly. Qualitative and quantitative assessment of changes in propagation of radiation within the SAS could become a source of information on changes in the geometry of this anatomical compartment playing a crucial role in cranio-spinal physiology and pathology. Essential for the idea of the possible noninvasive assessment of changes in width of the SAS by an optical method is the dependence of intensity of radiation reaching a photodetector located at a certain distance from the source on changes in the width of this fluid layer, which acts like a biological optical waveguide. Monte Carlo modeling and numerical analysis confirmed the feasibility of assessing changes in the width of the subarachnoid space optically. Presented here are details of the Monte Carlo simulation of light propagation in the tissues of human head and the results of such simulation as a function of the width of the subarachnoid space, calculated for different distances between the source and detector and for a few selected values of bone thickness. Results of numerical modeling were then compared with those of experiments on a mechanical-optical model.

[Evaluation of reproducibility of plethysmographic indices according to body posture, choice of forearm and method of measurement]. / Ocena powtarzalnosci wskazników pletyzmograficznych w zaleznosci od pozycji ciala, wyboru badanej konczyny oraz metody pomiaru.

The aim of the study was the analysis of the influence: a) body position (sitting vs. supine), b) choice of the arm (dominant vs. nondominant), c) variant of the method (classic vs. automatic) on plethysmographic indices describing forearm blood flow repeatability (arterial inflow--AI, fast blood flow--FBF, venous outflow VO, venous capacitance--VC). The study group included subjective healthy men, aged from 22 to 60 years. Individuals with body mass index (BMI) > 35 kg/m2 or treated pharmacologically weren't been included into examination. The indices of the forearm blood flow were calculated from the plethysmographic curve using the graphic technique: venous capacitance (VC), arterial inflow (AI), fast blood flow (FBF) and venous outflow (VO). We used relative repeatability coefficient (RRC) and coefficient of variation (CV) for assessing repeatability of obtained parameters. Repeatability of plethysmographic indices was better in supine position than in sitting one. RRC was respectively for supine vs. sitting position for VO: 0.41 vs. 0.68, for VC: 0.42 vs. 0.52 and for AI: 0.57 vs. 0.65. Plethysmographic indices VO, VC, AI were characterised by better repeatability when the exams were performed on the dominant arm. RRC was respectively for dominant vs. nondominant arm for VO: 0.68 vs. 0.71, for VC: 0.52 vs. 0.64 and for AI: 0.65 vs. 0.71. Coefficient of variation of arterial inflow assessed by conventional, automatic and by fast inflow was respectively 20%, 23% and 17%. The long-term repeatability of FBF estimated by RRC was 0.76 whereas CV yielded 17%. The same coefficient of variation was obtained when short-term repeatability was estimated-mean value CV was 17%, after rejection extremal values 11%. In conclusion the best repeatability was obtained when measurements were performed with automatic variant of method, in supine position, on dominant arm.

Modulation of cellular immunity by a lesion of the lateral hypothalamic area (LHA) in rats.

The subject of our investigation was the effect of bilateral damage to the lateral hypothalamic area (LHA) on the cellular immune response (CIR), assessed by measuring the diameter of skin infiltration (DSI) 24 and 48 h after intradermal administration of tuberculin in adult male Wistar rats, previously immunized with BCG vaccine. It has been shown that: 1) in the hypothalamo-lesioned rats (HLR) DSI measured 24 and 48 h after tuberculinization did not differ, 2) in the sham-operated rats (SOR) DSI measured 48 h after tuberculin injection was significantly higher (p < 0.02) than that found 24 h after the antigen administration, 3) the damage-induced loss of body weight and the 24-48 h difference in DSI in the responders of the HLR group correlated negatively with each other (r = -0.806, p < 0.05). These results indicate that damage to the LHA enhances the rate of formation, but not the maximal size, of the tuberculin-induced skin infiltration. Thus, the LHA seems to be involved in the regulation/modulation of processes engaged in the expression of tuberculin reaction.

New antagonists of the vasopressin receptor mediated contraction in rat tail artery.

A perfused isolated rat tail artery preparation was employed to study antagonistic properties of four newly synthesized arginine-vasopressin (AVP) analogues against the V1 receptor. The activity of the agents SCATyr(Me)AVP, OCATyr(Me)AVP, OCAAVP and SCAAVP was related to that of a recognized antagonist d(CH2)5Tyr(Me)AVP. SCATyr(Me)AVP elicited outstanding antagonistic properties by blocking at concentration of 10(-7) M nearly completely the constrictory activity of AVP. At concentration of 10(-9) M the agent inhibited the AVP-induced constriction of artery about 40 times more effectively than the oxytocin (OXT)-induced constriction. The results obtained prove the validity of the structure-activity relationship based search for new potent V1 receptor antagonists.

Constrictory activity of three new arginine-vasopressin (AVP) analogues (Ala-AVP, Ser-Ala-AVP, Thr-Ser-Ala-AVP) towards isolated rat tail artery as related to AVP alone.

The constrictory activity of vasopressin and its three novel analogues extended by 1-3 amino acids in accordance with the sequence of the bovine arginine-vasopressin neurophysin II precursor has been studied on isolated rat tail artery preparation. The analogues showed lower constrictory potency than AVP, but these agents strongly interacted with AVP. The net effect of interactions appeared complex and dependent on the nature and concentrations of the interacting agents. Basing on recent findings (Land et al. 1982) concerning the sequence of the bovine arginine-vasopressin neurophysin II precursor, Lammek et al. (1987) synthesized vasopressin analogues with primary structures derived from this precursor. Three such analogues, Ala-AVP, Ser-Ala-AVP, and Thr-Ser-Ala-AVP, showed pressor activity (147, 109, and 86 international units/mumol respectively) and antidiuretic activity (52, 130, and 48 international units/mumol, respectively) after intravenous administration to rats (Lammek et al. 1987). Having in view the possible clinical applications of vasopressin analogues and hormonogens in the treatment of bleeding disorders we were interested in the direct effect of the agents on isolated blood vessels. As the analogues considered may theoretically appear in a living system and interact with the native AVP, such interaction on the isolated rat tail artery preparation was analysed.

Carotid sinus reflexes in rats given small doses of lead.

The effect of low level lead poisoning on the carotid sinus reflex in rats was studied. The reflex was evoked by carotid artery clamping, in control and lead-poisoned animals. Wistar rats were given lead acetate trihydrate (50 mg/kg) via stomach tube once weekly for 5 weeks; control animals were given equimolar amounts of sodium acetate. Both groups were fed a regular animal food diet. At the end of the 6th week, and under urethane anesthesia, mean arterial blood pressure and heart rate were continuously recorded for both groups, before and after clamping, and after unclamping the left common carotid artery. In other experiments, some animals were pre-treated with dopamine, 0.040 mg/kg; practolol, 3 mg/kg; propranolol, 0.1 mg/kg; or atropine, 0.1 mg/kg. In animals not given drugs, lead produced a less pronounced rise in mean arterial blood pressure after clamping, and a more pronounced decrease in heart rate after unclamping, compared to control animals. Some drugs altered this response pattern. Atropine led to a more pronounced tachycardia in the lead-poisoned rats, whereas practolol led to a more pronounced bradycardia in the lead-poisoned rats. Propranolol pretreatment led to a less pronounced decrease in heart rate for lead-poisoned rats, again as compared to the controls. Atropine and beta-adrenergic receptor blocking agents produced similar carotid sinus reflex responses in control and lead-poisoned animals.

Response of the cardiovascular system to catecholamines in rats given small doses of lead.

Experiments were done in order to assess the influence of low level lead poisoning in rats upon the responses of the rat cardiovascular system to perturbation by norepinephrine, epinephrine, and isoproterenol administration. Wistar rats were given lead acetate (50.0 mg/kg) via stomach tube once weekly for 5 weeks. Control rats were given sodium acetate similarly; both groups of rats were on a regular animal food diet during the experiment. At the end of the sixth week 2 types of responses were determined. Under urethane anesthesia, the response of mean arterial pressure and heart rate in control and lead-poisoned animals to various catecholamines was measured. Also the response of perfusion pressure in isolated mesenteric vessels, to catecholamines, was measured for vessels having been obtained from control and lead poisoned animals. Our results indicate that lead-treated rats, as compared to controls, have augmented and prolonged pressor responses to epinephrine and norepinephrine; less pronounced depression of arterial pressure in response to epinephrine and isoproterenol; and more pronounced tachycardia in response to isoproterenol. In the lead-treated rats, more pronounced vasoconstriction was observed in the perfusion studies upon administration of exogenous norepinephrine. Small doses of lead intensified alpha receptor response, diminished beta receptor response in blood vessels, and increased positive chronotropic action of isoproterenol.

Dynamics of changes in the blood coagulation system, fibrynolysis and kallikreinogenesis in endotoxin shock in rabbits.

In rabbits intravenous injection of subtoxic doses of E. coli endotoxin serotype 02:K1 (10 micrograms/kg), resulted in the activation of intrinsic and extrinsic pathways of blood coagulation and, in some animals, simultaneous fall of the fibrinolytic activity which was manifested by hypercoagulability and intravascular coagulation already at the initial stage of the endotoxin shock. Among the earliest changes which appeared 30 min after endotoxin injection, was a decrease of blood platelets count and concentration of factor X in plasma. Out of the blood coagulation tests performed, most sensitive appeared the paracoagulation tests (particularly ethanol test). The second dose of endotoxin (20 micrograms/kg) injected 4 h later intensified the observed changes and autopsy of animals performed after further 4 h revealed extravasations of blood and hemorrhagic changes in the lungs, liver and occasionally in the kidneys. In spite of the above alterations, AT-III activity in the animals plasma decreased by ca. 30% as compared to the initial value.