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Biomedical subjects

M Stankiewicz

Publications and source records attributed to M Stankiewicz.

At least 19 recordsLinked to original sources
PubMed2006

Peptides of arachnid venoms with insecticidal activity targeting sodium channels.

Arachnids have a venom apparatus and secrete a complex chemical mixture of low molecular mass organic molecules, enzymes and polypeptide neurotoxins designed to paralyze or kill their prey. Most of these toxins are specific for membrane voltage-gated sodium channels, although some may also target calcium or potassium channels and other membrane receptors. Scorpions and spiders have provided the greatest number of the neurotoxins studied so far, for which, a good number of primary and 3D structures have been obtained. Structural features, comprising a folding that determines a similar spatial distribution of charged and hydrophobic side chains of specific amino acids, are strikingly common among the toxins from spider and scorpion venoms. Such similarities are, in turn, the key feature to target and bind these proteins to ionic channels. The search for new insecticidal compounds, as well as the study of their modes of action, constitutes a current approach to rationally design novel insecticides. This goal tends to be more relevant if the resistance to the conventional chemical products is considered. A promising alternative seems to be the biotechnological approach using toxin-expressing recombinant baculovirus. Spider and scorpion toxins having insecticidal activity are reviewed here considering their structures, toxicities and action mechanisms in sodium channels of excitable membranes.

Animals↗
PubMed2006

Usefulness of Toxoplasma gondii recombinant antigens (GRA1, GRA7 and SAG1) in an immunoglobulin G avidity test for the serodiagnosis of toxoplasmosis.

The precise diagnosis of an acute and recent Toxoplasma infection in pregnant women and the newborn child is important before treatment. This study describes a new Toxoplasma gondii IgG avidity test based on a combination of recombinant GRA1, GRA7 and SAG1 antigens and shows that this test is useful for diagnostic purposes and may replace the lysed, whole-cell antigens. Although more sera need to be tested, the results obtained here suggest that the IgG avidity test performed with rec-antigens correlated more with the stage of a T. gondii infection than the IgG avidity results obtained with the lysed, whole-cell antigen test, the VIDAS Toxo IgG avidity (bioMérieux).

Animals↗
PubMed2004

Studies using Teladorsagia circumcincta in an in vitro direct challenge method using abomasal tissue explants.

An in vitro direct challenge (IVDC) method in which abomasal tissue explants maintained at 37 degrees C in Hanks/Hepes solution (pH 7.6) in a high oxygen concentration (incubator gassed with pure oxygen and sealed prior to use) were-challenged with exsheathed third stage larvae of Teladorsagia circumcincta has been used to investigate the tissue association phase of larval establishment. Studies using naïve and recently challenged immune sheep and goat abomasal tissues have shown significant reductions (P < 0.05) in the numbers of tissue associated larvae in material from immune animals. The mechanisms that affect the tissue association process appear to be labile since more larvae were recovered from the tissue digests of previously infected sheep that had not been recently exposed to larval challenge in comparison to those that had been recently challenged (P < 0.05). The method has also been used to demonstrate the influence of protein nutrition on the establishment of larvae in abomasal tissues and region specific differences in the efficacy of exclusion mechanisms. The technique appears to have potential as a means of investigating the crucial first phase of the process of establishment.

Abomasum↗
PubMed2004

The sequential analysis of local inflammatory cells during abomasal nematode infection in periparturient sheep.

A technique to take sequential tissue biopsy samples in multiparous, periparturient ewes from the abomasal mucosa is described, developed in parallel in Scotland and New Zealand. Samples were extracted via abomasal cannulae inserted into the wall of the abomasum and exteriorised through dorso-ventral laparotomy. Animals recovered quickly post-surgery, and tolerated the cannula and sampling without any adverse signs of pain or discomfort. The technique was deployed in two pilot studies to investigate the sequential mucosal inflammatory cell responses in well-defined parasitological models, during the periparturient relaxation of immunity in ewes infected with gastrointestinal nematodes and subjected to different feeding treatments. One experiment (Moredun Research Institute, Scotland) involved the infection of twin-bearing ewes with Teladorsagia circumcincta L3 either before, or after lambing. By feeding ewes with different levels of protein supplementation, preliminary data on the impact of nutrition on the eosinophil, mucosal mast cell and globule leucocyte responses during this period were investigated. A similar study was also performed at Lincoln University, New Zealand, to investigate these cell responses in sheep fed relatively high or low protein diets during pregnancy, and infected with a combined immunisation regime of T. circumcincta and Trichostrongylus colubriformis L3. These studies confirmed the phenomenon termed the periparturient relaxation in immunity (PPRI) where a transitory increase in faecal egg counts is observed during late pregnancy and lactation, and this effect was exacerbated during protein undernutrition. Although the number of animals was low in each experiment and the cell responses variable, the results together suggest a reduction in the number of mucosal mast cells and globule leucocyte during the PPRI when protein supply was restricted. The present paper thus describes a successful technique to monitor ovine mucosal cell populations during local immune responses in normal and pregnant sheep. It is envisaged that this technique will be a powerful adjunct to investigations into mucosal immune mechanisms and disease pathogenesis, and will be employed to confirm the influence of dietary protein on the local inflammatory cell responses during the PPRI.

Abomasum↗
PubMed2003

The effect of Duddingtonia flagrans on trichostrongyle infections of Saanen goats on pasture.

Four groups of nine Saanen goat does with a naturally acquired mixed trichostrongylid infection were grazed on four paddocks. Two groups received a daily dose of Duddingtonia flagrans at the rate of 5 x 10(7) chlamydospores per animal per day for the 26-day grazing period. After a 19-day pasture resting period, 20 worm free 12-week-old tracer kids were introduced to the paddocks for 14 days prior to removal for worm burden analysis. Four groups of five does and four kids were drenched then turned out onto the paddocks and faecal egg count (FEC) monitored. The FEC between groups was comparable throughout the initial grazing period. There were significant reductions in number of Teladorsagia circumcincta (54.8%, P=0.004) and Haemonchus contortus (85.0%, P=0.02) worms recovered from tracer animals. FEC of animals subsequently grazing pasture were significantly reduced (P=0.036) with reductions of 44% observed 4 weeks post-turnout. No significant difference was observed after 6 weeks grazing. This trial has demonstrated the potential of D. flagrans to reduce larval numbers on pasture grazed by goats under New Zealand conditions.

Animals↗
PubMed2003

Primary structure and electrophysiological characterization of two almost identical isoforms of toxin from Isometrus vittatus (family: Buthidae) scorpion venom.

Two almost identical proteins with 70 amino acid residues each, closely packed by four disufide bridges, and molecular masses of 7899.5 and 7884.7 were isolated and sequenced from the venom of the scorpion Isometrus vittatus from Pakistan. They differ by an acidic amino acid residue (glutamic or aspartic) at the same position 55 of the peptide chain, however, they exhibit the same length, the same charge and are undistinguishable when separated by C(18) reverse phase HPLC. The mixture of the two proteins called IsomTx1 depolarizes the cockroach isolated axon; artificial repolarization is followed by sustained repetitive activity, artificial hyperpolarization facilitates bursting activity observed as an answer to rapid depolarization to -60 mV. The depolarization is antagonized by TTX. In voltage-clamp experiments IsomTx1 increases axonal sodium permeability which has a particular importance between resting and threshold potentials and moderately slows down the fast inactivation. These characteristics closely resemble those of other anti-insect scorpion toxins classified as contractive toxins from Androctonus and Buthotus venoms.

Amino Acid Sequence↗
PubMed2002

The toxin Tx4(6-1) from the spider Phoneutria nigriventer slows down Na(+) current inactivation in insect CNS via binding to receptor site 3.

Tx4(6-1) a neurotoxic peptide from the venom of the aggressive South American 'armed' spider Phoneutria nigriventer, has been previously isolated and sequenced. It shows no detectable activity in mice but affects the peripheral nervous system of insects by stimulating glutamate release at the neuromuscular junction. Here we investigate possible interactions of the toxin with voltage-activated sodium channels (Na(v)). We confirm that it is ineffective on mammalian Na(v) channels, and establish that it competes with the alpha-like toxin 125I-Bom IV, for binding on the site 3 of insect Na(v) channel (IC(50) value around 25nM). The physiological consequences of this binding to the insect Na(v) channel are shown by electrophysiology: Tx4(6-1) prolongs evoked axonal action potentials (APs) (<500&mgr;s duration in control). Prolonged 8-10ms or 'plateau' 500-800ms APs accompanied by repetitive firing at 80-150Hz are recorded after 4-8min of toxin action. This modification of evoked activity is due to a slowing down of sodium current inactivation. Effects of Tx4(6-1) on sodium current are compared with those of a typical scorpion alpha-toxin and of some other spider toxins active on insect Na(v) channels. At the end of long voltage pulses, the maintained inward sodium current may represent 50% of the peak current after scorpion alpha-toxin but only about 8-10% after spider toxins. To understand the slight differences in the effects of alpha-scorpion and spider toxins on the insect Na(v) channel, structural studies of toxin-channels interactions would be necessary.

Journal Article↗
PubMed2001

Anthelmintics for the control of nematode infections in the brushtail possum (Trichosurus vulpecula).

AIMS: To determine the efficacy of eprinomectin, doramectin and a combination of albendazole and levamisole in suppressing or eliminating nematode infections or faecal egg counts (FEC) in possums naturally or experimentally infected with Parastrongyloides trichosuri, Paraustrostrongylus trichosuri and Trichostrongylus colubriformis. METHODS: To establish an effective dose of eprinomectin, groups of naturally infected possums were treated with 0, 0.5, 2.5, 5.0 or 7.5 mg/kg liveweight (LW) eprinomectin pour-on (n=6 possums/group) and changes in FEC and nematode worm counts at necropsy determined, 18 days later. Efficacy of the 7.5 mg/kg dose was re-examined in a second group of naturally infected possums (n=12) by monitoring FEC weekly for 28 days post-treatment. Persistence of the anthelmintic effect of doramectin injection was tested using nematode-free possums treated with 0, 0.2, 0.4, 0.6 or 0.8 mg/kg LW (n=3 possums/ group), which were experimentally infected 14 days later with T. colubriformis, Paraustrostrongylus trichosuri and Parastrongyloides trichosuri infective larvae. Response to treatment was assessed by FEC and nematode worm counts at necropsy, 42 days posttreatment. Efficacy of a 1.0 mg/kg dose of doramectin was subsequently examined using naturally infected possums (n=11) by monitoring FEC weekly for 28 days post-treatment. To determine the efficacy of a levamisole-albendazole combination drench, possums with naturally acquired nematode infections (n=6) were treated orally with 37.5 mg/kg LW levamisole plus 23.75 mg/kg LW albendazole on 2 occasions, 7 days apart, and response to treatment was assessed by monitoring FEC for 57 days. RESULTS: Eprinomectin 7.5 mg/kg LW reduced Paraustrostrongylus trichosuri worm counts by 98 % (p<0.05). Doramectin 0.6 mg/kg LW reduced Parastrongyloides trichosuri and Trichostrongylus spp worm counts by 99% (p<0.05) and 0.8 mg/kg LW reduced Paraustrostrongylus trichosuri by 100% (p<0.05), in possums challenged with larvae 14 days after treatment. Treating possums with a levamisole-albendazole combination orally, twice, 7-days apart, reduced FEC by 99%. CONCLUSIONS: The doses of anthelmintics required to effectively control nematodes in possums were higher than those recommended for animals for which they are currently registered. Possums tolerate the high dose rates of anthelmintics used in this study without apparent adverse effects.

Journal Article↗
PubMed2001

Electrophysiological analysis of the neurotoxic action of a funnel-web spider toxin, delta-atracotoxin-HV1a, on insect voltage-gated Na+ channels.

The effects of delta-ACTX-Hv1a, purified from the venom of the funnel-web spider Hadronyche versuta, were studied on the isolated giant axon and dorsal unpaired median (DUM) neurones of the cockroach Periplaneta americana under current- and voltage-clamp conditions using the double oil-gap technique for single axons and the patch-clamp technique for neurones. In parallel, the effects of the toxin were investigated on the excitability of rat dorsal root ganglion (DRG) neurones. In both DRG and DUM neurones, delta-ACTX-Hv1a induced spontaneous repetitive firing accompanied by plateau potentials. However, in the case of DUM neurones, plateau action potentials were facilitated when the membrane was artificially hyperpolarized. In cockroach giant axons, delta-ACTX-Hv1a also produced plateau action potentials, but only when the membrane was pre-treated with 3-4 diaminopyridine. Under voltage-clamp conditions, delta-ACTX-Hv1a specifically affected voltage-gated Na+ channels in both axons and DUM neurones. Both the current/voltage and conductance/voltage curves of the delta-ACTX-Hv1a-modified inward current were shifted 10 mV to the left of control curves. In the presence of delta-ACTX-Hv1a, steady-state Na+ channel inactivation became incomplete, causing the appearance of a non-inactivating component at potentials more positive than -40 mV. The amplitude of this non-inactivating component was dependent on the holding potential. From this study, it is concluded that, in insect neurones, delta-ACTX-Hv1a mainly affects Na+ channel inactivation by a mechanism that differs slightly from that of scorpion alpha-toxins.

Action Potentials↗
PubMed2000

Sequence and electrophysiological characterization of two insect-selective excitatory toxins from the venom of the Chinese scorpion Buthus martensi.

The two insecticidal peptides Bm32-VI and Bm33-I, isolated from the venom of the Chinese scorpion Buthus martensi induce paralytical symptoms typical of insect contractive toxins. They show, respectively, 74% and 77% homology with AaIT from Androctonus australis, comparable insecticidal activity and no vertebrate toxicity. Under voltage-clamp conditions, both toxins induced (1) an increased fast Na(+) current, (2) a shift in voltage dependence of Na(+) current activation, (3) the occurrence of a delayed current, and (4) a slow development of a holding current. Increased Na(+) conductance at negative potential values is responsible for axonal hyperexcitability and the contractive paralysis of insect prey.

Amino Acid Sequence↗
PubMed2000

Immunisation of lambs with drug-abbreviated Haemonchus contortus infections: protection against homologous and heterologous challenge.

Haemonchus contortus is known as a prolific parasite, producing high numbers of eggs. It could therefore be used as a cheap source of larvae for immunisation of lambs. The value of immunisation would be improved if the immunity produced gave protection against not only homologous but also heterologous infections. Because antibody cross-reactivity between Haemonchus and Ostertagia has been well established, we wanted to know whether drug-abbreviated infections of H. contortus would stimulate production of antibodies that would cross-react with Trichostrongylus colubriformis. The results obtained from these trials indicated that H. contortus drug-abbreviated infections produced significant immunity against not only Haemonchus but also O. circumcincta. Immunisation increased the level of immunoglobulin E (IgE) and IgE-specific antibodies against T. colubriformis, but the differences between experimental and control animals were not statistically significant. Significantly higher levels of IgG-specific antibodies against T. colubriformis were observed.

Animals↗
PubMed2000

Immunomodulation of lambs following treatment with a proteasome preparation from infective larvae of Trichostrongylus colubriformis.

Proteinases are known to be capable of prolonging the survival of endoparasites in a host. We were therefore interested in knowing whether immunization of lambs against a proteasome (multisubunit proteinases) preparation obtained from Trichostrongylus colubriformis infective third-stage larvae (L3) would have any effect on the immune response to a single challenge infection with the same organism. A total of 21 penned lambs aged 8 months were divided into 3 equal groups. Group 1 was immunized on three occasions with increasing amounts of a proteasome-enriched fraction obtained from infective L3. Group 2 was given a similar amount of protein from the initial supernatant of homogenized larvae. Group 3 (controls) received adjuvant plus saline solution only. All groups were challenged with 60,000 infective T. colubriformis larvae at 28 days after the last immunization. Significant protection was obtained only when the initial supernatant extract was used to immunize lambs. The proteasome preparation seemed to have immunosuppressive effects through the stimulation of nonspecific IgE production. Significantly lower levels of specific IgE were observed in lambs immunized with the proteasome-enriched fraction, and levels of specific IgG antibodies were increased. We suggest that proteasome fractions of T. colubriformis may serve as useful preparations for the study of mechanisms of IgE production in parasitized sheep.

Animals↗
PubMed2000

Isolation, synthesis and pharmacological characterization of delta-palutoxins IT, novel insecticidal toxins from the spider Paracoelotes luctuosus (Amaurobiidae).

Four novel insecticidal toxins were isolated from the venom of the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) and named delta-palutoxins IT1 to IT4. The four toxins are homologous 36-37 amino acid peptides reticulated by four disulfide bridges and three have amidated C-terminal residues. The delta-palutoxins are highly homologous with the previously described mu-agatoxins and curtatoxins (77-97%). The four peptides demonstrated significant toxicity against larvae of the crop pest Spodoptera litura (Lepidoptera: Noctuidae) in a microinjection bioassay, with LD50 values in the 9-50 microg per g of insect range. This level of toxicity is equivalent to that of several of the most active scorpion toxins used in the development of recombinant baculoviruses, and the delta-palutoxins appear to be insect specific. Electrophysiological experiments demonstrated that delta-palutoxin IT1, the most active toxin acts by affecting insect sodium channel inactivation, resulting in the appearance of a late-maintained sodium current, in a similar fashion to insecticidal scorpion alpha and alpha-like toxins and is thus likely to bind to channel receptor site 3. However, delta-palutoxin IT1 was distinguished by its lack of effect on peak sodium conductance, on the early phase of sodium current inactivation and the absence of a shift in the activation voltage of the sodium channels. delta-Palutoxins are thus proposed as new insecticidal toxins related to the alpha and alpha-like scorpion toxins. They will be useful both in the development of recombinant baculoviruses in agrochemical applications and also as molecular probes for the investigation of molecular mechanisms of insect selectivity and structure and function of sodium channels.

Agatoxins↗
PubMed2000

A novel complex mutant beta-lactamase, TEM-68, identified in a Klebsiella pneumoniae isolate from an outbreak of extended-spectrum beta-lactamase-producing Klebsiellae.

Twenty-two Klebsiella pneumoniae and two K. oxytoca extended-spectrum beta-lactamase (ESBL)-producing isolates were collected in 1996 from patients in two pediatric wards of the University Hospital in Wrocław, Poland. Molecular typing has revealed that the K. pneumoniae isolates represented four different epidemic strains. Three kinds of enzymes with ESBL activity (pI values of 5.7, 6.0, and 8.2) were identified. The pI 6.0 beta-lactamases belonged to the TEM family, and sequencing of the bla(TEM) genes amplified from representative isolates revealed that these enzymes were TEM-47, previously identified in K. pneumoniae isolates from pediatric hospitals in Lódz and Warsaw. One of the TEM-47-producing strains from Wrocław was very closely related to the isolates from the other cities, and this indicated countrywide spread of the epidemic strain. The pI 5.7 beta-lactamase was produced by a single K. pneumoniae isolate for which, apart from oxyimino-beta-lactams, the MICs of beta-lactam-inhibitor combinations were also remarkably high. Sequencing revealed that this was a novel TEM beta-lactamase variant, TEM-68, specified by the following combination of mutations: Gly238Ser, Glu240Lys, Thr265Met, and Arg275Leu. The new enzyme has most probably evolved from TEM-47 by acquiring the single substitution of Arg275, which before was identified only twice in enzymes with inhibitor resistance (IR) activity. TEM-68 was shown to be a novel complex mutant TEM beta-lactamase (CMT-2) which combines strong ESBL activity with relatively weak IR activity and, when expressed in K. pneumoniae, is able to confer high-level resistance to a wide variety of beta-lactams, including inhibitor combinations. This data confirms the role of the Arg275Leu mutation in determining IR activity and documents the first isolation of K. pneumoniae producing the complex mutant enzyme.

Disease Outbreaks↗
PubMed2000

[Modification of bioelectric functions in the nervous system as a result of neurotoxin action].

Natural neurotoxins are promising molecules in the actual search for the development of alternative pest management since chemical insecticides pose unacceptable risks to the environment and to health. The aim of the article is to describe the application of two electrophysiological methods (double-oil-gap technique used on cockroach isolated giant axon and microelectrode technique used on cockroach neurosecretory DUM cells in situ) to study neurotoxin effects on insect nervous system function.

Animals↗
PubMed1999

The putative bioactive surface of insect-selective scorpion excitatory neurotoxins.

Scorpion neurotoxins of the excitatory group show total specificity for insects and serve as invaluable probes for insect sodium channels. However, despite their significance and potential for application in insect-pest control, the structural basis for their bioactivity is still unknown. We isolated, characterized, and expressed an atypically long excitatory toxin, Bj-xtrIT, whose bioactive features resembled those of classical excitatory toxins, despite only 49% sequence identity. With the objective of clarifying the toxic site of this unique pharmacological group, Bj-xtrIT was employed in a genetic approach using point mutagenesis and biological and structural assays of the mutant products. A primary target for modification was the structurally unique C-terminal region. Sequential deletions of C-terminal residues suggested an inevitable significance of Ile73 and Ile74 for toxicity. Based on the bioactive role of the C-terminal region and a comparison of Bj-xtrIT with a Bj-xtrIT-based model of a classical excitatory toxin, AaHIT, a conserved surface comprising the C terminus is suggested to form the site of recognition with the sodium channel receptor.

Amino Acid Sequence↗
PubMed1999

NMR structures and activity of a novel alpha-like toxin from the scorpion Leiurus quinquestriatus hebraeus.

NMR structures of a new toxin from the scorpion Leiurus quinquestriatus hebraeus (Lqh III) have been investigated in conjunction with its pharmacological properties. This toxin is proposed to belong to a new group of scorpion toxins, the alpha-like toxins that target voltage-gated sodium channels with specific properties compared with the classical alpha-scorpion toxins. Electrophysiological analysis showed that Lqh III inhibits a sodium current inactivation in the cockroach axon, but induces in addition a resting depolarization due to a slowly decaying tail current atypical to other alpha-toxin action. Binding studies indicated that radiolabeled Lqh III binds with a high degree of affinity (Ki=2.2 nM) on cockroach sodium channels and that the alpha-toxin from L quinquestriatus hebraeus highly active on insects (LqhalphaIT) and alpha-like toxins compete at low concentration for its receptor binding site, suggesting that the alpha-like toxin receptor site is partially overlapping with the receptor site 3. Conversely, in rat brain, Lqh III competes for binding of the most potent anti-mammal alpha-toxin from Androctonus australis Hector venom (AaH II) only at very high concentration. The NMR structures were used for the scrutiny of the similarities and differences with representative scorpion alpha-toxins targeting the voltage-gated sodium channels of either mammals or insects. Three turn regions involved in the functional binding site of the anti-insect LqhalphaIT toxin reveal significant differences in the Lqh III structure. The electrostatic charge distribution in the Lqh III toxin is also surprisingly different when compared with the anti-mammal alpha-toxin AaH II. Similarities in the electrostatic charge distribution are, however, recognized between alpha-toxins highly active on insects and the alpha-like toxin Lqh III. This affords additional important elements to the definition of the new alpha-like group of scorpion toxins and the mammal versus insect scorpion toxin selectivities.

Amino Acid Sequence↗