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Biomedical subjects

L Kaczmarek

Publications and source records attributed to L Kaczmarek.

At least 19 recordsLinked to original sources
PubMed2002

Anisomycin treatment paradigm affects duration of long-term potentiation in slices of the amygdala.

Anisomycin has been widely used as an inhibitor of protein synthesis in studies on learning and memory as well as synaptic plasticity. However, its mode of action is complicated. Besides the inhibition of translation, this drug displays other effects, most prominently on mitogen-activated protein kinases. In this report we analyze the effects of anisomycin on the late phase of long-term potentiation (LTP) in amygdala slices. The late phase of LTP was evoked by high-frequency repeated-train stimulation delivered to the external capsule and recorded in the lateral amygdaloid nucleus. In the no-drug condition, stimulation resulted in LTP lasting over 3 h without any sign of decay. Application of the drug 15 min before high-frequency stimulation (HFS) caused LTP decay to baseline within 1 h after induction. However, delivering the drug just after the first train of HFS resulted in LTP that returned to baseline level within 3 h since the onset of stimulation. These results show that the duration of the LTP in the amygdala depends on the anisomycin treatment paradigm and thus special caution should be exercised when interpreting the data obtained with this drug.

Action Potentials↗
PubMed2001

Kainate-induced genes in the hippocampus: lessons from expression patterns.

Kainate, the analog of the excitatory amino acid L-glutamate, upon binding to non-NMDA glutamate receptors, causes depolarization of neurons followed by severe status epilepticus, neurodegeneration, plasticity and gliosis. These events are best observed in hippocampus, the limbic structure implicated in learning and long-term memory formation. Neurons in all hippocampal structures undergo hyper-activation, however, whereas the cells in the CA subfields degenerate within 2--3 days following the application of kainate, the granule cells of the dentate gyrus are resistant to any form of neurodegeneration and even initiate new synaptic contacts. These physiological and histological changes are modulated by short-term and long-term alterations in gene expression. Perhaps close examination of the changing spatio-temporal patterns of mRNAs of various genes may help in generating a clearer picture of the molecular events leading to complex cognitive functions.

Animals↗
PubMed2001

Expression of c-Fos, Fos B, Jun B, and Zif268 transcription factor proteins in rat barrel cortex following apomorphine-evoked whisking behavior.

Apomorphine-evoked expression of transcription factor proteins: c-Fos, Fos B, Jun B, and Zif268 (also named Krox-24, NGFI-A, Egr-1), was investigated in rat somatosensory (barrel) cortex. The effect of the N-methyl-D-aspartate receptor antagonist MK-801 on their expression was also analyzed. Apomorphine is a dopamine receptor agonist, eliciting motor activity, including enhanced whisking leading to the activation of vibrissae representation in the barrel cortex. Rats had their whiskers clipped on one side of the snout. The Zif268 levels were markedly reduced by this procedure alone. In contrast, apomorphine (5.0 mg/kg) evoked marked c-Fos elevation, less pronounced changes in Jun B and Zif268 and no change in Fos B. The greatest apomorphine-evoked c-Fos accumulation was observed in layers IV and V/VI of non-deprived barrel cortex and was not significantly influenced by MK-801 injection at 0.1 mg/kg. A higher dose of MK-801 (1.0 mg/kg) produced abnormalities in locomotor behavior and diminished c-Fos levels on the non-deprived side to the ones observed in the sensory stimulus-deprived cortex. We conclude that the response of the somatosensory cortex is selective with respect to both the gene activated and its cortical layer localization. Furthermore, sensory stimulation provides a major but not the only component to apomorphine-evoked barrel cortex gene activation.

Afferent Pathways↗
PubMed2001

Complex formation in the indolo[2,3-b]quinolines--methylene blue systems in aqueous solutions.

Changes in the absorption spectra of methylene blue (MB) aqueous solutions containing an indoloquinoline can be described by assuming complex formation between MB and the indolinoquinoline. At the low concentration range of the two reactants a simple 1:1 complex stoichiometry was postulated on the basis of the Benesi-Hildebrand, Scott and Scatchard linearization of spectroscopic data and augmented by the formation of an isosbestic point. The estimated association constants were correlated with the ab initio calculated electronic properties of indolo[2,3-b]quinolines.

Ellipticines↗
PubMed2001

Two subtypes of G protein-coupled nucleotide receptors, P2Y(1) and P2Y(2) are involved in calcium signalling in glioma C6 cells.

1. In glioma C6 cells, the stimulation of P2Y receptors by ADP, ATP and UTP initiated an increase in the intracellular Ca2+ concentration, in a process that involved the release of Ca2+ from InsP(3)-sensitive store and the capacitative, extracellular Ca2+ entry. The presence of external Ca2+ was not necessary to elevate Ca(2+). 2. The rank order of potencies of nucleotide analogues in stimulating [Ca2+](i) was: 2MeSADP > ADP > 2MeSATP = 2ClATP > ATP > UTP. alpha,beta-Methylene ATP, adenosine and AMP were ineffective. 3. ADP and UTP effects were additive, while actions of ATP and UTP were not additive on [Ca2+](i) increase. Similarly, cross-desensitization between ATP and UTP but not between ADP and UTP occurred. 4. Suramin, a non-specific nucleotide receptors inhibitor, antagonized ATP-, UTP- and ADP-evoked Ca2+ responses. PPADS, a selective antagonist of the P2Y(1) receptor-generated InsP(3) accumulation, decreased ADP-initiated Ca2+ response with no effect on ATP and UTP. 5. Pertussis toxin (PTX) reduced ADP- and ATP-induced Ca2+ increases. Short-term treatment with TPA, inhibited both ATP and ADP stimulatory effects on [Ca2+](i). 6. ADP inhibited isoproterenol-induced cyclic AMP accumulation. PTX blocked this effect, but PPADS did not. 7. RT - PCR analysis revealed the molecular identity of P2Y receptors expressed by glioma C6 cells to be both P2Y(1) and P2Y(2). 8. It is concluded that both P2Y(1) and P2Y(2) receptors co-exist in glioma C6 cells. ADP acts as agonist of the first, and ATP and UTP of the second one. Both receptors are linked to phospholipase C (PLC).

Adenosine Diphosphate↗
PubMed2000

Efficient expression of tetracycline-responsive gene after transfection of dentate gyrus neurons in vitro.

Gene transfer into neurons both in vivo and in vitro may aid in understanding of gene regulation and function in nerve cells. Especially desirable is ability to control the gene expression. In this study we developed conditions for transfection of hippocampal dentate gyrus neurons in dissociated cultures in vitro by calcium-phosphate method. Furthermore, we describe an effective use of tetracycline responsive gene promoter (Tet-On) system for the controlled and very efficient expression of transfected genes. Under optimal conditions as established in this study, efficiency of transfection of neurons with green fluorescent protein (GFP) driven by constitutive cytomegalovirus (CMV) early promoter reached 2.7%. With tetracycline responsive promoter percentage of GFP-positive neurons raised in the presence of tetracycline analog, doxycycline up to 20%. Application of the Tet-On system resulted in almost 10-fold induction of GFP expression.

Animals↗
PubMed2000

Sequence-selectivity of 5,11-dimethyl-5H-indolo[2,3-b]quinoline binding to DNA. Footprinting and molecular modeling studies.

Indolo[2,3-b]quinolines are a new family of the DNA intercalators showing significant cytotoxic activity. The mechanism of their action is based on the inhibition of DNA topoisomerase II activity. It depends on their ability to induce and stabilize drug-topII-DNA cleavable complexes. Site-specific intercalation of 5,11-dimethyl-5H-indolo[2,3-b]quinoline (DiMIQ) was analyzed in vitro by DNaseI footprinting and by molecular modeling. To model the DNA-intercalator complex, use was made of the CVFF and ESFF force fields implemented in Insight 97.0 software. Experimental results were verified using a simple statistical model. The DiMIQ molecule was found to bind preferentially to the pBR322 DNA plasmid in the 5'-TGCTAACGC-3' region between adjacent adenine bases.

Base Sequence↗
PubMed2000

Estimation of the total range of 1J(CC) couplings in heterocyclic compounds: pyridines and their N-oxides, the experimental and DFT studies.

A very large set of one-bond spin-spin carbon carbon coupling constants, 1J(CC), has been measured for 32 variously mono- and disubstituted pyridine N-oxides and for 14 substituted pyridines. The N-oxides studied were 2-, 3- and 4-monosubstituted isomers, and a series of disubstituted compounds. A variety of substituents has been employed (CH3, COCH3, C5H4NO, CN, F, Br, Cl, OH, OCH3, NH2, N(CH3)2 and NO2), which allowed us to study substituent effects thoroughly. Good linear relationships between 1J(C3C4) in 3- and/or 4-substituted pyridine N-oxides and 1J(CipsoCortho) in benzenes and between 1J(C2C3) in 2- and/or 3-substituted pyridine N-oxides and 1J(CipsoCortho) in benzenes have been found. An analogous linear relationship has been observed between 1J(C3C4) in 3- and/or 4-substituted pyridines and 1J(CipsoCortho) in benzenes. It has been also concluded that, by analogy to 1J(CC) couplings in substituted benzenes, those in pyridines and their N-oxides are the substituent electronegativity dependent. The estimated total range covered by 1J(CC), couplings in substituted compounds varies, in the case of 1J(C2C3) couplings for example, from 25 Hz in 2-lithiopyridine N-oxide to ca. 100 Hz in 2,3-difluoropyridine N-oxide and from 18 Hz in 2-lithiopyridine to 92 Hz in 2,3-difluoropyridine. The DFT calculations have been carried out for the parent compounds and for a set of their 2-lithio, and variously substituted fluoro derivatives. The DFT data reproduced very well the experimental coupling values and revealed that the Fermi contact contribution is the dominating factor which governs the magnitude of the CC coupling across one bond.

Cyclic N-Oxides↗
PubMed2000

Precontact 50-kHz vocalizations in male rats during acquisition of sexual experience.

50-kHz ultrasonic vocalizations emitted by male rats during a 5-min period before introduction of a female (precontact vocalizations [PVs]) were analyzed in the context of acquisition of sexual experience. Changes in the main copulatory parameters and their N-methyl-D-aspartate (NMDA) receptor dependence, the role of contact with either anestrous or estrous females, and conditioning to odor and background cues were also investigated. Mount latency (ML) and intromission latency (IL) decreased after the 1st copulatory session, but ejaculation latency (EL) changed significantly only starting from the 4th session onward. The number of PVs gradually increased during the first 3-4 sessions. Blocking of NMDA receptors affected PVs and EL but not ML or IL. After a 5-month break in copulatory sessions, ML remained unchanged, whereas EL increased and the number of PVs decreased significantly. PVs were most robustly elevated by contact with estrous females. Exposure to background cues resulted in a linear decrease in number of PVs during 10 subsequent sessions without exposure to a female. The results suggest that, in the course of acquisition of a sexual experience, PVs reflect a learning process that depends on a rewarding value of sociosexual contact.

Animals↗
PubMed2000

Highly sensitive detection of hybridization of oligonucleotides to specific sequences of nucleic acids by application of fluorescence resonance energy transfer.

We show a new application of fluorescence resonance energy transfer (FRET) in two stages to detect specific sequences of nucleic acids. In the first stage, two fluorescently tagged oligonucleotides hybridize with a complementary target molecule to produce FRET. The sequences of the oligonucleotides and spectral properties of fluorophores are chosen to provide a basis for an efficient energy transfer. In the next step, the specificity of hybridization is tested by competition of labeled probes with an excess of unlabeled oligonucleotides of the same sequence. The resulting emission spectra, one obtained in the excess of unlabeled donor probe and the other produced in the excess of unlabeled acceptor probe, are compared with the spectrum from the first stage to look for differences in the emission pattern of the fluorescent labels. We show that it is possible to detect the existence of specific hybrids composed of the two probes and complementary target molecule even in very unfavorable conditions, such as the presence of unhybridized probes in the final reaction mixture, secondary nonacceptor quenching of donor probe fluorescence, and strong background emission of acceptor produced by its direct excitation with a donor excitation light.

Binding, Competitive↗
PubMed2000

Calcyclin (S100A6) binding protein (CacyBP) is highly expressed in brain neurons.

The expression of a novel calcyclin (S100A6) binding protein (CacyBP) in different rat tissues was determined by Western and Northern blotting. Polyclonal antibodies against recombinant CacyBP purified from E. coli exhibited the highest reaction in the brain and weaker reaction in liver, spleen, and stomach. CacyBP immunoreactivity was also detected in lung and kidney. Densitometric analysis showed that the concentration of CacyBP in the soluble fractions of total brain and cerebellum is approximately 0.17 and 0. 34 ng/microg protein, respectively. Northern blotting with a specific cDNA probe confirmed the high level of CacyBP expression in the rat brain and lower levels in other tissues examined. Immunohistochemistry and in situ hybridization of rat brain sections revealed strong expression of CacyBP in neurons of the cerebellum, hippocampus, and cortex. The in situ hybridization detected CacyBP in hippocampus as early as P7 (postnatal day 7) and a peak of expression at P21, and the expression signal was preserved until adulthood. In the entorhinal cortex, the peak of expression was observed at P7, whereas in the cerebellum it was seen at P21. The results presented here show that CacyBP is predominantly a neuronal protein. (J Histochem Cytochem 48:1195-1202)

Animals↗
PubMed2000

Gene expression in learning processes.

It has repeatedly been shown that long-term memory formation involves neuronal gene expression. In this article several different roles for neuronal gene function in a context of learning are considered: maintenance of neural functioning, replenishment of cellular elements that are exhausted in response to massive neuronal stimulation accompanying behavioral training, maintenance of the plastically reorganized neuronal connections, and finally integration of information at the level of transcription factor-promoter interaction. It is strongly advocated that only careful scrutiny of learning-related gene expression phenomena may aid in understanding of the complex learning process.

Animals↗
PubMed1999

Neuronal excitation-driven and AP-1-dependent activation of tissue inhibitor of metalloproteinases-1 gene expression in rodent hippocampus.

Understanding of biological function of AP-1 transcription factor in central nervous system may greatly benefit from identifying its target genes. In this study, we present several lines of evidence implying AP-1 in regulating expression of tissue inhibitor of metalloproteinases-1 (timp-1) gene in rodent hippocampus in response to increased neuronal excitation. Such a notion is supported by the findings that timp-1 mRNA accumulation occurs in the rat hippocampus after either kainate- or pentylenetetrazole-evoked seizures with a delayed, in comparison with AP-1 components, time course, as well as with spatial overlap with c-Fos protein (major inducible AP-1 component) expression. Furthermore, AP-1 sequence derived from timp-1 promoter is specifically bound by hippocampal AP-1 proteins after treating the rats with either pro-convulsive agent. Finally, timp-1 promoter responds to excitatory activation both in vivo, in transgenic mice harboring the timp-LacZ gene construct, and in vitro in neurons of the hippocampal dentate gyrus cultures. These findings suggest that the AP-1 transcription factor may exert its role in the brain through affecting extracellular matrix remodeling.

Animals↗
PubMed1999

Defensive conditioning-related increase in AP-1 transcription factor in the rat cortex.

In the studies reported herein, electrophoretic mobility shift assay (EMSA) and immunocytochemistry have been applied to document increased levels of AP-1 transcription factor, and its major component, c-Fos in the rat brain following behavioral training of two-way active avoidance. A single training session (50 trials) provoked elevation of AP-1 in the visual, sensory and limbic cortex but not in the hippocampus. A session following long term training (10 sessions, up to asymptotic level of performance) had much smaller effect on AP-1 levels in the visual cortex than single training session. The long term training was used to ensure that observed effects were related to acquisition of the reaction rather than simply to behavioral performance. Supershift EMSA analysis with antibodies directed at individual AP-1 components revealed that AP-1 extracted from the brains of trained as well as naive animals is composed of the same proteins, i.e., in order of relative level within the protein family: c-Fos, Fos B, Fra-2, and Jun D, Jun B, c-Jun. These studies reinforce the notion that transcription factors as regulators of gene expression-and AP-1 in particular-may respond to behavioral stimulation and furthermore may play a role in acquisition of behavioral reactions.

Animals↗
PubMed1999

Rapid phosphorylation of Elk-1 transcription factor and activation of MAP kinase signal transduction pathways in response to visual stimulation.

The AP-1 transcription factor, which is composed of various combinations of Fos and Jun proteins, is believed to be a key participant in molecular processes that guide activity-dependent changes in gene expression. In this study, we investigated the activity of different MAP kinases that have been implicated in AP-1 activation. We examined the activities of ERK, JNK/SAPK, and p38 MAPK along with their nuclear targets (Elk-1 and c-Jun) in rat visual cortex after light stimulation. The transcription factor Elk-1 (a possible regulator of c-fos expression) was found to be transiently modified by phosphorylation when visual stimulation was applied after a period of dark rearing. In vitro kinase assay with Elk-1 as substrate showed that light stimulation activated MAPK/ERK in visual cortex but not frontal cortex. Furthermore, ERK activation was temporally matched to onset of Elk-1 phosphorylation. The activity of JNK1 (c-Jun N-terminal kinase 1) was elevated at 2-6 h after visual exposure and was also temporally correlated to increase of endogenous P-c-Jun levels and its appearance within the AP-1 DNA-binding complex. The activities of p38 MAP kinases did not change significantly. These results demonstrate the differential engagement of MAPK signaling pathways following sensory stimulation and their relative effects upon AP-1 expression in the intact brain.

Animals↗
PubMed1999

Defensive conditioning-related functional heterogeneity among nuclei of the rat amygdala revealed by c-Fos mapping.

The amygdala is a complex forebrain structure proposed to play a pivotal role in fear conditioning circuitry. In this study, c-Fos immunomapping was applied to investigate the functional activation of particular amygdalar nuclei following a 50-trial training session of two-way active avoidance reaction. To dissect distinctive responses displayed by the animals and to cluster them into groups of correlated behaviors, factor analysis was employed. The training procedure resulted in an increase of c-Fos expression within the cortical, medial, lateral and basolateral, but not central, nuclei. The expression in the cortical nucleus correlated negatively with grooming behavior, whereas c-Fos immunolabeling of the other three subdivisions of the amygdala could be associated with the number of intertrial responses. No correlation was observed between c-Fos expression and avoidance reactions performed or the amount of shock received by the animal. The results obtained with c-Fos mapping of various regions of rat amygdala, combined with a fine dissection of behavioral repertoire, imply that there are specific functional links between particular parts of the structure and distinctive behaviors that reflect various emotional states of the animal.

Aggression↗