Variations in elemental compositions of rat hippocampal formation between acute and latent phases of pilocarpine-induced epilepsy: an X-ray fluorescence microscopy study.

There is growing experimental evidence that tracing the elements involved in brain hyperexcitability, excitotoxicity, and/or subsequent neurodegeneration could be a valuable source of data on the molecular mechanisms triggering or promoting further development of epilepsy. The most frequently used experimental model of the temporal lobe epilepsy observed in clinical practice is the one based on pilocarpine-induced seizures. In the frame of this study, the elemental anomalies occurring for the rat hippocampal tissue in acute and silent periods after injection of pilocarpine in rats were compared. X-ray fluorescence microscopy was applied for the topographic and quantitative elemental analysis. The differences in the levels of elements such as P, S, K, Ca, Fe, Cu, and Zn between the rats 3 days (SE72) and 6 h (SE6) after pilocarpine injection as well as naive controls were examined. Comparison of SE72 and control groups showed, for specific areas of the hippocampal formation, lower levels of P, K, Cu, and Zn, and an increase in Ca accumulation. These results as well as further analysis of the differences between the SE72 and SE6 groups confirmed that seizure-induced excitotoxicity as well as mossy fiber sprouting are the mechanisms involved in the neurodegenerative processes which may finally lead to spontaneous seizures in the chronic period of the pilocarpine model. Moreover, in the light of the results obtained, Cu seems to play a very important role in the pathogenesis of epilepsy in this animal model. For all areas analyzed, the levels of this element recorded in the latent period were not only lower than those for controls but were even lower than the levels found in the acute period. The decreased hippocampal accumulation of Cu in the phase of behavior and EEG stabilization, a possible inhibitory effect of this element on excitatory amino acid receptors, and enhanced seizure susceptibility in Menkes disease (an inherited Cu transport disorder leading to Cu deficiency in the brain) suggest a neuroprotective role rather than neurodegenerative and proconvulsive roles of Cu in pilocarpine-induced epilepsy.

Synchrotron radiation Fourier-transform infrared and Raman microspectroscopy study showing an increased frequency of creatine inclusions in the rat hippocampal formation following pilocarpine-induced seizures.

In the present work, synchrotron radiation Fourier-transform infrared (SRFTIR) and Raman microspectroscopies were used to evaluate a possible role of creatine in the pathogenesis and progress of pilocarpine-evoked seizures and seizure-induced neurodegenerative changes in the rat hippocampal tissue. The main goal of this study was to identify creatine deposits within the examined brain area, to analyze their frequency in epileptic animals and naive controls and to examine correlations between the number of inclusions in the hippocampal formation of epileptic rats and the quantitative parameters describing animal behavior during 6-h observation period after pilocarpine injection. The presence of creatine in the brain tissue was confirmed based on the vibrational bands specific for this compound in the infrared and Raman spectra. These were the bands occurring at the wavenumbers around 2800, 1621, 1398, and 1304 cm(-1) in IR spectra and around 1056, 908 and 834 cm(-1) in the Raman spectra. Creatine was detected in eight of ten analyzed epileptic samples and in only one of six controls under the study. The number of deposits in epileptic animals varied from 1 to 100 and a relative majority of inclusions were detected in the area of the Dentate Gyrus and in the multiform hippocampal layer. Moreover, the number of creatine inclusions was positively correlated with the total time of seizure activity.

Synchrotron FTIR micro-spectroscopy study of the rat hippocampal formation after pilocarpine-evoked seizures.

In the present work, synchrotron radiation Fourier transform infrared (SRFTIR) micro-spectroscopy and imaging were used for topographic and semi-quantitative biochemical analysis of rat brain tissue in cases of pilocarpine-induced epilepsy. The tissue samples were analyzed with a beam defined by small apertures and spatial resolution steps of 10 microm which allowed us to probe the selected cellular layers of hippocampal formation. Raster scanning of the samples has generated 2D chemical cartographies revealing the distribution of proteins, lipids and nucleic acids. Spectral analysis has shown changes in the saturation level of phospholipids and relative secondary structure of proteins. Special interest was put in the analysis of two areas of the hippocampal formation (sector 3 of the Ammon's horn, CA3 and dentate gyrus, DG) in which elemental abnormalities were observed during our previous studies. Statistically significant increase in the saturation level of phospholipids (increased ratio of the absorption intensities at around 2921 and 2958 cm(-1)) as well as conformational changes of proteins (beta-type structure discrepancies as shown by the increased ratio of the absorbance intensities at around 1631 and 1657 cm(-1) as well as the ratio of the absorbance at 1548 and 1657 cm(-1)) were detected in pyramidal cells of CA3 area as well as in the multiform and molecular layers of DG. The findings presented here suggest that abnormalities in the protein secondary structure and increases in the level of phospholipid saturation could be involved in mechanisms of neurodegenerative changes following the oxidative stress evoked in brain areas affected by pilocarpine-induced seizures.

Probing molecular interaction between concanavalin A and mannose ligands by means of SFM.

Recently, the scanning force microscope (SFM) has been widely used for direct monitoring of specific interactions between biologically active molecules. Such studies have employed the SFM liquid-cell setup, which allows measurements to be made in the native environment with force resolution down to a tenth of a picoNewton. In this study, the ligand-receptor strength of monoclonal anti-human prostatic acid phosphatase and prostatic acid phosphatase, representing an antigen-antibody system with a single type of interaction, was determined. Then, the interaction force occurring between concanavalin A and the carbohydrate component of the glycoproteins arylsulfatase A and carboxypeptidase Y was measured. High mannose-type glycans were sought on the human prostate carcinoma cell surface. Application of an analysis based on the Poisson distribution of the number of bonds formed in all these measured systems allowed the strength of the molecular interaction to be calculated. The values of the force acting between two single molecules were 530+/-25, 790+/-32, and 940+/-39 pN between prostatic acid phosphatase and monoclonal anti-human prostatic acid phosphatase, between concanavalin A and arylsulfatase A, and between concanavalin A and carboxypeptidase Y, respectively. The value calculated from data collected for the force between concanavalin A and mannose-containing ligands present on the surface of human prostate carcinoma cells was smaller, 116+/-17 pN. The different values of the binding force between concanavalin A and mannose-containing ligands were attributed to the structural changes of the carbohydrate components.

The effect of dihydrotestosterone on transcription of prostatic acid phosphatase mRNA in human hyperplastic gland.

The effect of 5alpha-dihydrotestosterone (DHT) on the level of human prostatic acid phosphatase (hPAP) mRNA was studied using tissue slices from various benign prostatic hyperplastic glands. The absence of DHT in the incubation medium led to a gradual, significant decrease of the hPAP mRNA level. Addition of the hormone induced hPAP mRNA in a time- and dose-dependent manner. The maximal 2-4-fold induction by 10(-9) M DHT was observed after 3-5 h of incubation, and then the hPAP mRNA level was 6-20-fold higher than that in a parallel sample incubated without DHT. The results suggest that DHT is necessary to sustain the expression of hPAP in hyperplastic prostates.

Stabilization of human prostatic acid phosphatase by coupling with chondroitin sulfate.

Human prostatic acid phosphatase (PAP) (EC 3.1.3.2) was covalently linked to chondroitin sulfate A from whale cartilage. In order to bind the protein amino groups with the preactivated carboxyl groups of chondroitin sulfate, 1-ethyl-3-(3'-dimethylaminepropyl)carbodiimide and N-hydroxysulfosuccinimide were used as coupling agents. The product was soluble and enzymatically active. The activity was on average 25% higher than that of the free enzyme. The product was heterogeneous in respect to charge and Mr (50-1500) kDa, as determined by chromatography on Sephacryl S 300 and polyacrylamide gel electrophoresis. The resulting polymers contained covalently bound chondroitin sulfate, as shown by the biotin-avidin test. The modified enzyme is more resistant against various denaturing agents, e.g., urea, ethanol, and heat. Thus covalent modification of PAP by cross-linking to chondroitin sulfate could be the preferred method for stabilization of its biological activity.

Immunoreactivity of proteolytic degradation products of human prostatic acid phosphatase.

Prostatic acid phosphatase (EC 3.1.3.2) was fragmented by trypsin and papain in the presence of sodium dodecyl sulphate. Trypsin-catalysed cleavage gave a peptide of 33 kDa which was subsequently trimmed to 18 kDa, 15 kDa and 13 kDa peptides. Even the small tryptic fragments reacted with antiphosphatase antibodies from rabbit serum and with monoclonal antibody mAb-14. Papain treatment under these conditions resulted in the release of a 40 kDa peptide which was gradually reduced to a 18 kDa peptide. The monoclonal antibody mAb-14 to the prostatic phosphatase was bound exclusively to the 50 kDa subunit of the phosphatase and to the 40 kDa peptide. The results suggest that the monoclonal antibody mAb-14 binding site represents a "local" sequence rather than a "conformational" one and does not require an extensive tertiary folding of the antigen molecule.

Stabilization of human prostate acid phosphatase by cross-linking with diimidoesters.

1. Modification of dimeric human prostate acid phosphatase (EC 3.1.3.2) by diimidoesters leads to the formation of water-soluble preparations of high enzymatic activity, resistant to denaturing agents. 2. Monomeric, dimeric, trimeric and tetrameric species were found in SDS-polyacrylamide gel electrophoresis of the phosphatase cross-linked with dimethyl-suberimidate, and dimeric, trimeric and tetrameric enzymatically active species on thin-layer Sephadex 200 gel filtration. This molecular pattern evidenced formation of the inter-subunit covalent linkages. All molecular forms are immunoreactive against the polyclonal rabbit anti-phosphatase antibodies. 3. The catalytic properties of the modified phosphatase are almost the same as those of the native enzyme. Differences in the optical properties between the modified and the native enzymes point to slight conformational transitions in the modified enzyme.