A control-based observer approach for estimating energy intake during pregnancy.

Gestational weight gain outside of Institute of Medicine guidelines poses a risk to both the mother and her unborn child. Behavioral interventions such as Healthy Mom Zone (HMZ) that aim to regulate gestational weight gain require self-monitoring of energy intake, which is often significantly under-reported by participants. This paper describes the use of a control systems approach for energy intake estimation during pregnancy. It relies on an energy balance model that predicts gestational weight based on physical activity and energy intake, the latter treated as an unmeasured disturbance. Two control-based observer formulations relying on Internal Model Control and Model Predictive Control, respectively, are presented in this paper, first for a hypothetical participant, then on data collected from four HMZ participants. Results demonstrate the effectiveness of the method, with generally best results obtained when estimating energy intake over a weekly time period.

Mutations in the tryptophan operon allow PurF-independent thiamine synthesis by altering flux in vivo.

Phosphoribosyl amine (PRA) is an intermediate in purine biosynthesis and also required for thiamine biosynthesis in Salmonella enterica. PRA is normally synthesized by phosphoribosyl pyrophosphate amidotransferase, a high-turnover enzyme of the purine biosynthetic pathway encoded by purF. However, PurF-independent PRA synthesis has been observed in strains having different genetic backgrounds and growing under diverse conditions. Genetic analysis has shown that the anthranilate synthase-phosphoribosyltransferase (AS-PRT) enzyme complex, involved in the synthesis of tryptophan, can play a role in the synthesis of PRA. This work describes the in vitro synthesis of PRA in the presence of the purified components of the AS-PRT complex. Results from in vitro assays and in vivo studies indicate that the cellular accumulation of phosphoribosyl anthranilate can result in nonenzymatic PRA formation sufficient for thiamine synthesis. These studies have uncovered a mechanism used by cells to redistribute metabolites to ensure thiamine synthesis and may define a general paradigm of metabolic robustness.

Salmonella enterica strains lacking the frataxin homolog CyaY show defects in Fe-S cluster metabolism in vivo.

In Salmonella enterica, the isc operon contains genes necessary for the synthesis of Fe-S clusters and strains lacking this operon have severe defects in a variety of cellular processes. Other cellular loci that impact Fe-S cluster synthesis to a lesser extent have been described. The cyaY locus encodes a frataxin homolog, and it is shown here that lesions in this locus affect Fe-S cluster metabolism. When present in combination with other lesions, mutations in cyaY can result in a strain with more severe defects than those lacking the isc locus.

Lack of YggX results in chronic oxidative stress and uncovers subtle defects in Fe-S cluster metabolism in Salmonella enterica.

As components involved in Fe-S cluster metabolism are described, the challenge becomes defining the integrated process that occurs in vivo based on the individual functions characterized in vitro. Strains lacking yggX have been used here to mimic chronic oxidative stress and uncover subtle defects in Fe-S cluster metabolism. We describe the in vivo similarities and differences between isc mutants, which have a known function in cluster assembly, and mutants disrupted in four additional loci, gshA, apbC, apbE, and rseC. The latter mutants share similarities with isc mutants: (i) a sensitivity to oxidative stress, (ii) a thiamine auxotrophy in the absence of the YggX protein, and (iii) decreased activities of Fe-S proteins, including aconitase, succinate dehydrogenase, and MiaB. However, they differ from isc mutants by displaying a phenotypic dependence on metals and a distinct defect in the SoxRS response to superoxides. Results presented herein support the proposed role of YggX in iron trafficking and protection against oxidative stress, describe additional phenotypes of isc mutants, and suggest a working model in which the ApbC, ApbE, and RseC proteins and glutathione participate in Fe-S cluster repair.

A mutation in the essential gene gmk (encoding guanlyate kinase) generates a requirement for adenine at low temperature in Salmonella enterica.

In Salmonella enterica serovar Typhimurium, gmk encodes guanylate kinase, an essential enzyme involved in the synthesis and salvage of guanine nucleotides. Here we report the isolation of a mutation in gmk that results in a nutritional requirement for adenine at low temperature. Comparisons of kinetic parameters from the wild-type and mutant Gmk enzymes revealed that the mutant enzyme had a more than 20-fold-higher Km for ATP than the wild-type enzyme. The growth dependence of the mutant on temperature and/or adenine could not be explained as a direct result of this kinetic difference. We propose a model in which previously described regulatory effects of GMP are responsible for these phenotypes.

Body image in middle school children.

The study purpose was to examine the relationship between sex, age, body mass index (BMI), and fitness level on the body image of 388 middle school children. Participants completed affective (social physique anxiety) and subjective evaluation (body dissatisfaction) body image measures and the 1-mile run/walk as an objective fitness measure. It was found that: (a) the females reported more social physique anxiety and body dissatisfaction than the males; (b) body dissatisfaction, especially for the females, was prevalent, (c) BMI was positively related to the body image measures; (d) for the males only, age was negatively related to the body image measures; (e) body dissatisfaction was negatively related to fitness levels; and (f) after controlling for BMI (and age for the males), fitness level was not a significant predictor of body dissatisfaction. The results indicate that body dissatisfaction was prevalent and early interventions with youth may be required.

Elevated levels of ketopantoate hydroxymethyltransferase (PanB) lead to a physiologically significant coenzyme A elevation in Salmonella enterica serovar Typhimurium.

Pantothenate is the product of the ATP-dependent condensation of pantoate and beta-alanine and is a direct precursor of coenzyme A. A connection exists between pantothenate biosynthesis and thiamine biosynthesis in Salmonella enterica serovar Typhimurium since derivatives of a purF mutant that can grow (on glucose medium) in the absence of thiamine excrete pantothenate. We show here that the causative mutation in three such mutants was the addition of a CG base pair upstream of the panB gene. This base addition brings the spacing between the -10 and -35 hexamers of the promoter to a consensus spacing of 17 bp and results in increased transcription of the pan operon. Furthermore, overexpression of PanB caused by this mutation, or by other means, was necessary and sufficient to increase pantothenate production and allow PurF-independent thiamine synthesis on glucose medium.

Children with intellectual disability in rural South Africa: prevalence and associated disability.

The objective of the present study was to determine the prevalence of intellectual disability (ID) and its associated disabilities in rural South African children aged 2-9 years. It was undertaken in eight villages in the district of Bushbuckridge, Northern Province, South Africa. A two-phase design was utilized. The first phase involved screening children on a house-to-house basis by interviewing mothers or caregivers using an internationally validated questionnaire for detecting childhood disability in developing countries. The second phase consisted of a paediatric/neurodevelopmental assessment of the children who screened positive. A total of 6692 children were screened; 722 (10.8%) had a paediatric evaluation and 238 children were diagnosed with ID, giving a minimum observed prevalence of 35.6 per 1000 children in this population. The prevalence of severe and mild ID was 0.64 per 1000 and 29.1 per 1000 children, respectively. The male:female ratio of children with ID was 3:2. In the affected children, a congenital aetiology for the ID was determined in 49 subjects (20.6%), an acquired aetiology in 15 (6.3%) and the aetiology was undetermined in 174 children (73.1%). Epilepsy (15.5%) and cerebral palsy (8.4%) were the commonest associated disabilities. The present study represents the first data on the prevalence of ID and associated disabilities in rural South African children. The prevalence of ID was comparable with results from a study performed in one other African country (Zambia) as well as those from other developing countries. The data provide an initial factual insight into ID and its associated disabilities for healthcare, social service and educational policy planners. This study provides a basis for the initiation and development of appropriate and integrated services for the best possible care of individuals affected with these disabilities, and for their possible prevention.

Neuropsychological impairment in soccer athletes.

BACKGROUND: Soccer reportedly places participants at risk for neuropsychological impairment, although it is unknown if the risk is associated primarily with concussion, subconcussive blows from heading the ball, or some combination thereof. Moreover, the extent to which younger versus older athletes are at risk for soccer-related cognitive impairment is unclear. We hypothesized that soccer athletes, especially older ones, would show poorer neuropsychological test performance than comparable swimmers. METHODS: Thirty-two soccer (26 college and 6 professional) and 29 swimmers (22 college and 7 masters level), all involved for at least 4 years in their sport at collegiate or national levels, participated. In a 2 X 2 (sport X age category) factorial design, all participants underwent 4 neuropsychological tests with 11 outcome measures assessing motor speed, attention, concentration, reaction time, and conceptual thinking. RESULTS: Soccer athletes performed worse than swimmers on measures of conceptual thinking. The older soccer group in particular performed poorly on measures of conceptual thinking, reaction time, and concentration. Among non-goaltender soccer athletes, estimates of career exposure to brain trauma (based on length of career and level of play) predicted significantly poorer test performance on 6 of 11 measures, even after statistically controlling for age. CONCLUSIONS: Results provide additional evidence that participation in soccer may be associated with poorer neuropsychological performance, although the observed pattern of findings does not specifically implicate heading as the cause. Although deficits were most apparent among older soccer players, serial neuropsychological testing for early detection of impairment is recommended for younger players as well.

Protection from superoxide damage associated with an increased level of the YggX protein in Salmonella enterica.

The deleterious effect of superoxide radicals on cell growth and survival is predominantly caused by rapid oxidation of labile [Fe-S] clusters in proteins. Oxidation of these clusters releases Fe(II) ions, which participate in Fenton chemistry that damages DNA. Here it is shown that elevated levels of the YggX protein increase the resistance of Salmonella enterica to superoxide stress, reverse enzymatic defects attributed to oxidized [Fe-S] clusters, and decrease the spontaneous mutation frequency. The data are consistent with a model in which YggX protects protein [Fe-S] clusters from oxidation.

Australian experience in general medical practice immunization--achievements and problems.

There are three essential components in the vaccination equation model: the vaccine giver, the vaccine receiver and the process facilitator. All three components must be considered if an immunization programme is to achieve its maximum potential. The author uses this model to explain the rationale behind strategies used in the Immunize Australia Campaign which began in 1997. The article discusses options used to overcome obstacles from the perspective of a general medical practitioner.

Altered pathway routing in a class of Salmonella enterica serovar Typhimurium mutants defective in aminoimidazole ribonucleotide synthetase.

In Salmonella enterica serovar Typhimurium, purine nucleotides and thiamine are synthesized by a branched pathway. The last known common intermediate, aminoimidazole ribonucleotide (AIR), is formed from formylglycinamidine ribonucleotide (FGAM) and ATP by AIR synthetase, encoded by the purI gene in S. enterica. Reduced flux through the first five steps of de novo purine synthesis results in a requirement for purines but not necessarily thiamine. To examine the relationship between the purine and thiamine biosynthetic pathways, purI mutants were made (J. L. Zilles and D. M. Downs, Genetics 143:37-44, 1996). Unexpectedly, some mutant purI alleles (R35C/E57G and K31N/A50G/L218R) allowed growth on minimal medium but resulted in thiamine auxotrophy when exogenous purines were supplied. To explain the biochemical basis for this phenotype, the R35C/E57G mutant PurI protein was purified and characterized kinetically. The K(m) of the mutant enzyme for FGAM was unchanged relative to the wild-type enzyme, but the V(max) was decreased 2.5-fold. The K(m) for ATP of the mutant enzyme was 13-fold increased. Genetic analysis determined that reduced flux through the purine pathway prevented PurI activity in the mutant strain, and purR null mutations suppressed this defect. The data are consistent with the hypothesis that an increased FGAM concentration has the ability to compensate for the lower affinity of the mutant PurI protein for ATP.

Crystal structure of the catalytic domain of human bile salt activated lipase.

Bile-salt activated lipase (BAL) is a pancreatic enzyme that digests a variety of lipids in the small intestine. A distinct property of BAL is its dependency on bile salts in hydrolyzing substrates of long acyl chains or bulky alcoholic motifs. A crystal structure of the catalytic domain of human BAL (residues 1-538) with two surface mutations (N186D and A298D), which were introduced in attempting to facilitate crystallization, has been determined at 2.3 A resolution. The crystal form belongs to space group P2(1)2(1)2(1) with one monomer per asymmetric unit, and the protein shows an alpha/beta hydrolase fold. In the absence of bound bile salt molecules, the protein possesses a preformed catalytic triad and a functional oxyanion hole. Several surface loops around the active site are mobile, including two loops potentially involved in substrate binding (residues 115-125 and 270-285).

Action of the thiamine antagonist bacimethrin on thiamine biosynthesis.

Bacimethrin is an analog of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine and inhibits the growth of Salmonella enterica serovar Typhimurium on a defined medium. Two classes of mutants that had increased bacimethrin resistance were isolated and characterized. Results showed that overexpression of the thi operon or specific lesions in thiD resulted in a bacimethrin-resistant phenotype. Phenotypic analyses of the thiD mutants suggested that they had a specific defect in one of the two kinase activities associated with this gene product and, further, that ThiD and not PdxK was primarily responsible for salvage of HMP from the medium.

Lesions in gshA (Encoding gamma-L-glutamyl-L-cysteine synthetase) prevent aerobic synthesis of thiamine in Salmonella enterica serovar typhimurium LT2.

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella enterica serovar Typhimurium and other bacteria. In addition to genes encoding enzymes in the biosynthetic pathway, mutations in other metabolic loci have been shown to prevent thiamine synthesis. The latter loci identify the integration of the thiamine biosynthetic pathway with other metabolic processes and can be uncovered when thiamine biosynthesis is challenged. Mutations in gshA, encoding gamma-L-glutamyl-L-cysteine synthetase, prevent the synthesis of glutathione, the major free thiol in the cell, and are shown here to result in a thiamine auxotrophy in some of the strains tested, including S. enterica LT2. Phenotypic characterization of the gshA mutants indicated they were similar enough to apbC and apbE mutants to warrant the definition of a class of mutants unified by (i) a requirement for both the hydroxymethyl pyrimidine (HMP) and thiazole (THZ) moiety of thiamine, (ii) the ability of L-tryosine to satisfy the THZ requirement, (iii) suppression of the thiamine requirement by anaerobic growth, and (iv) suppression by a second-site mutation at a single locus. Genetic data indicated that a defective ThiH generates the THZ requirement in these strains, and we suggest this defect is due to a reduced ability to repair a critical [Fe-S] cluster.

Metabolic defects caused by mutations in the isc gene cluster in Salmonella enterica serovar typhimurium: implications for thiamine synthesis.

The metabolic consequences of two insertions, iscR1::MudJ and iscA2::MudJ, in the isc gene cluster of Salmonella enterica serovar Typhimurium were studied. Each of these insertions had polar effects and caused a nutritional requirement for the thiazole moiety of thiamine. Data showed that IscS was required for the synthesis of nicotinic acid and the thiazole moiety of thiamine and that one or more additional isc gene products were required for a distinct step in the thiazole biosynthetic pathway. Strains with isc lesions had reduced succinate dehydrogenase and aconitase activities. Furthermore, isc mutants accumulated increased levels of pyruvate in the growth medium in response to exogenously added iron (FeCl(3)), and this response required a functional ferric uptake regulator, Fur.

Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein.

The cDNAs of two new human membrane-associated aspartic proteases, memapsin 1 and memapsin 2, have been cloned and sequenced. The deduced amino acid sequences show that each contains the typical pre, pro, and aspartic protease regions, but each also has a C-terminal extension of over 80 residues, which includes a single transmembrane domain and a C-terminal cytosolic domain. Memapsin 2 mRNA is abundant in human brain. The protease domain of memapsin 2 cDNA was expressed in Escherichia coli and was purified. Recombinant memapsin 2 specifically hydrolyzed peptides derived from the beta-secretase site of both the wild-type and Swedish mutant beta-amyloid precursor protein (APP) with over 60-fold increase of catalytic efficiency for the latter. Expression of APP and memapsin 2 in HeLa cells showed that memapsin 2 cleaved the beta-secretase site of APP intracellularly. These and other results suggest that memapsin 2 fits all of the criteria of beta-secretase, which catalyzes the rate-limiting step of the in vivo production of the beta-amyloid (Abeta) peptide leading to the progression of Alzheimer's disease. Recombinant memapsin 2 also cleaved a peptide derived from the processing site of presenilin 1, albeit with poor kinetic efficiency. Alignment of cleavage site sequences of peptides indicates that the specificity of memapsin 2 resides mainly at the S(1)' subsite, which prefers small side chains such as Ala, Ser, and Asp.

Lesions in the nuo operon, encoding NADH dehydrogenase complex I, prevent PurF-independent thiamine synthesis and reduce flux through the oxidative pentose phosphate pathway in Salmonella enterica serovar typhimurium.

In Salmonella enterica serovar Typhimurium, PurF-independent thiamine synthesis (or alternative pyrimidine biosynthesis) allows strains, under some growth conditions, to synthesize thiamine in the absence of the first step in the purine biosynthetic pathway. Mutations have been isolated in a number of loci that prevent this synthesis and thus result in an Apb(-) phenotype. Here we identify a new class of mutations that prevent PurF-independent thiamine synthesis and show that they are defective in the nuo genes, which encode the major, energy-generating NADH dehydrogenase of the cell. Data presented here indicated that a nuo mutant has reduced flux through the oxidative pentose phosphate pathway that may contribute to, but is not sufficient to cause, the observed thiamine requirement. We suggest that reduction of the oxidative pentose phosphate pathway capacity in a nuo mutant is an attempt to restore the ratio between reduced and oxidized pyridine nucleotide pools.

Reduced flux through the purine biosynthetic pathway results in an increased requirement for coenzyme A in thiamine synthesis in Salmonella enterica serovar typhimurium.

Work presented here establishes a connection between cellular coenzyme A (CoA) levels and thiamine biosynthesis in Salmonella enterica serovar Typhimurium. Prior work showed that panE mutants (panE encodes ketopantoate reductase) had a conditional requirement for thiamine or pantothenate. Data presented herein show that the nutritional requirement of panE mutants for either thiamine or pantothenate is manifest only when flux through the purine biosynthetic pathway is reduced. Further, the data show that under the above conditions it is the lack of thiamine pyrophosphate, and not decreased CoA levels, that directly prevents growth.

Impact of genomics and genetics on the elucidation of bacterial metabolism.

In the last few years, the emergence of complete genome sequences has had profound effects on all fields of biology. While the existence of these genome sequences has served to facilitate experimental work, it has also highlighted the gaps in our knowledge of bacterial metabolism. Our current knowledge of metabolism is primarily the result of data accumulated from decades of study by biochemists and geneticists. In general these studies focused on discrete pathways and their regulation. The technical innovations of the last decade, culminating with the sequencing of complete genomes, provide us with the ability to address the next frontier in physiology, metabolic integration. Herein we describe current approaches that can be used to complement classic genetic approaches and further our understanding of both novel metabolic functions and metabolic integration in microorganisms.