Modulation of GABAergic neurotransmission in the brain by dipeptides

The effects of endogenous and synthetic peptides containing GABA or its analogues on the GABA/benzodiazepine/chloride ionophore, complex, GABA_B receptor, Cl fluxes, GABA release and GABA uptake were studied using synaptic membranes, crude synaptoneurosomal preparations and slices prepared from the rat and mouse brain. The sodium-independent binding of GABA was strongly inhibited by GABA-histidine, followed by -glutamyl-homotaurine, GABA-glycine and -glutamyl-GABA. The binding of diazepam was slightly enhanced by the same peptides. The peptides alone had no effect on the chloride fluxes, but GABA-histidine, -glutamyl-GABA and GABA-glycine enhanced while -glutamyl-homotaurine and GABA-taurine inhibited GABA-stimulated chloride uptake. GABA-histidine was the most effective displacer of baclofen binding, but -glutamyl-homotaurine was entirely ineffective. The uptake of GABA was markedly inhibited in synaptosomal preparations by GABA-histidine, while all other peptides were less effective. -Glutamyl-taurine attenuated but -glutamyl-homotaurine and GABA-glycine enhanced the potassium-stimulated release of GABA. The present actions of GABA-histidine in vitro may be of significance for GABAergic neurotransmission in vivo.     

Helicobacter infection induces podosome assembly in primary hepatocytes in vitro

Helicobacter pylori (H. pylori) infection may contribute to many extragastric diseases including liver cirrhosis and hepatocellular carcinoma. However, the exact mechanism by which H. pylori induces the liver damage is largely unknown. We used cultured mouse primary hepatocytes as an in vitro model to investigate different aspects of liver physiology and pathology. In this study, we show that primary hepatocytes are able to assemble actin-based cytoskeletal structures called podosomes at the ventral plasma membrane. These structures are positive for podosome markers such as cortactin, vinculin and integrins and comprise proteolytic potential. Infection with the pathogen H. pylori further stimulates the formation of podosomes in primary hepatocytes. The use of pharmacological inhibitors reveals that this response is mediated, at least in part, by TGFβ, a cytokine known to regulate podosome formation in endothelial cells. Similar results are obtained with the hepatoma cell line Huh7. Podosome formation is associated with increased hepatocyte degrading capacities but also with reduced cell motility. Therefore, podosome assembly translates into hepatocyte malfunction. Our study supports the hypothesis that hepatocytes can also assemble podosomes under pathological conditions in vivo.     

Women With Hypertensive Pregnancies Have Difficulties in Regaining Pre‐pregnancy Weight and Show Metabolic Disturbances

The aim was to determine maternal weight gain and body composition during pregnancy and 3 months postpartum in women with uncomplicated singleton and twin pregnancies and in women with gestational diabetes (GDM) and gestational hypertension (GH). This prospective study includes four groups of subjects: those with an uncomplicated pregnancy (n = 32), those with a diagnosis of GH (n = 28), those with a diagnosis of GDM (n = 52), and those with twin pregnancy (n = 11). Their body compositions were estimated by a bioimpedance analysis and fasting lipids and glucose levels were determined during the pregnancy and 3 months after pregnancy. Women with GDM were 11.7 kg heavier than the reference group before pregnancy, whereas weight before pregnancy was not different in other investigated groups. Weight loss after delivery was attenuated in GH group. Percentage body fat remained elevated in women with GDM (34.1 ± 7.0%) and hypertension (31.5 ± 6.4%) at 3 months after pregnancy. Also their total cholesterol and low‐density lipoprotein (LDL)‐cholesterol levels as well as fasting glucose remained elevated in comparison to values of the reference group. In conclusion, women with hypertensive pregnancies, though not overweight before pregnancy, gain and retain excess gestational weight and this leads to metabolic abnormalities similar to those seen in women GDM. Thus, postpartum period appears to be critical for weight management and interventional programs are called for.     

Characterization of mutant xylanases using fourier transform ion cyclotron resonance mass spectrometry: stabilizing contributions of disulfide bridges and N-terminal extensions

Structural properties and thermal stability of Trichoderma reesei endo-1,4-beta-xylanase II (TRX II) and its three recombinant mutants were characterized using electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry and hydrogen/deuterium (H/D) exchange reactions. TRX II has been previously stabilized by a disulfide bridge C110-C154 and other site-directed mutations (TRX II mutants DS2 and DS5). Very recently, a highly thermostable mutant was introduced by combining mutations of DS5 with an N-terminal disulfide bridge C2-C28 (mutant DB1). Accurate mass measurements of TRX II, DS2, DS5, and DB1 verified the expected DNA-encoded protein sequences (average mass error 1.3 ppm) and allowed unequivocal assignment of the disulfides without chemical reduction and subsequent alkylation of the expected cross-links. Moreover, H/D exchange reactions provided means for the detection of a major heat-induced conformational change comprising two interconverting conformers of very different H/D exchange rates as well as allowed the apparent melting temperatures (T(m)) to be determined (62.6, 65.1, 68.0, and 82.2 degrees C for TRX II, DS2, DS5, and DB1, respectively). Residual activity measurements verified that the enzymes inactivated at significantly lower temperatures than expected on the basis of the apparent T(m) values, strongly suggesting that the inactivation takes place through minor conformational change other than observed by H/D exchange. ESI FT-ICR analyses also revealed molecular heterogeneity in DS5 and DB1 due to the propeptide incorporation. Resulting unintentional N-terminal extensions were observed to further improve the stability of the DB1 mutant. The extension of six amino acid residues upstream from the protein N-terminus increased stability by approximately 5 degrees C.     

Effect of Sulpiride on the Amphetamine-Induced Changes in Extracellular Dopamine,DOPAC, and Hydroxyl Radical Generation in the Rat Striatum

The neurotoxic effects of psychostimulants are mediated by several mechanisms, which together lead to neuronal damage. These mechanisms include an increase in the extracellular content of dopamine, stimulation of dopamine oxidation, accumulation of extracellular glutamate, and an increase in body temperature. In the present study, the dopamine receptor antagonist sulpiride proved able to prevent the delayed loss of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) and depressed the gradual generation of hydroxyl radicals induced in the rat striatum by D-amphetamine. However, sulpiride at a dose of 75 mg/kg × 2, coadministered with D-amphetamine (7.5 mg/kg × 4), potentiated the increase in extracellular dopamine and initially slightly enhanced D-amphetamine-induced stereotypy. The gradual increase in hydroxyl radical generation predicts the depletion of dopamine and DOPAC in the rat striatum after D-amphetamine administration, but the increase in extracellular dopamine is not a pivotal factor in the enhanced production of hydroxyl radicals.     

Cyclic AMP-mediated regulation of striatal glutamate release: interactions of presynaptic ligand- and voltage-gated ion channels and G-protein-coupled receptors

The presynaptic regulation of striatal glutamate transmission was investigated using D-[3H]aspartate and mouse striatal slices. Functional changes in voltage-dependent and glutamate receptor-gated ion channels were elicited by pharmacologically modifying intracellular cyclic AMP formation via G-protein-coupled receptor stimulation. The kainate (KA)-evoked release was potentiated by the stimulatory G-protein (G(s))-coupled beta-adrenoceptor agonist isoproterenol (ISO) in a concentration-dependent manner. This effect was mimicked by the specific calmodulin (CaM) antagonists trifluoperazine and calmidazolium. Tetrodotoxin (TTX), a blocker of Na(+) channels, did not affect the basal release but inhibited to the same degree the releases evoked by kainate alone and by kainate and isoproterenol together. Vinpocetine, a blocker of voltage-dependent Na(+) channels, did not alter the basal or the evoked release. The Na(+) channel activator veratridine enhanced the basal release in a concentration-dependent manner and isoproterenol attenuated this effect. The opposite effects of isoproterenol on the kainate- and veratridine-evoked releases may reflect prevention of the cyclic AMP-protein kinase A (PKA) phosphorylation cascade in striatal glutamatergic signal transduction. In addition, the calmidazolium-induced potentiation of kainate-evoked release was thwarted by LY354740 and L-2-amino-4-phosphonobutanoate, agonists of the inhibitory G-protein (G(i))-coupled metabotropic group II and III glutamate receptors (mGluRs). Vinpocetine, which inhibits the CaM-dependent phosphodiesterase (PDE1), was likewise inhibitory. In turn, selective agonists and antagonists of the G(q)-protein-coupled group I mGluRs and (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) and (RS)-1-aminoindan-1,5-dicarboxylate (AIDA), which modulate the intracellular Ca(2+) levels, did not alter the kainate-evoked release.The beta-adrenoceptor-mediated cyclic AMP accumulation seems to downregulate Na(+) channels but to enhance glutamate release by means of upregulation of kainate receptors. This regulation of presynaptic ligand- and voltage-gated ion channels is affected by the cAMP-protein kinase A-dependent phosphorylation cascade and controlled by G(i)-protein-coupled mGluRs.     

Chemical factors affecting the brown-rot decay resistance of Scots pine heartwood

The cell wall chemistry (amount of hemicellulose, f-cellulose, and total lignin) and the concentration of extractives (total acetone-soluble extractives, resin acids, pinosylvins and the total phenolics quantified as tannin acid equivalents) were studied in brown-rot resistant and susceptible juvenile heartwood of Scots pine (Pinus sylvestris L.). The study material consisted of a total of 18 trees from two 34-year-old progeny trials at Korpilahti and Kerimäki. The trees were selected from among 783 trees whose decay rate had previously been screened in a laboratory test using a brown-rot fungus, Coniophora puteana. Samples from neither location showed any significant difference in the concentration (mg/cm³) of hemicellulose, f-cellulose and total lignin between the decay resistant and susceptible trees. At both locations only the concentration of total phenolics was higher in the decay-resistant heartwood than in the decay-susceptible heartwood. At Korpilahti, the amount of acetone-soluble extractives and the concentration of pinosylvin and its derivatives were higher in the resistant than in the susceptible trees.     

Involvement of Nitric Oxide in Adenosine Release in the Developing and Adult Mouse Hippocampus

The novel type of neurotransmitter/neuromodulator nitric oxide (NO) is linked to activation of the N-methyl-D-aspartate (NMDA) class of glutamate receptors and has been shown to modify transmitter release in the brain. The inhibitory neuromodulator adenosine has been thought to act as an endogenous neuroprotectant against cerebral ischemia and neuronal damage. The effects of NO-generating compounds on the release of preloaded [3H]adenosine from hippocampal slices from developing (7-day-old) and adult (3-month-old) mice were investigated, using a superfusion system, under normal conditions and in vitro ischemia. The release of adenosine was markedly potentiated at both ages by the NO-producing compounds S-nitroso-N-acetylpenicillamine, sodium nitroprusside, and hydroxylamine. The evoked releases were reduced by the NO synthase inhibitors nitroarginine and 7-nitroindazole at both ages. They were also reduced by the inhibitor of soluble guanylyl cyclase 1H-(1,2,4-oxadiazolo(4,3a)quinoxalin-1-one (ODQ) in adults, indicating that the NO/cGMP pathway is involved in this release. Release of adenosine was also evoked when the cGMP levels were increased by superfusing slices with the phosphodiesterase inhibitor zaprinast. The markedly enhanced adenosine release under ischemic conditions was further potentiated by the ionotropic glutamate receptor agonists and NO-generating compounds, whereas zaprinast and ODQ had no effect, rendering unlikely the involvement of cGMP in the ischemic release. Moreover, NO was able to provoke substantial release of adenosine in the presence of NMDA under both normal and ischemic conditions, which could significantly add to the neuroprotective potential of this neuromodulator in both adult and developing hippocampus.     

Plasma and Cerebrospinal Fluid Amino Acids in Epileptic Patients

Altered plasma and cerebrospinal fluid amino acid levels may be associated with human epilepsy. We studied three groups of patients, those with a generalized epileptic syndrome, juvenile myoclonic epilepsy, patients with refractory localization-related epilepsies, and patients with acute seizures (within 24 h). Plasma levels of amino acids were studied in all patient groups, as were those in the cerebrospinal fluid (CSF) of patients with acute seizures. After acute seizures, the amino acid changes in the CSF were limited to a reduction in the level of taurine, whereas the levels of most amino acids in plasma were decreased. On the other hand, levels of the excitatory amino acids glutamate and aspartate were increased. The most notable finding in the juvenile myoclonic epilepsy patients was an increase in glutamate level in the plasma. Our study supports the conception of an altered metabolism of glutamate in generalized epilepsies.