Polyamine metabolism in transient focal ischemia of rat brain.

Polyamine metabolism was studied in rat brains subjected to 30 min transient cerebral ischemia by measuring the activity of the key enzyme ornithine decarboxylase (ODC) and levels of the polyamines putrescine, spermidine and spermine. A transient increase in ODC activity was apparent after 4 h of recirculation in the ipsilateral cortex and striatum (P less than 0.05). Putrescine levels were significantly increased in the ipsilateral striatum after 4 h of recirculation, and after 24 h of recirculation in both the ipsilateral cortex and striatum. During ischemia spermidine levels were significantly reduced in the ipsilateral hemisphere and spermine levels in the ipsilateral cortex. It is suggested that during ischemia polyamines are released from neurons into the extracellular compartment and cleared into the blood.     

Ausstattung von Rettungsmitteln

Ohne Zusammenfassung Dr. H.R. Paschen Abteilung für An?sthesiologie und Intensivmedizin, Evangelisches Amalie-Sieveking-Krankenhaus, Haselkamp 33, 22359 Hamburg, E-Mail: h.paschen@amalie.de     

Polyamine metabolism in gliomas

Summary Biosynthesis of the polyamines putrescine, spermidine, and spermine has been found to be activated in tissues with cellular proliferation. In the present study we have investigated polyamine levels and the activity of the first rate-limiting enzyme ornithine decarboxylase (ODC) in tumour samples obtained during operation of 202 patients with gliomas. Biochemical data were closely related to the grading of malignancy and to the morphological characteristics of each sample. Mean ODC activity was significantly higher in all gliomas as compared to peritumoural non-neoplastic brain. Furthermore, it was significantly higher (p 0.001) in anaplastic gliomas who grade III and IV (9.0 ± 9.6 nmol/g/h) than in gliomas WHO grade I and II (3.3 ± 4.2 nmol/g/h). Highest enzyme activity (58.5 nmol/g/h) was found in solid and vital parts of malignant tumours, whereas predominantly necrotic areas exhibited low ODC activity (< 1 nmol/g/h). Thus, intra- and interindividual variability of ODC activity corresponded well to histomorphological heterogeneity in high-grade gliomas. Putrescine levels also increased with rising grade of malignancy, whereas spermidine and spermine levels did not correlate with the histological grading. In conclusion, high ODC activity represents a biochemical marker of malignancy in gliomas, but low values do not prove benignity. The present study reinforces the need of further and more extensive tumour sampling closely related to follow-up investigations in the heterogeneous group of gliomas.     

Homocysteine-induced changes in mRNA levels of genes coding for cytoplasmic- and endoplasmic reticulum-resident stress proteins in neuronal cell cultures

Elevated homocysteine levels have been suggested to contribute to various pathological states of the brain. However, the basic mechanisms underlying homocysteine-induced neurotoxicity have not yet been fully elucidated. In the present series of experiments, we investigated the effect of homocysteine on mRNA levels of genes coding for cytoplasmic- or endoplasmic reticulum-resident stress proteins. Primary neuronal cell cultures were exposed to different homocysteine levels for 1-24 h. Cell injury was evaluated using the MTT assay, protein synthesis was studied by measuring the incorporation of L-[4,5-3H]leucine into proteins, mRNA levels of hsp70, gadd153, grp78, and grp94 were evaluated by quantitative PCR, and changes in protein levels of hsp70, grp78 and grp94 were analyzed by immunoblotting. Exposure of cells to 5 or 10 mM homocysteine for 24 h induced marked cell injury (decrease of viability to 58 or 45% of control respectively). After 6 h treatment, gadd153, grp78 and grp94 mRNA levels increased markedly, but only when cells were exposed to levels of homocysteine high enough to induce cell injury. In addition, hsp70 mRNA levels and protein synthesis were significantly reduced. At earlier (1 or 3 h) or later (12 or 24 h) time intervals, homocysteine exposure induced a marked increase in mRNA levels of all genes studied. GRP78 and GRP94 protein levels were increased in cells exposed to 5 mM homocysteine for 24 h but not in cells exposed to 10 mM homocysteine. HSP70 protein levels, in contrast, were decreased in cells exposed to homocysteine for different periods. The expression of genes coding for ER-resident stress proteins is specifically activated under conditions of ER stress. The close relationship between the extent of cell injury and increase in grp78 mRNA levels suggests that ER dysfunction may contribute to the pathological process. The results imply that the ER is an intracellular target of homocysteine toxicity.     

Detection of spontaneous CD4+ T-cell responses in melanoma patients against a tyrosinase-related protein-2-derived epitope identified in HLA-DRB1*0301 transgenic mice.

PURPOSE: The frequently expressed differentiation antigen tyrosinase-related protein-2 (TRP-2) has repeatedly been described as a target of spontaneous cytotoxic T-cell responses in melanoma patients, suggesting that it might be an ideal candidate antigen for T cell-based immunotherapy. As a prerequisite for immunization, T-cell epitopes have to be identified. Whereas a number of HLA class I-presented TRP-2-derived epitopes are known, information about HLA class II-presented antigenic ligands recognized by CD4+ T helper (Th) cells is limited. EXPERIMENTAL DESIGN: The search for TRP-2-derived Th epitopes was carried out by competitive in vitro peptide binding studies with predicted HLA-DRB1*0301 ligands in combination with peptide and protein immunizations of HLA-DRB1*0301 transgenic mice. In vivo selected candidate epitopes were subsequently verified for their immunogenicity in human T-cell cultures. RESULTS: This strategy led to the characterization of TRP-2(60-74) as an HLA-DRB1*0301-restricted Th epitope. Importantly, TRP-2(60-74)-reactive human CD4+ Th cell lines, specifically recognizing target cells loaded with recombinant TRP-2 protein, could be established by repeated peptide stimulation of peripheral blood lymphocytes from several HLA-DRB1*03+ melanoma patients. Even short-term peptide stimulation of patients' peripheral blood lymphocytes showed the presence of TRP-2(60-74)-reactive T cells, suggesting that these T cells were already activated in vivo. CONCLUSION: Peptide TRP-2(60-74) might be a useful tool for the improvement of immunotherapy and immune monitoring of melanoma patients.     

Genes associated with pro-apoptotic and protective mechanisms are affected differently on exposure of neuronal cell cultures to arsenite. No indication for endoplasmic reticulum stress despite activation of grp78 and gadd153 expression

The effect of arsenite exposure on cell viability, protein synthesis, energy metabolism and the expression of genes coding for cytoplasmic (hsp70) and endoplasmic reticulum (ER; gadd153, grp78, grp94) stress proteins was investigated in primary neuronal cell cultures. Furthermore, signs of ER stress were evaluated by investigating xbp1 mRNA processing. Arsenite levels of 30 and 100 microM induced severe cell injury. Protein synthesis was reduced to below 20% of control in cultures exposed to 30 and 100 microM arsenite for 1 h, and it remained markedly suppressed until 24 h of exposure. Arsenite induced a transient inhibition of energy metabolism after 1 h of exposure, but energy state recovered completely after 3 h. Arsenite exposure affected the expression and translation of genes coding for HSP70 and GRP78, GRP94, GADD153 to different extents. While hsp70 mRNA levels rose drastically, approximally 550-fold after 6 h exposure, HSP70 protein levels did not change over the first 6 h. On the other hand, gadd153 mRNA levels rose only approximately 14-fold after 6 h exposure, while GADD153 protein levels were markedly increased after 3 and 6 h exposure. HSP70 protein levels were markedly increased and GADD153 protein levels decreased to almost control levels in cultures left in arsenite solution for 24 h, i.e. when only a small fraction of cells had escaped arsenite toxicity. Arsenite exposure of neurons thus induced an imbalance between pro-apoptotic and survival-activating pathways. Despite the marked increase in gadd153 mRNA levels, we did not observe signs of xbp1 processing in arsenite exposed cultures, indicating that arsenite did not produce ER stress.     

Role of calcium in neuronal cell injury: which subcellular compartment is involved?

It is widely believed that calcium plays a primary role in the development of neuronal cell injury in different pathological states of the brain. Disturbances of calcium homeostasis may be induced in three different subcellular compartments, the cytoplasm, mitochondria or the endoplasmic reticulum (ER). The traditional calcium hypothesis holds that neuronal cell injury is induced by a marked increase in cytoplasmic calcium activity during stress (e.g., cerebral ischemia). Recently, this hypothesis has been modified, taking into account that under different experimental conditions the extent of cell injury does not correlate closely with calcium load or total calcium influx into the cell, and that neuronal cell injury has been found to be associated with both increases and decreases of cytoplasmic calcium activity. The mitochondrial calcium hypothesis is based on the observation that after a severe form of stress there is a massive influx of calcium ions into mitochondria, which may lead to production of free radicals, opening of the mitochondrial permeability transition (MPT) pore and disturbances of energy metabolism. However, it has still to be established whether drugs such as cyclosporin A are neuroprotective through their effect on MPT or through the blocking of processes upstream of MPT. The ER calcium hypothesis arose from the observation that ER calcium stores are depleted after severe forms of stress, and that the response of cells to disturbances of ER calcium homeostasis (activation of the expression of genes coding for ER resident stress proteins and suppression of the initiation of protein synthesis) resembles their response to a severe form of stress (e.g., transient ischemia) implying common underlying mechanisms. Elucidating the exact mechanisms of calcium toxicity and identifying the subcellular compartment playing the most important role in this pathological process will help to evaluate strategies for specific therapeutic intervention.