Synergistic induction of lipid catabolism and anti-inflammatory lipids in white fat of dietary obese mice in response to calorie restriction and n-3 fatty acids.

AIMS/HYPOTHESIS: Calorie restriction is an essential component in the treatment of obesity and associated diseases. Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) act as natural hypolipidaemics, reduce the risk of cardiovascular disease and could prevent the development of obesity and insulin resistance. We aimed to characterise the effectiveness and underlying mechanisms of the combination treatment with LC n-3 PUFA and 10% calorie restriction in the prevention of obesity and associated disorders in mice. METHODS: Male mice (C57BL/6J) were habituated to a corn-oil-based high-fat diet (cHF) for 2 weeks and then randomly assigned to various dietary treatments for 5 weeks or 15 weeks: (1) cHF, ad libitum; (2) cHF with LC n-3 PUFA concentrate replacing 15% (wt/wt) of dietary lipids (cHF + F), ad libitum; (3) cHF with calorie restriction (CR; cHF + CR); and (4) cHF + F + CR. Mice fed a chow diet were also studied. RESULTS: We show that white adipose tissue plays an active role in the amelioration of obesity and the improvement of glucose homeostasis by combining LC n-3 PUFA intake and calorie restriction in cHF-fed mice. Specifically in the epididymal fat in the abdomen, but not in other fat depots, synergistic induction of mitochondrial oxidative capacity and lipid catabolism was observed, resulting in increased oxidation of metabolic fuels in the absence of mitochondrial uncoupling, while low-grade inflammation was suppressed, reflecting changes in tissue levels of anti-inflammatory lipid mediators, namely 15-deoxy-Δ(12,15)-prostaglandin J(2) and protectin D1. CONCLUSIONS/INTERPRETATION: White adipose tissue metabolism linked to its inflammatory status in obesity could be modulated by combination treatment using calorie restriction and dietary LC n-3 PUFA to improve therapeutic strategies for metabolic syndrome.

The genetics of antiplatelet drug resistance.

Platelets have a central role in the development of arterial thrombosis and subsequent cardiovascular events. An appreciation of this complex process has made antiplatelet therapy the cornerstone of cardiovascular disease management. However, numerous patients will experience a recurrent atherothrombotic vascular event despite adequate antiplatelet therapy. Individual differences in the rate of platelet activation and reactivity markedly influence normal hemostasis and the pathological outcome of thrombosis. Such an individual variability is largely determined by environmental and genetic factors. These are known to either hamper platelets' response to agonists, and thereby mimic the pharmacological modulation of platelet function or mask therapy effect and sensitize platelets. In this article, we reviewed the antiplatelet mechanisms of aspirin and clopidogrel and the possible role of different polymorphisms, which may affect the efficacy of antiplatelet therapy. Heterogeneity in the way patients respond to aspirin and clopidogrel may in part reflect variation in cyclooxygenase (COX)-1, COX-2, glycoprotein (GP) Ib alpha, GP Ia/IIa, GP IIb/IIIa, UGT1A6*2, P2Y(1), P2Y(12), CYP2C9, CYP3A4 and CYP3A5 genotypes.

Relationship between neuronal vulnerability and potassium-chloride cotransporter 2 immunoreactivity in hippocampus following transient forebrain ischemia.

Cation chloride cotransporters have been reported to be expressed in neurons in the hippocampus and to regulate intracellular Cl(-) concentration. The neuron-specific K-Cl cotransporter 2 (KCC2) is necessary for maintaining the low intracellular chloride concentration required for the hyperpolarizing actions of GABA. In this study we examined the vulnerability of KCC2-containing neurons as well as the changes in the pattern of KCC2 distribution in the rat hippocampus following 15 min ischemia induced by four-vessel occlusion. Immunostaining for the 72 kDa heat shock protein (HSP-72) was used to investigate the extent of damage in neuronal populations previously shown to be vulnerable to ischemia. At 6-24 h after ischemia, when the pyramidal cells in the CA1 (subfield of cornu Ammonis) region showed no morphological signs of damage, a small rise of KCC2 immunoreactivity was already observed. After 2 days, when the CA1 pyramidal cells started to degenerate, a progressive downregulation of the KCC2 protein was visible. Interestingly, in the same areas, the parvalbumin containing interneurons showed no signs of ischemic damage, and KCC2 immunoreactivity was retained on their membrane surface. In CA1 pyramidal cells, the reduction in KCC2 expression may lead to an elevation of intracellular Cl(-) concentration, which causes a shift in equilibrium potential toward more positive levels. Consequently, the reduction of the inhibitory action of GABA through downregulation of KCC2 function may be involved in the pathomechanisms of delayed neuronal death in the CA1 subfield.

Signatures of the Dirac electron in the flux dependence of total persistent currents in isolated Aharonov-Bohm rings.

This paper deals with the total persistent current at T = 0 produced by the exact energy solution of the Dirac electron moving on isolated 1D Aharonov-Bohm rings. Leading contributions concerning the non-relativistic limit are written down for large values of the electron number. Usual non-relativistic currents get reproduced, but now in terms of a reversed parity of the electron number. Such an 'anomaly' is able to serve as a signature of the Dirac electron referred to above.

Facilitation of spike-wave discharge activity by lipopolysaccharides in Wistar Albino Glaxo/Rijswijk rats.

In normal rats the proinflammatory cytokines like interleukin-1beta, interleukin-6, which are induced by bacterial lipopolysaccharides, are able to control thalamo-cortical excitability by exerting strong effects on physiological synchronization such as sleep and on pathological synchronization like that in epileptic discharges. To investigate whether proinflammatory cytokines or lipopolysaccharides could modulate absence seizures resulting from a very different generator mechanism than the already investigated bicuculline-, kindling- and kainate-induced seizures, we used a genetically epileptic Wistar Albino Glaxo/Rijswijk rat strain, which is spontaneously generating high voltage spike-wave discharges. Wistar Albino Glaxo/Rijswijk rats responded with an increase of the number of spike-wave discharges to lipopolysaccharide injection (from 10 microg/kg to 350 microg/kg). Repetitive administration of 350 microg/kg lipopolysaccharides daily for 5 days increased the number of spike-wave discharges on the first, second and third days but the number of spike-wave discharges returned to the control value on day 5, at the 5th injection of lipopolysaccharides, showing a tolerance to lipopolysaccharides. The lipopolysaccharide-induced increase in spike-wave discharges was not directly correlated with the elevation of the core body temperature, as it is in febrile seizures, although lipopolysaccharide induced prostaglandin and is clearly pyrogenic at the doses used. Indomethacin, the prostaglandin synthesis inhibitor, efficiently blocked lipopolysaccharide-induced enhancement of spike-wave discharge genesis suggesting that the spike-wave discharge facilitating effect of lipopolysaccharides involves induction of cyclooxygenase 2 and subsequent synthesis and actions of prostaglandin E2. Low dose (40 mg/kg, i.p.) of competitive N-methyl-d-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid, and low dose of lipopolysaccharide (20 microg/kg) showed a synergistic interaction to increase the number of spike-wave discharges, whereas at supramaximal doses of lipopolysaccharide and the N-methyl-D-aspartate antagonist no synergy was present. The data reveal a functional connection between absence epileptic activity and lipopolysaccharide induction of prostaglandin synthesis and prostaglandin action and suggest some common cellular targets in epilepsy and lipopolysaccharide-induced inflammation.

Measurement of the optical parameters of purple membrane and plant light-harvesting complex films with optical waveguide lightmode spectroscopy.

Purple membrane (bacteriorhodopsin) and plant light-harvesting complexes (LHCII) were dried on the optical waveguide sensor with varying thicknesses in a wide range (from 20 to several hundreds of nanometers) and the optical parameters were studied with optical waveguide lightmode spectroscopy. It was found that applying the approximate 4-layer mode equations for the measured effective refractive indices resulted in unacceptable results for the optical parameters: with increasing thickness the refractive index decreased monotonously from 1.5 to 1.1. Therefore an inverse waveguide numerical method was developed and used to obtain reliable results from the experiments. The inverse method yielded an approximately constant (1.53) refractive index independently of the thickness for the purple membrane and LHCII films. Light-induced changes in the optical parameters of the purple membrane and LHCII films were also studied. For purple membrane films the most significant effect is the change in refractive index and absorption. For LHCII films prolonged illumination induced irreversible structural changes, most probably of thermo-optic origin.

Chemical chaperones: mechanisms of action and potential use.

An increasing number of studies indicate that low-molecular-weight compounds can help correct conformational diseases by inhibiting the aggregation or enable the mutant proteins to escape the quality control systems, and thus their function can be rescued. The small molecules were named chemical chaperones and it is thought that they nonselectively stabilize the mutant proteins and facilitate their folding. Chemical chaperones are usually osmotically active, such as DMSO, glycerol, or deuterated water, but other compounds, such as 4-phenylbutiric acid, are also members of the chemical chaperone group. More recently, compounds such as receptor ligands or enzyme inhibitors, which selectively recognize the mutant proteins, were also found to rescue conformational mutants and were termed pharmacological chaperones. An increasing amount of evidence suggests that the action of pharmacological chaperones could be generalized to a large number of misfolded proteins, representing new therapeutic possibilities for the treatment of conformational diseases. A new and exciting strategy has recently been developed, leading to the new chemical group called folding agonist. These small molecules are designed to bind proteins and thus restore their native conformation.

Electroconvulsive therapy in neuroleptic-induced parkinsonism.

The effect of electroconvulsive therapy (ECT) on the severity of neuroleptic-induced parkinsonism was studied in nine schizophrenic inpatients in a longitudinal triphasic design: neuroleptics-neuroleptics plus ECT-neuroleptics. The results suggest that ECT has a true antiparkinsonian potential. The role of ECT in the treatment of Parkinson's disease, especially with therapy-resistant patients complicated with on-off symptoms, is highlighted.

Glutathione status in retinopathy of prematurity.

This study examines the glutathione status of red blood cells in patients with retinopathy of prematurity (ROP) both in vivo and after an in vitro oxidative challenge. Fifty ROP patients of different ages (between 6 weeks and 6 years), born prematurely (gestational age: 28.7 +/- 1.3 weeks; birth weight: 1210 +/- 313 g; mean +/- SD) suffering either from active ROP (<3 months old; n = 12) or from a visual handicap due to preceding ROP (3 months-6 years; n = 38) as well as control patients of similar age and maturity (n = 56) were included. Infants with active disease have the lowest levels of reduced glutathione (GSH), the highest levels of oxidized form (GSSG), the highest GSSG/GSH ratios and the greatest fall in GSH after an in vitro oxidative challenge. After an in vitro oxidative stress, defective glutathione recycling was found in patients with preceding ROP and was suggested as a factor predisposing to oxidative hemolysis. The glutathione redox ratio was warranted as a biochemical screen for active ROP in premature infants.

Rationally designed inhibitors of inosine monophosphate dehydrogenase.

Functionalized 2-alkyl derivatives of inosinic acid have been synthesized to serve as reversible as well as irreversible inhibitors of the human type II enzyme inosine monophosphate dehydrogenase. These compounds were designed to react with Cys-331 of the enzyme to form covalent bonds so as to interfere with the normal enzyme mechanism which involves attack of Cys-331 at C-2 of the substrate. Mass spectrometric analysis of the reaction products after enzymatic degradation confirmed the appropriateness of the inhibitor design.

Structure-activity relationships for inhibition of inosine monophosphate dehydrogenase by nuclear variants of mycophenolic acid.

Structure-activity relationships in the region of the phthalide ring of the inosine monophosphate dehydrogenase inhibitor mycophenolic acid have been explored. Replacement of the lactone ring with other cyclic moieties resulted in loss of potency, especially for larger groups. Replacement of the ring by acyclic substituents also indicated a strong sensitivity to steric bulk. A phenolic hydroxyl group, with an adjacent hydrogen bond acceptor, was found to be essential for high potency. The aromatic methyl group was essential for activity; the methoxyl group could be replaced by ethyl to give a compound with 2-4 times the potency of mycophenolic acid in vitro and in vivo.

Development of the raphe-hippocampal projection in vitro.

In this study we examined whether the serotonergic raphe-hippocampal projection preserves its characteristic target selectivity for GABAergic interneurons when developing in vitro, in organotypic cultures. Hippocampal slices from one- to three-day-old rats were co-cultured with slices derived from the raphe nuclei of the same animals. After several weeks of in vitro incubation, a large number of raphe fibres--visualized by immunostaining for serotonin--were found to innervate the hippocampal tissue. In our random sample of over 250 serially sectioned boutons--52 of which were completely reconstructed from serial sections--only two were found to form conventional synapses in the electron microscope, and contacted dendritic spines. These results demonstrate that raphe-hippocampal serotonergic afferent are unable to form synaptic contacts with their normal targets in vitro, if explanted one to three days postnatally. Neurons in the afferent and/or target area may have passed a critical age when selective synaptic contacts can be formed, or unknown chemical or electrical signals may be missing under these conditions, which should serve to guide subcortical afferents to their synaptic target elements.

The apical shaft of CA1 pyramidal cells is under GABAergic interneuronal control.

Dendrites of pyramidal cells perform complex amplification and integration (reviewed in Refs 5, 9, 12 and 20). The presence of a large proximal apical dendrite has been shown to have functional implications for neuronal firing patterns (13) and under a variety of experimental conditions, the largest increases in intracellular Ca2+ occur in the apical shaft.(4,8,15,16,19,21-23) An important step in understanding the functional role of the proximal apical dendrite is to describe the nature of synaptic input to this dendritic region. Using light and electron microscopic methods combined with in vivo labeling of rat hippocampal CA1 pyramidal cells, we examined the total number of GABAergic and non-GABAergic inputs converging onto the first 200microm of the apical trunk. The number of spines associated with excitatory terminals increased from <0.2 spines/microm adjacent to the soma to 5.5 spines/microm at 200microm from the soma, whereas the number of GABAergic, symmetric terminals decreased from 0.8/microm to 0.08/microm over the same anatomical region. GABAergic terminals were either parvalbumin-, cholecystokinin- or vasointestinal peptide-immunoreactive. These findings indicate that the apical dendritic trunk mainly receives synaptic input from GABAergic interneurons. GABAergic inhibition during network oscillation may serve to periodically isolate the dendritic compartments from the perisomatic action potential generating sites.

Medial septal and median raphe innervation of vasoactive intestinal polypeptide-containing interneurons in the hippocampus.

Vasoactive intestinal polypeptide-immunoreactive interneurons are known to form three anatomically and neurochemically well-characterized neuron populations in the hippocampus. Two of these establish synaptic contacts selectively with other GABAergic cells (interneuron-selective cells), whereas the third type innervates pyramidal cell bodies and proximal dendrites like a conventional basket cell. Our aim was to examine which of the vasoactive intestinal polypeptide-containing interneuron populations are among the targets of GABAergic septohippocampal and serotonergic raphe-hippocampal pathways. Anterograde tracing with Phaseolus vulgaris leucoagglutinin combined with double immunocytochemistry for vasoactive intestinal polypeptide was used at the light and electron microscopic levels. Our results show that both interneuron-selective cells and vasoactive intestinal polypeptide-containing basket cells receive synaptic input from the medial septum and median raphe nucleus. The GABAergic component of the septohippocampal pathway establishes multiple contacts on both cell types. In the case of the raphe-hippocampal projection, single or double contacts were more frequent on vasoactive intestinal polypeptide-positive interneuron selective cells (76%), whereas multiple contacts predominated on basket cells (83%). The extrinsic GABAergic innervation of interneuron-selective cells in the hippocampus indicates a complex interaction among GABAergic systems, which might ensure the timing and rhythmic synchronization of inhibitory processes in the hippocampus. On the other hand, our results suggest that the serotonergic effect on perisomatic inhibition is exerted via vasoactive intestinal polypeptide-containing basket cells that are functionally distinct from their parvalbumin-positive relatives, which appear to escape control of serotonergic as well as local interneuron-selective cells.

Regeneration of the GABAergic septohippocampal projection in vitro.

The formation of the GABAergic septohippocampal projection was studied in vitro. Slice cultures of the septal complex from young postnatal rats were prepared and co-cultivated with hippocampal slices for up to four weeks. Then, the anterogradely transported tracer Phaseolus vulgaris leucoagglutinin was injected into the septal culture and the labeled fibers were traced into the hippocampal culture. Some fibers were identified as originating from GABAergic septal cells by double-labeling with an antiserum against GABA using the postembedding immunogold procedure. Our results showed that double-labeled terminals of GABAergic septohippocampal neurons established symmetric synapses exclusively with GABA-positive dendrites in one out of five co-cultures, but also contacted numerous GABA-negative structures in the remaining four co-cultures. These findings, together with light microscopic data from sections double-stained for Phaseolus and parvalbumin, indicate that the high target selectivity of the GABAergic septohippocampal pathway for GABAergic interneurons in vivo is lost in most cases, at least under the present in vitro conditions. It is hypothesized that this may be due to an immaturity of the connection, the lack of axon-guiding factors or an expansion of the septohippocampal GABAergic fibers in the absence of many extrinsic afferents, including GABAergic fibers. The simultaneous occurrence of anterogradely labeled, but GABA-negative, septohippocampal terminals in the hippocampal target culture also suggests that the septohippocampal cholinergic projection developed in vitro, as was shown before in other studies. Since most septohippocampal neurons have to be axotomized for culture preparation, the present results indicate that GABAergic septohippocampal neurons from young postnatal rats survive axotomy and are capable of regenerating a septohippocampal projection, including the formation of characteristic GABAergic synapses on co-cultured hippocampal neurons. However, the characteristic target selectivity is rarely preserved.

Distinct interneuron types express m2 muscarinic receptor immunoreactivity on their dendrites or axon terminals in the hippocampus.

In previous studies m2 muscarinic acetylcholine receptor-immunoreactive interneurons and various types of m2-positive axon terminals have been described in the hippocampal formation. The aim of the present study was to identify the types of interneurons expressing m2 receptor and to examine whether the somadendritic and axonal m2 immunostaining labels the same or distinct cell populations. In the CA1 subfield, neurons immunoreactive for m2 have horizontal dendrites, they are located at the stratum oriens/alveus border and have an axon that project to the dendritic region of pyramidal cells. In the CA3 subfield and the hilus, m2-positive neurons are multipolar and are scattered in all layers except stratum lacunosum-moleculare. In stratum pyramidale of the CA1 and CA3 regions, striking axon terminal staining for m2 was observed, surrounding the somata and axon initial segments of pyramidal cells in a basket-like manner. The co-localization of m2 with neurochemical markers and GABA was studied using the "mirror" technique and fluorescent double-immunostaining at the light microscopic level and with double-labelling using colloidal gold-conjugated antisera and immunoperoxidase reaction (diaminobenzidine) at the electron microscopic level. GABA was shown to be present in the somata of most m2-immunoreactive interneurons, as well as in the majority of m2-positive terminals in all layers. The calcium-binding protein parvalbumin was absent from practically all m2-immunoreactive cell bodies and dendrites. In contrast, many of the terminals synapsing on pyramidal cell somata and axon initial segments co-localized parvalbumin and m2, suggesting a differential distribution of m2 receptor immunoreactivity on the axonal and somadendritic membrane of parvalbumin-containing basket and axo-axonic cells. The co-existence of m2 receptors with the calcium-binding protein calbindin and the neuropeptides cholecystokinin and vasoactive intestinal polypeptide was rare throughout the hippocampal formation. Only calretinin and somatostatin showed an appreciable degree of co-localization with m2 (20% and 15%, respectively). Using retrograde tracing, some of the m2-positive cells in stratum oriens were shown to project to the medial septum, accouting for 38% of all projection neurons. The present results demonstrate that there is a differential distribution of m2 receptor immunoreactivity on the axonal vs the somadendritic membranes of distinct interneuron types and suggest that acetylcholine via m2 receptors may reduce GABA release presynaptically from the terminals of perisomatic inhibitory cells, while it may act to increase the activity of another class of interneuron, which innervates the dendritic region of pyramidal cells.

Hemangioblastoma of the optic nerve.

A 43-year-old woman presented with progressive loss of vision in the right eye. Magnetic resonance imaging (MRI) showed a prominently enhancing lesion of the optic nerve, thought preoperatively to represent an optic nerve meningioma or optic neuritis. Histological examination of the excised tumor showed this lesion to be hemangioblastoma. Her family history was unremarkable. However, subsequent review of the preoperative MRIs and postoperative imaging studies showed two small cerebellar lesions, probably hemangioblastomas, and renal, pancreatic, and adnexal cysts, establishing the diagnosis of von Hippel-Lindau disease.

Pregnancy associated with hypergonadotropic hypogonadism.

A case of a 30-year-old female with secondary amenorrhea and relative hypergonadotropic hypogonadism is presented. The patient demonstrated persistently elevated levels of gonadotropins in spite of clinical and laboratory evidence of estrogen production. Laparoscopic directed biopsy revealed a total absence of ova, but in spite of this finding, conception ensued. Variations in ovarian sensitivity throughout the female reproductive period and in certain pathologic states are discussed, as well as the apparent limitation of single laparoscopic directed biopsies for confirming the diagnosis of premature menopause. Finally, the theoretical use of exogenous estrogen to induce ovulation in patients with the ovarian insensitivity syndrome is discussed.

Application of the optical waveguide lightmode spectroscopy to monitor lipid bilayer phase transition.

An instrument for optical waveguide lightmode spectroscopy (OWLS) was designed and developed for measurements at different and controlled temperatures in a range of 15 degrees C around room temperature. The instrument allows to scan the waveguide modes at different wavelengths on the same optical chip using different lasers. This instrument was used to monitor DMPC lipid bilayer main phase transition around the critical temperature. The main problem in these experiments is that the OWLS measurements do not give enough information about an optically anisotropic system like a lipid bilayer. Experimental OWLS data at two different wavelengths can however approximately solve the problem. The temperature dependence of the thickness and the refractive indices (ordinary and extraordinary) for the lipid bilayer around the phase transition is presented. (A theoretical derivation of the extraordinary refractive index is given in.)

The effect of UV irradiation on uracil thin layer measured by optical waveguide lightmode spectroscopy.

The polycrystalline uracil thin-layer dosimeter is a well-established method to monitor the biological effects of the environmental ultraviolet (UV) radiation. It is based on the optical density (OD) decrease of the uracil layer in the UV absorption band due to photodimerization of the crystal caused by UV irradiation. In the present study, we report measurements made with optical waveguide lightmode spectroscopy (OWLS) to characterize the changes in the optogeometrical parameters of the uracil layer caused by an artificial UV source. It is shown that UV irradiation causes a decrease in the refractive index and an increase of the optical anisotropy. The determined kinetic parameters of the UV dose-sensor response curves correlate well with results of OD measurements, but the sensitivity of OWLS is about ten times higher. The results show that OWLS is capable of analyzing the UV response of the uracil layer and opens the way for dosimetrical applications.