Oxytocin action on components of endoplasmic reticulum in hippocampal neuronal cells.

Despite the known importance of the endoplasmic reticulum (ER) in protein synthesis and vesicular transport, it is not clear whether neuropeptide and neuromodulator oxytocin can directly affect components of the ER in neuronal cells. Therefore, in the present study, we hypothesize that incubation of hippocampal neuronal cells in a presence of oxytocin 1) plays a role in the regulation of the expression of selected ER chaperone components and molecules involved in unfolded protein response pathway 2) affects distribution of the intracellular fluorescence signal highly selective for the ER. We found that oxytocin (1 µM) after 60 min significantly decreased the gene expression of oxidoreductase Ero1ß, chaperone glucose-regulated proteins (Grp) 78 and Grp94. A significant decrease in GRP78 protein levels in response to oxytocin treatment occurred after 30, 60 and 120 min. We also observed a time-dependent increase in calreticulin protein levels with a statistically significant increase observed after 360 min. We found that the dynamics of the ER network changes significantly within 2 h of incubation under the influence of oxytocin. In conclusion we have shown that ER chaperones, oxidoreductases and trafficking molecules in neuronal cells are changing in response to oxytocin treatment in a short-term scenario potentially relevant for growth of dendrites and axons.

Chronic Atrophic Gastritis with Negative Intrinsic Factor and Parietal Cell Antibody Presenting as a Severe Hemolytic Anemia.

A 28-year-old Caucasian male with Hashimoto's disease and vitiligo presented with two weeks of dizziness on exertion following pharyngitis which was treated with prednisone 40 mg by mouth once a day for five days. Initial workup revealed anemia, elevated lactate dehydrogenase (LDH), and low haptoglobin. He underwent workup for causes of hemolytic anemia which was remarkable for a peripheral blood smear with hypersegmented neutrophils and low vitamin B12 levels concerning for pernicious anemia. Parietal cell and intrinsic factor antibodies were negative, and he then underwent an esophagogastroduodenoscopy with biopsy. The biopsy was negative for Helicobacter pylori, and the immunohistochemical stains were suggestive of chronic atrophic gastritis. He was started on vitamin B12 1,000 mcg intramuscular injections daily. His hemoglobin, LDH, and haptoglobin normalized. Given the absence of the parietal cell antibody and intrinsic factor antibody, this is a rare case of seronegative pernicious anemia.

Expression of synaptic proteins in the hippocampus is modulated by neonatal oxytocin treatment.

An alteration of oxytocin signaling during postnatal maturation of the brain could be associated with etiology of neurodevelopmental disorders among them autism. The aim of the present study was to examine the role of oxytocin in the regulation of expression of selected cell-adhesion molecules and scaffolding proteins in the hippocampus in early rat development. Oxytocin treatment (1 mg/ml, i.p., 50 µl/pup) at postnatal days P2-P3 resulted in the reduction of Neuroligin 3 gene expression, and was accompanied by lower SHANK1 and SHANK3 mRNA levels in the hippocampus at P5 day. Immunostaining revealed a clear trend for the lower density of Neuroligin 3 positive cells in the hippocampus and this trend has been significant in the CA3 hippocampal area. The significantly lower Neurexin 2ß mRNA levels were observed in response to oxytocin treatment, with no effect seen in the Neurexin 2α gene expression. No change has been observed in the gene expression of Neuroligin 1 and Neuroligin 2. Oxytocin induced an increase in the mRNA levels of Neuron-Specific Enolase (NSE) and a decrease in the mRNA levels of glial fibrillary acid protein (GFAP) - marker of astrocytes. Incubation of primary neuronal cells with oxytocin (1 µM, 48 h) stimulated a proliferation of NSE-positive cells. These results suggest that synaptic proteins could be under control of oxytocin in early stages of brain development. The changes of cell-adhesion molecule and scaffolding protein levels might be linked to the modulation of number of neuronal cells.

Neurite Outgrowth Stimulated by Oxytocin Is Modulated by Inhibition of the Calcium Voltage-Gated Channels.

Neuropeptide oxytocin contributes to the regulation of the neuron differentiation and cell morphology. However, the precise mechanisms are not yet fully understood. Oxytocin receptor function and its coupling to calcium entry are obvious objects of interest in relation to the neuron morphology. Postsynaptic scaffolding proteins including SHANK proteins interact with other synaptic molecules and change dendritic morphology. SH-SY5Y neuroblastoma cell line represents a useful neurobiological in vitro model to study the short-term oxytocin effects on neurite outgrowth and underlying mechanisms. In the present study, we show that oxytocin induces an increase in the intracellular calcium in SH-SY5Y cells. Specificity of the calcium influx was verified by blockade of the oxytocin receptors with oxytocin receptor antagonist L-371,257. Neurite outgrowth stimulated by oxytocin was inhibited by specific voltage-gated calcium channel blockers. The exposure of SH-SY5Y cells to oxytocin resulted in a significant increase in the gene expression of SHANK1 and SHANK3 proteins. Overall, the present data indicate that oxytocin may contribute to the regulation of scaffolding proteins expression known to be associated with clusters of calcium channels at the cell membrane. It appears that oxytocin stimulated neurite outgrowth is, at least, in part dependent on the voltage-gated calcium channels.

Oxytocin Modulates Expression of Neuron and Glial Markers in the Rat Hippocampus.

Neuropeptides including oxytocin belong to the group of factors that may play a role in the control of neuronal cell survival, proliferation and differentiation. The aim of the present study was to investigate potential contribution of oxytocin to neuronal differentiation by measuring gene and protein expression of specific neuron and glial markers in the brain. Neonatal and adult oxytocin administration was used to reveal developmental and/or acute effects of oxytocin in Wistar rats. Gene and protein expression of neuron-specific enolase (NSE) in the hippocampus was increased in 21-day and 2-month old rats in response to neonatal oxytocin administration. Neonatal oxytocin treatment induced a significant increase of gene and protein expression of the marker of astrocytes - glial fibrillary acid protein (GFAP). Oxytocin treatment resulted in a decrease of oligodendrocyte marker mRNA - 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) - in 21-day and 2-month old rats, while no change of CD68 mRNA, marker of microglia, was observed. Central oxytocin administration in adult rats induced a significant increase of gene expression of NSE and CNPase. The present study provides the first data revealing the effect of oxytocin on the expression of neuron and glial markers in the brain. It may be suggested that the oxytocin system is involved in the regulation of development of neuronal precursor cells in the brain.

Oxytocin Increases Neurite Length and Expression of Cytoskeletal Proteins Associated with Neuronal Growth.

Neuropeptide oxytocin acts as a growth and differentiation factor; however, its effects on neurite growth are poorly understood. The aims of the present study were (1) to evaluate time effects of oxytocin on expression of nestin and MAP2; (2) to measure the effect of oxytocin on gene expression of ß-actin, vimentin, cofilin, and drebrin; and (3) to measure changes in neurite length and number in response to oxytocin/oxytocin receptor antagonist L-371,257. Exposure of SH-SY5Y cells to 1 µM oxytocin resulted in a significant increase in gene expression and protein levels of nestin after 12, 24, and 48 h. Oxytocin treatment induced no changes in gene expression of MAP2; however, a decrease of protein levels was observed in all time intervals. Gene expression of ß-actin, vimentin, and drebrin increased in response to oxytocin. Oxytocin induced significant elongation of neurites after 12, 24, and 48 h. No change in neurite length was observed in the presence of the combination of retinoic acid and oxytocin receptor antagonist L-371,257. Oxytocin treatment for 12 h increased the number of neurites. Overall, the present data suggest that oxytocin contributes to the regulation of expression of cytoskeletal proteins associated with growth of neuronal cones and induces neurite elongation mediated by oxytocin receptors at least in certain types of neuronal cells.

Stereoselective coordination: a six-membered P,N-chelate tailored for asymmetric allylic alkylation.

Six-membered chelate complexes [Pd(1a-b)Cl2], (2a-b) and [Pd(1a-b)(η(3)-PhCHCHCHPh)]BF4, (3a-b) of P,N-type ligands 1a, ((2S,4S)-2-diphenyl-phosphino-4-isopropylamino-pentane) and 1b, ((2S,4S)-2-diphenyl-phosphino-4-methylamino-pentane) have been prepared. The Pd-complexes have been characterized in solution by 1D and 2D NMR spectroscopy. The observed structures were confirmed by DFT calculations and in the case of 2a also by X-ray crystallography. Unexpectedly, the coordination of the all-carbon-backbone aminophosphine 1a resulted in not only a stereospecific locking of the donor nitrogen atom into one of the two possible configurations but also the conformation of the six-membered chelate rings containing three alkyl substituents was forced into the same single chair structure showing the axially placed isopropyl group on the coordinated N-atom. The stereodiscriminative complexation of 1a led to the formation of a palladium catalyst with a conformationally rigid chelate having a configurationally fixed nitrogen and electronically different coordination sites due to the presence of P and N donors. The stereochemically fixed catalyst provided excellent ee's (up to 96%) and activities in asymmetric allylic alkylation reactions. In contrast, the chelate rings formed by 1b exist in two different chair conformations, both containing axial methyl groups, but with the opposite configurations of the coordinated N-atom. Pd-complexes of 1b provided low enantioselectivities in similar alkylations, therefore emphasizing the importance of the stereoselective coordination of N-atoms in analogous P-N chelates. The factors determining the coordination of the ligands were also studied with respect to the chelate ring conformation and the nitrogen configuration.

Intracellular distribution of 3,6-bis(3-alkylguanidino)acridines determines their cytotoxicity.

UNLABELLED: Cytotoxicity of two derivatives of 3,6-bis(3-alkylguanidino)acridines (GNDAs; pentyl- and hexyl-GNDA) was determined against three cell lines: a murine immortalized fibroblast cell line NIH-3T3, a human ovarian carcinoma cell line A2780, and a human neuroblastoma cell line SH-SY5Y. We found out that these GNDAs were cytotoxic against A2780 and NIH-3T3 cells but they showed only a marginal cytotoxicity against neuroblastoma cells SH-SY5Y. To explain differences in cytotoxicity, intracellular distribution of GNDAs was monitored. GNDAs were accumulated in A2780 and NIH-3T3 cells in the nuclei (fluorescence microscopy). In contrast to these cell lines, in SH-SY5Y cells, GNDAs were localized outside of the nuclei, at the plasma membrane and surroundings, extending also to the cytosol. This distribution of GNDAs was confirmed by an ImageStream Flow Cytometer. Acetylcholinesterase (AChE) activity in the SH-SY5Y cells decreased upon incubation with GNDAs. Kinetic studies showed that GNDAs were able to inhibit AChE by the same mode as tacrine (9-amino-1,2,3,4-tetrahydroacridine), a known inhibitor of AChE. A low cytotocity of GNDAs against SH-SY5Y cells could be caused by their affinity to AChE (the enzyme is localized mainly at the plasma membrane). The interaction of GNDAs with AChE may affect their intracellular distribution and consequently the cytotoxicity. KEYWORDS: acetylcholinesterase, acridine, neuroblastoma cell line SH-SY5Y.

Increased plasma levels of the high mobility group box 1 protein (HMGB1) are associated with a higher score of gastrointestinal dysfunction in individuals with autism.

Autism is a disorder of neural development characterized by impairments in communication, social interaction, restricted interests and repetitive behavior. The etiology of autism is poorly understood, the evidence indicates that inflammation may play a key role. In autism a high prevalence of gastrointestinal disturbances is reported, that are linked to a low-grade chronic inflammation of the intestinal mucosa. High mobility group box 1 protein (HMGB1) is an intranuclear protein that can be passively released from necrotic cells or actively secreted under inflammatory conditions as alarmin or late proinflammatory cytokine. The objective of this study was to measure plasma levels of HMGB1 in individuals with autism and to analyze their association with gastrointestinal symptoms. The study involved 31 subjects with low-functioning autistic disorder aged 2-22 years and 16 healthy controls. Plasma HMGB1 levels were significantly higher in individuals with autism than in controls (13.8+/-11.7 ng/ml vs. 7.90+/-4.0 ng/ml, p<0.02). In subjects with plasma HMGB1 levels higher than 11 ng/ml severe forms of GI disorders were more prevalent (83.3 %) than in subjects with lower levels (38.9 %, p<0.04). Results of the study support the involvement of the systemic low-grade inflammation in the pathomechanisms of autism and its possible association with GI symptoms.

Prolactin increases expression of cytoskeletal proteins in SK-N-SH cells.

Although many studies have demonstrated the role of prolactin in the central nervous system, there is a considerable lack of known effects of prolactin on the parameters of neurogenesis and neuronal differentiation. The aim of the present study was to test whether prolactin changes gene expression and protein levels of nestin and microtubule-associated protein 2 (MAP2) in neuroblastoma (SK-N-SH) and glioblastoma (U-87MG) cells. Nestin and MAP2 represent cytoskeletal proteins associated with neuronal differentiation and they contribute to radial growth of the axons, dendrites and glial processes. SK-N-SH and U-87MG cells were exposed to prolactin (10 nM) for 48 h. Total mRNA was extracted. After reverse transcription, qPCR with specific primers for nestin and MAP2 was performed. The levels of proteins were measured by the In-Cell Western assay. Mitochondrial activity test was used to evaluate the viability of cells under the influence of prolactin. Incubation with 10 nM prolactin did not change the viability, either in SK-N-SH or in U-87MG cells. Prolactin significantly increased the gene expression and protein levels of both nestin and MAP2 in SK-N-SH cells, while no significant changes were observed in U-87MG cells. The presented data suggest that prolactin is linked to the regulation of cytoskeletal proteins in the neuronal type of cells and might be important for their differentiation.

[Mechanisms of growth of neuronal axons and dendrites]. / Mechanizmy zmien dlzky axónov a dendritov neurónu.

Brain development is determined by neuronal differentiation including changes of cell polarity and asymetric growth of neuronal processes. Although, there are many unkown factors contributing to changes of lenght of neuronal cones, mounting experimental and review papers focus on changes of growth conus and role of axonal transport. In particular, mechanisms of actin/microtubule polymerisation and depolymerisation are important. Role of intracellular calcium is also significant. Normal and properly timed changes of lenght of axons and dendrites are dependent on interaction of neurons and glia. Moreover, regeneration of injured axons is dependent on growth factors secreted from glial cells. The aim of the present study is characterisation of the most important mechanisms underlying changes of lenght of neurites.

Cell proliferation in the hippocampus and in the heart is modified by exposure to repeated stress and treatment with memantine.

The present studies were aimed to verify the hypothesis that treatment with memantine, a low affinity NMDA glutamate receptor antagonist, can reduce possible stress-induced alterations in cell proliferation in the hippocampus and in the heart and has consequences on stress hormone release. Adult male Wistar rats were exposed to repeated hypokinesis (movement restraint, 2 h daily) or remained undisturbed and they were treated with memantine (5 mg/kg/day, s.c.) or vehicle for 8 days. On the day 7, all animals were injected with 5-bromo-2'-deoxyuridine (BrdU), a marker of cell proliferation. The mild form of chronic stress used resulted only in moderate decrease in BrdU incorporation into DNA in the hippocampus, while the same stimulus caused a pronounced reduction of the new cells formed in left heart ventricle. In both tissues, stress-induced reduction in cell proliferation was more evident in memantine-treated rats. Memantine failed to modify hormones of the hypothalamic-pituitary-adrenocortical axis, while the treatment increased plasma renin activity. The present study demonstrates that treatment with memantine potentiated rather than prevented stress-induced reduction of cell proliferation. We have shown that stress exposure may induce a reduction in cell proliferation in the heart, even in a higher extent than that in the hippocampus. Effects of memantine under stress conditions might be relevant with respect to clinical use of memantine, which is being used in the treatment of neurodegenerative diseases.

An extremely robust strong-field control of atomic coherence.

We propose and analyze a scheme for adiabatic creation of maximum coherence of 0.5 with a controllable phase between a ground state and the excited state in a model Λ-structured atom using two short laser pulses. One of the pulses has constant carrier frequency quasi-resonant with transition between an initially empty ground and the excite states. The frequency of the second pulse is swept through the resonance with the adjacent transition between the initially populated ground state and the common excited state of the atom. We demonstrate high degree of robustness of the scheme against variation of parameters of the laser radiation in relatively broad region of values. The proposed scheme may find practical applications in the field of multi-photon ionization, high-order harmonics and Raman sideband generation, as well as in nonlinear wave mixing in coherently prepared media.

Neuroendocrine and cardiovascular parameters during simulation of stress-induced rise in circulating oxytocin in the rat.

Physiological functions of oxytocin released during stress are not well understood. We have (1) investigated the release of oxytocin during chronic stress using two long-term stress models and (2) simulated stress-induced oxytocin secretion by chronic treatment with oxytocin via osmotic minipumps. Plasma oxytocin levels were significantly elevated in rats subjected to acute immobilization stress for 120 min, to repeated immobilization for 7 days and to combined chronic cold stress exposure for 28 days with 7 days immobilization. To simulate elevation of oxytocin during chronic stress, rats were implanted with osmotic minipumps subcutaneously and treated with oxytocin (3.6 microg/100 g body weight/day) or vehicle for 2 weeks. Chronic subcutaneous oxytocin infusion led to an increase in plasma oxytocin, adrenocorticotropic hormone, corticosterone, adrenal weights and heart/body weight ratio. Oxytocin treatment had no effect on the incorporation of 5-bromo-2-deoxyuridine into DNA in the heart ventricle. Mean arterial pressure response to intravenous phenylephrine was reduced in oxytocin-treated animals. Decrease in adrenal tyrosin hydroxylase mRNA following oxytocin treatment was not statistically significant. Oxytocin treatment failed to modify food intake and slightly increased water consumption. These data provide evidence on increased concentrations of oxytocin during chronic stress. It is possible that the role of oxytocin released during stress is in modulating hypothalamic-pituitary-adrenocortical axis and selected sympathetic functions.

Enriched environment influences hormonal status and hippocampal brain derived neurotrophic factor in a sex dependent manner.

The present study is aimed at testing the hypothesis that an enriched environment (EE) induces sex-dependent changes in stress hormone release and in markers of increased brain plasticity. The focus was on hypothalamic-pituitary-adrenocortical (HPA) axis activity, plasma levels of stress hormones, gene expression of glutamate receptor subunits and concentrations of brain-derived neurotrophic factor (BDNF) in selected brain regions. Rats exposed to EE were housed in groups of 12 in large cages with various objects, which were frequently changed, for 6 weeks. Control animals were housed four per cage under standard conditions. In females the EE-induced rise in hippocampal BDNF, a neurotrophic factor associated with increased neural plasticity, was more pronounced than in males. Similar sex-specific changes were observed in BDNF concentrations in the hypothalamus. EE also significantly attenuated oxytocin and aldosterone levels only in female but not male rats. Plasma testosterone positively correlated with hippocampal BDNF in female but not male rats housed in EE. In male rats housing in EE led to enhanced levels of testosterone and adrenocorticotropic hormone (ACTH), this was not seen in females. Hippocampal glucocorticoid but not mineralocorticoid receptor levels decreased in rats housed in EE irrespective of sex. Housing conditions failed to modify mRNA levels of glutamate receptor type 1 (Glur1) and metabotropic glutamate receptor subtype 5 (mGlur5) subunits of glutamate receptors in the forebrain. Moreover, a negative association between corticosterone and BDNF was observed in both sexes. The results demonstrate that the association between hormones and changes in brain plasticity is sex related. In particular, testosterone seems to be involved in the regulatory processes related to neuroplasticity in females.

Oxytocin levels in the posterior pituitary and in the heart are modified by voluntary wheel running.

We hypothesized that voluntary wheel running results in increased secretion of oxytocin, a peptide involved in the stress response. An additional hypothesis was that prolonged exercise affects oxytocin levels in the heart, which is in line with the potential role of oxytocin in cardiovascular functions. Voluntary wheel running lasted 3 weeks and daily running distances increased progressively reaching maximum levels about 8 km (Sprague-Dawley rats) and 4 km (Lewis strain). The exercise resulted in significant reduction of epididymal fat, slight increase in glucose transporter GLUT4 mRNA levels and significant enhancement of plasma density. Voluntary exercise failed to influence plasma oxytocin levels either in Lewis or Sprague-Dawley rats, but it resulted in a significant decrease of oxytocin concentrations in the posterior pituitary. Plasma oxytocin concentrations were not modified even if the measurements were made in the dark phase of the day. In voluntary wheel running Sprague-Dawley rats, the content of oxytocin in the right heart atrium was lower than in controls. Thus, the present findings demonstrate that prolonged voluntary wheel running results in a decrease in pituitary oxytocin content without evident changes in hormone concentrations in peripheral blood. However, prolonged exercise used has a significant impact on oxytocin levels in the heart.

Ascites reinfusion dialysis (ARD) for renal failure with refractory ascites.

BACKGROUND: Dialysis is difficult for patients who have simultaneous liver and kidney failure. Effective mobilization of ascites is rare, and hypotension is common. Combining repeated paracentesis with continuous renal replacement therapy can achieve effective volume removal with hemodynamic stability, but requires intensive care unit resources. Large amounts of albumin are lost from the body in the drained ascites. Combining ascites reinfusion with hemodialysis is a potential alternative therapy. METHODS: Eight treatments were undertaken in 3 patients with refractory ascites in the setting of acute onset renal failure. Hemodialysis was unsuccessful due to hypotension in each case. Two patients were treated twice, and 1 patient was treated 4 times. Each patient underwent hemodialysis with reinfusion of ascites directly into the blood inlet of the dialysis machine. Weight, blood urea nitrogen, albumin and platelet counts were measured before and after treatment. Hemodynamic tolerance was assessed, and patients were observed for the development ofencephalopathy, disseminated intravascular coagulation, infection and hemodynamic decompensation. RESULTS: All patients survived. There was 1 episode of transient hemoperitoneum, but no encephalopathy, GI bleeding or infection. One patient recovered renal function, and the other 2 were discharged ambulatory to chronic hemodialysis programs. Blood pressure was supported easily during therapy, despite removal of 3-8 kg of fluid. Platelet counts decreased by 27,000 +/- 13,000, and albumin increased by 0.5 +/- 0.2 g/dl. All values returned to baseline over the next 1-4 days. CONCLUSIONS: Ascites recirculation with dialysis is a safe and effective therapy for patients with refractory ascites and severe renal failure, which can be carried out in routine inpatient and outpatient settings. Hemodynamic tolerance was good and thrombocytopenia was modest.

Non-local-thermodynamical-equilibrium effects in the x-ray emission of radiatively heated materials of different atomic numbers.

X-ray self-emission of radiatively heated materials with different values of Z has been investigated. Thin foils were uniformly heated by a 120-eV Hohlraum radiation of 400-ps duration in order to study the self-emission of a homogeneous, optically thin material. The x-ray emission spectra were followed for more than 2 ns. The spectrally integrated emission shows not only a strong Z dependence, but different temporal behaviors for different values of Z. The lower is the value of Z of the x-ray heated matter, the longer is the duration of self-emission. Theoretical comparison with a hydrocode and FLY post-processing shows a non-local-thermal equilibrium behavior caused by direct photoionization due to the thermal pumping radiation, which has a higher brightness temperature than the matter temperature of the heated material.

Coherent writing and reading of information using frequency-chirped short bichromatic laser pulses.

We propose to use the sensitivity of the population transfer in three-level L-atoms to the relative phases and amplitudes of frequency-chirped short bichromatic laser pulses for coherent, fast and robust storage and processing of phase or intensity optical information. The information is being written into the excited state population which in a second step is transferred in a fast and robust way into a nondecaying storage level. It is shown that an arbitrary superposition of the ground states can be generated by controlling the relative phase between the laser pulses.

Urinary 6-sulphatoxymelatonin excretion is increased in rats after 24 hours of exposure to vertical 50 Hz, 100 microT magnetic field.

The effect of exposure to a 50 Hz, vertical magnetic field on the excretion of urinary 6-sulphatoxymelatonin (aMT6s) of rats was studied in a self-controlled experiment. Ten male Wistar rats were kept under 9:15 h light: dark conditions in metabolic cages. The rats were exposed to 1.0 or 100 microT flux density for 24 h. The excretion of aMT6s, which is the primary metabolite of melatonin in the urine, did not show a statistically significant decrease, as measured by 125I radioimmunoassay, during or after magnetic field exposure of rats to either flux density. At 100 microT flux density, the increase of aMT6s excretion on the day after exposure was statistically significant (P < .02), compared with the value under exposure, but was not significant compared with the baseline values before exposure.