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M Stankiewicz

Publications and source records attributed to M Stankiewicz.

At least 37 records · Page 2Linked to original sources
PubMed1999

Effects of a centipede venom fraction on insect nervous system, a native Xenopus oocyte receptor and on an expressed Drosophila muscarinic receptor.

Centipede venoms are complex protein mixtures; very few is known about their pharmacological actions. Application of a Scolopendra sp. venom fraction (SC1) on the cockroach giant axon induced an increase in the leak current correlated with a decrease in the membrane resistance, suggesting the presence in SC1 of components opening non-specific pores in the axonal membrane. On a cockroach central cholinergic synapse, microinjection of SC1 induced a small transient depolarization of the postsynaptic membrane, followed by a slow stable depolarization and a drastic decrease in the evoked subthreshold excitatory postsynaptic potential amplitude. A pretreatment of the ganglion with atropine or scopolamine reduced the amplitude of the SC1-induced depolarizing wave, suggesting a possible cholinergic muscarinic target. On control Xenopus oocytes, SC1 induced an inward oscillatory Ca2(+)-dependent Cl- current mediated through the activation of native lysophosphatidic acid receptors (LPAr). Indeed, pretreatment of oocytes with 1 microM N-palmitoyl-tyrosine phosphoric acid, a selective competitive antagonist of LPAr, decreased responses to SC1 by 70%. Application of SC1 to oocytes expressing a cloned Drosophila muscarinic receptor (Dml) induced a biphasic response comprising: (1) a large fast Cl- current that was abolished by pretreatment with atropine and scopolamine and (2) a slow and small oscillating Cl- current corresponding to the response observed in control oocytes. These observations confirm the presence of muscarinic agonists in SCI and reveal their direct action on an insect muscarinic receptor subtype homologous to mammalian M1-M3 receptors.

Animals↗
PubMed1999

Scorpion alpha-like toxins, toxic to both mammals and insects, differentially interact with receptor site 3 on voltage-gated sodium channels in mammals and insects.

alpha-Like toxins, a unique group designated among the scorpion alpha-toxin class that inhibit sodium channel inactivation, are highly toxic to mice but do not compete for alpha-toxin binding to receptor site 3 on rat brain sodium channels. We analysed the sequence of a new alpha-like toxin, which was also highly active on insects, and studied its action and binding on both mammalian and insect sodium channels. Action of the alpha-like toxin on isolated cockroach axon is similar to that of an alpha-toxin, and the radioactive toxin binds with a high affinity to insect sodium channels. Other sodium channel neurotoxins interact competitively or allosterically with the insect alpha-like toxin receptor site, similarly to alpha-toxins, suggesting that the alpha-like toxin receptor site is closely related to receptor site 3. Conversely, on rat brain sodium channels, specific binding of 125I-alpha-like toxin could not be detected, although at high concentration it inhibits sodium current inactivation on rat brain sodium channels. The difficulty in measuring binding to rat brain channels may be attributed to low-affinity binding due to the acidic properties of the alpha-like toxins that also impair the interaction with receptor site 3. The results suggest that alpha-like toxins bind to a distinct receptor site on sodium channels that is differentially related to receptor site 3 on mammalian and insect sodium channels.

Animals↗
PubMed1999

Biophysical properties of scorpion alpha-toxin-sensitive background sodium channel contributing to the pacemaker activity in insect neurosecretory cells (DUM neurons).

A scorpion alpha-toxin-sensitive background sodium channel was characterized in short-term cultured adult cockroach dorsal unpaired median (DUM) neurons using the cell-attached patch-clamp configuration. Under control conditions, spontaneous sodium currents were recorded at different steady-state holding potentials, including the range of normal resting membrane potential. At -50 mV, the sodium current was observed as unclustered, single openings. For potentials more negative than -70 mV, investigated patches contained large unitary current steps appearing generally in bursts. These background channels were blocked by tetrodotoxin (TTX, 100 nm), and replacing sodium with TMA-Cl led to a complete loss of channel activity. The current-voltage relationship has a slope conductance of 36 pS. At -50 mV, the mean open time constant was 0.22 +/- 0.05 ms (n = 5). The curve of the open probability versus holding potentials was bell-shaped, with its maximum (0.008 +/- 0.004; n = 5) at -50 mV. LqhalphaIT (10-8 m) altered the background channel activity in a time-dependent manner. At -50 mV, the channel activity appeared in bursts. The linear current-voltage relationship of the LqhalphaIT-modified sodium current determined for the first three well-resolved open states gave three conductance levels: 34, 69 and 104 pS, and reversed at the same extrapolated reversal potential (+52 mV). LqhalphaIT increased the open probability but did not affect either the bell-shaped voltage dependence or the open time constant. Mammal toxin AaHII induced very similar effects on background sodium channels but at a concentration 100 x higher than LqhalphaIT. At 10-7 m, LqhalphaIT produced longer silence periods interrupted by bursts of increased channel activity. Whole-cell experiments suggested that background sodium channels can provide the depolarizing drive for DUM neurons essential to maintain beating pacemaker activity, and revealed that 10-7 m LqhalphaIT transformed a beating pacemaker activity into a rhythmic bursting.

Animals↗
PubMed1998

Superoxide dismutase and total antioxidant status of larvae and adults of Trichostrongylus colubriformis, Haemonchus contortus and Ostertagia circumcincta.

Superoxide dismutase (SOD), a cytosolic enzyme that is specific for scavenging superoxide radicals, is involved in protective mechanism(s) in tissue injury following oxidative processes and phagocytosis. The presence of SOD activity in larval and adult Trichostrongylus colubriformis, Haemonchus contortus and Ostertagia circumcincta was examined using a xanthine-xanthine oxidase assay and by polyacrylamide gel electrophoresis (PAGE) and non-denaturing sodium dodecyl sulfate (SDS)-PAGE followed by specific enzyme staining. Total antioxidant status was determined using the Randox Laboratories kit. The infective larval stages (L3) of the three species contained 8-10 times more activity than the corresponding adults. SOD activity from adult parasites was sensitive to KCN and SDS and may therefore belong to a Cu/Zn and Mn class of enzymes. SOD from the larvae was sensitive only to KCN, suggesting that it may belong to a Cu/Zn class of enzymes. Insignificant interspecies variation was observed when SOD isozyme profiles of larvae were compared. PAGE showed at least five bands of SOD activity with molecular weights of between 18 and 205 kDa. Examination of total antioxidant status showed that non-enzymatic antioxidant potential was also present, but only in the infective larvae. The level of antioxidants in the three genera of larvae studied was similar and amounted to about 0.33-1.07 microM/mg of protein.

Animals↗
PubMed1998

[Mycoplasma hominis and Ureaplasma urealyticum of cervical canal and in the pouch of Douglas in infertile women: a preliminary report].

Detection of Mycoplasma hominis and Ureaplasma urealyticum in the samples obtained from the cervical canal of the uterus and from the pouch of Douglas in 71 women in whom underwent diagnostic and operative laparoscopy is presented. Ureaplasma urealyticum in 33 cases diagnosed for infertility was present in 6 (18.2%) patients. In cervical canal and the pouch of Douglas in 5 and 2 women respectively. In 1 (3.0%) woman Ureaplasma urealyticum was present in both places. In the women from the control group U. urealyticum was detected in 6 (15.5%) cases only in cervical canal. Mycoplasma hominis was present only in cervical canal more frequently in the control group 4 (cases) than among infertile women 1 (3.0%).

Adult↗
PubMed1998

[Anti-insect scorpion toxins: historical account, activities and prospects].

Some toxins from scorpion venoms, much more toxic to insects than to other animal classes, possess high affinity to Na+ channels. These anti-insect scorpion toxins have been divided into: 1) alpha toxins which lack strict selectivity for insects, do not compete with following groups of anti-insect toxins, resemble other alpha scorpion toxins by their structure and their ability, as alpha anemone toxins, to prolong insect axonal action potential durations through a drastic slowing down of the Na+ current inactivation, 2) excitatory insect selective scorpion toxins which induce in blowfly larvae an immediate fast paralysis; in isolated cockroach axons, they depolarize and induce a sustained repetitive activity of short (normal) action potentials through a shift of Na+ activation mechanism towards more negative potentials and some decrease of inactivation at these potential values, 3) depressant insect selective neurotoxins which cause a slow progressive flaccid paralysis of larvae, depolarize insect axons and reduce or even suppress evoked action potentials; resting depolarizations which are antagonized by a post-application of TTX, are due to the opening of sodium channels at very negative potential values and to the suppression of their inactivation mechanism. The decrease of the maximal Na+ conductance following flaccid toxin action may be understood if toxin-modified channels opened at very negative potentials values remain open (or re-open) for much longer times than in control conditions and pass by substate less conductant states. Anti-insect scorpion toxins become of major interest into insect neurophysiology and also into insect pest control, due to their specific target sites and to the recent constructions of insecticidal baculovirus expressions of several of these toxins.

Animals↗
PubMed1997

In vitro folding and functional analysis of an anti-insect selective scorpion depressant neurotoxin produced in Escherichia coli.

The selective toxicity of depressant scorpion neurotoxins to insects is useful in studying insect sodium channel gating and has an applied potential. In order to establish a genetic system enabling a structure-activity approach, the functional expression of such polypeptides is required. By engineering the cDNA encoding the depressant scorpion neurotoxin, LahIT2, behind the T7 promoter, large amounts of recombinant insoluble and nonactive toxin were obtained in Escherichia coli. Following denaturation and reduction, the recombinant protein, constructed with an additional N-terminal methionine residue, was subjected to renaturation. Optimal conditions for reconstitution of a functional toxin, having a dominant fold over many other possible isoforms, were established. The recombinant active toxin was purified by RP-HPLC and characterized. Toxicity (ED50) to insects, binding affinity (IC50) to an insect receptor site, and electrophysiological effect on an insect axonal preparation were found to be similar to those of the native toxin. Substitution of the C-terminal glycine by a Gly-Lys-Lys triplet did not abolish folding but affected toxicity (3.5-fold decrease) of LqhIT2. Apparently, this efficient bacterial expression system (500 micrograms HPLC-purified toxin/1 liter E. coli culture) provides the means for studying structure/ activity relationship and the molecular basis for the phylogenetic selectivity of scorpion depressant neurotoxins.

Animals↗
PubMed1997

Proteolytic enzymes of infective larvae and adults of Trichostrongylus colubriformis and Haemonchus contortus.

The aim of this study was to acquire preliminary characterisation of the proteolytic enzymes of Trichostrongylus colubriformis and Haemonchus contortus so as to obtain a better understanding of the parasites' defence mechanisms against the host immune response. The proteinase band-pattern activity obtained using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed nine bands in T. colubriformis and H. contortus L3 larvae and four bands in adult T. colubriformis at alkaline pH. Similar testing of adult H. contortus at acidic pH gave four bands. Within the same species the same proteinase may occur in both larvae and adults. Some enzymes of similar molecular weight occurred in both species, but their relative activities differed. The majority of proteinases of T. colubriformis and H. contortus larvae and adult forms are generally of high molecular weight (35-200 kDa). Because proteinase of T. colubriformis and H. contortus have been difficult to classify on the basis of their enzymatic activity, we suggest they may be proteasomes.

Animals↗
PubMed1997

Refined electrophysiological analysis suggests that a depressant toxin is a sodium channel opener rather than a blocker.

The effects of a recombinant depressant insect toxin from Leiurus quinquestriatus hebraeus, Lqh IT2-r, have been studied in current and voltage-clamp conditions on the isolated axonal and DUM neuron preparations of the cockroach Periplaneta americana. Lqh IT2-r depolarizes the axon, blocks the evoked action potentials, and modifies the amplitude and the kinetics of the sodium current. The inward transient peak current is greatly decreased and is followed by a maintained slow activating-deactivating sodium current. The slow component develops at membrane potentials more negative than the control, and has a time constant of activation of several tens of milliseconds. The flaccid properties of Lqh IT2-r do not correspond to a blockage of the Na+ channels, but may be attributed to modified Na+ channels which open at more negative potential, activate slowly and do not inactivate normally.

Animals↗
PubMed1997

Purification, structure and activity of three insect toxins from Buthus occitanus tunetanus venom.

One contractive and two depressant toxins active on insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to insects (LD50 to Blatella germanica = 110 ng/100 mg body weight). They show a strong antigenic cross-reaction with a depressive toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory insect toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant toxin BotIT1 is highly toxic to B. germanica (LD50 = 60 ng/ 100 mg body weight) and barely toxic to mice (LD50 = 1 microgram/20 g body weight) when injected intracerebroventricularly. It does not compete with 125I-AaHIT for its receptor site on P. americana synaptosomal membranes. On cockroach axon, BotIT1 develops plateau potentials and slows down the inactivation mechanism of the Na+ channels. Thus, BotIT1 belongs to the group of alpha insect-selective toxins and shows a strong sequence identity (> 90%) with Lqh alpha IT and LqqIII, two insect alpha-toxins previously purified from the venom of L. q. hebraeus and L. q. quinquestriatus. respectively.

Action Potentials↗
PubMed1997

Action of babycurus-toxin 1 from the east African scorpion Babycurus centrurimorphus on the isolated cockroach giant axon.

A toxin named babycurus-toxin 1 (mol. wt 8191), from telson extracts of the scorpion Babycurus centrurimorphus, was found to depolarize the cockroach giant axon. It progressively blocked the evoked action potentials after a short period of limited repetitive activity and after 30 min of toxin action it became impossible to evoke responses to current stimulations. Voltage-clamp experiments on the sodium current indicated that the toxin in micromolar concentrations progressively decreased the transient inward peak sodium current, but also slowed the activation phase of this sodium current and maintained an inward current during the voltage pulses, which deactivated slowly. The toxin also induced in the insect axon a slowly activating-deactivating component of the sodium current. This suggests that the toxin modifies both activation and inactivation mechanisms of sodium channels. Thus there is some similarity in the electrophysiological effects between BcTx1 and the beta-toxins active on mammals.

Amino Acid Sequence↗
PubMed1997

Internal parasites of possums (Trichosurus vulpecula) from Kawau Island, Chatham Island and Stewart Island.

As part of a search for pathogens that might be useful agents for biological control of possums, the three largest offshore islands of New Zealand that still have possums were surveyed to determine the pathogens present in these isolated populations. Brushtail possums from Kawau Island (n = 158), Chatham Island (n = 214) and Stewart Island (n = 194) were examined for internal parasites. Possums from Kawau Island were infected with Eimeria spp. (16.7%), Bertiella trichosuri (5.2%) and Purustrongyloides trichosuri (15.5%). No Paraustrostrongylus trichosuri or Trichostrongylus spp. were found. Possums from Chatham Island were infected with Eimeria spp. (10.9%). Bertiella trichosuri (3.6%), T colubriformis (6.6%), T retortaeformis (1%) and T. vitrinus (0.5%). No Parastrongyloides or Paraustrostrongylus spp. were found. Possums from Stewart Island were infected only with Eimeria spp. (4.6%). Because of their paucity of some parasites, the opportunity exists to use these offshore islands to study the introduction and spread of a parasite into a possum population, and what technology would be required to bring it to hyperendemicity.

Journal Article↗
PubMed1997

Endoparasites of brushtail possums (Trichosurus vulpecula) from the South Island, New Zealand.

As part of a study to assess whether endoparasites could assist in the biological control of brushtail possums in New Zealand, we investigated the composition and distribution of possum endoparasites in the South Island. Possums were collected near five of the original release areas in the South Island : Banks Peninsula, Hokitika, Nelson, Dunedin and Invercargill. Among the nematodes, those most frequently encountered were Trichostrongylus spp., which were present in possums from all five study areas. Trichostrongylus species from possums in the Invercargill area comprised 4.5% T. colubriformis, 0.9% T. vitrinus and 11.3% T. retortaeformis. Paraustrostnmgylus trichosuri and Parastrongyloides trichosuri were found only in the Invercargill area, where they infected 1.4% and 14% of possums respectively. The cestode Bertiella trichosuri was present in possums from all locations except Dunedin. The protozoan Eimeria spp. occurred in all areas. These are the first records of Parastrongyloides trichosuri, Paraustrostrongylus trichosuri, T. vitrinus, T. retortaeformis and Eimeria spp. in South Island possums. The prevalence of endoparasites and the intensity of infection was very low compared to the lower North Island of New Zealand. Endoparasites at the existing levels in the South Island probably have very little effect on possum populations.

Journal Article↗
PubMed1997

The results of anthelmintic-abbreviated infections of Trichostrongylus colubriformis and Teladorsagia circumcincta on fecal egg counts in goats on pasture.

Twenty, 2-yr-old angora goats kept on nematode larvae-contaminated pasture since birth were divided into 2 equal groups. Goats from group 1 were immunized by drug-abbreviated infection, a procedure that gave high protection against field challenge in 12-moold sheep. Group 1 was orally dosed 3 times with increasing numbers of Trichostrongylus colubriformis and Teladorsagia circumcincta infective larvae. Each time, the infection was abbreviated with Oxfendazole (OXF) 15 days after dosing. Group 2 received only OXF. After the third dose of OXF, the goats were grazed together on the same pasture and fecal egg counts determined. No protection in immunized goats was achieved. In fact, immunized goats produced significantly more nematode eggs than the nonimmunized group.

Analysis of Variance↗
PubMed1996

Immunisation of sheep by drug-abbreviated infections of Ostertagia circumcincta and Trichostrongylus colubriformis against field challenge of gastro-intestinal nematodes.

A very high level of protection was achieved against homologous (up to 97%) and heterologous (up to 87%) infections in 12-month-old Romney sheep immunised with oxfendazole-abbreviated infections of Ostertagia circumcincta and Trichostrongylus colubriformis. No significant protection occurred following ivermectin-abbreviated infections. None of the immunised sheep showed an increase in antibody level against excretory-secretory antigen of T. colubriformis infective larvae. The immunisation procedures did not cause a decrease in wool production, or liveweight gains compared with non-immunised controls.

Animal Feed↗
PubMed1996

A new scorpion venom toxin paralytic to insects that affects Na+ channel activation. Purification, structure, antigenicity and mode of action.

A new toxin, BotIT2, with a unique mode of action on the isolated giant axon of the cockroach Periplaneta americana and DUM (dorsal unpaired median) neurons, has been purified from the venom of the scorpion Buthus occitanus tunetanus. Its structural, antigenic and pharmacological properties are compared to those of three other groups of neurotoxins found in Buthidae scorpion venoms. Like excitatory, depressant and alpha-type insect-selective neurotoxins, BotIT2 is toxic to insects, but shows the following common and distinctive characteristics. (a) As alpha-type toxins, BotIT2 lack strict selectivity to insects; they have measurable but low toxicity to mice. (b) As depressant toxins and unlike alpha-type toxins, BotIT2 is able to displace iodinated AaHIT from its binding sites in insect neuronal membranes. This indicates that the binding site for BotIT2 is identical, contiguous or in allosteric interaction with that of AaHIT and depressant toxins. (c) The BotIT2 amino acid sequence shows strong similarity to depressant toxins. However, unexpectedly, despite this high sequence similarity, BotIT2 shares moderate cross-antigenic reactivity with depressant toxins. (d) Voltage and current-clamp studies show that BotIT2 induces limited depolarization concomitantly with the development of depolarizing after potential, repetitive activity and later plateau potentials terminated by bursts. Under voltage-clamp conditions, BotIT2 specifically acts on Na+ channels by decreasing the peak Na+ current and by simultaneously inducing a new current with very slow activation/deactivation kinetics. The voltage dependence of this slow current is not significantly different from that of the control current. These observations indicate that BotIT2 chiefly modifies the kinetics of axonal and DUM neuronal membrane Na(+)-channel activation.

Amino Acid Sequence↗