Department of Clinical Pharmacology

Sialylated glycoconjugates seem to play crucial role in the mechanisms that control the most important functions of the body. Sialylation is an important mechanism for the regulation of intercellular interactions that underlie neuronal plasticity as well as immune defense in the central nervous system (CNS). In this study, we analyzed the effect of lipopolysaccharide (LPS) on sialylation pattern in several regions of CNS. Additionally, we tested the effects of inflammatory stimulation on Siglec-F expression in microglial cells. Using lectin blotting with Maackia amurensis and Sambucus nigra agglutinins and immunostaining with antibody directed against PSA-NCAM we demonstrated altered expression of sialylated glycoconjugates differentially due to LPS-induced inflammation. We found that LPS caused significant increase of α2,3and α2,6-linked sialic acids in the hippocampus and spinal cord. In the prefrontal cortex, the level of α2,3-linked sialic acids in selected glycoconjugates tended to be increased (p>0.05), while α2,6-linked sialic acids were reduced (p<0.05), while the expression of PSA-NCAM in all analyzed structures were significantly higher in comparison to the control group. The expression of Siglec-F in microglial cells stimulated with LPS remained unchanged. Given the significance of glycans in the brain biology we can conclude that sialic acids and their receptors Siglec may be crucial regulators of immune response in the CNS.

It has been shown that free radical dam age m ay be involved in eth anol-induced cytotox icity in cultured neural cells. Since changes in oxidative m eta bolism and the resultin g lipid peroxidation readily m odify biological mem branes and alter cell function s we studied the eOE ect of eth anol and its m etabolite acetaldehyde on rat astroglial fatty acids pro® les in the m ost com m on lipid classes of mitochondrial and m icrosomal m em branes, i.e. phosphatidylch oline (PC ) and phosphatidyletha nola mine (PE). Rat astroglia l cells were grown for 1 week in the presence of 50 m M or 100 m M ethanol. To exam ine aceta ldehyde eOE ects we used a 4-day co-cultu re model consisting of astroglia l cells and alcohol dehydrogenase-transfected Chinese hamster ovar y (CH O) cells. Aceta ldehyde produced by these cells reached 172 l M and 265 l M , respectively, for eth anol concent rations of 10 and 20 m M . M itochon drial and m icrosom al m embranes were prepared by diOE erential centr ifugation, phosphatidylch oline and phosphatidyletha nola m ine were separated using thin layer chromatog raphy and fatty acid quantitation was perform ed by G LC. Neither eth anol nor acetaldehyde changed the m itochon drial phosphatidylch oline or phosphatidylethanolam ine pro® les of tota l saturated, mono-un saturated or polyuns aturated fatty acids. H owever, som e signi® cant alterations in particula r fatty acids appeared especially after acetaldehyde but also after the highest eth anol dose. In microsom al phosphatidylch oline monouns aturated fatty acids were signi® cantly increa sed after both , etha nol and acetaldehyde exposure. Am ong polyuns aturated fatty acids, arachidonic acid was found to be especially aOE ected by both etha nol and acetaldehyde. Sim ilar decreases were obser ved in adrenic, docosa pentaenoic and docosa hexaenoic acids in the groups treated with eth anol. In m icrosom al phosphatidyletha nola m ine, eth anol and acetaldehyde decreased m onounsa turated and som e polyunsaturated fatty acids. These data sup port the role of peroxidative processes in cultured rat astroglia exposed to etha nol and point to the role of aceta ldehyde in this m ech anism.

The central nervous system (CNS) is a complex and precise mechanism that controls the most highest functions of the body. All of them depend on the cellular and molecular interactions called by neurobiologists "cellular plasticity". The CNS is a flexible structure but its regeneration after damage is strongly limited. Better understanding of cellular and molecular basis of brain repair can open new way in the development of therapeutic tools for neurodegeneration. Among many molecules that participate in the formation of neuronal networks, neural cell adhesion molecule (NCAM) and its sialylated derivative seem to play crucial role in the life of brain. In particular, polysialylated cell adhesion molecule (PSA-NCAM) is proposed to participate in the neuroprotective response in neurodegeneration by reducing of AMPA/NMDA receptors sensitivity to glutamate and facilitating disconnection of cell-cell interactions. These mechanisms protect from excitotoxic damage and promote dendritic/ spine re-growth. This review briefly focuses on the expression and role of PSA-NCAM in neurodegenerative diseases and its potential application in therapy.

St. John's wort (Hypericum perforatum) is one of the leading psychotherapeutic phytomedicines. Beneficial effects of this herb in the treatment of mild to moderate depression are well known. In this study we tested a hypothesis that St. John's wort alleviates age-related memory impairments by increasing the levels of cyclic adenosine 3', 5'-monophosphate response element binding protein (CREB) and phosphorylated CREB (pCREB) in hippocampus. Middleaged rats (18 month-old) displayed a decline in the acquisition of spatial working memory (p < 0.001) in the Morris water maze (MWM). Chronic administration of Hypericum perforatum (HP) (350 mg/kg for 21 days), potently and significantly improved the processing of spatial information in the aged rats (p < 0.001). Also the herb increased the levels of pCREB in the aged rat's hippocampus (p < 0.01) as measured by western immunoblotting. Aging caused significant locomotor impairments as tested in the open field (p < 0.001) but not in the MWM test. However, these were unaffected by treatment with HP. Thus, this study indicates that St. John's wort effectively prevents aging-induced deterioration of spatial memory in 18 month-old rats, possibly by the activation of CREB regulated genes associated with memory formation. It appears that mechanism is probably inactive in young rats.

Sialidases are acid exoglycosidases that catalyse the removal of sialic acid from non-reducing end of sialoglucoconjugated substrates. Synaptic plasticity depends on sialylation state of proteins and lipids mediated by sialic acid-metabolizing enzymes. Since chronic stress causes both, hippocampal atrophy and impairment of learning, it is reasonable to investigate whether sialidase is implicated in these processes. In this study, we tested effects of chronic stress (immobilization, 2 h daily, 21 days) or chronic corticosterone administration (5 mg/kg, sc, daily) on sialidase activity and sialylated NCAMs expression in rat hippocampus.

Tamoxifen can exert its effects through the competitive inhibition of estrogen receptors or other mechanisms. HepG2 cells lacking estrogen receptors and engineered to overexpress CYP3A4, the most important CYP to metabolize the drug, appear to be a good model to study the effects of tamoxifen metabolites. Tamoxifen altered cell cycle of transduced HepG2 cells, decreased G0/G1 cell numbers, diminished proliferation index and induced cell death mostly in cells overexpressing CYP3A4 but was without significant effect on cytotoxicity or proliferation of cells engineered to overexpress CYP2E1 or on empty vector transfected cells. Tamoxifen did not change MDR1 levels irrespectively on CYP450s expression, but inhibited by approximately 50% p-gp functions in all cell types. Drug treatment significantly increased dehydroepiandrosterone sulfotransferase activity and sulfotransferase inhibition significantly decreased tamoxifen cytotoxicity. Our results support the view that metabolic activation of tamoxifen in liver cells may proceed via CYP450-mediated metabolism and subsequent sulfotransferase-mediated activation and point to the role of CYP3A4 and dehydroepiandrosterone sulfotransferase in adverse tamoxifen effects. #

The effect of acetaldehyde on astrocytes have been investigated because not only do they play an important role in brain maturation but also recent reports have shown their delayed proliferation following both 'in vivo' and 'in vitro' ethanol exposure. Biochemical parameters related to apoptotic and necrotic processes were examined in primary cultures of rat astrocytes exposed for 4 days to acetaldehyde generated from ethanol by co-cultured alcohol dehydrogenase-transfected Chinese hamster ovary cells. Acetaldehyde levels in the culture media attained concentrations of approximately 450 mM. To study ethanol effects, alcohol oxidation was inhibited by Ž . 4-methylpyrazole an inhibitor of alcohol dehydrogenase . Acetaldehyde but not ethanol increased intracellular calcium levels by 155%. Moreover, significant DNA fragmentation was detected using a random oligonucleotide primed synthesis assay, by flow cytometry and when using agar gel electrophoresis. Transglutaminase activity was elevated in the cells treated with acetaldehyde but when acetaldehyde formation was inhibited by 4-methylpyrazole the enzyme activity was unaffected. Nitrate levels in the culture media were unchanged. Additionally, microscopic examination of cell nuclei revealed chromatin condensation in astrocytes exposed to acetaldehyde. It can be concluded, that in 'in vitro' acetaldehyde exposed rat astrocytes apoptotic pathways are activated. q

Since it is known that sialic acid participates in neuronal plasticity, it is resonable to investigate its role in microglia-neuron interactions. In this study, we tested the effects of enzymatic removal of sialic acid on neurite and cell body density in microglia-neuron co-cultures. Additionaly, we analyzed the expression of Siglec-F protein, putative receptor for sialic acids, in microglial cells as well as its affinity to neurons. The results showed that removal of sialic acids affects neuronal integrity and changes microglial morphology. In presence of microglial cells, endoneuraminidase and a-neuraminidase significantly reduced neurite density (p < 0.05). Endoneuraminidase (p < 0.05) and a-neuraminidase (p > 0.05) decreased the number of neuronal cell bodies in comparison to control co-cultures. Neuraminidases-treated neurons showed reduced binding of Siglec-F protein, which we found in microglial cells. Our results suggest that interactions between sialic acids and Siglec receptors may protect neuronal integrity during neurodegenerative processes.

The serum levels of matrix metalloproteinase-9 (MMP-9) in neuropsychiatric disorders of adults have been widely investigated. So far, no studies have been conducted on the relationship of MMP-9 and cognitive domains in children with two phenotype models, attention deficit/ hyperactivity disorder and hyperkinetic disorder (ADHD/ HKD). The aim of this research was to evaluate and test the hypothesis that serum MMP-9 levels are associated with the severity of symptoms in children with ADHD/HKD and to compare the results in two models of this disorder. The study group comprised 37 Caucasian boys aged 7-12 years with HKD, being a subset of the combined ADHD subtype. Intellectual functions were measured using Wechsler Intelligence Scale for Children-Revised. The analysis of serum concentrations of MMP-9 was based on a quantitative sandwich ELISA. The statistical regression analysis revealed a correlation between increased serum MMP-9 levels and severity of symptoms in the ADHD (b = 0.33; p = 0.043) and HKD (b = 0.34, p = 0.037) model. According to the results, elevated levels of serum MMP-9 in boys with HKD may be associated with clinical impulsivity domain (b = 0.38; p = 0.019).

Aim of the study: Cirsium rivulare (Jacq.) All. (Asteraceae) is a herbaceous perennial plant occurring in Central Europe. It has been traditionally used in Polish folk medicine to treat anxiety. In the present study methanolic extracts from flowers and leaves of Cirsium rivulare containing flavonoid compounds linarin, pectolinarin, apigenin, hispidulin, their glycosides and a newly isolated compound isokaemferide 7-O-(6 -methylglucuronide) were studied for anxiolytic and pro-cognitive properties. Materials and methods: Male Wistar rats (150-160 g) were used. They were treated orally with standardized methanol extracts of flowers and leaves of Cirsium rivulare and subsequently tested for memory in passive avoidance (PA) and object recognition (OR) tests. Auxiliary tests for motor (open field, OF) and emotional (elevated 'plus' maze, EPM) effects of the above treatments were also employed. Results: We found that the extract from flowers of Cirsium rivulare, in addition to its anxiolytic effects as measured in the EPM, improves memory of the appetitively (by curiosity, OR) and aversively (by footshook, PA) motivated tasks. This is in contrast to classical anxiolytics as for example benzodiazepines that typically impair memory. The extract from leaves of Cirsium rivulare showed some anxiolytic properties in the EPM, and no effect in both cognitive tests. The examined extracts of Cirsium rivulare did not affect psychomotor exploratory activity of rats tested in the OF. Conclusions: These results suggest that the flavonoids from Cirsium rivulare possess anxiolytic and procognitive effects, the extract from flowers being more pro-cognitive and that from the leaves more anxiolytic.

Although a number of studies confirm the important role of metabolites in the cytotoxicity of acetaminophen, its precise mechanisms remain unknown. Acetaminophen is metabolized by microsomal enzymes. Cytochrome P4502E1 (CYP2E1) mediated N-hydroxy-lation results in the formation of N-acetyl-benzo-quinoneimine, a highly reactive intermediate. We examined biochemical parameters related to necrotic and apoptotic processes in acetaminophen-exposed PC12 cells is and in a PC12 cell line genetically engineered to express human CYP2E1. Both the [ 3 H]thymidine incorporation test and the protein assay uniformly showed dose-and timerelated significant growth retardation in both cell lines exposed to the drug. This was more evident in CYP2E1transfected cells. Moreover, the cytotoxic effect of acetaminophen was increased as evidenced by lactate dehydrogenase activity in the culture medium. Both random oligonucleotide primed synthesis assay and enzymelinked immunosorbent assay revealed significant DNA fragmentation in both cell lines, which was greater in transfected cells, reaching about 11% of total cellular DNA. These results were confirmed by flow cytometry and microscopic examination of cell nuclei. Intracellular calcium levels were increased only in transfected cells, approximately threefold when 5 mM acetaminophen was administered for 48 h. These results indicate the cytotoxic effects of acetaminophen via apoptosis, necrosis, and growth retardation. While the precise mechanism remains obscure, it seems that DNA fragmentation and apoptotic cascade represent a preliminary biochemical event in acute cell death, and that acetaminophen biotransformation by CYP2E1 stimulates this pathway.

It is recognized that chronic stress is an important risk factor for the development of several cognitive impairments involving working memory. Working memory refers to the memory in which the information to be remembered changes from trial-to-trial and should be assessed in a task able to detect retrieval of that information. In the present study, we tested the hypothesis that preventive administration of Hypericum perforatum (also named St John's wort) may counteract the working memory impairments caused by repeated stress. Specifically, we attempted to characterize the preventive action of longlasting treatment with St John's wort (350 mg/kg, p.o.) on the spatial working memory impairments caused by chronic restraint stress (2 h daily for 21 days) or durable medication with exogenous corticosterone (5 mg/kg, s.c.) in male Wistar rats. Spatial working memory was tested in Barnes maze (BM) and in the Morris water maze (MWM). We found that H. perforatum prevented the deleterious effects of both chronic restraint stress and prolonged corticosterone on working memory measured in both tests. The herb significantly improved hippocampus dependent spatial working memory in comparison with control (p<0.01) and alleviated some other negative effects of stress on cognitive functions.

Angiotensin peptides produced by the brain reninangiotensin system have established roles in cognition, but there is no mechanistic basis of angiotensin effects on memory. Astroglial cells present throughout the whole brain, synthesize all the components of the renin-angiotensin system and express angiotensin receptors; therefore our aim was to assess changes in intracellular signalling pathways related to memory formation, particularly the activation of CREB and ERK1/2 in astroglial cells grown in the presence of angiotensin peptides. Cultured rat astroglial cells were treated for 24 h with 10 μM angiotensin II and/or 10 μM angiotensin IV in the presence or absence of 100 μM losartan (AT1receptor antagonist) or 100 μM PD123319 (AT2-receptor antagonist). Both angiotensin peptides alone were without effect on culture protein levels and cell viability and did not induce oxidative stress, but both peptides together slightly elevated cell growth rates and increased damaged, apoptotic cell numbers. This effect was most probably mediated by the AT1 receptor. Angiotensin II but not angiotensin IV increased intracellular calcium via activation of AT1 receptor. Angiotensin IV but not angiotensin II increased extracellularregulated protein kinases 1 and 2 (ERK1/2) by 65% and T202, T204 phosphorylated ERK1/2 levels by 36%; this effect was blocked in part by both losartan and PD123319. Angiotensin II but not angiotensin IV increased cyclic AMPresponsive element binding protein (CREB) expression by almost 100% and elevated Ser 133-phosphorylated CREB levels by 56%. These effects were also inhibited in part by both losartan and PD123319. Our results indicate that CREB activation in cultured rat glial cells is mediated mostly by angiotensin II. Angiotensin IV appears to affect the ERK1/2 pathway.

Our previous studies showed that D 1 and D 2 dopamine receptors are indispensable for the cognitive effects of angiotensin IV (Ang IV) and its des-Phe 6 derivative des-Phe 6 -Ang IV to occur. As most neuroleptics currently used in the treatment of schizophrenia have variable D 2 /D 3 dopaminolytic selectivity, in this study we searched for the role of the D 3 dopamine receptors in facilitating learning and improving memory actions of Ang IV and des-Phe 6 -Ang IV in rats.

The physiological effects of physical exercise have been extensively studied. Nevertheless, its influence on cognitive functioning remains a matter of controversy. In this study we have attempted to assess the effects of repeated exercise (6 weeks of daily treadmill running, meant to resemble human physical training), on parallel learning of a complex task [delayed non matching to sample (DNMS)] by rats. The trained rats appeared to learn the procedure slower and made a significantly lower percentage of correct choices (PB0.01) as compared to those in the non-trained control group. However, when only those rats, from both the groups, which reached the criterion of 80% correct choices in two consecutive sessions were compared, no significant differences were observed. These results suggest an adverse influence of long term physical exercise on rats' ability to learn complex tasks, but only by the 'poor performers'. The 'good performers' were insensitive to the deleterious effects of the exercise.