Biomedical subjects

T Zalewska

Publications and source records attributed to T Zalewska.

At least 19 recordsLinked to original sources

PubMed2006

Contamination of the southern Baltic Sea with 137Cs and 90Sr over the period 2000-2004.

Changes of the (137)Cs activity concentrations in the southern Baltic Sea waters were investigated over the period 2000-2004 and distribution of the (90)Sr concentrations was determined in 2004. In the discriminated period further decrease of (137)Cs concentrations was observed. The average activity concentration of (137)Cs declined from 59.4Bq/m(3) in 2000 to 45.1Bq/m(3) in 2004. The greatest changes of (137)Cs activities occurred in 2003 as a consequence of the medium-size inflow of a saline waters from the North Sea. This inflow affected (137)Cs activities as well as its distribution in the Baltic Sea. Changes in (90)Sr activity concentrations proceeded at much slower rate. The average activity concentration of (90)Sr was equal to 8.7Bq/m(3) in 2004. The annual fluvial (137)Cs fluxes from the Vistula river were also estimated. In 2000, the (137)Cs load from Vistula was of an order of 0.15TBq/year, and in 2004 0.05TBq/year.

Baltic States↗

PubMed2003

Decrease of PKC precedes other cellular signs of calpain activation in area CA1 of the hippocampus after transient cerebral ischemia.

One of the specific features of severe brain injury is an activation of calcium-dependent proteolysis by calpains. We have observed a significant increase of activity as early as 3 h after the insult in a well defined model of delayed ischemic neuronal death in gerbil hippocampus. At 24 h, the enzymatic activity transiently normalized, then increased again, following the place and time of selective cellular death in the CA1 region of hippocampus. The enhanced postischemic proteolysis resulted in concomitant cleavage of calpain-specific endogenous substrates like protein kinase C (PKC), fodrin and microtubule-associated protein-2 (MAP2). These effects were also time-dependent and restricted to the vulnerable, CA1 pyramidal neurons-containing the dorsal part (DP) of the hippocampus. We have also characterized the postischemic changes of six different isoforms of PKC. The vulnerable dorsal part of the hippocampus, but not its relative resistant abdominal part (AbP), exhibited a loss of PKCalpha, beta, gamma, and delta isoforms as early as 3 h after ischemic insult. However, at this time, solely in the soluble fraction of homogenate. Later (72 h), a further loss of the enzyme proteins, comprised the particulate fraction as well and resulted in an about 50% decrease of total PKCs in the vulnerable DP region. In the case of PKCalpha, the immunostaining pattern showed, in addition to the disappearance of the enzyme from the injured area, an extensive translocation into nuclei of the survived, ischemia-resistant neurones. The early decreases of PKC isoforms in the cytosol paralleled the transient calpain activation at 3h postischemia but substantially preceded the proteolysis of any other classical calpain substrates, such as fodrin and MAP2, being evidenced not earlier than 48-72 h after the insult and restricted also to the vulnerable dorsal part. In conclusion, our results of the time-dependent effects of transient global cerebral ischemia on the calpain activity, levels and localization of its several substrates suggest, that calpain-mediated proteolysis is specifically involved in the early (induction) as well as in the late (execution) phases of delayed ischemic neuronal death in the CA1 hippocampus.

Animals↗

PubMed1999

Ischemia-induced modifications of protein components of rat brain postsynaptic densities.

Considering that postsynaptic densities (PSD) are a functionally active zone involved in excitatory synaptic transmission we evaluated the influence of global, postdecapitative cerebral ischemia of 15 min duration on characteristic protein constituents of PSD in rats. Ischemia induced changes in the assembly and function of calcium, calmodulin-dependent kinase II (CaMKII), calpains and a novel, 85 kDa/RING3 kinase but to different extents. CaMKII is translocated toward the PSD very rapidly and extensively after the first seconds of ischemia. Concomitantly, the total phosphorylating potency of this kinase with endogenous, as well as exogenous, substrates was elevated but to a lower extent than suggested by the increased protein content. Of the two brain-specific isoforms of calpain (mu and m), only recently recognized in PSD, the proteolytically activated, 76 kDa subunit of mu-calpain was significantly down-regulated after 15 min of brain ischemia. However, this effect is coupled with the decline of fodrin, the only calpain substrate that has been demonstrated to be a calpain target in vivo. Together, these findings may suggest that calpains, primarily activated by calcium in ischemic PSD, are subsequently degraded. A new observation is the relatively high phosphorylating activity of a novel, 85 kDa/RING3 kinase in the PSD which independently of other kinase systems, was greatly enhanced after ischemia. These data provide evidence that the signal transduction processes could be rapidly altered by short-term (15 min) brain ischemia due to changes in the assembly and function of PSD connected proteins.

Animals↗

PubMed1998

Expression of Ca2+-dependent (classical) PKC mRNA isoforms after transient cerebral ischemia in gerbil hippocampus.

Cerebral ischemia is known to modify the expression of genetic information in the brain. To complement this knowledge, in the present study we have estimated the expression of calcium- and phospholipid-dependent (classical) protein kinase C (c PKC) isoform mRNAs (alpha, beta1 and gamma) at different time following ischemia. Forebrain cerebral ischemia was performed on Mongolian gerbils by 5 minutes bilateral occlusion of common carotid arteries. At the pointed time the cytoplasmic RNA was extracted from hippocampus and the expression of PKC mRNA quantified by RT PCR technique using GAPDH expression as an internal standard. Results indicate that only one gamma isoform of cPKC mRNA expression becomes significantly modified in postischemic hippocampus. A transient increase up to 145% of control within the first 3 h was followed by its decline to 60-65% at a longer recirculation period. This lowered levels returned back to control at 72 h postischemic recovery. This result indicates that gamma PKC could be particularly sensitive to ischemic insult and would react in accordance with the other early signals determining ischemic outcome.

Analysis of Variance↗

PubMed1998

Dual response of calpain to rat brain postdecapitative ischemia.

Calpains, Ca(2+)-dependent neutral proteinases (microM and mM Ca(2+)-sensitive), and their endogenous inhibitor calpastatin were examined in rat brain. Specific activity of m-calpain exceeded almost 10 times that of mu-calpain, and the both isoforms of calpain together with calpastatin were mainly located in the soluble fraction of homogenate. Acute postdecapitative ischemia of 15 min duration resulted in a gradual, time-dependent decrease of total mu-calpain activity (to 60% of control values) and in the moderate elevation of calpastatin activity (by 28%). The decrease of total mu-calpain activity coincided with its remarkable increase (above 300% of control values) in particulate fraction. In the case of m-calpain, the only observed effect of ischemia was its redistribution and, as a consequence, the elevation of activity in particulate fraction. The accumulation of breakdown products, resulting from calpain-catalyzed proteolysis of fodrin (as revealed by Western blotting) indicated activation of calpain under ischemia. The findings suggest that this rapid activation involves partial enzyme translocation toward membranes, and is followed (at least in acute phase) by mu-calpain downregulation and increased calpastatin activity.

Animals↗

PubMed1998

Phosphorylation of protein kinase C substrate proteins in rat hippocampal slices--effect of calpain inhibition.

Incubation of the acutely dissected rat hippocampal slices in calcium-containing media resulted in spontaneous activation-translocation of classical PKC isoforms and their subsequent (especially gamma-type) proteolytic degradation. These changes were blocked by calpain inhibitor MDL 28 170 in 100 microM concentration. Rat hippocampal slices were metabolically prelabelled with 32Pi and stimulated with NMDA/glycine, depolarization or phorbol dibutyrate (PDBu) treatment. The basal phosphorylation of specific PKC substrates (MARCKS, neuromodulin and neurogranin) was significantly reduced in non-stimulated slices by MDL pretreatment. In contrast, only the slices where calpain activity was inhibited responded to further NMDA or phorbol dibutyrate stimulation by a substantial increase of PKC-dependent protein phosphorylation. It is concluded that the PKC phosphorylation system is severely affected by non-specific activation and a subsequent, calpain-dependent proteolysis in the acutely prepared hippocampal slices. Calpain inhibition by 100 microM MDL partially prevented these changes and increased stimulus-dependent phosphorylation of PKC-specific protein substrates.

Animals↗

PubMed1997

Cyclic enkephalin analogues with exceptional potency and selectivity for delta-opioid receptors.

Superpotent and highly delta-opioid receptor selective cyclic peptides of the general formula H-Tyr-c[D-Pen-Gly-Phe(p-X)-Pen]-Phe-OH (where X = hydrogen or halogen) have been synthesized. In the binding assays the most selective and most potent compound is the p-bromophenyl-alanine-4 analogue (IC50 value = 0.19 nM, selectivity ratio = 21,000 for delta vs mu). In the GPI and MVD bioassays the most selective and most potent analogue is the p-fluoro-substituted analogue Tyr-[D-Pen-Gly-Phe(p-F)-Pen]-Phe-OH. In the MVD assay it has an exceptionally low IC50 value of 0.016 nM and a delta vs mu selectivity ratio of 45,000.

Amino Acid Sequence↗

PubMed1997

Para-substituted phenylalanine-4 analogues of [L-Ala3]DPDPE: highly selective delta opioid receptor ligands.

Several para-substituted Phe4 analogues of the delta 1-selective antagonist [L-Ala3]DPDPE (DPADPE) were prepared and evaluated for their brain-binding and in vitro pharmacological effects. Unlike the p-haloPhe4 analogues of DPDPE and the deltorphins, similar analogues of DPADPE with electron-withdrawing groups substituted at the para-position of the Phe4 aromatic ring did not all have increased potency and selectivity for delta opioid receptors, but all retained high potency and selectivity for delta opioid receptors greater than DPDPE.

Animals↗

PubMed1996

The use of topographical constraints in receptor mapping: investigation of the topographical requirements of the tryptophan 30 residue for receptor binding of Asp-Tyr-D-Phe-Gly-Trp-(N-Me)Nle-Asp-Phe-NH2 (SNF 9007), a cholecystokinin (26-33) analogue that binds to both CCK-B and delta-opioid receptors.

The cholecystokinin (26-33) [CCK (26-33)] octapeptide analog Asp-Tyr-D-Phe-Gly-Trp(N-Me)-Nle-Asp-Phe-NH2 (SNF 9007) is a potent and selective ligand for both the CCK-B and delta-opioid receptors. Pharmacological studies of SNF 9007 suggest a relationship between the ligand requirements of CCK-B and delta-opioid receptors, which further implies a possible structural relationship between these receptors. We have utilized topographical constrainment of the important Trp30 residue to investigate structural features of SNF 9007 that would distinguish between binding requirements in this region for the CCK-B and delta-opioid receptors. Thus, the four optically pure isomers of beta-MeTrp were substituted for L-Trp30 of SNF 9007. Receptor binding results suggest that the preferred topography of the Trp30 residue for CCK-B receptor binding may be the 2S,3S (erythro-L) configuration whereas for the delta-opioid receptor it may be the 2S,3R (threo-L) configuration. Molecular modeling studies of these ligands further support the recently revised receptor-bound model for CCK-B octapeptide ligands (Kolodziej et al. J. Med. Chem. 1995, 38, 137-149) and are in good agreement with the DPDPE-delta opioid receptor "template" model (Nikiforovich et al. Biopolymers 1991, 31, 941-955).

Amino Acid Sequence↗

PubMed1996

delta-Opioid receptor: the third extracellular loop determines naltrindole selectivity.

Human delta/mu-opioid receptor chimeras were constructed to determine the role of the second and third extracellular loops in alkaloid ligand selectivity. Exchanging the third extracellular loop of the delta-opioid receptor with that of the mu-opioid receptor dramatically decreased the affinity of naltrindole, but not that of morphine. The results suggest that different domains of the opioid receptor are involved in the binding of naltrindole and morphine.

Animals↗

PubMed1996

Brain ischaemia transiently activates Ca2+/calmodulin-independent protein kinase II.

The activity of Ca2+/calmodulin-dependent protein kinase II (CaM-KII) during short-term global ischaemia was investigated in the gerbil brain hippocampus and cortex. Ischaemia of 0.5 min duration significantly stimulated Ca(2+)-independent 'autonomous' activity, indicating activation of the first step of intramolecular enzyme phosphorylation just after ischaemia has developed. Prolongation of the ischaemic period up to 5 min inhibited both Ca(2+)-dependent and, to a lesser extent, Ca(2+)-independent activities of CaM-KII. These last events coincide with an extensive translocation of CaM-KII protein from the soluble to the membranous fraction. In effect, in spite of inhibition of total CaM-KII activity, its Ca(2+)-independent, persistently active component can still remain more abundant at specific membrane regions.

Animals↗

PubMed1996

Autophosphorylation as a possible mechanism of calcium/calmodulin-dependent protein kinase II inhibition during ischemia.

Cardiac arrest induced rat brain ischemia of 15 min duration produces a rapid and profound decrease in activity of calcium/calmodulin stimulated protein kinase (CaM-KII). In contrast to that, the total amount of enzyme protein remains stable as revealed by Western blotting (alpha subunit specific) analysis. Ischemic insult also results in translocation of the enzyme toward plasmatic membranes, reducing its content in soluble (cytosolic) fraction down to 7% with respect to 50% of control. The qualitatively similar translocation can be achieved by autophosphorylation of the control enzyme in vitro. Moreover, severely reduced response of immunoprecipitated enzyme to autophosphorylation observed after ischemia ex vivo probably reflects the higher level of its endogenous phosphorylation during the insult. The results strongly suggest that among various possible mechanisms of postischemic CaM-KII inhibition the most probable would be that involving abnormal or irreversible phosphorylation of the enzyme molecule. It would consequently block or inhibit the autophosphorylation/dephosphorylation cycle of endogenous CaM-KII interconversion necessary for its full catalytic activity.

Animals↗

PubMed1996

Changes of Ca2+/calmodulin-dependent protein kinase-II after transient ischemia in gerbil hippocampus.

Transient cerebral ischemia induces, besides delayed neurodegeneration in selected brain structures, a number of early responses which may mediate ischemic injury/repair processes. Here we report that 5 min exposure to cerebral ischemia in gerbils induces a rapid inhibition and subsequent translocation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). These changes were partially reversible during a 24 h post-ischemic recovery. Concomitantly the total amount of the enzyme protein, as revealed by Western blotting (alpha-subunit specific), remained stable. This is consistent with our previous hypothesis, that the mechanism of ischemic CaMKII down-regulation involves a reversible posttranslational modification-(auto)phosphorylation, rather than the degradation of enzyme protein. The effectiveness of known modulators of post-ischemic outcome in counteracting CaMKII inhibition was tested. Three of these drugs, namely dizocilpine (MK-801), N-nitro-L-arginine methyl ester (L-NAME) and ginkgolide (BN52021), all significantly attenuated the enzyme response to ischemia, whereas an obvious diversity in the time-course of their actions implicates different mechanisms involved.

Animals↗

PubMed1996

Calpain as proposed target for neuroprotective treatment of brain ischemia.

Increasing evidence now suggests that excessive activation of calcium-dependent neutral proteases, calpains, could play a key or contributory role in the pathology of cerebral ischemia. This assumption has been supported in part by the suppressive or neuroprotective effects of calpain inhibitors on post-ischemic damage. Targeting calcium-activated proteolysis could be therefore an alternative strategy for protecting neurons against post-ischemic injury. The data of this review indicate that unregulated activation of calcium-dependent proteolysis plays a significant role in the brain damage that occurs following an ischemic insult and that selective and permanent calpain inhibitors may provide a powerfully effective therapeutic means of limiting neuronal damage.

Brain Ischemia↗

PubMed1996

The third extracellular loop of the human delta-opioid receptor determines the selectivity of delta-opioid agonists.

In the present study, we replaced the third extracellular loop of the human delta-opioid receptor with that of the human mu-opioid receptor. A modified polymerase chain reaction overlap extension method was used to achieve the exact splicing in the chimera to show the importance of the extracellular loop in ligand binding without interference from transmembrane substitutions. The replacement of the third extracellular loop did not alter the affinity of [3H]diprenorphine but caused a dramatic decrease in the affinity of both the delta-selective peptide agonists cyclo[D-Pen2,4'Cl-Phe4,D-Pen5]enkephalin and deltorphin II and the delta-selective nonpeptide agonists SNC 121 and (-)TAN 67. The affinities of the mu-selective peptide agonist [D-Ala2-MePhe4-Gly-ol5]enkephalin and the mu-preferring nonpeptide agonist morphine were not affected. Site-directed mutagenesis studies show that the mechanism of ligand recognition might be different for each structural class of opioid ligands.

Amino Acid Sequence↗

PubMed1994

Design, synthesis, and biological properties of highly potent cyclic dynorphin A analogues. Analogues cyclized between positions 5 and 11.

We have recently reported the synthesis of several cyclic disulfide bridge-containing peptide analogues of dynorphin A (Dyn A), which were conformationally constrained in the putative address segment of the opioid ligand. Several of these analogues, bridged between positions 5 and 11 of Dyn A1-11-NH2, exhibited unexpected selectivities for the kappa and mu receptors of the central over the peripheral nervous systems. In order to further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, we have synthesized a systematic series of Dyn A1-11-NH2 analogues incorporating the sulfydryl containing amino acids L- and D-Cys and L- and D-Pen in positions 5 and 11, thus producing 16 cyclic peptides. In addition, Dyn A1-11-NH2, [D-Leu5]Dyn A1-11-NH2, and [D-Lys11]Dyn A1-11-NH2 were synthesized as standards. Several of these cyclic analogues, especially c[Cys5,D-Cys11] Dyn A1-11-NH2, c[Cys5, L- or D-Pen11]Dyn A1-11-NH2, c[Pen5, L-Pen11]Dyn A1-11-NH2 and c[Pen5, L- or D-Cys11]Dyn A1-11-NH2, retained the same affinity and selectivity (vs the mu and delta receptors) as the parent compound Dyn A1-11-NH2 in the guinea pig brain (GPB). These same analogues and most others exhibited a much lower activity in the guinea pig ileum (GPI), thus leading to centrally vs peripherally selective peptides, but showed a different structure-activity relationship than found previously. In a wider scope, this series of analogues also provided new insights into which amino acids (and their configurations) may be used in positions 5 and 11 of Dyn A analogues for high potency and good selectivity at kappa opioid receptors. The results obtained in the GPB suggest that requirements for binding are not the same for the kappa, mu, or delta central receptors.

Amino Acid Sequence↗

PubMed1994

[N-methylnorleucine-(28,31)]cholecystokinin-(26-33) (SNF 8702) activity at a cloned rat CCKB receptor.

[N-methyl-Nle 28,31)]cholecystokinin-(26-33) (SNF 8702) is a highly selective ligand for the CCKB type of receptor present in the vertebrate central nervous system. Radioligand binding data showing that SNF 8702 binding affinity is reduced by the GTP analog guanylyl-imidodiphosphate suggest that SNF 8702 is an agonist but the ability of SNF 8702 to activate CCKB receptors has not been demonstrated. The present study shows that SNF 8702 is a highly potent agonist at rat CCKB receptors expressed on COS-7 cells and that these receptors are coupled to the mobilization of intracellular calcium. The A50 measured for SNF 8702-induced calcium mobilization (66 pM) is over 6-fold less than that of cholecytstokinin octapeptide (420 pM). Data are also presented showing that SNF 8702 has high binding affinity for these receptors with a Kd value (760 pM) consistent with previous measurements using guinea pig brain tissue preparations.

Animals↗

PubMed1994

Newly discovered stereochemical requirements in the side-chain conformation of delta opioid agonists for recognizing opioid delta receptors.

Topographic design of peptide ligands using specialized topographically constrained amino acids can provide new insights into the stereochemical requirements for delta opioid receptors. A highly constrained tyrosine derivative, (2S,3S)-beta-methyl-2',6'-dimethyltyrosine [(2S,3S)-TMT], was prepared by asymmetric synthesis and incorporated in [D-Pen2,D-Pen5] enkephalin (delta 1) and Deltorphin I (delta 2). The results of binding assays and bioassays showed that the two analogues (3 and 4) acted very differently at delta opioid receptors. Further pharmacological evaluations suggested that they actually interact primarily with the delta 1 and delta 2 receptor subtypes, respectively. These results, and conformational studies using NMR and computer-assisted modeling, provided insights into the different stereochemical requirements for these two delta opioid ligands to recognize the delta opioid receptor and its subtypes.

Amino Acid Sequence↗