Loss of glial glutamate transporters and induction of neuronal expression of GLT-1B in the hypoxic neonatal pig brain

The homeostasis of glutamate is critical to normal brain function; deficiencies in the regulation of extracellular glutamate are thought to be a major determinant of damage in hypoxic brains. Extracellular levels of glutamate are regulated mainly by plasmalemmal glutamate transporters. We have evaluated the distribution of the glutamate transporter GLAST and two splice variants of GLT-1 in the hypoxic neonatal pig brain using this as model of neonatal humans. In response to severe hypoxic insults, we observe a rapid loss of two glial glutamate transporters from specific brain regions, such as the CA1 region of the hippocampus, but not the dentate gyrus. The spatial distribution of loss accords with patterns of damage in these brains. Conversely, we demonstrate that hypoxia evokes the expression of a splice variant of GLT-1 in neurons. We suggest that this expression may be induced in response to elevated extracellular glutamate around these neurons, and that this splice variant may represent a useful marker for direct quantification of the extent of likely neuronal damage in hypoxic brains. © 2004 Elsevier B.V. All rights reserved.

Lysine requirements of pre-lay broiler breeder pullets: Determination by indicator amino acid oxidation

The indicator amino acid oxidation (IAAO) method allows the determination of amino acid requirements under conditions of low growth rate as found in pre-laying broiler breeder pullets. Cobb 500 breeder pullets (20 wk old; 2290 +/- 280 g, n = 4) were adapted (6 d) to a pelleted, purified control diet containing all nutrients at greater than or equal to 110% of NRC recommendations. After recovery from surgery for implantation of a jugular catheter, each bird was fed, in random order, test diets containing one of nine levels of lysine (0.48, 0.96, 1.92, 2.88, 3.84, 4.80, 7.68, 9.60 and 14.40 g/kg of diet). Indicator oxidation was determined during 4-h primed (74 kBq/kg body), constant infusions (44 kBq (.) h(-1) (.) kg body(-1)) of L-[1-C-14]phenylalanine. Using the breakpoint of a one-slope broken-line model, the lysine requirement was determined to be 4.88 +/- 0.96 g/kg of diet or 366 +/- 72 mg (.) hen(-1) (.) d(-1) with an upper 95% Cl of 6.40 g/kg of diet or 480 mg (.) hen(-1) (.) d(-1). IAAO allows determination of individual bird amino acid requirements for specific ages and types of birds over short periods of time and enables more accurate broiler breeder pullet diet formulation.

Comparison of apparent ileal amino acid digestibility of feed ingredients measured with broilers, layers, and roosters

The apparent ileal digestibility of amino acids in 7 feed ingredients was determined using broilers, layers, and roosters. The ingredients included 3 cereals (wheat, sorghum, and corn), 3 oilseed meals (canola, cottonseed, and soybean meals), and 1 animal protein meal (meat and bone meal). Dietary protein in the assay diets was supplied solely by the test ingredient. All diets contained 20 g/kg of acid-insoluble ash as an indigestible marker, and each diet was offered ad libitum in mash form to 5 replicate pens of broilers and layers, and 4 replicate pens of roosters. The digestibility coefficients of individual amino acids for wheat, corn, and sorghum were higher (P < 0.05) in broilers than in layers and roosters. The digestibility of most amino acids for corn and sorghum was higher (P < 0.05) in roosters compared with those in layers, whereas the digestibility for wheat in layers and roosters was similar. In general, the digestibility of amino acids in canola meal, cottonseed meal, and meat and bone meal was similar among the 3 classes of chickens. The digestibility of amino acids in soybean meal was higher (P < 0.05) for layers compared with those for broilers and roosters but similar between broilers and roosters. These results suggest that the class of chickens significantly influenced the apparent ileal digestibility of amino acids in some feed ingredients.

Deduced amino acid sequences surrounding the fusion glycoprotein cleavage site and of the carboxyl-terminus of haemagglutinin-neuraminidase protein of the avirulent thermostable vaccine strain I-2 of Newcastle disease virus

A single-tube RT-PCR technique generated a 387 bp or 300 bp cDNA amplicon covering the F-0 cleavage site or the carboxyl (C)-terminus of the HN gene, respectively, of Newcastle disease virus (NDV) strain 1-2. Sequence analysis was used to deduce the amino acid sequences of the cleavage site of F protein and the C-terminus of HN protein, which were then compared with sequences for other NDV strains. The cleavage site of NDV strain 1-2 had a sequence Motif of (112)RKQGRLIG(119), consistent with an avirulent phenotype. Nucleotide sequencing and deduction of amino acids at the C-terminus of HN revealed that strain 1-2 had a 7-amino-acid extension (VEILKDGVREARSSR). This differs from the virulent viruses that caused outbreaks of Newcastle disease in Australia in the 1930s and 1990s, which have HN extensions of 0 and 9 amino acids, respectively. Amino acid sequence analyses of the F and HN genes of strain 1-2 confirmed its avirulent nature and its Australian origin.

Interactions between serotonergic genotype and amino acid transmitter receptor expression in human alcoholics

Serotonin can modulate the activity of neural reward pathways that are strongly implicated in mediating the effects of chronic alcohol misuse, and its treatment, in human subjects. In previous work and as discussed elsewhere at this meeting, we and others have found consistent differences in the parameters of GABA and glutamate receptors, and the expression of their component subunit transcripts and proteins, in areas of the alcoholic brain that are altered by alcoholism. We did not fi nd clear changes in GABA and glutamate transport function in such samples, but a series of microarray analyses showed consistent upregulation of the presynaptic GABA/betaine transporter SLC6A12. Microarray studies showed no signifi cant differences in the expression of transcripts associated with 5HT transmission; however, only a small number of such elements were present on the arrays. Here we partitioned GABAA and NMDA pharmacology, and subunit mRNA and protein expression, measured in samples of frontal and motor cortex obtained at autopsy from alcoholics without comorbid disease, alcoholics with liver cirrhosis, and controls, according to 5HTTLPR (SLC6A4) and 5HT1B (HTR1B) polymorphisms. We found no effect of these genotypes on the expression of GABAA receptor gene products, but there was a signifi cant mRNA Transcript X Area X Group X 5HTTLPR Interaction with NMDA subunit isoform expression measured by Real Time PCR with GAPDH normalization. Further analysis showed the effect to be selective for alcoholics with cirrhosis, to be most marked in the pathologically vulnerable frontal cortex, and to vary with subunit transcript (F2,76 = 6.545, P = 0.002). NR1 expression was most affected, followed by NR2A, with NR2B expression least altered. Pilot data suggest 5HT1B genotype may also modulate NMDA subunit expression. Interactions between amino acid and serotonin transmission may infl uence susceptibility to alcohol dependence or pathogenesis

Endogenous amino acid flow in the avian ileum: quantification using three techniques

The aim of the present study was to compare the protein-free diet, guanidinated casein (GuC) and enzyme hydrolysed casein (EHC) methods for the quantification of endogenous amino acid (AA) flow in the avian ileum. Growing broiler chickens (5 weeks old) were used. All three assay diets were based on dextrose, and in the GuC and EHC diets GuC or EHC were the sole source of N. Endogenous AA flows determined with the use of protein-free diet were considerably lower (P < 0.05) than those determined by the GuC and EHC methods. The, total endogenous AA flows determined by the GuC and EHC methods were almost 3-fold greater (P < 0.05) than those determined by the protein-free diet. The endogenous AA values obtained from GuC and EHC methods were similar (P >0.05), except for the flow of arginine, which was lower (P < 0.05) in the EHC method. Glutamic acid, aspartic acid, threonine and glycine were the predominant endogenous AA present in digesta from the distal ileum. The contents of methionine, histidine and cystine were lower compared with other AA. The method of determination had no effect on the AA composition of endogenous protein, except for threonine, glutamic acid, lysine, arginine and cystine. The concentrations of threonine and arginine were lower (P < 0.05) and that of lysine was higher (P < 0.05) with the EHC method compared with the other two methods. The concentration of glutamic acid was greater (P < 0.05) and that of cystine was lower (P < 0.05) in the EHC and GuC methods compared with the protein-free diet method. The results showed that the ileal endogenous flows of N and AA are markedly enhanced by the presence of protein and peptides, above those determined following feeding of a protein-free diet. It is concluded that the use of EHC and GuC methods enables the measurement of ileal endogenous losses in chickens under normal physiological conditions.

Glutamate transporter localization does not correspond to the temporary functional recovery and late degeneration after acute ocular ischemia in rats

Purpose: To determine whether the localization of retinal glutamate transporters is affected by retinal ischaemia and whether their ability to transport glutamate decreases with the progression of ischemic retinal and optic nerve degeneration. Methods: Retinal ischemia was induced in rats by acutely increasing the intraocular pressure (IOP, 110 mmHg/60 min). Reperfusion was permitted for periods up to 60 days post-ischemia. Functional evaluation was performed by monitoring the pupil light reflexes (PLRs) and electroretinograms (flash, flicker ERG and oscillatory potentials). Glutamate transporter localization and D-aspartate (glutamate analogue) uptake were assessed by immunohistochemistry. Results: Intense immunoreactivity for the retinal glutamate transporters (GLAST, GLT1, EAAC1 and EAAT5) was observed at all time points after the insult, despite severe retinal degeneration. D-aspartate was also normally accumulated in the ischemic retinas. Ten days post-operatively the PLR ratio (ratio = indirect/direct PLR = 34 +/- 7(.)5%) was significantly less than the pre-operative value (pre-op = 76(.)7 +/- 2 (.)6%, p < 0(.)05). However, 25 and 35 days post-operatively PLR ratios did not differ significantly from pre-operative values (44(.)4 +/- 6(.)9 and 53(.)8 +/- 9(.)6%, p > 0(.)05). Forty-five and 60 days post-operatively the PLR ratio declined again and was significantly lower than the pre-operative value (33(.)8 + 8(.)7 and 26(.)2 + 8(.)9%, p < 0(.)05). Statistical analysis revealed that all tested ERG components had significantly higher values at 32, but not at 42 and 58 days post-operatively when compared to the first time point recorded post-operatively (10 days). Conclusions: While retinal glutamate transport is compromised during an acute ischemic insult, consequent retinal recovery and degeneration are not due to a change in the excitatory amino acid transporter localization or D-aspartate (glutamate analogue) uptake. Rat retina and optic nerve are capable of spontaneous, but temporary, functional recovery after an acute ischemic insult. (C) 2004 Elsevier Ltd. All rights reserved.

Increased duodenal expression of divalent metal transporter 1 and iron-regulated gene 1 in cirrhosis

Hepatic hemosiderosis and increased iron absorption are common findings in cirrhosis. It has been proposed that a positive relation exists between intestinal iron absorption and the development of hepatic hemosiderosis. The current study investigated the duodenal expression of the iron transport molecules divalent metal transporter 1 (DMT1 [IRE]), iron-regulated gene 1 (Ireg1 [ferroportin]), hephaestin, and duodenal cytochrome b (Dyctb) in 46 patients with cirrhosis and 20 control subjects. Total RNA samples were extracted from duodenal biopsy samples and the expression of the iron transport genes was assessed by ribonuclease protection assays. Expression of DMT1 and Ireg1 was increased 1.5 to 3-fold in subjects with cirrhosis compared with iron-replete control subjects. The presence of cirrhosis per se and serum ferritin (SF) concentration were independent factors that influenced the expression of DMT1. However, only SF concentration was independently associated with Iregl expression. In cirrhosis, the expression of DMT1 and Iregl was not related to the severity of liver disease or cirrhosis type. There was no correlation between the duodenal expression of DMT1 and Iregl and the degree of hepatic siderosis. In conclusion, the presence of cirrhosis is an independent factor associated with increased expression of DMT1 but not Iregl. The mechanism by which cirrhosis mediates this change in DMT1 expression has yet to be determined. Increased expression of DMT1 may play an important role in the pathogenesis of cirrhosis-associated hepatic iron overload.

Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site

Sulfadoxine is predominantly used in combination with pyrimethamine, commonly known as Fansidar, for the treatment of Plasmodium falciparum. This combination is usually less effective against Plasmodium vivax, probably due to the innate refractoriness of parasites to the sulfadoxine component. To investigate this mechanism of resistance by P. vivax to sulfadoxine, we cloned and sequenced the P. vivax dhps (pvdhps) gene. The protein sequence was determined, and three-dimensional homology models of dihydropteroate synthase (DHPS) from P. vivax as well as P. falciparum were created. The docking of sulfadoxine to the two DHPS models allowed us to compare contact residues in the putative sulfadoxine-binding site in both species. The predicted sulfadoxine-binding sites between the species differ by one residue, V585 in P. vivax, equivalent to A613 in P. falciparum. V585 in P. vivax is predicted by energy minimization to cause a reduction in binding of sulfadoxine to DHPS in P. vivax compared to P. falciparum. Sequencing dhps genes from a limited set of geographically different P. vivax isolates revealed that V585 was present in all of the samples, suggesting that V585 may be responsible for innate resistance of P. vivax to sulfadoxine. Additionally, amino acid mutations were observed in some P. vivax isolates in positions known to cause resistance in P. falciparum, suggesting that, as in P. falciparum, these mutations are responsible for acquired increases in resistance of P. vivax to sulfadoxine.

Better prediction of protein contact number using a support vector regression analysis of amino acid sequence

Background: Protein tertiary structure can be partly characterized via each amino acid's contact number measuring how residues are spatially arranged. The contact number of a residue in a folded protein is a measure of its exposure to the local environment, and is defined as the number of C-beta atoms in other residues within a sphere around the C-beta atom of the residue of interest. Contact number is partly conserved between protein folds and thus is useful for protein fold and structure prediction. In turn, each residue's contact number can be partially predicted from primary amino acid sequence, assisting tertiary fold analysis from sequence data. In this study, we provide a more accurate contact number prediction method from protein primary sequence. Results: We predict contact number from protein sequence using a novel support vector regression algorithm. Using protein local sequences with multiple sequence alignments (PSI-BLAST profiles), we demonstrate a correlation coefficient between predicted and observed contact numbers of 0.70, which outperforms previously achieved accuracies. Including additional information about sequence weight and amino acid composition further improves prediction accuracies significantly with the correlation coefficient reaching 0.73. If residues are classified as being either contacted or non-contacted, the prediction accuracies are all greater than 77%, regardless of the choice of classification thresholds. Conclusion: The successful application of support vector regression to the prediction of protein contact number reported here, together with previous applications of this approach to the prediction of protein accessible surface area and B-factor profile, suggests that a support vector regression approach may be very useful for determining the structure-function relation between primary sequence and higher order consecutive protein structural and functional properties.

D-amino acid residue in a defensin-like peptide from platypus venom: effect on structure and chromatographic properties

The recent discovery that the natriuretic peptide OvCNPb (Ornithorhynchus venom C-type natriuretic peptide B) from platypus (Ornithorynchus anatinus) venom contains a D-amino acid residue suggested that other D-amino-acid-containing peptides might be present in the venom. In the present study, we show that DLP-2 (defensin-like peptide-2), a 42-amino-acid residue polypeptide in the platypus venom, also contains a D-amino acid residue, D-methionine, at position 2, while DLP-4, which has an identical amino acid sequence, has all amino acids in the L-form. These findings were supported further by the detection of isomerase activity in the platypus gland venom extract that converts DLP-4 into DLP-2. In the light of this new information, the tertiary structure of DLP-2 was recalculated using a new structural template with D-Met(2). The structure of DLP-4 was also determined in order to evaluate the effect of a D-amino acid at position 2 on the structure and possibly to explain the large retention time difference observed for the two molecules in reverse-phase HPLC. The solution structures of the DLP-2 and DLP-4 are very similar to each other and to the earlier reported structure of DLP-2, which assumed that all amino acids were in the L-form. Our results suggest that the incorporation of the D-amino acid at position 2 has minimal effect on the overall fold in solution.

A gradient descent algorithm for minimizing amino acid coupling reactions when synthesizing cyclic-peptide libraries

Combinatorial chemistry has become an invaluable tool in medicinal chemistry for the identification of new drug leads. For example, libraries of predetermined sequences and head-to-tail cyclized peptides are routinely synthesized in our laboratory using the IRORI approach. Such libraries are used as molecular toolkits that enable the development of pharmacophores that define activity and specificity at receptor targets. These libraries can be quite large and difficult to handle, due to physical and chemical constraints imposed by their size. Therefore, smaller sub-libraries are often targeted for synthesis. The number of coupling reactions required can be greatly reduced if the peptides having common amino acids are grouped into the same sub-library (batching). This paper describes a schedule optimizer to minimize the number of coupling reactions by rotating and aligning sequences while simultaneously batching. The gradient descent method thereby reduces the number of coupling reactions required for synthesizing cyclic peptide libraries. We show that the algorithm results in a 75% reduction in the number of coupling reactions for a typical cyclic peptide library.