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Naturally occurring three-dimensional (3D) biopolymer-based matrices that can be used in different biomedical applications are sustainable alternatives to various artificial 3D materials. For this purpose, chitin-based structures from marine sponges are very promising substitutes. Marine sponges from the order Verongiida (class Demospongiae) are typical examples of demosponges with well-developed chitinous skeletons. In particular, species belonging to the family Ianthellidae possess chitinous, flat, fan-like fibrous skeletons with a unique, microporous 3D architecture that makes them particularly interesting for applications. In this work, we focus our attention on the demosponge Ianthella flabelliformis (Linnaeus, 1759) for simultaneous extraction of both naturally occurring (&ldquo, ready-to-use&rdquo, ) chitin scaffolds, and biologically active bromotyrosines which are recognized as potential antibiotic, antitumor, and marine antifouling substances. We show that selected bromotyrosines are located within pigmental cells which, however, are localized within chitinous skeletal fibers of I. flabelliformis. A two-step reaction provides two products: treatment with methanol extracts the bromotyrosine compounds bastadin 25 and araplysillin-I N20 sulfamate, and a subsequent treatment with acetic acid and sodium hydroxide exposes the 3D chitinous scaffold. This scaffold is a mesh-like structure, which retains its capillary network, and its use as a potential drug delivery biomaterial was examined for the first time. The results demonstrate that sponge-derived chitin scaffolds, impregnated with decamethoxine, effectively inhibit growth of the human pathogen Staphylococcus aureus in an agar diffusion assay.

Introduction: Nowadays, the study of biological safety of modern cationic surface-active antiseptics with a wide antimicrobial spectrum has acquired particular importance. The aim was to study antimicrobial effectiveness of antiseptics decamethoxin, miramistin and their influence on nuclear DNA fragmentation and cellular cycle.Materials and methods: A comparative microbiological study of antimicrobial efficacy and a cytometric study of the effect of decamethoxin 0,02% and miramistin 0,01% on the cellular cycle were carried out. Antimicrobial activity of decamethoxin and miramistin was estimated by their minimal inhibitory and minimal microbicidal concentrations against opportunistic microorganisms using serial double dilution technique. Decamethoxin and miramistin cytotoxicity on anterior corneal epithelial cells, after their two-week daily instillation into the eyes of a Vistar line male rats was studied using flow cytometry. The parameters of epithelial cellular cycle, nuclear DNA fragmentation and apoptosis under the influence of antiseptics were registered.Results: High antimicrobial effect of decamethoxin and miramistin against Gram-positive, Gram-negative bacteria with the significant advantages of decamethoxin were found (р0,001). Decamethoxin caused minimal influence on anterior corneal epithelial cells, the insignificant decrease of their proliferation index, low increase of apoptosis (0.68%), no difference of mitotic activity (p0.05). But the use of miramistin resulted in the significant increase of nuclear DNA fragmentation, decrease of proliferative activity (р0.05).Conclusions: Higher antimicrobial effect against a wide range of opportunistic pathogens is proved in decamethoxin 0,02% comparably to miramistin 0,01% (р0,001). In prolonged antiseptic use of the first one there were found no cytotoxic and no pro-apoptotic effects on the epithelium (р0,05).

The possibility of one-step creating of pH-sensitive mesostructured silica-based nanocontainers with exceptionally high payload using associates of two antiseptics (including hydrolyzable one) as templates is demonstrated. The effects of the template nature and the conditions of the sol-gel process on the porous structure of silica nanocontainers are studied and discussed. The kinetics of the templating drug release from such containers is studied and some features of this process are analyzed. It is shown that the drug release rate can be tuned by varying the medium pH. The bactericidal activity of two encapsulated antiseptics against the Staphylococcus aureus is evaluated in vitro by agar diffusion method with replacement of agar with agarose. The diameters of the inhibition zones for silica-based containers loaded with antiseptics increased with the pre-diffusion time at 4 °C. At the same time, empty containers (after elimination of antiseptics by etching) did not reveal any bactericidal properties.

The effect of d-tubocurarine on the kinetics of the decamethonium uptake by mouse kidney slices was studied. Slices were incubated (1 hour) in a Krebs-Ringer bicarbonate medium (37°, pH 7.4) in the presence of 14C-decame-thonium and d-tubocurarine. With increasing d-tubocurarine concentration the 14C-slice-to-medium concentration ratio decreased towards a constant value (1.3) regardless of the decamethonium concentration used. The decamethonium uptake can thus be divided into two components, one which is sensitive to d-tubocurarine (curare-sensitive) and one which is not (curare-insensitive). The latter component increased proportionally with the external decamethonium concentration. The curare-sensitive component, which showed saturation with increasing decamethonium concentration, had a maximum capacity of 1100–1280 μM kg -1, h -1 and a half saturation concentration of 175–180 μM. The latter component was inhibited by d-tubocurarine with an inhibitor constant of 12 μM and the inhibition was in accordance with competitive antagonism. The result suggest that decamethonium is taken up by two distinct processes, one mediated by a carrier and one by a passive diffusion into the cells. Furthermore the carrier is common to both decamethonium and d-tubocurarine.

An investigation was performed to determine whether decamethonium and carbamoylcholine share a common transport mechanism with tetraethyl-ammonium and N1-methylnicotinamide in mouse kidney slices. Slices were incubated (1 hour) in Krebs-Ringer bicarbonate medium (37°, pH 7.4) containing 2 × 10-6M 14C-labelled decamethonium or carbamoylcholine both in the absence and presence of the drugs. Tetraethylammonium and N1-methylnicotinamide stimulated decamethonium uptake at relatively low concentrations (5 × 10-5M and 10-4M tetraethylammonium, 10-4M and 5 × 10-4M N1-methylnicotinamide), whereas both agents at higher concentrations (10-3M and 10-2M tetraethylammonium, 2 × 10-2M N1-methylnicotinamide) depressed the uptake. Tetraethylammonium (10-4M, 10-3M) and N1-methylnicotinamide (10-3M) also inhibited the uptake of carbamoylcholine. The initial decamethonium influx (3 minutes incubation) could be stimulated by pre-incubating slices (1 hour) with 3 × 10-4M tetraethylammonium or 5 × 10-4M N1-methylnicotinamide (in the absence of decamethonium) before being transferred to a final medium containing only decamethonium. Stimulation can thus be interpreted as an example of accelerative exchange diffusion, which should indicate that efflux of tetraethylammonium or N1-methylnicotinamide accumulated by the slices accelerates the influx of decamethonium. It is concluded that decamethonium and carbamoylcholine use a carrier mechanism which is also involved in tetraethylammonium and N1-methylnicotinamide transport. This transport mechanism is most likely in part at least, identical with that responsible for the secretion of tetraethylammonium and N1-methylnicotinamide (and other organic cations) by the intact kidney.

The dimeric form of Torpedo californica acetylcholinesterase provides a valuable experimental system for studying transitions between native, partially unfolded, and unfolded states since long-lived partially unfolded states can be generated by chemical modification of a nonconserved buried cysteine residue, Cys 231, by denaturing agents, by oxidative stress, and by thermal inactivation. Elucidation of the 3D structures of complexes of Torpedo californica acetylcholinesterase with a repertoire of reversible inhibitors permits their classification into three categories: (a) active-site directed inhibitors, which interact with the catalytic anionic subsite, at the bottom of the active-site gorge, such as edrophonium and tacrine; (b) peripheral anionic site inhibitors, which interact with a site at the entrance to the gorge, such as propidium and d-tubocurarine; and (c) elongated gorge-spanning inhibitors, which bridge the two sites, such as BW284c51 and decamethonium. The effects of these three categories of reversible inhibitors on the stability of Torpedo californica acetylcholinesterase were investigated using spectroscopic techniques and differential scanning calorimetry. Thermodynamic parameters obtained calorimetrically permitted quantitative comparison of the effects of the inhibitors on the enzyme's thermal stability. Peripheral site inhibitors had a relatively small effect, while gorge-spanning ligands and those binding at the catalytic anionic site, had a much larger stabilizing effect. The strongest effect was, however, observed with the polypeptide toxin, fasciculin II (FasII), even though, in terms of its binding site, it belongs to the category of peripheral site ligands. The stabilizing effect of the ligands binding at the anionic subsite of the active site, like that of the gorge-spanning ligands, may be ascribed to their capacity to stabilize the interaction between the two subdomains of the enzyme. The effect of fasciculin II may be ascribed to the large surface area of interaction (2000 A(2)) between the two proteins. Stabilization of Torpedo californica acetylcholinesterase by both divalent cations and chemical chaperones was earlier shown to be due to a shift in equilibrium between the native state and a partially unfolded state ( Millard et al. ( 2003 ) Protein Sci. 12 , 2337 - 2347 ). The low molecular weight inhibitors used in the present study may act similarly and can thus be considered as pharmacological chaperones for stabilizing the fully folded native form of the enzyme.

Sepsis is a severe generalised infection caused usually by pathogenic bacteria. It is often the cause of hospitalization and death in patients treated in intensive care and other hospital wards. Latest research brought to better understanding of patomechanisms, took place significant development of therapy heading to improvement of general patients condition treated as a basis and additionally supported by local therapy.was to evaluate the possibility of using the solution Decasan in the comprehensive treatment of patients necrosis of soft tissues.The study included 192 patients (W/M 103/89; average -aged 53.35 ± 5.36 years). According to the classification of septic states (Chicago, 1991), patients were divided into three groups: first – patients to the local form of the infection, the second – with Systemic Inflammatory Response Syndrome (SIRS), which lasted up to 72 hours and the third – patients with various forms of sepsis, SIRS in which lasted 72 hours.As a result of our studies carried out in patients where the wound was made decontamination solution Decasanu, received: pain reduction, decrease tissue swelling, early debridement of the wound and the appearance in the wound granulation, to reduce delays wound healing. Proposed algorithms treatment of various forms of sepsis, pointed out the essential elements, ie.: a comprehensive approach to the treatment of infection by early surgical intervention, intensive supportive therapy (fluid resuscitation), antibiotic therapy directed to microorganisms that cause infections and topical antiseptics therapy (solution Decasanu).The preparation antiseptic Decasan can be safely used for disinfection of skin, mucous membranes and wounds in the foci of infections caused by bacteria, fungi and protozoa.

It has long been appreciated that studying the embryonic chick in ovo provides a variety of advantages, including the potential to control the embryo's environment and its movement independently of maternal influences. This allowed early workers to identify movement as a pivotal factor in the development of the locomotor apparatus. With an increasing focus on the earliest detectable movements, we have exploited this system by developing novel models and schemes to examine the influence of defined periods of movement during musculoskeletal development. Utilizing drugs with known neuromuscular actions to provoke hyperactivity (4-aminopyridine, AP) and either rigid (decamethonium bromide, DMB) or flaccid (pancuronium bromide, PB) paralysis, we have examined the role of movement in joint, osteochondral and muscle development. Our initial studies focusing on the joint showed that AP-induced hyperactivity had little, if any, effect on the timing or scope of joint cavity elaboration, suggesting that endogenous activity levels provide sufficient stimulus, and additional mobilization is without effect. By contrast, imposition of either rigid or flaccid paralysis prior to cavity formation completely blocked this process and, with time, produced fusion of cartilaginous elements and formation of continuous single cartilaginous rods across locations where joints would ordinarily form. The effect of these distinct forms of paralysis differed, however, when treatment was initiated after formation of an overt cavity; rigid, but not flaccid, paralysis partly conserved precavitated joints. This observation suggests that 'static' loading derived from 'spastic' rigidity can act to preserve joint cavities. Another facet of these studies was the observation that DMB-induced rigid paralysis produces a uniform and specific pattern of limb deformity whereas PB generated a diverse range of fixed positional deformities. Both also reduced limb growth, with different developmental periods preferentially modifying specific osteochondral components. Changes in cartilage and bone growth induced by 3-day periods of flaccid immobilization, imposed at distinct developmental phases, provides support for a diminution in cartilage elaboration at an early phase and for a relatively delayed influence of movement on osteogenesis, invoking critical periods during which the developing skeleton becomes receptive to the impact of movement. Immobilization also exerts differential impact along the proximo-distal axis of the limb. Finally, our preliminary results support the possibility that embryonic hyperactivity influences the potential for postnatal muscle growth.

This study determined the effect of decamethonium bromide (DMBr), a non-competitive blocker of the neuromuscular junction, on skeletal muscle development during chick embryogenesis. Decamethonium bromide caused generalized edema and high mortality with treated embryos rarely surviving beyond day 16 of incubation. Muscle degeneration was grossly evident on the muscles of abdomen, pectoral girdle, and leg. Semi-thin sections showed a high infiltration of macrophages in treated embryos and a massive degenerative process. Electron microscopy showed that both fast and slow fibers formed in the control and treated embryos, but those of the treated embryos failed to form myofibrils. Other organ systems, such as the heart and the gut, appeared histologically normal throughout the course of treatment. To investigate possible nerve independent action of DMBr on muscle development we determined the effect of this compound on the growth and differentiation of the C2C12 skeletal muscle cell line. DMBr treatment of C2C12 cell cultures did not affect the growth or survival of the cells, even at a tenfold higher concentration than that used in ovo, but myosin heavy chain expression was dramatically inhibited. We conclude that DMBr has a nerve independent blocking inhibition effect on myosin heavy chain synthesis in the developing avian embryo besides the recognized role as a non-competitive post-synaptic blocker of the neuromuscular junction.

The mechanical behavior of cartilage is intimately related to its biochemical composition, and tissue composition is known to be influenced by its local mechanical loading environment. Although this phenomenon has been well-studied in adult cartilage, few investigations have examined such structure-function relationships in embryonic cartilage. The goal of this work was to elucidate the role of mechanical loading on the development of cartilage composition during embryogenesis. Using an embryonic chick model, cartilage from the tibiofemoral joints of immobilized embryos was compared to that of controls. The normal time course of changes in glycosaminoglycan/DNA and hydroxyproline/DNA were significantly influenced by loading history, with the most pronounced effects observed between days 9 and 14 during the period of most rapid increase in motility in control embryos. Stress-relaxation tests conducted on samples from day 14 indicate that the effects of embryonic immobilization on cartilage matrix composition have direct consequences for the mechanical behavior of the tissue, resulting in compromised material properties (e.g. 50% reduction in E(inst)). Because embryogenesis provides a unique model for identifying key factors which influence the establishment of functional biomechanical tissues in the skeleton, these data suggest that treating mechanical loading as an in vitro culture variable for tissue engineering approaches to cartilage repair is likely to be a sound approach.

We have studied the action of some membranotropic agents (MTAs) on the parameters of mono- and multilayers of dipalmitoylphosphatidylcholine (DPPC). The MTAs used included an antimicrobial drug, decamethoxinum, the model amphiphilic agent stearoyl-L-alpha-alanine, and cholesterol as a reference substance. Using differential scanning calorimetry and the Langmuir monolayer technique, we measured the temperature and enthalpy of the main phase transition of DPPC, the mean molecular area, the collapse pressure and the free energy of the mixed monolayers of DPPC and MTA. A good correlation has been obtained between the structure of the MTA used and changes in the parameters of both mono- and multilayers. Thus, for cholesterol, its well-known condensing effect in the L alpha phase correlates with its behavior in the mixed monolayers. The disturbing action of decamethoxinum (depression of the phase transition in DPPC multilayers and relatively high free energy of mixing in monolayers) is presumably connected with interaction of its charged ammonium moieties with polar phospholipid heads. At the same time, stearoyl-L-alpha- alpha-alanine condensed the lipid layers and increased the melting point of DPPC, owing to its interaction with both polar and non-polar lipid moieties. One can conclude that the three MTAs used can really be considered as representative examples of three different types of behavior in mono- and multilayers.

The kinetics of decamethoxine liberation from medical antimicrobial textiles was studied. The elution of decamethoxine was shown to be a complicated diffusive-kinetic process dependent on the exposure and concentration of decamethoxine.

Experience of treatment of 17 patients, suffering cholangitis of various genesis, using antiseptic Decasan, is presented. Clinical efficacy of the preparation in complex treatment of cholangitis, confirmed by results of the bile bacteriological investigation, was noted.

Experience in treatment of 91 patients with peritonitis on various genesis using antiseptic Dekasan are presented. A marked clinical efficacy compared with that of other antiseptics in the complex treatment of peritonitis was noted.

Antimicrobial properties of a composite based on decamethoxine and modified polysaccharides (carboxymethylamylum, oxyethyl-cellulose) were studied. The composite was shown to have high antimicrobial activity against grampositive and gramnegative bacteria under different conditions of the experiment.

The objective of this study was to investigate how temporal and spatial patterns of characteristic extracellular matrix molecules are altered in the absence of normal functional skeletal muscle contractions during avian synovial joint development. By using in situ detection of protein and mRNA expression in developing avian feet and femorotibial joints from a wide range of developmental stages, we demonstrate that the morphological abnormalities that result from embryonic immobilization are associated with altered patterns of tenascin-C and collagen-XII expression within developing joint structures. As the joints fuse in immobilized embryos, the cells of the presumptive articular surface differentiate from flattened fibroblasts to more rounded chondrocytes and collagens XII and I are no longer detected at sites of complete joint fusion. Although the expression of collagen XII persists at normal levels elsewhere within the immobilized joint, tenascin-C expression is diminished within the chondroepiphysis, synovium, and tendons, as well as within the remains of the fibrous articular surface. This effect is most notable for the shortest tenascin variant (Tn 190) within the chondroepiphysis and the largest variant (Tn230) within tendons, synovium, and the fibrous surface layer of the joint. This study thus provides in vivo support of previous in vitro work that suggests that tenascin expression is sensitive to external changes in mechanical loading environment. However, these data do not support a similar conclusion for collagen XII during early development.

The crystal structure of Torpedo californica (Tc) acetylcholinesterase (AChE) carbamoylated by the physostigmine analogue 8-(cis-2,6-dimethylmorpholino)octylcarbamoyleseroline (MF268) is reported at 2.7 A resolution. In the X-ray structure, the dimethylmorpholinooctylcarbamic moiety of MF268 is covalently bound to the catalytic serine, which is located at the bottom of a long and narrow gorge. The alkyl chain of the inhibitor fills the upper part of the gorge, blocking the entrance of the active site. This prevents eseroline, the leaving group of the carbamoylation process, from exiting through this path. Surprisingly, the relatively bulky eseroline is not found in the crystal structure, thus implying the existence of an alternative route for its clearance. This represents indirect evidence that a "back door" opening may occur and shows that the release of products via a "back door" is a likely alternative for this enzyme. However, its relevance as far as the mechanism of substrate hydrolysis is concerned needs to be established. This study suggests that the use of properly designed acylating inhibitors, which can block the entrance of catalytic sites, may be exploited as a general approach for investigating the existence of "back doors" for the clearance of products.

The nicotinic acetylcholine receptor (AChR) from the electric organ of Torpedo species is an oligomeric protein composed of alpha2 beta gamma delta subunits. Although much is known about its tertiary and quaternary structure, the conformation of the large extracellular domains of each of the subunits has not been analysed in detail. In order to obtain information about the spatial structure of the extracellular domain, we have expressed the N-terminal fragment 1-209 of the Torpedo californica AChR alpha-subunit in Escherichia coli. Two vectors coding for a (His)6 tag, either preceding or following the 1-209 sequence, were used and the recombinant proteins obtained (designated alpha1-209pET and alpha1-209pQE, respectively) were purified by affinity chromatography on a Ni2+-agarose column. The chemical structure of both proteins was verified by Edman degradation and mass spectrometry. The proteins were soluble in aqueous buffers but to make possible a comparison with the whole AChR or its isolated subunits, the recombinant proteins were analyzed both in aqueous solution and with the addition of detergents. The two proteins bound [125I]alpha-bungarotoxin with equal potency (KD approximately 130 nm, Bmax approximately 10 nmol.mg-1). Both were shown to interact with several monoclonal antibodies raised against purified Torpedo AChR. The circular dichroism (CD) spectra of the two proteins in aqueous solution revealed predominantly beta-structure (50-56%), the fraction of alpha-helices amounting to 32-35%. Nonionic (beta-octylglucoside) and zwitterionic (CHAPS) detergents did not appreciably change the CD spectra, while the addition of SDS or trifluoroethanol decreased the percentage of beta-structure or increased the alpha-helicity, respectively. The predominance of beta-structure is in accord with recent data on the N-terminal domain of the mouse muscle AChR alpha-subunit expressed in the mammalian cells [West et al. (1997) J. Biol. Chem. 272, 25 468]. Thus, expression in E. coli provides milligram amounts of the protein that retains several structural characteristics of the N-terminal domain of the Torpedo AChR alpha-subunit and appears to share with the latter a similar secondary structure. The expression of recombinant polypeptides representing functional domains of the AChR provides an essential first step towards a more detailed structural analysis.

We investigated the functional relationship between nuclear topology, as expressed by degree and type of nuclear aggregation, and appearance of acetylcholine receptor (AChR) subunit mRNAs. Embryonic chick muscle cell cultures treated with the muscle activity blocking agents decamethonium (DCM), d-tubocurare (TBC), and tetrodotoxin (TTX) or co-cultured with cholinergic neurons were examined for the influence of muscle activity on nuclear aggregation and its effects on AChR α-, γ-, and δ-subunit message expression. mRNA was measured by in situ hybridization and nuclei were visualized by bis-benzimide DNA staining. DCM and TBC treatments, as well as neuronal co-culture, resulted in increased nuclear clustering within myotubes and a per nucleus upregulation in mRNA expression relative to control for each subunit. The pattern of nuclear aggregation was treatment dependent, with more and larger aggregates found when myotubes were co-cultured with neurons. Moreover, as nuclear aggregates became larger: (1) nearly all nuclei within active aggregates expressed mRNA and (2) local accumulation (mRNA per unit area) was elevated relative to single nuclei, while per nucleus mRNA production decreased. To determine whether mRNA expression was transient and did not result in steady-state upregulation of AChR receptor protein, we performed a double labeling of surface AChRs with 125 I -α-bungarotoxin ( 125 I -α-BTX) concomitant to the in situ hybridization for mRNA quantification on TTX treated muscle cells. Surface receptor expression tracked mRNA expression for all types of nuclear topology observed, indicating that message levels are in fact reliable indicators of receptor population on the plasma membrane surface in myotubes. We propose that nuclear clustering is an organelle-level, accessory mechanism whereby cells concentrate relatively large amounts of AChR mRNA/protein in specific myotube regions.

Most BK-type voltage- and Ca(2+)-dependent K+ channels in rat chromaffin cells exhibit rapid inactivation. This inactivation is abolished by brief trypsin application to the cytosolic face of membrane patches. Here we examine the effects of cytosolic channel blockade and pore occupancy on this inactivation process, using inside-out patches and whole-cell recordings. Occupancy of a superficial pore-blocking site by cytosolic quaternary blockers does not slow inactivation. Occupancy of a deeper pore-blocking site by cytosolic application of Cs+ is also without effect on the onset of inactivation. Although the rate of inactivation is relatively unaffected by changes in extracellular K+, the rate of recovery from inactivation (at -80 and -140 mV with 10 microM Ca2+) is faster with increases in extracellular K+ but is unaffected by the impermeant ion, Na+. When tail currents are compared after repolarization, either while channels are open or after inactivation, no channel reopening is detectable during recovery from inactivation. BK inactivation appears to be mechanistically distinct from that of other inactivating voltage-dependent channels. Although involving a trypsin-sensitive cytosolic structure, the block to permeation does not appear to occur directly at the cytosolic mouth or inner half of the ion permeation pathway.

Morphological investigation for studying of a local impact on the tissues, localized in the antiseptic textile implantation zone, was conducted. The textile was impregnated by composition of decametoxine with modified polysaccharides. Basing on the investigation result there was established the absence of a toxic impact of antiseptic medical textile on the macroorganism tissues, the regenerative processes course, the wounds epithelization, antioedematous and anti-inflammatory effects.

We have almost no understanding of how our joints take on their range of distinctive shapes, despite the clinical relevance of joint morphogenesis to postnatal skeletal malformations such as developmental dysplasia of the hip (DDH). In this study, we investigate the role of spontaneous prenatal movements in joint morphogenesis using pharmacological immobilization of developing chicks, and assess the system as a suitable model for early-onset hip dysplasia. We show that, prior to joint cavitation, the lack of dynamic muscle contractions has little impact on the shape of the hip joint. However, after the timepoint at which cavitation occurs, a dramatic effect on hip joint morphogenesis was observed. Effects in the immobilized chicks included flattening of the proximal femur, abnormal orientation of the pelvis relative to the femur and abnormal placement and coverage of the acetabulum. Although many clinical case studies have identified reduced or restricted movement as a risk factor for DDH, this study provides the first experimental evidence of the role of prenatal movements in early hip joint development. We propose that the immobilized chick embryo serves as a suitable model system for the type of early-onset DDH which arises due to neuromuscular conditions such as spinal muscular atrophy.

Basing on analysis of results of clinical and experimental investigations, there was established, that application of the cationic antiseptics solution (including 0.02% solution of decametoxin) for the abdominal cavity sanation permits to reduce the microbal soiling while sanation performance and as well so on--the postoperative complications rate and mortality in surgical peritonitis.

An analytical method has been developed for the analysis of chemical warfare agents by capillary zone electrophoresis (CZE). Sorbate anion has been selected as indirect UV-absorbing agent and decamethonium cation reduces the electroosmotic flow. The optimized electrolyte (5 mM sorbic acid, 0.1 mM decamethonium bromide pH 6) allows the simultaneous separation of alkylphosphonic acids and their monoester derivatives in less than 15 min. The measurement of phosphonic solutes has been achieved in spiked soils using calibration curves in the 5-50 mg/l concentration range.

We used isobolographic analysis to investigate the interaction of decamethonium with either hexamethonium or vecuronium in the rat phrenic nerve hemidiaphragm preparation. EC50 values of decamethonium, hexamethonium, and vecuronium were (mean +/- SEM) 47.36 +/- 9.58 microM, 4.27 +/- 0.53 mM, and 5.19 +/- 1.17 microM, respectively. Combinations of drugs in concentrations corresponding to the 1:2, 1:1, and 2:1 ratios of their EC50 values were used to determine three points of each isobole. Decamethonium and hexamethonium showed antagonism: significant deviations from the line of additivity were found at EC50 ratios of 2:1 and 1:1 (P < 0.01 and P < 0.05, respectively) indicating that hexamethonium is a potent antagonist of decamethonium. For decamethonium and vecuronium none of the three points on the isobole was significantly different from the corresponding point on the line of additivity. Hexamethonium is known to be a weak antagonist at the postsynaptic nicotinic acetylcholine receptor but a potent antagonist at the presynaptic nicotinic receptor. Vecuronium is a more potent antagonist at the postsynaptic nicotinic receptor but a much weaker antagonist at the presynaptic site. It was postulated that in the rat the primary site of action of decamethonium is at the presynaptic nerve terminal. Our findings suggest that presynaptic rather than postsynaptic potency of a nondepolarizing drug determines ability to antagonize the effect of a depolarizing drug in the rat.

This report documents slow changes in cochlear responses produced by electrical stimulation of the olivocochlear bundle (OCB), which provides efferent innervation to the hair cells of the cochlea. These slow changes have time constants of 25–50 sec, three orders of magnitude slower than those reported previously. Such “slow effects” are similar to classically described “fast effects” in that (1) they comprise a suppression of the compound action potential (CAP) of the auditory nerve mirrored by an enhancement of the cochlear microphonic potential (CM) generated largely by the outer hair cells; (2) the magnitude of suppression decreases as the intensity of the acoustic stimulus increases; (3) they share the same dependence on OCB stimulation rate; (4) both are extinguished upon cutting the OCB; and (5) both are blocked with similar concentrations of a variety of cholinergic antagonists as well as with strychnine and bicuculline. These observations suggest that both fast and slow effects are mediated by the same receptor and are produced by conductance changes in outer hair cells. Slow effects differ from fast effects in that (1) fast effects are greatest for acoustic stimulus frequencies between 6 and 10 kHz, whereas slow effects peak for frequencies from 12 to 16 kHz, and (2) fast effects persist over long periods of OCB stimulation, whereas slow effects diminish after 60 sec of stimulation. The time course of the slow effects can be described mathematically by assuming that each shock-burst produces, in addition to a fast effect, a small decrease in CAP amplitude that decays exponentially with a time constant that is long relative to the intershock interval. The long time constant of the slow effect compared to the fast effect suggests that it may arise from a distinct intracellular mechanism, possibly mediated by second- messenger systems.

We previously demonstrated that ammonium- or guanidinium- phosphate interactions are key to forming non-covalent complexes (NCXs) through salt bridge formation with G-protein coupled receptors (GPCR), which are immersed in the cell membrane's lipids. The present work highlights MALDI ion mobility coupled to orthogonal time-of-flight mass spectrometry (MALDI IM oTOF MS) as a method to determine qualitative and relative quantitative affinity of drugs to form NCXs with targeted GPCRs' epitopes in a model system using, bis-quaternary amine based drugs, α- and β- subunit epitopes of the nicotinic acetylcholine receptor' (nAChR) and phospholipids. Bis-quaternary amines proved to have a strong affinity for all nAChR epitopes and negatively charged phospholipids, even in the presence of the physiological neurotransmitter acetylcholine. Ion mobility baseline separated isobaric phosphatidyl ethanolamine and a matrix cluster, providing an accurate estimate for phospholipid counts. Overall this technique is a powerful method for screening drugs' interactions with targeted lipids and protein respectively containing quaternary amines and guanidinium moieties.

Dose-dependent inhibitory effect of chlorhexidine (2.5 x 10(-5)-5.0 x 10(-4) g/ml on neuromuscular transmission were localized by tension and electromyogram recording during indirect stimulation. Subsequent direct stimulation showed an additional inhibition which might be located to the sarcolemma. In addition, contracture was observed at the highest concentrations. Microelectrode experiments showed a miniature endplate potential frequency increase (at 1.0 x 10(-6) g/ml), suggesting a presynaptic action. Increasing the dose to 5.0 x 10(-6) g/ml disclosed a decrease of the miniature endplate potential amplitude, indicating a reduction of the postsynaptic receptor sensitivity, which might cause neuromuscular inhibition. This was confirmed by endplate potential recording in cut preparations. The endplate potential showed a moderate degree of use-dependent inhibition which did not usually cause a tetanic fade. Experiments with curare, decamethonium and neostigmine indicated that chlorhexidine probably caused a decamethonium-like inhibition. A small depolarization of the sarcolemma was probably not the cause of the directly elicited inhibition or contracture. The contracture was potentiated in preparations made myotonic by pretreatment with para-hydroxy-mercuribenzoate. It could not be inhibited by the excitation-contraction uncoupler dantrolene, but it was dependent on extracellular Ca2+. Chlorhexidine-induced influx of Ca2+ might thus cause the contracture.

Several organophosphate triesters are widely used as flame retardants and can be metabolized to dibutyl (DBP), diphenyl (DPhP), di(2-ethylhexyl) (DEHP) and di(1,3-dichloro-2-propyl) (or bis(1,3-dichloro-2-propyl); DDCPP) phosphoric acid, respectively. A highly sensitive liquid chromatography-electrospray ionization(+)-triple quadrupole mass spectrometry (LC-ESI(+)-QQQ-MS/MS) based analysis method was presently developed. In this method the target compounds were separated with a C(18)-based reversed phase LC column, and decamethonium hydroxide (dicatonic reagent) was introduced post-LC to form ion-pairs, which were subsequently detected by ESI(+). For the phosphate acid diester ion-pairs, the mass spectra showed the most abundant ion to be [(CH(3))(2)N(CH(2))(10)N(CH(3))(3)](+), with lesser abundances of [[M-H](-)[(CH(3))(3)N(CH(2))(9)CH(2)](2+)](+) and [CH(2)CH(CH(2))(8)N(CH(3))(3)](+). For DDCPP, the fragment ions of [[Cl](-)[(CH(3))(3)N(CH(2))(10)N(CH(3))(3)](2+)](+) and [[Cl](-)[(CH(3))(3)N(CH(2))(9)CH(2)](2+)](+) could also be observed. The limits of quantitation (LOQs) by LC-ESI(+)-MS/MS (based on multiple reaction monitoring) were 0.14, 0.03, 0.14 and 0.02 ng/mL for DPhP, DBP, DDCPP and DEHP, respectively. The response was highly linearly correlated (r0.995) with concentration over the range of the LOD to 1000 ng/mL. The matrix effect on ESI+ was negligible for the samples in experiment of in vitro metabolism using rat liver microsomes.

The results of studying of antiseptic preparation Decasan for the treatment of patients, suffering purulent infections of pararectal region, are adduced. Efficacy of Decasan was analyzed in 102 patients. The results of investigation obtained are trusting hig antiseptic efficacy of preparation, witnessing possibility of its application as a preparation of choice for the treatment of patients, suffering purulent infections of pararectal region.

Prostate-specific antigen (PSA) is the usual biomarker for prostate cancer (PCa). However, its lack of selectivity has lead to the search for new biomarkers. PSA glyco- sylation seems to depend on the pathophysiological conditions of the individual. Thus, methods to separate PSA isoforms (peaks) to study their role as PCa markers are needed. In this work, CE methods for PSA isoforms separation, based on the use of different dynamic coatings, are developed using UV detection. Three complementary CE methods allowing the separation of 8 or 9 PSA isoforms are selected. The longest method takes only 17 min, while the shortest one separates 9 isoforms in o 8 min. Depending on the isoforms of interest for their use as PCa biomarker, the CE method to be used can be chosen or various of them can be combined. A remarkable aspect of these methods is that the BGEs employed are devoid of compounds with primary amino groups, making the CE methods compatible with fluorescent on-column derivatization through amino residues. As a proof-of-concept, a preliminary result shows that LIF detection of labeled PSA analyzed by one of the three developed methods permits detection of glycoprotein isoforms.

Binding sites of Torpedo acetylcholinesterase (EC 3.1.1.7) for quaternary ligands were investigated by x-ray crystallography and photoaffinity labeling. Crystal structures of complexes with ligands were determined at 2.8-A resolution. In a complex with edrophonium, and quaternary nitrogen of the ligand interacts with the indole of Trp-84, and its m-hydroxyl displays bifurcated hydrogen bonding to two members of the catalytic triad, Ser-200 and His-440. In a complex with tacrine, the acridine is stacked against the indole of Trp-84. The bisquaternary ligand decamethonium is oriented along the narrow gorge leading to the active site; one quaternary group is apposed to the indole of Trp-84 and the other to that of Trp-279, near the top of the gorge. The only major conformational difference between the three complexes is in the orientation of the phenyl ring of Phe-330. In the decamethonium complex it lies parallel to the surface of the gorge; in the other two complexes it is positioned to make contact with the bound ligand. This close interaction was confirmed by photoaffinity labelling by the photosensitive probe 3H-labeled p-(N,N-dimethylamino)benzenediazonium fluoroborate, which labeled, predominantly, Phe-330 within the active site. Labeling of Trp-279 was also observed. One mole of label is incorporated per mole of AcChoEase inactivated, indicating that labeling of Trp-279 and that of Phe-330 are mutually exclusive. The structural and chemical data, together, show the important role of aromatic groups as binding sites for quaternary ligands, and they provide complementary evidence assigning Trp-84 and Phe-330 to the "anionic" subsite of the active site and Trp-279 to the "peripheral" anionic site.

1. Using whole-cell and single channel recordings, we have examined the properties of acetylcholine (ACh)-activated currents in neurones from larval and pupal Drosophila melanogaster (fruit fly), larval and embryonic Musca domestica (house fly), and nymphal Schistocerca gregaria (locust). 2. In all preparations, single channel recordings revealed two major classes of ACh-activated channels, with average conductances of approximately 32 and 59 pS. 3. At ACh concentrations from 1 to 10 microM, channel activity in Drosophila larval neurones occurs in bursts with an average of 1-2 openings. Open times and burst durations are described by one or two exponentials. Burst durations for the 32 pS channel (approximately 3 ms, slow component) were longer than those for the 59 pS channel (approximately 1.0 ms). The mean open interval duration for the 32 pS channel (slow component) was also longer than that of the 59 pS channel. 4. At high ACh (20-200 microM) concentrations, bursts of the smaller conductance channel occur in clusters separated by long-lived periods without channel activity. Considerable kinetic heterogeneity was observed among clusters. 5. The whole-cell dose-response curve suggests that activation of current by ACh increases up to at least 100 microM and that multiple ligand binding steps are involved. 6. Drosophila and Musca larval neuronal ACh-activated channels show some unique features in their cholinergic pharmacological properties: (a) they are only weakly activated by the potent neuromuscular nicotinic agonist suberyldicholine, (b) hexamethonium and decamethonium are weak, but approximately equi-effective blockers, and (c) alpha- and kappa-bungarotoxin (BTX) both blocked reversibly, though alpha-BTX appears to be the more potent inhibitor.

The experience of the use of domestic antiseptic dekasan for topical use in patients over the infected pancreatic necrosis and its complications. There was significant improvement in the results of patients treatment.

Transgenic mouse models with nicotinic acetylcholine receptor (nAChR) knockouts and knockins have provided important insights into the molecular substrates of addiction and disease. However, most studies of heterologously expressed neuronal nAChR have used clones obtained from other species, usually human or rat. In this work, we use mouse clones expressed in Xenopus oocytes to provide a relatively comprehensive characterization of the three primary classes of nAChR: muscle-type receptors, heteromeric neuronal receptors, and homomeric alpha7-type receptors. We evaluated the activation of these receptor subtypes with acetylcholine and cytisine-related compounds, including varenicline. We also characterized the activity of classic nAChR antagonists, confirming the utility of mecamylamine and dihydro-beta-erythroidine as selective antagonists in mouse models of alpha3beta4 and alpha4beta2 receptors, respectively. We also conducted an in-depth analysis of decamethonium and hexamethonium on muscle and neuronal receptor subtypes. Our data indicate that, as with receptors cloned from other species, pairwise expression of neuronal alpha and beta subunits in oocytes generates heterogeneous populations of receptors, most likely caused by variations in subunit stoichiometry. Coexpression of the mouse alpha5 subunit had varying effects, depending on the other subunits expressed. The properties of cytisine-related compounds are similar for mouse, rat, and human nAChR, except that varenicline produced greater residual inhibition of mouse alpha4beta2 receptors than with human receptors. We confirm that decamethonium is a partial agonist, selective for muscle-type receptors, but also note that it is a nondepolarizing antagonist for neuronal-type receptors. Hexamethonium was a relatively nonselective antagonist with mixed competitive and noncompetitive activity.

Intradermal testing and RIA testing for specific IgE antibodies to neuromuscular blocking drugs (NMBDs) were performed in patients referred to an Anaesthetic Allergy Clinic. Six patients were initially investigated four to 29 years after clinical anaphylaxis during anaesthesia and two of these patients and sixteen others were investigated by intradermal testing on two occasions at least four years apart. Seven patients had RIA tests for NMBD-specific IgE antibodies on two occasions at the time of skin testing. In all but two patients the evidence for drug-specific antibodies persisted 4–29 years after the reactions. In one patient all tests became negative and in another the skin test became negative but the positive RIA persisted. Evidence of antibodies to NMBDs persisted in 21 of 22 patients who had had anaphylactic reactions to these drugs during anaesthesia. In the absence of evidence of allergy diminishing with time in the majority of patients it would seem wise to avoid drugs responsible for reactions for the rest of the patient's life.

Introduction Exposure to irreversible cholinesterase (ChE)-inhibiting compounds, such as organophosphates may produce neuromuscular dysfunction. However, less is known about changes in neuromuscular transmission after treatment with reversible ChE-inhibitors. These studies adapted single fiber electromyography (SFEMG) techniques to quantify neuromuscular jitter in adult and juvenile rats after treatment with agents that alter cholinergic neurotransmission. Methods Anesthetized adult and juvenile rats were tested using stimulation SFEMG, recorded in the gastrocnemius muscle, after stimulation in the sciatic notch region. The influence of electrode placement, treatment with decamethonium (to disrupt neuromuscular transmission) or physostigmine (reversible ChE inhibitor), and the impact of varied stimulation frequency were quantified. Results No significant effects of needle placement or criterion amplitude were observed when calculating the mean consecutive difference (MCD). Treatment with decamethonium did not alter MCD values in adult or juvenile rats. However, decamethonium produced an increased blocking of muscle action potentials (MAP) in juveniles. Also, when stimulated at 9.09 Hz, both adult and juvenile animals had a greater decrease in MAP amplitude between the first and fourth responses (T1–T4 decrement) after treatment with decamethonium. Prior to treatment with decamethonium, the T1–T4 decrement averaged about 3 fold greater in juveniles than adults, and was larger at 3.03 and 9.09 Hz than with 0.91 Hz stimulation. Treatment with physostigmine resulted in at least 50% inhibition of muscle ChE activity, but produced minimal changes in the MCD values in adults or juveniles. Combined over treatments and stimulation frequencies, the median MCD for juveniles (11.6 µs) was less than that for of adults (18.8 µs). In juveniles, the median MCD increased from 9.3 µs to 13.9 µs as the stimulation rate was increased from 0.91 to 9.09 Hz. This stimulus-dependant change was more evident in juveniles than in adults. Discussion A technique was developed to record stimulation SFEMG and neuromuscular jitter, in vivo, in adult and juvenile rats. The method was sufficiently sensitive to detect age-related differences, potentially allowing developmental processes to be examined. Based on the literature and the current data, the technique appears to be more sensitive to prolonged inhibition of ChE enzymes than the reversible inhibition produced by physostigmine.

The effects of a depolarizing neuromuscular blocker on electrically evoked acetylcholine (ACh) release were studied using a mouse phrenic nerve-diaphragm muscle preparation preloaded with [3H]-choline, and the changes in muscle tension were recorded simultaneously. Succinylcholine at a low concentration (1 microM) enhanced evoked [3H]-ACh release, which tended to follow the increase in peak amplitude of tetanic tension; whereas at high concentrations (10 and 30 microM), it simultaneously reduced both release and tension. Decamethonium even at 10 and 30 microM had little effect on [3H]-ACh release despite producing a significantly greater reduction in tension compared with succinylcholine. (+)-Tubocurarine (5 microM) prevented the enhancing effect of [3H]-ACh release induced by 1 microM, but not the decreasing effect induced by 10 microM succinylcholine. These results suggest that succinylcholine induced acceleration at low concentrations due to a positive feedback mechanism through presynaptic nicotinic ACh receptors and the inhibition of ACh release at high concentration contributes in part to the neuromuscular blockade.

We have previously used MALDI mass spectrometry to highlight ammonium- or guanidinium-aromatic interactions via cation-pi bonding and ammonium- or guanidinium-phosphate interactions through salt bridge formation. In the present work, the gas-phase stability and dissociation pathways of the interaction between phosphorylated peptides and compounds containing quaternary amines are demonstrated using electrospray ionization mass spectrometry. The presence of one quaternary amine in a compound is enough to form a noncovalent complex with a phosphorylated residue. However, if two quaternary amines are present in one molecule, the electrostatic interactions of the quaternary amines with the phosphate results in a "covalent-like" stability, and these bonds can withstand fragmentation by collision-induced dissociation at energies similar to those that fragment covalent bonds. Such interactions are important in accounting for physiological, pathophysiological, and pharmacological effects of many therapeutic compounds and small molecules containing quaternary amines or phosphates.

Nicotinic acetylcholine receptor was synthesized in Xenopus oocytes after injection of the mRNA purified from Electrophorus electricus electroplax. Nicotine, succinylcholine, and decamethonium (agonist)-elicited membrane currents in the injected oocytes were measured electrophysiologically by the voltage-clamping method. The following four different measurements were made to establish the relationship between the agonist concentration and the membrane current: 1) the agonist-induced membrane current before desensitization, 2) the agonist-induced membrane current after desensitization equilibrium, 3) the fraction of the active form of the receptors after desensitization equilibrium, 4) the rate of recovery of desensitized receptors upon removal of the agonist. These results were analyzed on the basis of the minimal model proposed from receptor-mediated ion translocation measurements. The equilibrium and rate constants of the model were evaluated for nicotine, succinylcholine, and decamethonium, and could explain the observed electrical responses in the injected oocyte, i.e. the characteristics of the receptor response caused by these agonists.