Biomedical subjects

T Nagy

Publications and source records attributed to T Nagy.

At least 19 recordsLinked to original sources

PubMed2004

Integrative plasmid vector for constructing single-copy reporter systems to study gene regulation in Rhizobium meliloti and related species.

The integrative system of phage 16-3 of Rhizobium meliloti 41 was shown to function in several bacterial species belonging to the Rhizobium, Bradyrhizobium, Azorhizobium, and Agrobacterium genera. It might also function in many other bacterial species provided that both the target site (attB) and the required host factor(s) are present. Here we report on the construction of a new integrative vector that can be utilized in gene regulation studies. It provides an opportunity to create a single-copy set-up for characterizing DNA-protein interactions in vivo, in a wide range of bacteria. To demonstrate the usefulness of the vector, transcription repression by binding of the C repressor protein of phage 16-3 to wild type operators was studied. The assay system provided highly reproducible quantitative data on repression.

Agrobacterium tumefaciens↗

PubMed2002

Cyclophosphamide, etoposide and G-CSF to mobilize peripheral blood stem cells for autologous stem cell transplantation in patients with lymphoma.

We aimed to assess the effectiveness of cyclophosphamide, etoposide and G-CSF (C+E) to mobilize peripheral blood stem cells for autologous stem cell transplantation in patients with lymphoma. A matched cohort study was performed comparing patients mobilized with C+E to patients mobilized with cyclophosphamide and G-CSF (C alone). Patients were matched for disease, prior radiotherapy and a chemotherapy score reflecting the amount and type of prior chemotherapy. Thirty-eight consecutive patients mobilized with C+E were compared with 38 matched controls. C+E was equivalent to C alone in terms of numbers of patients achieving a minimum threshold of > or =2 x 10(6)/kg CD34(+)cells (82% vs 79%, P = 0.74). C+E was superior, however, in terms of total CD34(+) yield (6.35 vs 3.3 x 10(6)/kg, P < 0.01), achieving a target graft of > or =5 x 10(6)/kg (55% vs 34%, P = 0.04) and obtaining both a minimum (61% vs 32%, P < 0.01) and target (45% vs 13%, P < 0.01) graft in one apheresis. This superiority was largely confined to patients with lower chemotherapy scores. There was no difference in neutrophil and platelet recovery or transfusion requirements for those who subsequently received high-dose therapy and stem cell transplantation. Thus, C+E improves the efficiency of peripheral blood stem cell collection, but does not increase the number of patients who can proceed to transplantation. Most of the benefit of the regimen was confined to patients who had not received extensive prior therapy. Novel strategies are required to increase the collection efficiency of 'hard to mobilize' patients.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2002

Ultrastructure of neuromuscular contacts in the embryonic pond snail Lymnaea stagnalis L.

Ultrastructural characteristics of muscle fibers and neuromuscular contacts were investigated during two stages of embryogenesis of the pulmonate snail Lymnaea stagnalis. The first muscle cells appear as early as during metamorphosis (50-55% of embryonic development), whereas previously, in the trochophore/veliger stages (25-45%), muscular elements cannot be detected at all. The first muscle fibers contain large amounts of free numbers, a well-developed rER system and only a few irregularly arranged contractile elements. The nucleus is densely packed with heterochromatine material. At 75% adult-like postmetamorphic stage, the frequency of muscle fibers increases significantly, but, bundles of muscle fibers cannot yet be observed. Furthermore the muscle cells are characterized by large numbers of free ribosomes and numerous rER elements. Fine axon bundles and single axon processes, both accompanied by glial elements, can already be found at this time. Axon varicosities with different vesicle and/or granule contents form membrane contacts with muscle fibers, but without revealing membrane specialization on the pre- or postsynaptic side. The late development of the muscle system and neuromuscular contacts during Lymnaea embryogenesis correlates well with the maturation of different forms of behavior of adult, free-living life, and also with the peripheral appearance of chemically identified components of the embryonic nervous system of central origin.

Animals↗

PubMed2001

Association of Na(+)-H(+) exchanger isoform NHE3 and dipeptidyl peptidase IV in the renal proximal tubule.

In an attempt to identify proteins that assemble with the apical membrane Na(+)-H(+) exchanger isoform NHE3, we generated monoclonal antibodies (mAbs) against affinity-purified NHE3 protein complexes isolated from solubilized renal microvillus membrane vesicles. Hybridomas were selected based on their ability to immunoprecipitate NHE3. We have characterized in detail one of the mAbs (1D11) that specifically co-precipitated NHE3 but not villin or NaPi-2. Western blot analyses of microvillus membranes and immunoelectron microscopy of kidney sections showed that mAb 1D11 recognizes a 110-kDa protein highly expressed on the apical membrane of proximal tubule cells. Immunoaffinity chromatography was used to isolate the antigen against which mAb 1D11 is directed. N-terminal sequencing of the purified protein identified it as dipeptidyl peptidase IV (DPPIV) (EC ), which was confirmed by assays of DPPIV enzyme activity. We also evaluated the distribution of the NHE3-DPPIV complex in microdomains of rabbit renal brush border. In contrast to the previously described NHE3-megalin complex, which principally resides in a dense membrane population (coated pits) in which NHE3 is inactive, the NHE3-DPPIV complex was predominantly in the microvillar fraction in which NHE3 is active. Serial precipitation experiments confirmed that anti-megalin and anti-DPPIV antibodies co-precipitate different pools of NHE3. Taken together, these studies revealed an unexpected association of the brush border Na(+)-H(+) exchanger NHE3 with dipeptidyl peptidase IV in the proximal tubule. These findings raise the possibility that association with DPPIV may affect NHE3 surface expression and/or activity.

Amino Acid Sequence↗

PubMed2001

Role of hydrogen bonding in the interaction between a xylan binding module and xylan.

NMR studies of the internal family 2b carbohydrate binding module (CBM2b-1) of Cellulomonas fimi xylanase 11A have identified six polar residues and two aromatic residues that interact with its target ligand, xylan. To investigate the importance of the various interactions, free energy and enthalpy changes have been measured for the binding of xylan to native and mutant forms of CBM2b-1. The data show that the two aromatic residues, Trp 259 and Trp 291, play a critical role in the binding, and similarly that mutants N264A and T316A have no affinity for the xylose polymer. Interestingly, mutations E257A, Q288A, N292A, E257A/Q288A, E257A/N292A, and E257A/N292A/Q288A do not significantly diminish the affinity of CBM2b-1 for the xylose polymers, but do influence the thermodynamics driving the protein-carbohydrate interactions. These thermodynamic parameters have been interpreted in light of a fresh understanding of enthalpy-entropy compensation and show the following. (1) For proteins whose ligands are bound on an exposed surface, hydrogen bonding confers little specificity or affinity. It also displays little cooperativity. Most specificity and affinity derive from binding between the face of sugar rings and aromatic rings. (2) Loss of hydrogen bonding interactions leads to a redistribution of the remaining bonding interactions such that the entropic mobility of the ligand is maximized, at the expense (if necessary) of enthalpically favorable bonds. (3) Changes in entropy and enthalpy in the binding between polysaccharide and a range of mutants can be interpreted by considering changes in binding and flexibility, without any need to consider solvent reorganization.

Actinomycetales↗

PubMed2001

Characterization of a cellulosome dockerin domain from the anaerobic fungus Piromyces equi.

The recycling of photosynthetically fixed carbon in plant cell walls is a key microbial process. In anaerobes, the degradation is carried out by a high molecular weight multifunctional complex termed the cellulosome. This consists of a number of independent enzyme components, each of which contains a conserved dockerin domain, which functions to bind the enzyme to a cohesin domain within the protein scaffoldin protein. Here we describe the first three-dimensional structure of a fungal dockerin, the N-terminal dockerin of Cel45A from the anaerobic fungus Piromyces equi. The structure contains a novel fold of 42 residues. The ligand binding site consists of residues Trp 35, Tyr 8 and Asp 23, which are conserved in all fungal dockerins. The binding site is on the opposite side of the N- and C-termini of the molecule, implying that tandem dockerin domains, seen in the majority of anaerobic fungal plant cell wall degrading enzymes, could present multiple simultaneous binding sites and, therefore, permit tailoring of binding to catalytic demands.

Amino Acid Sequence↗

PubMed2000

High dose chemotherapy and autologous blood stem cell support in women with breast carcinoma and isolated supraclavicular lymph node metastases.

BACKGROUND: The prognosis of patients with isolated supraclavicular lymph node (SCN) metastases is similar to patients with metastatic breast carcinoma involving other sites. Because these patients have a lower disease burden compared with women with distant metastases, their outcome after high dose chemotherapy (HDCT) may be superior. METHODS: The authors evaluated event free survival (EFS) and overall survival in a series of 20 consecutive women with SCN metastases as the only site of metastatic disease who were treated with HDCT and peripheral blood stem cell transplantation at The Toronto Hospital. All patients had responded to 4-6 cycles of induction CT using either an anthracycline-containing regimen or a single agent taxane, and received intensive therapy comprised of mitoxantrone, 64 mg/m(2); cyclophosphamide, 6000 mg/m(2); and carboplatin, 800-2000 mg/m(2), each divided over 4 days followed by the infusion of autologous peripheral blood stem cells. Involved field radiation therapy (RT) was administered when possible after transplantation to the supraclavicular fossa and tamoxifen was given to previously untreated patients if they were hormone receptor positive or if their hormone receptor status was unknown. RESULTS: At a median follow-up of 28 months, 13 of the 20 women were alive, 11 of whom (55%) remained in continuous complete remission. There were no treatment-related deaths. The median overall survival was 37 months and the median progression free survival was 32 months from the date of transplantation. Consolidative RT was delivered to 11 women and on univariate analysis was found to be significantly associated with better EFS (P = 0.02). CONCLUSIONS: The long term outcome of women with breast carcinoma and isolated SCN metastases whose disease is sensitive to CT appears to be favorable; whether this result is superior to that achieved with standard therapy alone remains to be confirmed in prospective, randomized trials.

Adult↗

PubMed2000

Solution structure of the CBM10 cellulose binding module from Pseudomonas xylanase A.

Plant cell wall hydrolases generally have a modular structure consisting of a catalytic domain linked to one or more noncatalytic carbohydrate-binding modules (CBMs), whose common function is to attach the enzyme to the polymeric substrate. Xylanase A from Pseudomonas fluorescens subsp. cellulosa (Pf Xyn10A) consists of a family 10 catalytic domain, an N-terminal family IIa cellulose-binding module, and an internal family 10 cellulose-binding module. The structure of the 45-residue family 10 CBM has been determined in solution using NMR. It consists of two antiparallel beta-sheets, one with two strands and one with three, with a short alpha-helix across one face of the three-stranded sheet. There is a high density of aromatic residues on one side of the protein, including three aromatic residues (Tyr8, Trp22, and Trp24), which are exposed and form a flat surface on one face, in a classical polysaccharide-binding arrangement. The fold is closely similar to that of the oligonucleotide/oligosaccharide-binding (OB) fold, but appears to have arisen by convergent evolution, because there is no sequence similarity, and the presumed binding sites are on different faces.

Amino Acid Sequence↗

PubMed2000

Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the family 10 cellulose-binding module from Pseudomonas xylanase A to insoluble ligands.

Aromatic amino acids are believed to play a pivotal role in carbohydrate-binding proteins, by forming hydrophobic stacking interactions with the sugar rings of their target ligands. Family 10 cellulose-binding modules (CBM10s), present in a number of cellulases and xylanases expressed by Pseudomonas fluorescens subsp. cellulosa, contain two tyrosine and three tryptophan residues which are highly conserved. To investigate whether these amino acids play an important role in the interaction of CBM10 from P. fluorescens subsp. cellulosa xylanase A (Pf Xyn10A) with cellulose, each of these residues was changed to alanine in CBM10 expressed as a discrete module or fused to the catalytic domain of Pf Xyn10A (CBM10-CD), and the capacity of the mutant proteins of CBM10-CD to bind the polysaccharide was evaluated. The data showed that W22A, W24A, and Y8A bound very weakly to cellulose compared to the wild-type protein, while Y12A retained its capacity to interact with the glucose polymer. When the W7A mutation was introduced into CBM10 the protein domain did not accumulate in Escherichia coli. In contrast, the W7A mutant of CBM10-CD was efficiently expressed in E. coli, although the protein bound very weakly to cellulose. NMR spectra of wild-type CBM10, W22A, and W24A were very similar, suggesting that the mutations did not significantly affect the protein fold. Titration of wild-type CBM10, W22A, and W24A with N-bromosuccinimide indicated that Trp22 and Trp24 were on the surface of the protein, while Trp7 was buried. Collectively, these data indicate that Trp22, Trp24, and Tyr8 play a direct role in the binding of Pf Xyn10A CBM10 to cellulose. The results are discussed in the light of the three-dimensional structure of CBM10 [Raghothama, S., Simpson, P. J., Szabó, L., Nagy, T., Gilbert, H. J., and Williamson, M. P. (2000) Biochemistry 39, 978-984].

Bromosuccinimide↗

PubMed2000

Embryogenesis of the central nervous system of the pond snail Lymnaea stagnalis L. An ultrastructural study.

The ultrastructural characteristics of the developing CNS of the pond snail, Lymnaea stagnalis, were investigated, with special attention paid to three specific stages of embryonic development, representing distinctly different phases of both the body morphogenesis and gangliogenesis. These were the 35% (veliger), the 50-55% (metamorphic), and the 75% (post-metamorphic, adult-like) stages of embryonic development. Also, a brief comparison was done with the CNS of hatchlings (100% of embryonic development). During embryogenesis specialized axo-axonic synapses and elements of the glial system, including the ganglionic (neural) sheath, were rarely observed, whereas the frequent occurrence of unspecialized axo-somatic contacts could be demonstrated. Synapse-like axo-axonic connections could be found first in 75% embryos, showing asymmetric vesicle clustering on the "presynaptic" side and increased electron density of the apposed membranes. These phenomena may reflect the dominance of modulatory processes in the CNS during embryogenesis, and the absence of the neural sheath may facilitate trophic and/or hormonal influences within the developing ganglia. The gradual increase in the size of ganglia and the diameter of their neuropils was not accompanied by any widening of the cell body layer or increasing diameter of the nerve cell bodies until the very end of embryogenesis. With respect to the ultrastructural organization of the neuropil, and possibly the entire CNS, a determining stage seems to be that of metamorphosis. Two types of neuropil could be observed at this time; the metamorphosing neuropil with an irregular organization of wavy axon profiles, and well-structured neuropil with a regular organization of axon profiles. Ganglia with irregular or regular neuropil occurred simultaneously in the developing CNS.

Animals↗

PubMed2000

A novel Cellvibrio mixtus family 10 xylanase that is both intracellular and expressed under non-inducing conditions.

Hydrolysis of the plant cell wall polysaccharides cellulose and xylan requires the synergistic interaction of a repertoire of extracellular enzymes. Recently, evidence has emerged that anaerobic bacteria can synthesize high levels of periplasmic xylanases which may be involved in the hydrolysis of small xylo-oligosaccharides absorbed by the micro-organism. Cellvibrio mixtus, a saprophytic aerobic soil bacterium that is highly active against plant cell wall polysaccharides, was shown to express internal xylanase activity when cultured on media containing xylan or glucose as sole carbon source. A genomic library of C. mixtus DNA, constructed in lambdaZAPII, was screened for xylanase activity. The nucleotide sequence of the genomic insert from a xylanase-positive clone that expressed intracellular xylanase activity in Escherichia coli revealed an ORF of 1137 bp (xynC), encoding a polypeptide with a deduced M(r) of 43413, defined as xylanase C (XylC). Probing a gene library of Pseudomonas fluorescens subsp. cellulosa with C. mixtus xynC identified a xynC homologue (designated xynG) encoding XylG; XylG and xynG were 67% and 63% identical to the corresponding C. mixtus sequences, respectively. Both XylC and XylG exhibit extensive sequence identity with family 10 xylanases, particularly with non-modular enzymes, and gene deletion studies on xynC supported the suggestion that they are single-domain xylanases. Purified recombinant XylC had an M(r) of 41000, and displayed biochemical properties typical of family 10 polysaccharidases. However, unlike previously characterized xylanases, XylC was particularly sensitive to proteolytic inactivation by pancreatic proteinases and was thermolabile. C. mixtus was grown to late-exponential phase in the presence of glucose or xylan and the cytoplasmic, periplasmic and cell envelope fractions were probed with anti-XylC antibodies. The results showed that XylC was absent from the culture media but was predominantly present in the periplasm of C. mixtus cells grown on glucose, xylan, CM-cellulose or Avicel. These data suggest that C. mixtus can express non-modular internal xylanases whose potential roles in the hydrolysis of plant cell wall components are discussed.

Cell Wall↗

PubMed2000

Phase I/II trial of cyclophosphamide, mitoxantrone, and escalated doses of carboplatin supported by peripheral-blood stem cells in women with metastatic breast cancer.

PURPOSE: To intensify a regimen of high-dose cyclophosphamide, mitoxantrone, and carboplatin that had previously produced high complete and overall response rates in metastatic breast cancer (MBC). PATIENTS AND METHODS: Forty-four patients with a median age of 43 years (range, 25 to 57 years) and previously untreated MBC who were responding to anthracycline-based or single-agent taxane chemotherapy received cyclophosphamide 1.5 g/m(2)/d and mitoxantrone 16 mg/m(2)/d combined with escalating doses of carboplatin 200 to 500 mg/m(2)/d, each given daily from days -6 to -3. Hematopoiesis was supported by mobilized peripheral-blood stem cells infused on day 0 and by use of granulocyte-macrophage colony-stimulating factor 300 microg/d subcutaneously starting on day 1. RESULTS: A total of six dose levels of carboplatin were examined. Grades 3 and 4 toxicity occurred in 10 patients and one patient, respectively, with grade 3 toxicity occurring in only five of 31 patients treated with < or = 400 mg/m(2) of carboplatin. Major dose-limiting toxicities were cardiac, pulmonary, and renal. Four patients developed congestive heart failure: two had persistently low ejection fraction 11 and 36 months after peripheral-blood stem-cell transplantation (PBSCT), and two recovered. Hematologic recovery to an absolute neutrophil count of greater than 0.5 x 10(9)/L occurred at a median of 11 days (range, 8 to 25 days) and to a platelet count of greater than 20 x 10(9)/L at a median of 10.5 days (range, 6 to 60 days). There were two toxic deaths from sepsis: one on day 27 (level 5) and one from cardiac arrest on day 42 (level 6). CONCLUSION: The maximum-tolerated dose of carboplatin was 400 mg/m(2)/d in combination with mitoxantrone 16 mg/m(2)/d and cyclophosphamide 1,500 mg/m(2), all drugs given over 4 days. This regimen is being tested in a phase III trial of high-dose chemotherapy and PBSCT versus standard treatment.

Adolescent↗

PubMed1999

Specific association of megalin and the Na+/H+ exchanger isoform NHE3 in the proximal tubule.

We investigated whether the renal brush border Na+/H+ exchanger NHE3 exists in assemblies with other proteins in native kidney membranes. To this end we generated monoclonal antibodies (mAbs) against affinity purified NHE3 protein complexes. Hybridomas were selected based on ability to immunoprecipitate NHE3. One of the resulting mAbs (10A3) labeled a high molecular mass (>200 kDa) protein and stained primarily the coated pit region of the proximal tubule in a manner similar to that described for megalin (gp330). We then confirmed that both mAb 10A3 and a known anti-megalin mAb immunoprecipitated and immunoblotted the same protein, namely megalin. mAb 10A3 specifically co-precipitated NHE3 but not villin or NaPi-2 from solubilized renal membranes, indicating specificity of the NHE3-megalin interaction. When immunoprecipitations were performed using either 10A3 or anti-NHE3 mAb 2B9 after separation of solubilized renal proteins by sucrose velocity gradient centrifugation, we found that NHE3 exists in two states with distinct sedimentation coefficients, a 9.6 S megalin-free form and a 21 S megalin-bound form, and that when NHE3 assembles with megalin, epitopes within the carboxyl-terminal 131 amino acids of NHE3 are blocked. Taken together, these findings indicate that a significant pool of NHE3 exists as a multimeric complex with megalin in the brush border of the proximal tubule.

Animals↗

PubMed1999

Pathophysiological effects of human TNF-alpha-producing tumor xenografts in immunosuppressed mice.

Groups of CBA mice immunosuppressed with anti-thymocyte serum (ATS) treatment were xeno-transplanted with either HeLa human cervical carcinoma cells or genetically modified cells expressing the human tumor necrosis factor-alpha (TNF) gene (All cells). Both cell lines were highly resistant to the cytotoxic effects of TNF. If 3 x 10(6) tumor cells were inoculated s.c. into female mice, HeLa cells grew progressively into large tumors and killed 74% of the recipients, while TNF-expressing All cells caused fatal tumor growth only in 22% of the mice. 3 x 10(6) or 1.5 x 10(7). All cells produced progressive tumor growth and lethality in all male recipients. In sera of all the A11-cell-transplanted mice, biologically active TNF was detected shortly (4.5 h) after tumor inoculation (6 39 U/ml), decreasing to below detection level in the circulation by day 3. In recipients of 15 million A11 cells, circulating TNF reappeared and reached high levels (12-1000 U/ml) 3 to 7 weeks later, when the animals bore large tumors (14-23 mm). Generally, such mice became cachectic, severely anemic, hypothermic, and soon died. On account of calcium mobilization from bones, their serum Ca levels were high. Electron microscopy revealed severe liver damage, but there were no signs of chronic arthritis. These results suggest that ATS-treated mice xenotransplanted with TNF-gene-transfected A11 human tumor cells provide a new model for studying the pathophysiological and anti-tumor effects of TNF.

Anemia↗

PubMed1999

MIP-immunoreactive innervation of the snail, Helix pomatia, heart. An ultrastructural study.

The ultrastructural characteristics of the innervation established by MIP-(Mytilus inhibitory peptide) immunoreactive neurons was investigated in the heart of the snail, Helix pomatia, applying correlative light- and electron microscopic pre-embedding immunocytochemistry on Vibratome-slices. In both the auricle and ventricle, the muscle fibers receive a rich innervation by MIP-immunoreactive (IR) varicose fibers. However, the innervation is seasonally changing in the two parts of the heart. The varicosities, containing a morphologically uniform population of large (120-150 nm) electron-dense granules, can be found in three different positions in relation to the muscle fibers: (i) close (15-20 nm) but unspecialized membrane connections between MIP-(IR) varicosities and muscle fibers; (ii) MIP-IR varicosities located relatively far (0.5-several microm) from the muscles fibers; (iii) MIP-IR profiles localized freely in the extracellular space among the loosely arranged muscle fibers. A general modulatory role of MIP in regulating the heart activity of Helix is suggested.

Animals↗

PubMed1998

All three surface tryptophans in Type IIa cellulose binding domains play a pivotal role in binding both soluble and insoluble ligands.

The three surface tryptophans of the Type IIa cellulose binding domain of Pseudomonas fluorescens subsp. cellulosa xylanase A (CBD(XYLA)) were independently mutated to alanine, to create the mutants W13A, W49A and W66A. The three mutant proteins were purified, and their capacity to bind to a variety of ligands was determined. The mutant proteins have native-like structures but exhibited much weaker affinity for crystalline and amorphous cellulose and for cellohexaose than the wild type. These data indicate that all three tryptophans are important for binding to cellulose, and support a model in which the three tryptophans form an aromatic strip on the surface of the protein that binds to a single cellulose.

Bacterial Proteins↗

PubMed1998

Integrative promoter cloning plasmid vectors for Rhizobium meliloti.

A set of integrative 'promoter probe' plasmids were constructed for both translational and transcriptional fusions. The vectors are based on the broad host range, low copy number plasmid pRK290 (IncPl) in which the attachment site of Rhizobium phage 16-3 and the lacZ gene of Escherichia coli were combined. The vectors integrate into the chromosome of Rhizobium meliloti, providing also the advantages of the single copy promoter probe cassettes. Thus they fulfil the prerequisite of the systems used for investigating gene regulation. The plasmids were applied for the study of the transcription regulation of the 16-3 phage. Their versatile use is also demonstrated.

Amino Acid Sequence↗

PubMed1997

Parvalbumin- and calbindin-containing neurons express c-fos protein in primary and secondary (mirror) epileptic foci of the rat neocortex.

The present experiments aimed at the description and further immunocytochemical characterization of activated neocortical neurons expressing the c-fos gene. Focal seizures were induced by the topical application of isotonic, isohydric 4-aminopyridine solution to the frontal neocortex of adult anesthetized Wistar rats. The EEG of both hemispheres was recorded from the surface of the skull. The animals were perfused with fixative, coronal plane vibratome sections were cut and stained with cocktails containing polyclonal c-fos and monoclonal calbindin or parvalbumin antibodies. The polyclonal c-fos antibody was tested with Western blotting and the diffusion of 4-aminopyridine investigated with autoradiography of [3H]4-aminopyridine. The c-fos protein was detected in every layer of the neocortex (primary focus) and in some allocortical areas of the treated hemisphere. Scattered immunostained nuclei were observed in layers II, III, IV and VI of the contralateral neocortex (mirror focus). Several parvalbumin- and calbindin-positive neurons contained the c-fos protein in both foci. The medium-sized non-pyramidal parvalbumin neurons were found in layers II-IV and VI of the neocortex and in stratum multiforme of the prepiriform cortex. The c-fos protein was colocalized with calbindin mainly in layers II and III in small and medium-sized non-pyramidal neurons. The results prove that focal epileptiform activity of the neocortex activates diverse inhibitory neuronal populations. As concluded, the inhibitory control is probably more effective in the contralateral hemisphere (mirror focus) than on the side of 4-APY treatment (primary focus).

Animals↗