Nitrogen shuttling between neurons and glial cells during glutamate synthesis

The relationship between neuronal glutamate turnover, the glutamate/glutamine cycle and de novo glutamate synthesis was examined using two different model systems, freshly dissected rat retinas ex vivo and in vivo perfused rat brains. In the ex vivo rat retina, dual kinetic control of de novo glutamate synthesis by pyruvate carboxylation and transamination of alpha-ketoglutarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide the alpha-amino group to glutamate. Measurements of synthesis of (14)C-glutamate and of (14)C-glutamine from H(14)CO(3) have shown that (14)C-amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mM. The specific radioactivity of (14)C-glutamine indicated that approximately 30% of glutamine was derived from (14)CO(2) fixation. Using gabapentin, an inhibitor of the cytosolic branched-chain aminotransferase, synthesis of (14)C-glutamate and (14)C-glutamine from H(14)CO(3)(-) was inhibited by 31%. These results suggest that transamination of alpha-ketoglutarate to glutamate in Müller cells is slow, the supply of branched-chain amino acids may limit flux, and that branched-chain amino acids are an obligatory source of the nitrogen required for optimal rates of de novo glutamate synthesis. Kinetic analysis suggests that the glutamate/glutamine cycle accounts for 15% of total neuronal glutamate turnover in the ex vivo retina. To examine the contribution of the glutamate/glutamine cycle to glutamate turnover in the whole brain in vivo, rats were infused intravenously with H(14)CO(3)(-). (14)C-metabolites in brain extracts were measured to determine net incorporation of (14)CO(2) and specific radioactivity of glutamate and glutamine. The results indicate that 23% of glutamine in the brain in vivo is derived from (14)CO(2) fixation. Using published values for whole brain neuronal glutamate turnover, we calculated that the glutamate/glutamine cycle accounts for approximately 60% of total neuronal turnover. Finally, differences between glutamine/glutamate cycle rates in these two model systems suggest that the cycle is closely linked to neuronal activity.     

The 3' end of Norwalk virus mRNA contains determinants that regulate the expression and stability of the viral capsid protein VP1: a novel function for the VP2 protein

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3' untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3' terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3' elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3'UTR (ORF2+3+3'UTR). In contrast, expression of VP1 from constructs that lacked the 3'UTR (ORF2+3), ORF3 (ORF2+3'UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a cis role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3'UTR revealed that the 3'UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in cis was also observed in mammalian cells.     

Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase

The human mitochondrial branched chain aminotransferase isoenzyme (hBCATm) must be stored in a reducing environment to remain active. Oxidation or labeling of hBCATm with sulfhydryl reagents results in enzyme inhibition. In this study, we investigated both the structural and biochemical basis for the sensitivity of hBCATm to these reagents. In its native form, hBCATm has two reactive cysteine residues which were identified as Cys315 and Cys318 using iodinated beta-(4-hydroxyphenyl)ethyl maleimide. These are located in the large domain of the homodimer, about 10 A from the active site. The crystal structures show evidence for a thiol-thiolate hydrogen bond between Cys315 and Cys318. Under oxidizing conditions, these cysteine residues can reasonably form a disulfide bond because of the short distance between the sulfur atoms (3.09-3.46 A), requiring only a decrease of 1.1-1.5 A. In addition to Cys315 playing a structural role by anchoring Tyr173, which in the ketimine form increases access to the active site, our evidence indicates that these cysteine residues act as a redox switch in hBCATm. Electrospray ionization mass spectrometry analysis and UV-Vis spectroscopic studies of 5,5'-dithiobis(2-nitrobenzoic acid) labeled hBCATm showed that during labeling, an intrasubunit disulfide bond was formed in a significant portion of the protein. Furthermore, it was established that reaction of hBCATm with H2O2 abolished its activity and resulted in the formation of an intrasubunit disulfide bond between Cys315 and Cys318. Addition of dithiothreitol completely reversed the oxidation and restored activity. Therefore, the results demonstrate that there is redox-linked regulation of hBCATm activity by a peroxide sensitive CXXC center. Future studies will determine if this center has an in vivo role in the regulation of branched chain amino acid metabolism.     

Bacteriology of the Labrador dog gut: a cultural and genotypic approach

AIMS: To carry out an extensive study of the microflora composition of the Labrador dog gut. METHODS AND RESULTS: Faecal specimens from four Labradors were collected and plated onto growth media designed to recover total anaerobes, bacteroides, bifidobacteria, lactobacilli, clostridia, Gram-positive cocci, total aerobes and coliforms. Morphologically different isolates were collected from all agars inoculated with faeces from one canine individual (repeated four times). A total of 157 out of 171 isolates were identified using 16S rRNA gene sequencing. Sequence analysis showed that agar selectivity was poor, especially when bacteroides and Gram-positive cocci were the targets. Bifidobacteria were not detected in any of the samples analysed, indicating their presence at low or negligible levels. The gene sequences of many of the isolates (n=45, representing 29% of the total) did not correlate with known species in the Ribosomal Database Project and EMBL databases, suggesting the presence of novel gut diversity. CONCLUSIONS: Traditional culture methods fail to reflect the bacterial diversity present in Labrador dog faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown the value of molecular-based methodologies for determining bacterial profiles in the Labrador dog gut microbiota, but has also exposed the limitations of purportedly selective agars.     

Testosterone regulates 25-hydroxycholesterol production in testicular macrophages

Recently, we found that testicular macrophages produce 25-hydroxycholesterol (25-HC) and express 25-hydroxylase, the enzyme that converts cholesterol to 25-HC. In addition, 25-HC may be an important paracrine factor mediating the known interactions between macrophages and neighboring Leydig cells, because it is efficiently converted to testosterone by Leydig cells. The purpose of the present study was to determine if testosterone can regulate the production of 25-HC in rat testicular macrophages, representing a potential negative-feedback loop from Leydig cells. We found that expression of 25-hydroxylase mRNA and production of 25-HC by cultured testicular macrophages were significantly inhibited by testosterone at 10 micro g/ml. This dose of testosterone did not have an effect on cell viability and did not change the rate of mRNA degradation in the presence of actinomycin D. These studies indicate that production of 25-HC is negatively regulated by testosterone, which may be representative of a paracrine negative-feedback loop.     

Neural crest and cardiovascular development: a 20-year perspective

Background

Twenty years ago this year was the first publication describing a region of neural crest cells necessary for normal cardiovascular development. Ablation of this region in chick resulted in persistent truncus arteriosus, mispatterning of the great vessels, outflow malalignments, and hypoplasia or aplasia of the pharyngeal glands.

Methods

We begin with a historical perspective and then review the progress that has been made in the ensuing 20 years in determining the direct and indirect contributions of the neural crest cells, now termed cardiac neural crest cells, in cardiovascular and pharyngeal arch development. Many of the molecular pathways that are now known to influence the specification, migration, patterning and final targeting of the cardiac neural crest cells are also reviewed.

Results

Although much knowledge has been gained by using many genetic manipulations to understand the cardiac neural crest cells' role in cardiovascular development, most models fail to explain the phenotypes seen in syndromic and non‐syndromic human congenital heart defects, such as the DiGeorge syndrome.

Conclusions

We propose that the cardiac neural crest exists as part of a larger cardiocraniofacial morphogenetic field and describe several human syndromes that result from abnormal development of this field. Birth Defects Research (Part C) 69:2–13, 2003. © 2003 Wiley‐Liss, Inc.

     

Liquid chromatographic determination of hydroxyproline in tissue samples

We describe a reversed-phase assay of hydroxyproline in rat lung tissue using sarcosine for the internal standard and pre-injection reaction with both o-phthalaldehyde (OPA) and 9-fluorenylmethylchloroformate (FMOC). Intra-assay variability in the concentration range of 25-500 microM hydroxyproline was less than 1%. Normal rat (left) lung was found to have a hydroxyproline content of 1.08+/-0.18 mg/lung. This ability to measure minute amounts of hydroxyproline is being applied to the measure of collagen and pathological fibrosis.     

The regulation of <Emphasis Type="Italic">yp3</Emphasis> expression in the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> fat body

The regulation of the Drosophila melanogaster yolk protein genes 1 and 2 have been well characterised. Cis-acting DNA elements and trans-acting factors regulating ovarian fat body and sex-specific expression have been identified. In this paper we have analysed the regulation of yolk protein 3, which is separated from the other two genes on the X-chromosome. We have separated sex-specific control from fat body control in some constructs in transgenic flies. We propose that the organisation of the regulatory elements in yp3 differs from yp1 and yp2 for control of fat body expression and that it closely resembles the regulation of a reporter gene using Musca and Calliphora yp promoter enhancer sequences in transgenic Drosophila.     

Use of sulfhydryl reagents to investigate branched chain alpha-keto acid transport in mitochondria

The goal of this paper was to determine the contribution of the mitochondrial branched chain aminotransferase (BCATm) to branched chain alpha-keto acid transport within rat heart mitochondria. Isolated heart mitochondria were treated with sulfhydryl reagents of varying permeability, and the data suggest that essential cysteine residues in BCATm are accessible from the cytosolic face of the inner membrane. Treatment with 15 nmol/mg N-ethylmaleimide (NEM) inhibited initial rates of alpha-ketoisocaproate (KIC) uptake in reconstituted mitochondrial detergent extracts by 70% and in the intact organelle by 50%. KIC protected against inhibition suggesting that NEM labeled a cysteine residue that is inaccessible when substrate is bound to the enzyme. Additionally, the apparent mitochondrial equilibrium KIC concentration was decreased 50-60% after NEM labeling, and this difference could not be attributed to effects of NEM on matrix pH or KIC oxidation. In fact, NEM was a better inhibitor of KIC oxidation than rotenone. Measuring matrix aspartate and glutamate levels revealed that the effects of NEM on the steady-state KIC concentration resulted from inhibition of BCATm catalyzed transamination of KIC with matrix glutamate to form leucine. Furthermore, circular dichroism spectra of recombinant human BCATm with liposomes showed that the commercial lipids used in the reconstituted transport assay contain BCAT amino acid substrates. Thus BCATm is distinct from the branched chain alpha-keto acid carrier but may interact with the inner mitochondrial membrane, and it is necessary to inhibit or remove transaminase activity in both intact and reconstituted systems prior to quantifying transport of alpha-keto acids which are transaminase substrates.     

The influence of barley food rewards on sheep movement through a handling system

The use of barley food rewards to improve sheep handling was evaluated in two experiments. In Experiment 1, six groups of sheep were trained to run through a race system and into a sheep-handling machine. Three groups were rewarded and three were not. After 10 days of training over 1 month, rewarded and unrewarded groups were either not handled, clamped in the machine, or clamped and inverted in the machine, and the efficiency of handling was measured. Food rewards significantly reduced the amount of labour required for sheep handling. Less time was spent pushing up rewarded sheep than unrewarded sheep for all handling treatments, and the race system was more than 90% efficient in 9 out of 15 tests with rewarded sheep compared with 1 out of 15 for unrewarded sheep.In Experiment 2, long-term memory was assessed by re-testing the same sheep 1 year later. Similar results to the first test were obtained for control (not handled) and clamped groups, but for inverted sheep there was no significant difference in push-up time between rewarded and unrewarded groups. These results suggest that sheep have excellent long-term memories of sheep handling procedures, both when rewarded and unrewarded. Although the effectiveness of food rewards was diminished as the severity of the handling treatment increased, the results indicate that rewarding sheep could lead to long-term improvement in sheep handling efficiency, especially when severe treatments are performed rarely.