Repeated injection of MK801: An animal model of schizophrenia?

Glutamate-induced neurotoxicity plays an important role in neurological and psychiatric diseases. Thus, much attention has been given to the potential neuroprotective role of glutamate receptor antagonists, especially to those acting on the N-methyl-d-aspartate (NMDA) subtype. However, in addition to their neuroprotective potential, these compounds have also neurotoxic and psychotogenic properties. In the present study we used repeated injections of MK801 to examine if this non-competitive NMDA receptor antagonist could be used to produce schizophrenia-like alterations in behavior and brain metabolism in animals. Rats were given injections of MK801 (0.1 mg/kg) on six consecutive days, the last dose together with [1-(13)C]glucose and [1,2-(13)C]acetate, to probe neuronal and astrocytic metabolism, respectively. Analyses of extracts from parts of the frontal cortex plus cingulate and retrosplenial cortices and temporal lobes were performed using (13)C and (1)H magnetic resonance spectroscopy. Changes in glutamate and glutamine were restricted to the temporal lobe, in which amounts and labeling from [1-(13)C]glucose and [1,2-(13)C]acetate were increased compared to control. Locomotor activity was slightly higher in rats treated with MK801 compared to untreated animals. Metabolic changes did not resemble the alterations occurring in schizophrenia and those after repeated high dose (0.5 mg/kg) [Kondziella, D., Brenner, E., Eyjolfsson, E.M., Markinhuhta, K.R., Carlsson, M., Sonnewald, U., 2005. Glial-neuronal interactions are impaired in the schizophrenia model of repeated MK801 exposure. Neuropsychopharmacology, Epub ahead of print] but rather those caused by MK801 seen after a single high dose (0.5 mg/kg) [Brenner, E., Kondziella, D., Haberg, A., Sonnewald, U., 2005. Impaired glutamine metabolism in NMDA receptor hypofunction induced by MK801. J. Neurochem. 94, 1594-1603.].     

Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons

Pyruvate recycling was studied in primary cultures of mouse cerebrocortical astrocytes, GABAergic cerebrocortical interneurons, and co-cultures consisting of both cell types by measuring production of [4-¹³C]glutamate from [3-¹³C]glutamate by aid of nuclear magnetic resonance spectroscopy. This change in the position of the label can only occur by entry of [3-¹³C]glutamate into the tricarboxylic acid (TCA) cycle, conversion of labeled -ketoglutarate to malate or oxaloacetate, malic enzyme-mediated decarboxylation of malate to pyruvate or phosphoenolpyruvate carboxykinase-mediated conversion of oxaloacetate to phosphoenolpyruvate and subsequent hydrolysis of the latter to pyruvate, and introduction of the labeled pyruvate into the TCA cycle, i.e., after exit of the carbon skeleton of pyruvate from the TCA cycle followed by re-entry of the same pyruvate molecules via acetyl CoA. In agreement with earlier observations, pyruvate recycling was demonstrated in astrocytes, indicating the ability of these cells to undertake complete oxidative degradation of glutamate. The recycled [4-¹³C]glutamate was not further converted to glutamine, showing compartmentation of astrocytic metabolism. Thus, absence of recycling into glutamine in the brain in vivo cannot be taken as indication that pyruvate recycling is absent in astrocytes. No recycling could be demonstrated in the cerebrocortical neurons. This is consistent with a previously demonstrated lack of incorporation of label from glutamate into lactate, and it also indicates that mitochondrial malic enzyme is not operational. Nor was there any indication of pyruvate recycling in the co-cultures. Although this may partly be due to more rapid depletion of glutamate in the co-cultures, this observation at the very least indicates that pyruvate recycling is not up-regulated in the neuronal-astrocytic co-cultures.     

Kinetics control preferential heterodimer formation of platelet-derived growth factor from unfolded A- and B-chains

The folding and assembly of platelet-derived growth factor (PDGF), a potent mitogen involved in wound-healing processes and member of the cystine knot growth factor family, was studied. The kinetics of the formation of disulfide-bonded dimers were investigated under redox reshuffling conditions starting either from unfolded and reduced PDGF-A- or B-chains or an equimolar mixture of both chains. It is shown that in all cases the formation of disulfide-bonded dimers is a very slow process occurring in the time scale of hours with a first-order rate-determining step. The formation of disulfide-bonded PDGF-AA or PDGF-BB homodimers displayed identical kinetics, indicating that both monomeric forms as well as the dimerized homodimer have similar folding and assembly pathways. In contrast, the formation of the heterodimer occurred three times more rapidly compared with the formation of the homodimers. As both monomeric forms revealed similar renaturation kinetics, it can be concluded that the first-order rate-determining folding step does not occur during monomer folding but must be attributed to conformational rearrangements of the dimerized, not yet disulfide-bonded protein. These structural rearrangements allow a more rapid formation of intermolecular disulfide bonds between the two different monomers of a heterodimer compared with the formation of the disulfide bonds between two identical monomers. The preferential formation of disulfide-bonded heterodimers from an equimolar mixture of unfolded A- and B-chains is thus a kinetically controlled process. Moreover, similar activation enthalpies for the formation of all different isoforms suggest that faster heterodimerization is controlled by entropic factors.     

Triacylglycerol fatty acid composition in diet-induced weight loss in subjects with abnormal glucose metabolism--the GENOBIN study

Background

The effect of weight loss on different plasma lipid subclasses at the molecular level is unknown. The aim of this study was to examine whether a diet-induced weight reduction result in changes in the extended plasma lipid profiles (lipidome) in subjects with features of metabolic syndrome in a 33-week intervention.

Methodology/Principal Findings

Plasma samples of 9 subjects in the weight reduction group and 10 subjects in the control group were analyzed using mass spectrometry based lipidomic and fatty acid analyses. Body weight decreased in the weight reduction group by 7.8±2.9% (p<0.01). Most of the serum triacylglycerols and phosphatidylcholines were reduced. The decrease in triacylglycerols affected predominantly the saturated short chain fatty acids. This decrease of saturated short chain fatty acid containing triacylglycerols correlated with the increase of insulin sensitivity. However, levels of several longer chain fatty acids, including arachidonic and docosahexanoic acid, were not affected by weight loss. Levels of other lipids known to be associated with obesity such as sphingolipids and lysophosphatidylcholines were not altered by weight reduction.

Conclusions/Significance

Diet-induced weight loss caused significant changes in global lipid profiles in subjects with abnormal glucose metabolism. The observed changes may affect insulin sensitivity and glucose metabolism in these subjects.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00621205","term_id":"NCT00621205"}}NCT00621205     

The Caenorhabditis elegans Elongator Complex Regulates Neuronal α-tubulin Acetylation

Although acetylated α-tubulin is known to be a marker of stable microtubules in neurons, precise factors that regulate α-tubulin acetylation are, to date, largely unknown. Therefore, a genetic screen was employed in the nematode Caenorhabditis elegans that identified the Elongator complex as a possible regulator of α-tubulin acetylation. Detailed characterization of mutant animals revealed that the acetyltransferase activity of the Elongator is indeed required for correct acetylation of microtubules and for neuronal development. Moreover, the velocity of vesicles on microtubules was affected by mutations in Elongator. Elongator mutants also displayed defects in neurotransmitter levels. Furthermore, acetylation of α-tubulin was shown to act as a novel signal for the fine-tuning of microtubules dynamics by modulating α-tubulin turnover, which in turn affected neuronal shape. Given that mutations in the acetyltransferase subunit of the Elongator (Elp3) and in a scaffold subunit (Elp1) have previously been linked to human neurodegenerative diseases, namely Amyotrophic Lateral Sclerosis and Familial Dysautonomia respectively highlights the importance of this work and offers new insights to understand their etiology.     

Probing the activation site of ribonuclease L with new N6-substituted 2',5'-adenylate trimers

2-5A trimer [5'-monophosphoryladenylyl(2'-5')adenylyl(2'-5')adenosine] activates RNase L. While the 5'-terminal and 2'-terminal adenosine N(6)-amino groups play a key role in binding to and activation of RNase L, the exocyclic amino function of the second adenylate (from the 5'-terminus) plays a relatively minor role in 2-5A's biological activity. To probe the available space proximal to the amino function of the central adenylate of 2-5A trimer during binding to RNase L, a variety of substituents were placed at that position. To accomplish this, the convertible building block 5'-O-dimethoxytrityl-3'-O-(tert-butyldimethylsilyl)-6-(2,4-dinitrophenyl)thioinosine 2'-(2-cyanoethylN,N-diisopropylphosphoramidite) was prepared as a synthon to introduce 6-(2,4-dinitrophenyl)thioinosine into the middle position of the 2-5A trimer during automated synthesis. Post-synthetic treatment with aqueous amines transformed the (2,4-dinitrophenyl)thioinosine into N(6)-substituted adenosines. Assays of these modified trimers for their ability to bind and activate RNase L showed that activation activity could be retained, albeit with some sacrifice compared to unmodified p5'A2'p5'A2'p5'A. Thus, the spatial domain about this N(6)-amino function could be available for modifications to enhance the biological potency of 2-5A analogues and to ligate 2-5A to targeting vehicles such as antisense molecules.     

Substrate binding analysis of the 23S rRNA methyltransferase RrmJ

The 23S rRNA methyltransferase RrmJ (FtsJ) is responsible for the 2'-O methylation of the universally conserved U2552 in the A loop of 23S rRNA. This 23S rRNA modification appears to be critical for ribosome stability, because the absence of functional RrmJ causes the cellular accumulation of the individual ribosomal subunits at the expense of the functional 70S ribosomes. To gain insight into the mechanism of substrate recognition for RrmJ, we performed extensive site-directed mutagenesis of the residues conserved in RrmJ and characterized the mutant proteins both in vivo and in vitro. We identified a positively charged, highly conserved ridge in RrmJ that appears to play a significant role in 23S rRNA binding and methylation. We provide a structural model of how the A loop of the 23S rRNA binds to RrmJ. Based on these modeling studies and the structure of the 50S ribosome, we propose a two-step model where the A loop undocks from the tightly packed 50S ribosomal subunit, allowing RrmJ to gain access to the substrate nucleotide U2552, and where U2552 undergoes base flipping, allowing the enzyme to methylate the 2'-O position of the ribose.     

True absorption and endogenous fecal excretion of manganese in relation to its dietary supply in growing rats

A conventional balance study with 48 male weanling rats was conducted to determine true absorption and endogenous fecal excretion of manganese (Mn) in relation to dietary Mn supply, following the procedures of a previously adapted isotope dilution technique. After 10 d on a diet with 1.5 ppm Mn, eight animals each were assigned to diets containing 1.5, 4.5, 11.2, 35, 65, or 100 ppm Mn on a dry-matter basis. Three days later, each rat was given an intramuscular⁵⁴Mn injection and kept on treatment for a balance period of 16 d. Apparent Mn absorption assessed for the final 8 d, averaged 8.6 μg/d without significant treatment effects, although Mn intake ranged from 18.6 to 1200 μg/d, in direct relation to dietary Mn concentrations. Mean fecal excretion of endogenous Mn for the six treatments was 0.9, 2.7, 7.4, 11.0, 16.3, and 17.7 μg/d, respectively. These values delineate the rates to which true absorption exceeded apparent rates. True absorption, as percent of Mn intake, averaged 28.7, 15.9, 11.7, 6.1, 3.4, and 2.0, respectively, as compared with mean values of 23.9, 10.9, 6.2, 3.4, 1.2, and 0.5 for percent apparent absorption. It was concluded that both true absorption and endogenous fecal excretion markedly responded to Mn nutrition and that the reduction in the efficiency of true absorption was quantitatively the most significant homeostatic response for maintaining stable Mn concentrations in body tissues.     

Simultaneous determination of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in biological samples using solid-phase extraction and high-performance liquid chromatography

A sensitive and rapid method for measuring simultaneously adenosine, S-adenosylhomocysteine and S-adenosylmethionine in renal tissue, and for the analysis of adenosine and S-adenosylhomocysteine concentrations in the urine is presented. Separation and quantification of the nucleosides are performed following solid-phase extraction by reversed-phase ion-pair high-performance liquid chromatography with a binary gradient system. N⁶-Methyladenosine is used as the internal standard. This method is characterized by an absolute recovery of over 90% of the nucleosides plus the following limits of quantification: 0.25–1.0 nmol/g wet weight for renal tissue and 0.25–0.5 μM for urine. The relative recovery (corrected for internal standard) of the three nucleosides ranges between 98.1±2.6% and 102.5±4.0% for renal tissue and urine, respectively (mean±S.D., n=3). Since the adenosine content in kidney tissue increases instantly after the onset of ischemia, a stop freezing technique is mandatory to observe the tissue levels of the nucleosides under normoxic conditions. The resulting tissue contents of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in normoxic rat kidney are 5.64±2.2, 0.67±0.18 and 46.2±1.9 nmol/g wet weight, respectively (mean±S.D., n=6). Urine concentrations of adenosine and S-adenosylhomocysteine of man and rat are in the low μM range and are negatively correlated with urine flow-rate.