Stephen P. J. Brooks

Bound and determined : a computer program for making buffers of defined ion concentrations

A computer program that allows the preparation of buffers containing known concentrations of metal-ligand complexes at defined pH values and temperatures is described. Ligands are defined as compounds that bind metals and may include AMP, ADP, ATP, GMP, GDP, GTP, EGTA, EDTA, BAPTA, phosphate, sulfate, chloride, monocarboxylic acids, dicarboxylic acids, organophosphates, and/or citric acid. Metals may include sodium, potassium, magnesium, calcium, and/or manganese. The program uses association constants corrected for temperature and ionic strength so that solutions between 0 and 40 degrees C and between pH values of 4 and 10 can be defined. The program can perform the following: (i) calculate the concentration of all metal-ligand complexes when total metal and total ligand concentrations are known, (ii) calculate the concentration of metal ion required to make a solution of known free metal ion concentration when total ligand concentrations are known, (iii) calculate the concentration of ligand required to make a solution of known free metal ion concentration when total metal concentrations are known, and (iv) calculate the total concentrations of metal and ligand required to make a buffer of known metal-ligand concentration. Options i-iii are useful for making buffers of defined free metal ion concentrations; option iv is useful for making buffers of defined metal-nucleotide concentrations.

Glycolytic controls in estivation and anoxia: a comparison of metabolic arrest in land and marine molluscs.

Facultative metabolic rate depression is the common adaptive strategy underlying various animal mechanisms for surviving harsh environmental conditions. This strategy is common among molluscs, enabling animals to survive over days or even months in the absence of oxygen or under extremely dry conditions. The large reductions in metabolic rate during estivation and anoxia can translate into considerable energy savings when dormant animals are compared to active animals. A complex metabolic coordination is required during the transition into the dormant state to maintain cellular homeostasis and involves both energy-consuming and energy-producing pathways. With regard to energy-producing pathways, several different mechanisms have been identified that participate in controlling flux. One such mechanism, enzyme phosphorylation, can have a wide-ranging effect. For example, phosphorylated enzymes exhibit altered substrate, activator, and inhibitor affinities. This effect may be magnified by changes in the concentrations of allosteric effectors, such as fructose 2,6-bisphosphate, that occur during hypometabolic states. Changes in fructose 2,6-bisphosphate are related to changes in enzyme phosphorylation through changes in the relative activity of phosphofructokinase-2. Alterations in glycolytic enzyme binding can also be brought about through changes in enzyme phosphorylation. The present review focuses on identifying hypometabolism-related changes in enzyme phosphorylation as well as characterizing the mechanisms involved in mediating these phosphorylation events.

Metabolic adjustments during daily torpor in the Djungarian hamster

Djungarian hamsters (Phodopus sungorus) acclimated to a short photoperiod (8:16-h light-dark cycle) display spontaneous daily torpor with ad libitum food availability. The time course of body temperature (Tb), metabolic rate, respiratory quotient (RQ), and substrate and enzyme changes was measured during entrance into torpor and in deep torpor. RQ, blood glucose, and serum lipids are high during the first hours of torpor but then gradually decline, suggesting that glucose is the primary fuel during the first hours of torpor, with a gradual change to lipid utilization. No major changes in enzyme activities were observed during torpor except for inactivation of the pyruvate dehydrogenase (PDH) complex in liver, brown adipose tissue, and heart muscle. PDH inactivation closely correlates with the reduction of total metabolic rate, whereas in brain, kidney, diaphragm, and skeletal muscle, PDH activity was maintained at the initial level. These findings suggest inhibition of carbohydrate oxidation in heart, brown adipose tissue, and liver during entrance into daily torpor.Djungarian hamsters ( Phodopus sungorus) acclimated to a short photoperiod (8:16-h light-dark cycle) display spontaneous daily torpor with ad libitum food availability. The time course of body temperature (Tb), metabolic rate, respiratory quotient (RQ), and substrate and enzyme changes was measured during entrance into torpor and in deep torpor. RQ, blood glucose, and serum lipids are high during the first hours of torpor but then gradually decline, suggesting that glucose is the primary fuel during the first hours of torpor, with a gradual change to lipid utilization. No major changes in enzyme activities were observed during torpor except for inactivation of the pyruvate dehydrogenase (PDH) complex in liver, brown adipose tissue, and heart muscle. PDH inactivation closely correlates with the reduction of total metabolic rate, whereas in brain, kidney, diaphragm, and skeletal muscle, PDH activity was maintained at the initial level. These findings suggest inhibition of carbohydrate oxidation in heart, brown adipose tissue, and liver during entrance into daily torpor.

Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells

Ctr1 (copper transporter 1) mediates high-affinity copper uptake. Ctr2 (copper transporter 2) shares sequence similarity with Ctr1, yet its function in mammalian cells is poorly understood. In African green monkey kidney COS-7 cells and rat tissues, Ctr2 migrated as a predominant band of approximately 70 kDa and was most abundantly expressed in placenta and heart. A transiently expressed hCtr2-GFP (human Ctr2-green fluorescent protein) fusion protein and the endogenous Ctr2 in COS-7 cells were mainly localized to the outer membrane of cytoplasmic vesicles, but were also detected at the plasma membrane. Biotinylation of Ctr2 with the membrane-impermeant reagent sulfo-NHS-SS-biotin [sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate] confirmed localization at the cell surface. Cells expressing hCtr2-GFP hyperaccumulated copper when incubated in medium supplemented with 10 microM CuSO(4), whereas cells depleted of endogenous Ctr2 by siRNAs (small interfering RNAs) accumulated lower levels of copper. hCtr2-GFP expression did not affect copper efflux, suggesting that hCtr2-GFP increased cellular copper concentrations by promoting uptake at the cell surface. Kinetic analyses showed that hCtr2-GFP stimulated saturable copper uptake with a K(m) of 11.0+/-2.5 microM and a K(0.5) of 6.9+/-0.7 microM when data were fitted to a rectangular hyperbola or Hill equation respectively. Competition experiments revealed that silver completely inhibited hCtr2-GFP-dependent copper uptake, whereas zinc, iron and manganese had no effect on uptake. Furthermore, increased copper concentrations in hCtr2-GFP-expressing cells were inversely correlated with copper chaperone for Cu/Zn superoxide dismutase protein expression. Collectively, these results suggest that Ctr2 promotes copper uptake at the plasma membrane and plays a role in regulating copper levels in COS-7 cells.

Mechanisms of glycolytic control during hibernation in the ground squirrel Spermophilus lateralis

SummaryThe mechanisms of glycolytic rate control during hibernation in the ground squirrel Spermophilus lateralis were investigated in four tissues: heart, liver, kidney, and leg muscle. Overall glycogen phosphorylase activity decreased significantly in liver and kidney to give 50% or 75% of the activity found in the corresponding euthermic organs, respectively. The concentration of fructose-2,6-bisphosphate (F-2,6-P2) decreased significantly in heart and leg muscle during hibernation to 50% and 80% of euthermic tissue concentrations, respectively, but remained constant in liver and kidney. The overall activity of pyruvate dehydrogenase (PDH) in heart and kidney from hibernators was only 4% of the corresponding euthermic values. Measurements of phosphofructokinase (PFK) and pyruvate kinase (PK) kinetic parameters in euthermic and hibernating animals showed that heart and skeletal muscle had typical rabbit skeletal M-type PFK and M1-type PK. Liver and kidney PFK were similar to the L-type enzyme from rabbit liver, whereas liver and kidney PK were similar to the M2 isozyme found primarily in rabbit kidney. The kinetic parameters of PFK and PK from euthermic vs hibernating animals were not statistically different. These data indicate that tissue-specific phosphorylation of glycogen phosphorylase and PDH, as well as changes in the concentration of F-2,6-P2 may be part of a general mechanism to coordinate glycolytic rate reduction in hibernating S. lateralis.

Differential survival of Venus gallina and Scapharca inaequivalvis during anoxic stress: Covalent modification of phosphofructokinase and glycogen phosphorylase during anoxia

SummaryBiochemical mechanisms underlying anaerobiosis were assessed in two Mediterranean bivalve species, Scapharca inaequivalvis and Venus gallina, with widely differing tolerances for oxygen lack. These species displayed LT50 values for anoxic survival at 17–18°C of 17 and 4 d, respectively. Succinate and alanine were the major products of 24 h anaerobic metabolism in both species but only S. inaequivalvis further metabolized succinate to propionate. Both species reduced metabolic rate while anoxic but metabolic arrest was more pronounced in S. inaequivalvis. Calculated ATP turnover rate (MATP) during exposure to N2-bubbled seawater was only 4.51% of the aerobic rate in S. inaequivalvis but was 12.68% in V. gallina. To counteract a greater load of acid end products, V. gallina foot showed a significantly greater buffering capacity, 23.38±0.20 slykes, compared to 19.6±0.79 slykes in S. inaequivalvis. The two species also differed distinctly in the enzymatic regulation of anaerobiosis. In V. gallina anoxia exposure caused only a small change in PFK kinetic parameters (a decrease in Ka AMP) and had no effect on glycogen phosphorylase. By contrast, S. inaequivalvis foot showed a strong modification of enzyme properties in anoxia. The percentage of glycogen phosphorylase in the a form dropped significantly only in S. inaequivalvis. Other changes included alterations in the properties of PFK leading to a less active enzyme form in anoxia. Compared to the aerobic enzyme form, PFK from anoxic foot showed a reduced affinity for fructose-6-P (Km increased 2.4-fold), greater inhibition by ATP (I50 decreased 6.8-fold), and an increase in sensitivity to AMP activation (Ka decreased by 50%). These enzyme changes appear to be key to a glycolytic rate depression during anaerobiosis in S. inaequivalvis foot muscle.

Diets Enriched in Oat Bran or Wheat Bran Temporally and Differentially Alter the Composition of the Fecal Community of Rats

A clear understanding of how diet alters gastrointestinal communities is important given the suggested link between gut community composition and a wide variety of disease pathologies. To characterize this link for commonly consumed dietary fiber sources, we investigated the change in the fecal community of rats fed diets containing 5% nonnutritive fiber (control), 3% (wt:wt) oat bran plus 2% nonnutritive fiber (OB), or 5% (w/w) wheat bran (WB) over a 28-d feeding trial using both molecular- and cultivation-based methodologies. Pooled fecal samples from 8 rats fed the same diet were analyzed at 4 time points. On d 28, bran-fed rats had approximately twice the total cultivable bacteria than rats fed the control diet. Over the course of feeding, the cultivable community was initially dominated by bacteroides, then by bifidobacteria, lactobacilli, enterococci, and various enterics. In contrast, molecular analysis revealed the appearance of new operational taxonomic units (phylotypes) that were both temporally and inequitably distributed throughout the fecal community. The majority of change occurred in 2 major lineages within the Firmicutes: the Clostridium coccoides group and the Clostridium leptum subgroup. The time course of change depended on the source of bran, with the majority of new phylotypes appearing by d 14 (OB) or d 28 (WB), although adaptation of the fecal community was slow and continued over the entire feeding trial. Bacterial community richness was higher in bran-fed rats than in those fed the control diet. Change within the C. coccoides and C. leptum lineages likely reflect their high abundance within the gut bacterial community and the role of clostridia in fiber digestion. The results illustrate the limitations of relying solely on cultivation to assess bacterial changes and illustrate that community changes are complex in an ecosystem containing high numbers of interdependent and competing species of bacteria.

Promotion of Autoimmune Diabetes by Cereal Diet in the Presence or Absence of Microbes Associated With Gut Immune Activation, Regulatory Imbalance, and Altered Cathelicidin Antimicrobial Peptide

We are exposed to millions of microbial and dietary antigens via the gastrointestinal tract, which likely play a key role in type 1 diabetes (T1D). We differentiated the effects of these two major environmental factors on gut immunity and T1D. Diabetes-prone BioBreeding (BBdp) rats were housed in specific pathogen-free (SPF) or germ-free (GF) conditions and weaned onto diabetes-promoting cereal diets or a protective low-antigen hydrolyzed casein (HC) diet, and T1D incidence was monitored. Fecal microbiota 16S rRNA genes, immune cell distribution, and gene expression in the jejunum were analyzed. T1D was highest in cereal-SPF (65%) and cereal-GF rats (53%) but inhibited and delayed in HC-fed counterparts. Nearly all HC-GF rats remained diabetes-free, whereas HC-fed SPF rats were less protected (7 vs. 29%). Bacterial communities differed in SPF rats fed cereal compared with HC. Cereal-SPF rats displayed increased gut CD3+ and CD8α+ lymphocytes, ratio of Ifng to Il4 mRNA, and Lck expression, indicating T-cell activation. The ratio of CD3+ T cells expressing the Treg marker Foxp3+ was highest in HC-GF and lowest in cereal-SPF rats. Resident CD163+ M2 macrophages were increased in HC-protected rats. The cathelicidin antimicrobial peptide (Camp) gene was upregulated in the jejunum of HC diet–protected rats, and CAMP+ cells colocalized with CD163. A cereal diet was a stronger promoter of T1D than gut microbes in association with impaired gut immune homeostasis.

Summary of an NIH Workshop to Identify Research Needs to Improve the Monitoring of Iodine Status in the United States and to Inform the DRI

The Office of Dietary Supplements (ODS) at the NIH sponsored a workshop on May 12-13, 2011, to bring together representatives from various NIH institutes and centers as a first step in developing an NIH iodine research initiative. The workshop also provided an opportunity to identify research needs that would inform the dietary reference intakes for iodine, which were last revised in 2001. Iodine is required throughout the life cycle, but pregnant women and infants are the populations most at risk of deficiency, because iodine is required for normal brain development and growth. The CDC monitors iodine status of the population on a regular basis, but the status of the most vulnerable populations remains uncertain. The NIH funds very little investigator-initiated research relevant to iodine and human nutrition, but the ODS has worked for several years with a number of other U.S. government agencies to develop many of the resources needed to conduct iodine research of high quality (e.g., validated analytical methods and reference materials for multiple types of samples). Iodine experts, scientists from several U.S. government agencies, and NIH representatives met for 2 d to identify iodine research needs appropriate to the NIH mission.

Where is the glycolytic complex? A critical evaluation of present data from muscle tissue

Assocaiations between glycolytic enzymes and subscellular structures have been interpreted as presenting a novel mechanism of glycolytic control; reversible enzymes binding to subcellular structural components is believed to regulate enzymes activity in vivo through the formation of a multi‐enzyme complex. However, three lines of evidence suggest that eyzyme binding to cellular structures is not involved in the control of glycolsis, (i) Calculations of the distribution of glycolytic enzymes under the physiological cellular conditions of higher ionic strenght and higher enzyme concentrations indicate that a large multi‐enzyme complex would not exist, (ii) In many eases, binding to subcellular structures is accompanied by changes in enzyme kinetic parameters brought about by allosteric modification, but these changes often inhibit enzyme activity, (iii) In the case where formation of binary enzyme/enzyme complexes activates enzymes, the overall increase in flux through the enzyme reaction is negible.