Participation of metabolism in stem cell differentiation has been largely disregarded until recently. Here, functional proteomics and metabolomics were performed to unveil the mitochondrial and nuclear metabolism during dimethyl sulfoxide (DMSO)-induced differentiation of P19 cells. DMSO-treated cells were shown to exhibit increased glycolytic enzymes activities and fuel pyruvate into oxidative phosphorylation. Subsequently, enzymes of electron transport chain also had elevated activities upon differentiation. These changes in mitochondrial metabolism were concomitant with increased mitochondrial biogenesis as PGC-1α expression was higher in the differentiated cells. To study nuclear metabolism, particular focus was placed on delineating a potential role of nuclear lactate dehydrogenase (LDH). Nuclear LDH was found to exhibit higher expression in pluripotent cells. NAD+ generated from LDH reaction was discovered to promote histone deacetylation via sirtuin-1 (SIRT1). Drastic alterations in mitochondrial and nuclear metabolism during differentiation point to a pivotal role of metabolism in deciding the final destination of stem cells.

Human skin fibroblasts derived from patients with all 7 known inborn errors of vitamin B$ sb{12}$ metabolism have been studied for functional integrity of methylmalonyl CoA mutase and methionine synthase. Cocultivation of cblC and cblF fibroblasts in the absence of polyethylene glycol resulted in a twofold increase over the expected in both ($ sp{14}$C) propionate and ($ sp{14}$C) methyltetrahydrofolate incorporation into acid-precipitable material, suggesting that metabolic cooperation between cells occurs. CblD fibroblasts, which are biochemically similar to cblC cells (Goodman et al, 1970; Willard et al, 1977), do not cooperate metabolically when mixed with cblF cells. Partial correction in phenotype was seen in mixtures of cblD and cblG cells, but not cblC and cblG cells. These observations lend further support for the division of cblC and cblD disease into two discrete complementation classes. Cocultivation of cblF fibroblasts with both cblE and cblG cells also resulted in partial correction in phenotype. / ($ sp{14}$C) Propionate incorporation in both cblC and cblF cells exposed to conditioned medium from control cells was increased more than twofold. ($ sp{14}$C) methyltetrahydrofolate incorporation in cblC cells exposed to conditioned medium from cblF cells was increased twofold. This suggests the presence of a diffusible factor correcting the defect in the mutant cell lines.

In order to test the hypotheses: (1) that the digestive absorption of fluoride (F) is affected by the presence of aluminum (Al), and (2) that the distribution of F absorbed among organs/tissues is affected by Al, 36 American kestrels (Falco sparverius) were randomly divided into 6 groups of 6, and given oral doses daily for 30 days according to the following scheme: (1) deionized water only, (2) 30 mg/kg F$ sp-$, (3) 24 mg/kg F$ sp-$, (4) 24 mg/kg Al$ sp{3+}$, (5) 30 mg/kg F$ sp-$ + 24 mg/kg Al$ sp{3+},$ (6) 24 mg/kg F$ sp-$ + 24 mg/kg Al$ sp{3+}$. Excreta was collected every 24 hours between dosing. Femurae, kidneys, hearts, alimentary canals, skeletal muscle, and livers were obtained from all birds at the end of experiment. All samples were analyzed for F, Al, total phosphate (P) and calcium (Ca). F excretion was significantly higher in birds given 30 mg/kg F$ sp-$ + 24 mg/kg Al$ sp{3+}$ than in their counterparts which received the same amount of either F or Al alone (p $<$ 0.05). Excretion of Al was also elevated in all groups (except those only given water) from the level of excretion before the experiment commenced (p $<$ 0.05), despite the fact that 2 groups out of those 5 received F only. P and Ca contents of excreta were not affected by the oral dose. F contents in femurae from groups receiving both F and Al were significantly lower compared to those levels in those birds which were given F only (p $<$ 0.05). Significantly more Al was found in kidneys from the group receiving 30 mg/kg F$ sp-$ + 24 mg/kg Al$ sp{3+}$ than in those groups given only 1 of the 2 ions (p $<$ 0.05).

Biofilm communities were cultivated in rotating annular bioreactors using water from the South Saskatchewan River. The impacts of seasonal variations, nutrients, pollutants and dissolved oxygen on the activity and composition of the biofilms were assessed by using a combination of microcosm assays and molecular biology techniques. / The seasonal pattern in nitrification, denitrification and hexadecane mineralization, and in the occurrence of nirK in the South Saskatchewan River biofilms was: fall greater than winter, which was equivalent to spring. Hexadecane mineralization was higher in fall 1999 than in fall 2001, denitrification was similar in these two years, and no seasonal pattern of nitrification was observed. / The addition of combined nutrients (C, N, and P) resulted in significant increases in the measured bacterial activities and in the predominance of alkB, nirS and nirK in all seasons and years. The addition of individual nutrients did not stimulate hexadecane mineralization, denitrification, and the PCR amplification of nirS and nirK. In fall 1999, CNP and, to a lesser extent P, stimulated nitrification, whereas in fall 2001, no pattern was observed. The results showed that nutrients, especially P, were limiting for bacterial activities, and that the biofilm activities and composition varied with nutrient availability and time of year. / At the concentration assessed (1 ppb), hexadecane partially inhibited denitrification to similar extents in both years, had a negative impact on nitrification and hexadecane mineralization in fall 1999, and a positive impact on these two latter activities in fall 2001. Nickel (0.5 mg liter-1 ) negatively affected denitrification but had no effect on hexadecane mineralization. The alkB and nirS genes were less predominant and absent, respectively, in biofilms grown in the presence of nickel. DGGE analyses indicated that nickel reduced the biofilm bacterial diversity. / The results presented herein provide much needed information on the microbial ecology of river biofilms, and on the impact and interactive effects of pollutant and nutrient inputs on these biofilms. These results and the techniques used in this project can be applied to monitor environmental effects of anthropogenic activities on aquatic biofilms, and can contribute to establish or revise environmental regulations.

Methylmalonic aciduria (MMA) is an autosomal recessive metabolic disorder with an incidence of 1 in 48,000, which may be due to a defect in the mitochondrial homodimeric enzyme methylmalonyl CoA mutase (mut MMA). mut MMA is subdivided into $mut sp circ$ and $mut sp-$ subclasses on the basis of complementation analysis; $mut sp circ$ cell lines have very low incorporation of ($ sp{14}$C) from propionate into acid precipitable material while incorporation in $mut sp-$ cells is increased when cells are incubated in cobalamin. Intragenic complementation was first observed with WG 1130, a $mut sp circ$ fibroblast line with a homozygous R93H mutation, that is capable of complementing MCM activity when fused with some $mut sp circ$ and some $mut sp-$ cells (1). Extensive intragenic complementation in mut MMA was subsequently observed. Fibroblasts cultured from thirteen unrelated patients (6 $mut sp-$, 7 $mut sp circ$) were fused in all possible pairwise combination and MCM activity was assayed in the heterokaryons by measuring the incorporation of ($ sp{14}$C) from propionate into acid precipitable material. Intragenic complementation, indicated by stimulation of ($ sp{14}$C) -propionate incorporation following cell fusion with polyethylene glycol, was observed in fusions involving twelve of the thirteen strains. Of these thirteen strains, mutations have been identified in six; four have a homozygous mutation (WG 1130 (R93H), WG 1511 (H678R), WG 1610 (G717V), WG 1609 (G630E)), and two cell lines are compound heterozygous (WG 1681 (G623R and G703R), WG 1607 (W105R and A377E)); the remainders are yet to be determined. These intragenic complementations will provide information for grouping the mutations in defined domains in order to correlate structure and function of MCM.

Methylmalonic aciduria is an autosomal recessive metabolic disorder, which may be due to a defect in the methylmalonyl CoA mutase (MCM) apoenzyme. The mut$ sp circ$ mutation is characterized by undetectable enzyme activity in cell extracts, and by the low incorporation of ($ sp{14}$C) propionate in the presence of hydroxocobalamin in culture. A mut$ sp circ$ fibroblast cell line, WG 1681, from an African-American male infant was shown to complement another mut$ sp circ$ cell line, WG 1130. Subsequent cloning and sequencing of cDNA from WG 1681 identified two previously described homozygous polymorphisms: H532R and V671I(1). In addition, compound heterozygosity was observed for two novel changes at highly conserved sites: G623R and G703R. Hybridization of allele specific oligonucleotides to PCR amplified MCM exons from WG 1681 and family members identified a clinically normal mother, sister and half-brother as carriers of the G703R change in cis with both polymorphisms. The putative father was not identified as a carrier of the G623R change. transfection of each change, singly and in cis with both polymorphisms, into GM1673 cells demonstrated a lack of stimulation of ($ sp{14}$C) propionate uptake in the absence and presence of OH-Cbl, in comparison to controls. Co-transfection of each separate mutation with the previously identified R93H mutation of WG 1130 (2) stimulated propionate uptake. These results indicate that G623R and G703R are novel mutations responsible for deficient MCM activity and the mut$ sp circ$ phenotype in WG 1681, and both mutations are independently capable of complementing the R93H mutation of WG 1130.

The purpose of this investigation was to determine the relationship between calcium and glucose uptake following muscle contraction with the use of the calcium channel blocker dantrolene. In previous studies an exercise model has been used to investigate the role of calcium during post-exercise glucose uptake. This study utilized electrical stimulation. It has been shown that exercise-induced glucose uptake is calciummediated, but to date no one has shown that glucose transport induced by electrical stimulation is calcium-mediated. Twenty four male Sprague Dawley rats weighing 140 g were sacrificed and their epitrochlearis muscles were removed. Four treatment groups were established: control, muscle incubated in glucose (4mM); insulin, muscles incubated in glucose (4mM) and insulin (1000uU/ml); electrical stimulation, at 50 Hz for two five minute intervals separated by one minute rest periods; insulin (1000uU/ml) and electrical stimulation at 50 Hz for two five minute intervals separated by one minute intervals. Each group consisted of contain 8-10 muscle preparations. Glucose uptake was measured through the use of a double label of radioactive mannitol and 3-O-methylglucose and analyzed using liquid scintillation. This project followed a randomized group design. Treatments were measured with a one way ANOVA. / School of Physical Education

Seven strains of Actinobacillus suis (ATCC 15557, B49, C84, H89-1173, H91-0380, SO4 and VSB 3714) were investigated with respect to iron acquisition from animal transferrins (Tfs) and haemoglobins (Hbs). Growth assays with porcine, bovine and human Tfs and Hbs revealed that all seven strains could use porcine (but not human or bovine) Tf and all three Hbs as iron sources. In solid phase binding assays, membranes derived from all strains exhibited strong binding of porcine Tf and each of the Hbs. Competition binding assays indicated that all three Hbs were bound by the same receptor(s). Affinity procedures allowed the isolation and identification of iron repressible Tf-binding (~100 kDa and ~63 kDa) and Hb-binding (~105 kDa) polypeptides from all strains. Nucleotide sequence analyses revealed that A. suis strains SO4 and C84 possess genes that encode homologues of the Actinobacillus pleuropneumoniae Tf-binding proteins, TbpA and TbpB, and Hb-binding protein, HgbA. In both strains, tbpB was located immediately upstream of tbpA and was shown to be preceded by tonB, exbB and exbD homologues; hgbA was shown to be preceded by a hugZ homologue. Putative promoter and Fur box sequences were located upstream of tonB and hugZ and RT-PCR revealed that the genes in each of these clusters (tonB-exbB-exbD-tbpB-tbpA; hugZ-hgbA) are co-transcribed and iron-repressible. The molecular masses of the predicted mature TbpA, TbpB and HgbA proteins were calculated to be 104.3, 63.4 and 105.0 kDa, respectively, suggesting that the affinity-isolated, ~100 kDa and ~63 kDa Tf-binding polypeptides represent TbpA and TbpB, respectively, and that the ~105 kDa Hb-binding polypeptide represents HgbA. TbpB of A. suis was expressed in Escherichia coli and the recombinant TbpB (rTbpB) was identified by immunoblotting using swine sera raised against recombinant TbpB (A. pleuropneumoniae). It is envisaged that the acquisition of Tf- and Hb-bound iron by A. suis involves mechan

No description available.

A model was developed in growing piglets to study the use of urinary total sulfur excretion as an indicator of sulfur amino acid (SAA) catabolism and the nitrogen (N)/sulfur (S) balance ratio as an indicator of non-protein SAA storage. The recovery of administrated methionine as urinary total S over 48 hours was 106% in well-nourished piglets, but only 69% in protein malnourished piglets. The N/S balance ratio of protein malnourished piglets was lower than that of well-nourished piglets, and this ratio further decreased after methionine administration. We conclude that in a protein malnourished state, relatively more S than N is retained and a significant portion of the S derived from administrated methionine is retained in non-protein pools. These results demonstrate that urinary total S excretion can provide an accurate measure of SAA catabolism; and the N/S balance ratio can provide valuable information about non-protein SAA storage in growing piglets.