Biomedical subjects

L Kaczmarek

Publications and source records attributed to L Kaczmarek.

At least 55 records · Page 3Linked to original sources

PubMed1996

Spatio-temporal pattern of N-methyl-D-aspartate receptor NR1 mRNA expression during postnatal development of visual structures of the rat brain.

N-methyl-D-aspartate (NMDA) excitatory amino acid receptors (NMDAR) play a pivotal role in various physiological and pathological responses of the central nervous system (CNS), including such phenomena of neuronal plasticity as cortical development. Hence, it could be suggested that expression of genes coding for NMDAR components can be differentially regulated throughout the development. To test this notion we analyzed with in situ hybridization mRNA levels of NMDAR subunit designated as NR1, during time of development (1-6 weeks postnatally) spanning the critical period of functional formation of major visual structures of the rat brain, namely visual cortex (VC), superior colliculus (SC), and lateral geniculate nucleus (LGN). The highest levels of NR1 mRNA were found in the VC and SC at the onset of the critical period, i.e., 21 days postnatally, whereas in the LGN a tendency toward a similar pattern of expression was observed. Analysis of spatial distribution of NR1 mRNA in the SC and VC revealed that an adult-like laminar pattern of expression was also achieved between 14 and 21 days postnatally. This pattern of expression corroborates with developmentally regulated changes in an overall density of cell bodies in these areas.

Animals↗

PubMed1996

A solution and solid state 15N NMR study of hydrogen bonding in a Schiffs base.

The 15N results reveal a difference in the extent of intramolecular hydrogen bond formation exhibited by the Schiffs base, compound 2, in solution and the solid state. Comparison of the 15N shielding changes observed, with those arising from protonation, provides the basis for a quantitative estimate of the strength of the hydrogen bonds formed.

Hydrogen Bonding↗

PubMed1996

c-Fos expression as a tool to search for the neurobiological base of the sexual behaviour of males.

Elevated expression of c-fos mRNA and/or its protein (c-Fos) has been repeatedly observed following activation of brain cells with various stimuli. As a consequence it has been proposed that elevated c-fos expression may serve as a marker of neuronal activation reflecting a predisposal to long-term changes in cell functioning (neuronal plasticity). In studies on copulatory behaviour of male animals, elevated Fos immunoreactivity (related most probably to c-Fos) has been used by many researchers as a tool to reveal functional neuroanatomy of the behaviour. They found that brain regions such as medial preoptic area (MPOA), bed nucleus of stria terminalis (BNST), medial amygdala (MeAm) and central tegmental field (CTF) are always activated in a copulation-dependent manner in various species (rats and hamsters were the most often studied in this regard). This paper reviews the data gathered in this field, and discusses them in the context of a role of elevated Fos expression in acquisition vs. performance of the sexual behaviour.

Animals↗

PubMed1995

Kainate-evoked secondary gene expression in the rat hippocampus.

Kainate treatment provides a model to study elevated expression of genes whose function may be related to neuronal plasticity. In particular, expression of components of AP-1 transcription factor, i.e. Fos and Jun proteins, has been widely investigated in this system. While AP-1 has been repeatedly implicated in various plasticity-related phenomena, very little is known about its downstream gene targets. In the experiments reported here we have analyzed whether genes recently identified as kainate-induced in the rat dentate gyrus and coding for secretogranin II, clathrin heavy chain and heat shock cognate protein 70 can be characterized by a secondary, i.e. possibly inducible transcription factor-dependent mode of activation. Using in situ hybridization and northern studies we have found that expression of all three genes occurs in all hippocampal regions activated following kainate treatment, the time-course of this activation is delayed when compared to mRNA accumulation of AP-1 components, and finally the expression of all three genes is significantly blocked by a cycloheximide-protein synthesis inhibitor. These results suggest that indeed the genes examined are characterized by their secondary mode of activation.

Animals↗

PubMed1995

Elevated cathepsin D expression in kainate-evoked rat brain neurodegeneration.

Expression patterns of cathepsin D (lysosomal aspartic protease) and glial fibrillary acidic protein (GFAP, a marker of reactive astroglia) were determined by Northern blot analysis and immunohistochemistry in the rat brain during neurodegeneration accompanying kainate-evoked seizures. The level of cathepsin D mRNA in the hippocampus, limbic cortex, and temporo-parieto-occipital neocortex was shown to increase, starting at 6 h after kainate treatment, and reaching peak values at 3-7 days after the neurotoxin administration. A similar time course of elevated accumulation was noted for GFAP mRNA in these structures. Immunohistochemical analysis performed 3 days after kainate treatment showed that the increased cathepsin D levels were confined mainly to the degenerating neurons in the susceptible brain areas, while the elevated GFAP immunoreactivity was observed in reactive astrocytes. Although cathepsin D and GFAP expression levels were elevated by kainate administration, their expression patterns revealed significant differences with regard to both intensity and site of induction.

Animals↗

PubMed1995

Antisense oligodeoxyribonucleotides: stability and distribution after intracerebral injection into rat brain.

As a prerequisite for blocking specific gene expression in the brain, the pharmacokinetics of two radiolabelled analogs of antisense oligodeoxyribonucleotides (unmodified O-ODN and nuclease resistant phosphorothioate S-ODN) were examined by infusion into the baso-lateral nucleus of amygdala. Both ODN analogs were found to penetrate at restricted distances into the brain tissue. Rapidly after injection, O-ODN was almost completely degraded, while S-ODN remained intact up to 24 h following administration as examined by gel electrophoresis of nucleic acids recovered from the injection site. The tissue clearance of the radioactivity delivered in a form of O-ODN and S-ODN was also different, the former characterized by much better tissue retention. Microscopic studies suggested that S-ODN can apparently penetrate across the cell membrane and accumulate both in the cytoplasm in the cell nucleus. In situ hybridisation histochemistry experiments (antisense probe to injected ODN) revealed that injected S-ODN was present in a form available for annealing with the complementary strand. Our results provide a basic description of the distribution, retention, and stability of antisense oligonucleotides injected into brain tissue.

Amygdala↗

PubMed1995

Inducible and constitutive transcription factor NF-kappa B-like DNA binding activities in rat brain cells cultured in vitro.

Description of constitutive and inducible transcription factors in brain cells is a prerequisite for understanding phenomena of long-term neuronal plasticity. In this report we report that basal levels of NF-kappa B-like transcription factor DNA binding activity (measured by electrophoretic mobility shift assay) are observed in two kinds of cultured rat brain cells, namely neurons of fetal cerebral hemispheres and astroglia derived from newborn brains. In the latter culture, phorbol ester induces additional forms of DNA binding activity, whereas L-glutamate fails to do so. In neurons, neither treatment is effective in inducing NF-kappa B-like DNA binding activity over the basal level. These results are in contrast to the fact that L-glutamate in both neurons and glia elevates DNA binding activity of AP-1, another transcription factor. These data, besides describing behavior of NF-kappa B-like DNA binding activities, also provide some evidence that L-glutamate exerts its modulatory functions on neurons and glia through specific transcription factors like AP-1.

Animals↗

PubMed1995

Seizure related changes in the regulation of opioid genes and transcription factors in the dentate gyrus of rat hippocampus.

An in situ hybridization study showed that limbic seizures induced by kainate strongly augmented the prodynorphin and proenkephalin messenger RNA levels in granular cells of the rat hippocampal dentate gyrus. Pentylenetetrazole increased the level of proenkephalin messenger RNA, but slightly decreased that of prodynorphin messenger RNA in the dentate gyrus. Administration of kainate to rats caused a profound increase in messenger RNAs of the transcription factor genes c-fos and c-jun in the dentate gyrus, followed by an increase in the level of the transcriptional complex activator protein-1 in hippocampal neurons. Pentylenetetrazole also elevated the formation of activator protein-1, but the effect appeared earlier than that induced by kainate. Thus, recurrent limbic seizures activate both prodynorphin and proenkephalin genes, whereas generalized clonic-tonic seizures seem to decrease the prodynorphin and increase the proenkephalin gene expression in the dentate gyrus. Furthermore, our present results suggest that the transcription factors, c-fos, c-jun and activator protein-1 complex may be involved in the process of inducing the hippocampal proenkephalin gene, while these factors might be differently involved in regulation of prodynorphin gene expression.

Animals↗

PubMed1995

Towards understanding of the role of transcription factors in learning processes.

Recent advances in application of molecular biology to studies on learning and memory formation suggest that understanding of these seemingly elusive phenomena may be within our reach. This mini-review summarizes the present knowledge on activation and possible functions of transcription factors in learning processes with a focus on studies performed in the author's laboratory.

Animals↗

PubMed1994

Synthesis and structure-activity relationship of methyl-substituted indolo[2,3-b]quinolines: novel cytotoxic, DNA topoisomerase II inhibitors.

In furtherance of our SAR study on the chemistry and antitumor activity of fused nitrogen heteroaromatic compounds, a series of linear, methyl-substituted derivatives of 5H- and 6H-indolo[2,3-b]quinolines were synthesized according to the modified Graebe-Ullmann reaction. To establish the relationship between the physicochemical and biological activities of indolo[2,3-b]quinolines, their lipophilic properties, cytotoxic and antimicrobial activity, and ability to induce topoisomerase II dependent pSP65 DNA cleavage in vitro were investigated. We found that the antimicrobial and cytotoxic activity of indolo[2,3-b]quinolines was strongly influenced by the position, and the number of methyl substituents and the presence of methyl group at pyridine nitrogen was essential for the cytotoxicity of these compounds. All indolo[2,3-b]quinolines belonging to the 5H series, i.e., bearing a methyl group on the pyridine nitrogen, showed significant activity against procaryotic and eucaryotic organisms. They inhibited the growth of Gram-positive bacteria and pathogenic fungi at MIC range 3 x 10(-2) to 2.5 x 10(-1) mumol/mL, displayed cytotoxicity against KB cells ID50 in the range 2 x 10(-3) to 9 x 10(-3) mumol/mL, and stimulated the formation of calf thymus topoisomerase II mediated DNA cleavage at concentration between 0.4 and 10 microM. None of the indolo[2,3-b]quinolines belonging to the 6H series, i.e., lacking a methyl group on the pyridine nitrogen, was active in analogous tests. Of the investigated compounds, the most active was 2,5,9,11-tetramethyl-5H-indolo[2,3-b]quinoline, a compound bearing the highest number of symmetrically distributed methyl groups. The interaction of indolo[2,3-b]quinolines with DNA was studied by measuring the increase of calf thymus DNA denaturating temperature (Tm). The delta Tm values for the 5H series were found to be about 10 times as high as those for the 6H compounds. Indolo[2,3-b]quinolines with the highest number of methyl groups had the greatest contribution to the increase in the Tm of calf thymus DNA. The values of delta Tm reached 19 degrees C and 1.6 degrees C for the most substituted compounds of both series.

Animals↗

PubMed1994

Dynamic changes in the composition of the AP-1 transcription factor DNA-binding activity in rat brain following kainate-induced seizures and cell death.

Kainate, a potent excitatory and neurotoxic agent, has also proved useful in studies on other glutamate-driven phenomena, such as neuronal plasticity. Long-term effects of kainate are apparently dependent on its influence on the expression of various genes, including those encoding the AP-1 transcription factor, consisting of proteins belonging to the Fos and Jun families. In our studies we analysed c-fos, fos B, c-jun, jun B and jun D mRNA levels as well as a functional feature of AP-1, its DNA-binding activity, in the rat brain following systemic injection of kainate. Two phases of elevated AP-1 DNA-binding activity were observed in the hippocampus and entorhinal cortex, and were correlated with period of seizures (2 and 6 h after kainate injection) and neuron damage (48-72 h). At 72 h after kainate treatment DNA fragmentation, believed to be diagnostic of apoptotic processes typical of programmed cell death phenomena, was noted. Two and six hours after the treatment, AP-1 consisted predominantly of Fos B, c-Fos, Fra-2 and Jun B, while at 72 h Jun D constituted the major AP-1 component in place of Jun B, and no c-Fos was detected. Only a slight AP-1 increase was seen 24 h after kainate treatment. In the sensory cortex, only the late phase of AP-1 elevation was detected. Contrary to AP-1, no effect of kainate on levels of two other transcription factors, CREB/ATF (cAMP-responsive element binding proteins) and OCT (octamer element DNA-binding activity) was seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

DNA fragmentation in rat brain after intraperitoneal administration of kainate.

Cell death occurs in many neuropathological conditions. However, the mechanisms governing this process(es) remain generally unknown. In this report we studied whether excitotoxic neuronal death evoked by kainic acid (KA) in rat brain is associated with ladder-like DNA fragmentation. DNA was isolated from hippocampi, entorhinal and sensory cortices at various times following intraperitoneal KA (10 mg kg-1) injections. Typical oligonucleosome-sized DNA fragmentation was observed in all three structures at 18 h and 72 h following KA administration. These findings were further confirmed by in situ nick-translation. DNA fragmentation is believed to be diagnostic for apoptosis. The clear ladders of DNA fragmentation appeared after 18 h, although slight degradation was observed as early as 12 h after KA administration.

Animals↗

PubMed1994

AP-1 and CRE DNA binding activities in rat brain following pentylenetetrazole induced seizures.

Pentylenetetrazole (PTZ) evoked seizures, known to be dependent on stimulation of excitatory amino acids (EAA) receptors, serve as a useful model to study genomic responses to increased brain activity. It is believed that these responses form the basis for long term modifications in neuronal functions. Formation of the AP-1 transcription factor genes and proteins in hippocampal cells is the best known example of a genomic response to PTZ seizures and to an activation of the EAA receptors. In the studies reported herein electrophoretic mobility shift assay (EMSA) was employed to investigate levels of AP-1 DNA binding activity in various regions of the rat brain following PTZ seizures and these levels were compared to the cyclic AMP responsive element (CRE) DNA binding activity. A dramatic increase of the AP-1 DNA binding activity was observed in the hippocampus and in sensory and limbic cortices, and to much lesser extent in the cerebellum. The EMSA supershift method provided an evidence that Jun B and c-Fos and probably Fos B are major components of AP-1 at 2 h after the seizures. In none of the structure investigated, clear modulation of CRE DNA binding activity was noted. These data are discussed in the context of CRE and AP-1 DNA binding crossreactivity.

Activating Transcription Factor 2↗

PubMed1994

Glutamate receptor-driven activation of transcription factors in primary neuronal cultures.

We have used primary neuronal cultures prepared from fetal cerebral hemispheres to investigate the effects of different glutamate receptor agonists and antagonists on the expression of transcription factor encoding genes, such as c-fos, fosB, c-jun, junB, junD, c-myc, and zif/268. The addition of glutamate (100 microM) to the culture medium rapidly activated c-fos, fosB, c-jun, junB and zif/268 gene expression, reaching the maximal level at 30-60 minutes for zif/268 and at 60 minutes for the other genes. The onset of fosB mRNA accumulation was slightly delayed in comparison to the other genes. No clear induction was found for junD and c-myc. Different glutamate receptor agonists, such as NMDA, kainate, quisqualate, trans-(+/-)-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) were able to increase c-fos, c-jun, and zif-268 mRNA levels with rapid and transient kinetics similar to those observed after glutamate treatment. Similar results were obtained for junB and fosB after kainate and quisqualate stimulation. Pretreatment with MK-801, a non competitive NMDA antagonist, produced an almost complete inhibition of glutamate-driven expression of transcription factor genes, thus suggesting that NMDA receptor plays a major role in glutamate induced-gene expression. On the contrary the kainate/AMPA receptor antagonist, DNQX, did not influence glutamate induced-gene expression. Under the conditions used in the present study, NMDA was effective in inducing the simultaneous activation of several IEGs even when added to the culture medium containing millimolar concentration of magnesium. When experiments were performed in Krebs solution, NMDA was effective in stimulating zif/268 and c-fos mRNAs only in the absence of Mg2+, while glutamate activated c-fos and zif/268 both in the presence and absence of magnesium ions. As expected, NMDA effect was fully inhibited by MK-801. The level of AP-1 DNA binding activity, as measured by electrophoretic mobility shift assay, increased after addition of glutamate and NMDA to cultured neurons and such increase was antagonized by the pretreatment with MK-801.

Animals↗

PubMed1994

Disruption of two-way active avoidance behavior produced by nimodipine.

Nimodipine, a voltage-sensitive calcium channel blocker, has been suggested to be a procognitive drug. In the studies reported herein, we found that low doses of IP-injected nimodipine (0.5 and 0.05 mg/kg) impaired two-way active avoidance behavior. The acquisition phase of the training was the same for drug-treated and control animals. However, the nimodipine-injected rats achieved a significantly lower level of performance. The no-shock tests revealed much faster extinction of the learned behavior in drug-treated vs. control animals. These results could be interpreted as indicating learning-disruptive effects of nimodipine.

Animals↗

PubMed1994

Antibody raised to the short sequence from the zinc-finger domain of the EGR-1 recognizes 102 KD protein in mouse fibroblasts.

EGR (early growth response) genes represent a family of the proteins whose structure includes a zinc finger domain(s) interacting with the specific site in regulatory sequences of different genes. Using as an antigen the short sequence RSNHLTTHIR from the middle of the zinc finger domain of EGR-1 we have generated a clone of the hybridoma cells producing the monoclonal antibody (mAb) that binds to 102 kD and 58 kD proteins, but apparently does not bind EGR-1 protein. The 102 kD protein is probably made of the 58 kD subunit and another one of 44 kD. Expression of these proteins is strongly induced in serum stimulated mouse fibroblasts.

3T3 Cells↗

PubMed1994

Mutagenicity of carboline methyl derivatives in Salmonella.

A series of linear, methyl-substituted derivatives of 5H-indolo[2,3-b] quinolines was tested for mutagenic activity with the battery of Ames tester strains. Mutagenic activity of indoloquinolones was strongly influenced by the position and a number of methyl groups. All compounds tested, with one exception, act like frame-shift mutagens. Only two compounds among them were mutagenic in the strain detecting oxidative and cross-linking mutagens.

Carbolines↗

PubMed1994

[The antibodies obtained by immunization with a peptide identical to a section of the zinc-binding domain of the early growth response 1 factor (EGR-1) interact with a 102-kDa protein from mouse fibroblasts].

The early growth response (EGR) genes represent a family of proteins whose structure includes zinc finger domain(s) interacting with a specific site in promotors of different genes to regulate their transcriptional activity. Using as an antigen a short sequence RSDHLTTHIR from the middle zinc finger sequence of EGR-1 we have generated a clone of hybridoma cells producing a monoclonal antibody (mAb) that binds a 102 kDa protein, but does not bind EGR-1 protein. The 102 kDa protein consists apparently of 2 polypeptides, of 58 kDa and 44 kDa, and its expression is strongly stimulated in serum-induced mouse fibroblasts.

3T3 Cells↗