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Background & Aims: We recently showed that the functional capacity for ureagenesis is deficient in non-alcoholic fatty liver disease (NAFLD) patients. The aim of this study was to assess expression of urea cycle-related genes to elucidate a possible gene regulatory basis to the functional problem. Methods: Liver mRNA expression analyses within the gene pathway governing hepatic nitrogen conversion were performed in 20 non-diabetic, biopsy-proven NAFLD patients (8 simple steatosis; 12 non-alcoholic steatohepatitis [NASH]) and 12 obese and 14 lean healthy individuals. Sixteen NAFLD patients were included for gene expression validation. Relationship between gene expressions and functional capacity for ureagenesis was described. Results: Gene expression of most urea cycle-related enzymes were downregulated in NAFLD vs both control groups; markedly so for the urea cycle flux-generating carbamoyl phosphate synthetase (CPS1) (~3.5-fold, P 1.5-fold (P ≤.03), inversely related to CPS1 expression (P =.004). Conclusions: NAFLD downregulated the expression of urea cycle-related genes. Downregulation of urea cycle flux-generating CPS1 correlated with the loss of functional capacity for ureagenesis in NASH. On gene level, these changes coincided with an increase in the major ammonia scavenging enzyme GS. The effects seemed related to a fatty liver as such rather than NASH or obesity. The findings support gene regulatory mechanisms involved in the deficient ureagenesis of NAFLD, but it remains unexplained how hepatocyte fat accumulation exerts these effects.

The interaction between host and external environment mainly occurs in the gastrointestinal tract, where the mucosal barrier has a critical role in many physiologic functions ranging from digestion, absorption, and metabolism. This barrier allows the passage and absorption of nutrients, but at the same time, it must regulate the contact between luminal antigens and the immune system, confining undesirable products to the lumen. Diet is an important regulator of the mucosal barrier, and the cross-talk among dietary factors, the immune system, and microbiota is crucial for the modulation of intestinal permeability and for the maintenance of gastrointestinal tract (GI) homeostasis. In the present review, we will discuss the role of a number of dietary nutrients that have been proposed as regulators of inflammation and epithelial barrier function. We will also consider the metabolic function of the microbiota, which is capable of elaborating the diverse nutrients and synthesizing products of great interest. Better knowledge of the influence of dietary nutrients on inflammation and barrier function can be important for the future development of new therapeutic approaches for patients with mucosal barrier dysfunction, a critical factor in the pathogenesis of many GI and non-GI diseases. ispartof: NUTRIENTS vol:12 issue:4 ispartof: location:Switzerland status: published

The human insulin receptor (IR) exists in two isoforms that differ by the absence (IR-A) or the presence (IR-B) of a 12-amino acid segment encoded by exon 11. Both isoforms are functionally distinct regarding their binding affinities and intracellular signalling. However, the underlying mechanisms related to their cellular functions in several tissues are only partially understood. In this review, we summarize the current knowledge in this field regarding the alternative splicing of IR isoform, tissue-specific distribution and signalling both in physiology and disease, with an emphasis on the human placenta in gestational diabetes mellitus (GDM). Furthermore, we discuss the clinical relevance of IR isoforms highlighted by findings that show altered insulin signalling due to differential IR-A and IR-B expression in human placental endothelium in GDM pregnancies. Future research and clinical studies focused on the role of IR isoform signalling might provide novel therapeutic targets for treating GDM to improve the adverse maternal and neonatal outcomes. Copyright (c) 2015 John Wiley & Sons, Ltd.

Conventional anatomical imaging with CT and MRI has limitations in the evaluation of prostate cancer. PET is a powerful imaging technique, which can be directed toward molecular targets as diverse as glucose metabolism, density of prostate-specific membrane antigen receptors, and skeletal osteoblastic activity. Although 2-deoxy-2- 18 F-FDG-PET is the mainstay of molecular imaging, FDG has limitations in typically indolent prostate cancer. Yet, there are many useful and emerging PET tracers beyond FDG, which provide added value. These include radiotracers interrogating prostate cancer via molecular mechanisms related to the biology of choline, acetate, amino acids, bombesin, and dihydrotestosterone, among others. Choline is used for cell membrane synthesis and its metabolism is upregulated in prostate cancer. 11 C-choline and 18 F-choline are in wide clinical use outside the United States, and they have proven most beneficial for detection of recurrent prostate cancer. 11 C-acetate is an indirect biomarker of fatty acid synthesis, which is also upregulated in prostate cancer. Imaging of prostate cancer with 11 C-acetate is overall similar to the choline radiotracers yet is not as widely used. Upregulation of amino acid transport in prostate cancer provides the biologic basis for amino acid–based radiotracers. Most recent progress has been made with the nonnatural alicyclic amino acid analogue radiotracer anti -1-amino-3- 18 F-fluorocyclobutane-1-carboxylic acid (FACBC or fluciclovine) also proven most useful for the detection of recurrent prostate cancer. Other emerging PET radiotracers for prostate cancer include the bombesin group directed to the gastrin-releasing peptide receptor, 16β- 18 F-fluoro-5α-dihydrotestosterone (FDHT) that binds to the androgen receptor, and those targeting the vasoactive intestinal polypeptide receptor 1 (VPAC-1) and urokinase plasminogen activator receptor (uPAR), which are also overexpressed in prostate cancer.

Hyperaminoacidemia following protein ingestion enhances the anabolic effect of resistance-type exercise by increasing the stimulation of muscle protein synthesis and attenuating the exercise-mediated increase in muscle protein breakdown rates. Although factors such as the source of protein ingested and the timing of intake relative to exercise can impact post-exercise muscle protein synthesis rates, the amount of protein ingested after exercise appears to be the key nutritional factor dictating the magnitude of the muscle protein synthetic response during post-exercise recovery. In younger adults, muscle protein synthesis rates after resistance-type exercise respond in a dose-dependent manner to ingested protein and are maximally stimulated following ingestion of ~20 g of protein. In contrast to younger adults, older adults are less sensitive to smaller doses of ingested protein (less than ~20 g) after exercise, as evidenced by an attenuated increase in muscle protein synthesis rates during post-exercise recovery. However, older muscle appears to retain the capacity to display a robust stimulation of muscle protein synthesis in response to the ingestion of greater doses of protein (~40 g), and such an amount may be required for older adults to achieve a robust stimulation of muscle protein synthesis during post-exercise recovery. The aim of this article is to discuss the current state of evidence regarding the dose-dependent relationship between dietary protein ingestion and changes in skeletal muscle protein synthesis during recovery from resistance-type exercise in older adults. We provide recommendations on the amount of protein that may be required to maximize skeletal muscle reconditioning in response to resistance-type exercise in older adults.

Phenylketonuria (PKU) is a metabolic disorder that results in significant brain dysfunction if untreated. Although phenylalanine restricted diets instituted at birth have clearly improved PKU outcomes, neuropsychological deficits and neurological changes still represent substantial problems. The specific mechanisms by which Phe affects the brains of individuals with PKU are yet fully determined. The use of animal models in PKU research significantly broadens the possibilities for investigating these mechanisms. This report presents an overview of findings from animal studies on the mechanisms of Phe action in the PKU brain, discussing the importance of changes in protein synthesis, transport of large neutral amino acids across the blood-brain barrier, synthesis of monoamine neurotransmitters, activity Of glutamate receptors, animal behavior, and translation of animal behavioral data to patients with PKU. This report shows that great progress has been made in past years and demonstrates the importance of further animal research to understand the neuropathological mechanisms underlying brain dysfunction in PKU. A better understanding of these mechanisms will guide the development of optimal treatment strategies for PKU. (C) 2009 Elsevier Inc. All rights reserved.

The cytoplasmic membrane of bacteria and archaea determine to a large extent the composition of the cytoplasm. Since the ion and in particular the proton and/or the sodium ion electrochemical gradients across the membranes are crucial for the bioenergetic conditions of these microorganisms, strategies are needed to restrict the permeation of these ions across their cytoplasmic membrane. The proton and sodium permeabilities of all biological membranes increase with the temperature. Psychrophilic and mesophilic bacteria, and mesophilic, (hyper)thermophilic and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains low and constant (homeo-proton permeability). Thermophilic bacteria, however, have more difficulties to restrict the proton permeation across their membrane at high temperatures and these organisms have to rely on the less permeable sodium ions for maintaining a high sodium-motive force for driving their energy requiring membrane-bound processes. Transport of solutes across the bacterial and archaeal membrane is mainly catalyzed by primary ATP driven transport systems or by proton or sodium motive force driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary ATP-driven uptake systems for their carbon and energy sources. Several high-affinity ABC transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments.

The radiolabelled amino acid analogue L-3-[I-125]iodo-alpha-methyl-tyrosine (IMT) is under evaluation in brain tumours, where it reflects amino acid transport activity, but is also taken up in many other tumour types. This study investigated the uptake mechanism of IMT in tumour cells not derived from brain tumours, in comparison with the native amino acid C-14-tyrosine (Tyr) from which IMT is derived. Human GLC4 small-cell lung cancer cells in log-phase were incubated with IMT and Tyr. Tracer uptake was determined in various buffers, incubation periods, concentrations of specific amino acid transport blockers, pH and temperature. IMT uptake was very fast, reaching a plateau within 5 min, while Tyr kept on accumulating for > 60 min. Based on steady-state experiments, > 90% of IMT uptake could be attributed to amino acid transport activity. The L transport system was the most important, both fur IMT and Tyr. IMT uptake into GLC4 tumour cells is almost completely the result of amino acid transport activity (especially the L system) and is very similar to Tyr uptake. Therefore, also outside the brain, IMT is a metabolic tracer that may reflect the increased amino acid transport that is characteristic for malignant tumours. ((C) 2001 Lippincott Williams & Wilkins).

The V-type Na+-ATPase of the thermophilic, anaerobic bacterium Caloramator fervidus was purified to homogeneity. The subunit compositions of the catalytic V-1 and membrane-embedded V-0 parts were determined and the structure of the enzyme complex was studied by electron microscopy. The V-1 headpiece consists of seven subunits present in one to three copies, and the V-0 part of two subunits in a ratio of 5:2. An analysis of over 7500 single particle images obtained by electron microscopy of the purified V1V0 enzyme complex revealed that the stalk region, connecting the V-1 and V-0 parts, contains two peripheral stalks in addition to a central stalk. One of the two is connected to the V-0 part, while the other is connected to the first via a bar-like structure that is positioned just above V-0, parallel with the plane of the membrane. Ln projection, this bar seems to contact the central stalk. The data show that the stator structure that prevents rotation of the static part of V-0 relative to V-1 in the rotary catalysis mechanism of energy coupling in ATPases/ATPsynthases is more complex than previously thought. (C) 2000 Academic Press.

BackgroundIn phenylketonuria (PKU), elevated blood phenylalanine (Phe) concentrations are considered to impair transport of large neutral amino acids (LNAAs) from blood to brain. This impairment is believed to underlie cognitive deficits in PKU via different mechanisms, including reduced cerebral protein synthesis. In this study, we investigated the hypothesis that impaired LNAA influx relates to reduced cerebral protein synthesis.MethodsUsing positron emission tomography, L-[1-11C]-tyrosine (11C-Tyr) brain influx and incorporation into cerebral protein were studied in 16 PKU patients (median age 24, range 16 – 47 years), most of whom were early and continuously treated. Data were analyzed by regression analyses, using either11C-Tyr brain influx or11C-Tyr cerebral protein incorporation as outcome variable. Predictor variables were baseline plasma Phe concentration, Phe tolerance, age, and11C-Tyr brain efflux. For the modelling of cerebral protein incorporation,11C-Tyr brain influx was added as a predictor variable.Results11C-Tyr brain influx was inversely associated with plasma Phe concentrations (median 512, range 233 – 1362 μmol/L; delta adjusted R2=0.571, p=0.013). In addition,11C-Tyr brain influx was positively associated with11C-Tyr brain efflux (delta adjusted R2=0.098, p=0.041). Cerebral protein incorporation was positively associated with11C-Tyr brain influx (adjusted R2=0.567, pConclusionsOur data favour the hypothesis that an elevated concentration of Phe in blood reduces cerebral protein synthesis by impairing LNAA transport from blood to brain. Considering the importance of cerebral protein synthesis for adequate brain development and functioning, our results support the notion that PKU treatment be continued in adulthood. Future studies investigating the effects of impaired LNAA transport on cerebral protein synthesis in more detail are indicated.

Transport of acidic amino acids in Bacillus subtilis is an electrogenic process in which L-glutamate or L-aspartate is symported with at least two protons. This is shown by studies of transport in membrane vesicles in which a proton motive force is generated by oxidation of ascorbate-phenazine methosulfate or by artificial ion gradients. An inwards directed sodium gradient had no (stimulatory) effect on proton motive force-driven L-glutamate uptake. The transporter is specific for L-glutamate and L-aspartate. L-Glutamate transport is inhibited by beta-hydroxyaspartate and cysteic acid but not by alpha-methyl-glutamate. The gene encoding the L-glutamate transport protein of B. subtilis (gltP(Bsu)) was cloned by complementation of Escherichia coli JC5412 for growth on glutamate as the sole source of carbon, energy, and nitrogen, and its nucleotide sequence was determined. Putative promoter, terminator, and ribosome binding site sequences were found in the flanking regions. UUG is most likely the start codon, gltP(Bsu) encodes a polypeptide of 414 amino acid residues and is homologous to several proteins that transport glutamate and/or structurally related compounds such as aspartate, fumarate, malate, and succinate. Both sodium- and proton coupled transporters belong to this family of dicarboxylate transporters. Hydropathy profiling and multiple alignment of the family of carboxylate transporters suggest that each of the proteins spans the cytoplasmic membrane 12 times with both the amino and carboxy termini on the inside.

The applicability of protein synthesis rate (PSR) determination with L-[1-C-11]tyrosine (C-11-TYR) and PET was assessed in patients suspected of a primary or recurrent brain tumor. Methods: Simultaneous to intravenous injection of 265 MBq of C-11-TYR, dynamic PET acquisition was started and continued for 50 min. Arterial samples were taken and analyzed for C-11-TYR and metabolites. Based on this data, a model was proposed and the corresponding PSR calculated. Results: Plasma metabolites were (CO2)-C-11, C-11-proteins and C-11-L-DOPA, constituting more than 50% of total plasma radioactivity at 40 min postinjection. Plasma (CO2)-C-11 reached a plateau of around 25% of total plasma radioactivity at 20 min postinjection. Plasma C-11-protein was not detected before 10 min postinjection, but increased exponentially afterwards to 20% at 40 min postinjection. Plasma C-11-L-DOPA was the only acid-soluble radioactive metabolite detected and was less than 8% at 40 min postinjection. Using a five-compartment model, it was shown that while the net PSR was dependent on the recycling of amino acids from protein, the amino acid incorporation was not, which was thus used for subsequent analysis. It was found that our curve-fitting results were unreliable due to the exchange of C-11-TYR between plasma and erythrocytes whereas the graphical Patlak-Gjedde analysis is hardly influenced by this transport phenomenon. The average amino acid incorporation rate thus calculated was 0.7 nmole/ml . min for nontumor tissue with a tumor versus nontumor average ratio of 1.7. Conclusion: The assessment of the PSR with TYR-PET is valuable and relatively simple to implement clinically.

For microoganisms to live under extreme physical conditions requires important adaptations of the cells. In many organisms the use of Na+ instead of protons as coupling ion in energy transduction is associated with such adaptation. This review focuses on the enzymes that are responsible for the generation and utilization of Na+ gradients in extremophilic microorganisms. Aspects that are dealt with include: bioenergetics and ion homeostasis in extremophilic Bacteria and Archaea; the molecular mechanism of Na+ translocation; and (dis)advantages of Na+ as coupling ion in energy transduction.

For microorganisms to live under extreme physical conditions requires important adaptations of the cells. In many organisms the use of Na(+) instead of protons as coupling ion in energy transduction is associated with such adaptation. This review focuses on the enzymes that are responsible for the generation and utilization of Na(+) gradients in extremophilic microorganisms. Aspects that are dealt with include: bioenergetics and ion homeostasis in extremophilic Bacteria and Archaea; the molecular mechanism of Na(+) translocation; and (dis)advantages of Na(+) as coupling ion in energy transduction.

Portal vein glucose delivery (the portal glucose signal) stimulates glucose uptake and glycogen storage by the liver, whereas portal amino acid (AA) delivery (the portal AA signal) induces an increase in protein synthesis by the liver. During a meal, both signals coexist and may interact. In this study, we compared the protein synthesis rates in the liver and muscle in response to portal or peripheral glucose infusion during intraportal infusion of a complete AA mixture. Dogs were surgically prepared with hepatic sampling catheters and flow probes. After a 42-h fast, they underwent a 3-h hyperinsulinemic (4× basal) hyperglucagonemic (3× basal) hyperglycemic (≈160 mg/dl) hyperaminoacidemic (hepatic load 1.5× basal; delivered intraportally) clamp (postprandial conditions). Glucose was infused either via a peripheral (PeG; n = 7) or the portal vein (PoG; n = 8). Protein synthesis was assessed with a primed, continuous [14C]leucine infusion. Net hepatic glucose uptake was stimulated by portal glucose infusion (+1 mg·kg−1·min−1, P < 0.05) as expected, but hepatic fractional AA extraction and hepatic protein synthesis did not differ between groups. There was a lower arterial AA concentration in the PoG group (−19%, P < 0.05) and a significant stimulation (+30%) of muscle protein synthesis associated with increased expression of LAT1 and ASCT2 AA transporters and p70S6 phosphorylation. Concomitant portal glucose and AA delivery enhances skeletal muscle protein synthesis compared with peripheral glucose and portal AA delivery. These data suggest that enteral nutrition support may have an advantage over parenteral nutrition in stimulating muscle protein synthesis.

Amino acid transport in right-side-out membrane vesicles of Acinetobacter johnsonii 210A was studied. L-Alanine, L-lysine, and L-proline were actively transported when a proton motive force of -76 mV was generated by the oxidation of glucose via the membrane-bound glucose dehydrogenase. Kinetic analysis of amino acid uptake at concentrations of up to 80 microM revealed the presence of a single transport system for each of these amino acids with a Kt of less than 4 microM. The mode of energy coupling to solute uptake was analyzed by imposition of artificial ion diffusion gradients. The uptake of alanine and lysine was driven by a membrane potential and a transmembrane pH gradient. In contrast, the uptake of proline was driven by a membrane potential and a transmembrane chemical gradient of sodium ions. The mechanistic stoichiometry for the solute and the coupling ion was close to unity for all three amino acids. The Na+ dependence of the proline carrier was studied in greater detail. Membrane potential-driven uptake of proline was stimulated by Na+, with a half-maximal Na+ concentration of 26 microM. At Na+ concentrations above 250 microM, proline uptake was strongly inhibited. Generation of a sodium motive force and maintenance of a low internal Na+ concentration are most likely mediated by a sodium/proton antiporter, the presence of which was suggested by the Na(+)-dependent alkalinization of the intravesicular pH in inside-out membrane vesicles. The results show that both H+ and Na+ can function as coupling ions in amino acid transport in Acinetobacter spp.

International audience; Intrauterine growth restriction (IUGR), along with postnatal growth trajectory, is closely linked with metabolic diseases and obesity at adulthood. The present study reports the time-dependent metabolomic response of male offspring of rat dams exposed to maternal adequate protein diet during pregnancy and lactation (CC) or protein deprivation during pregnancy only (IUGR with rapid catch-up growth, RC) or through pregnancy and lactation (IUGR with slow postnatal growth, RR). Plasma LC-HRMS metabolomic fingerprints for 8 male rats per group, combined with multivariate statistical analysis (PLS-DA and HCA), were used to study the impact of IUGR and postnatal growth velocity on the offspring metabolism in early life (until weaning) and once they reached adulthood (8 months). Compared with CC rats, RR pups had clear-cut alterations in plasma metabolome during suckling, but none at adulthood; in contrast, in RC pups, alterations in metabolome were minimal in early life but more pronounced in the long run. In particular, our results pinpoint transient alterations in proline, arginine, and histidine in RR rats, compared to CC rats, and persistent differences in tyrosine and carnitine, compared to RC rats at adulthood. These findings suggest that the long-term deregulation in feeding behavior and fatty acid metabolism in IUGR rats depends on postnatal growth velocity

To investigate whether hepatobiliary transport of organic cations is under regulatory control, we studied transport of tri-n-butylmethylammonium in the isolated perfused rat liver and in isolated rat hepatocytes. Transport was investigated in the presence of modulators of the protein kinase C and the cyclic AMP second-messenger system. In the isolated perfused rat liver, it was observed that compounds modulating protein kinase C activity clearly affected the biliary excretion process of the cation tri-n-butylmethylammonium. Phorbol 12-myristate 13-acetate, a compound that directly stimulates protein kinase C, elevated the biliary excretion rate of tri-n-butylmethylammonium in a concentration-dependent manner, reaching a twofold increase at 60 nmol/L of the phorbol ester. The inactive derivative 4alpha-phorbol 12, 13-didecanoate (60 nmol/L) did not show any effect. Vasopressin (48 nmol/L), a receptor-mediated activator of protein kinase C, stimulated the excretion rate of the cation by about 50%. Staurosporin (1 mumol/L), an inhibitor of protein kinase C, clearly decreased the biliary excretion rate of the cation and also blocked its stimulation by phorbol 12-myristate 13-acetate. Neither phorbol 12-myristate 13-acetate nor vasopressin (at concentrations ranging from 10(-9) to 10(-6) mol/L) affected the initial uptake velocity of tri-n-butylmethylammonium in isolated hepatocytes and isolated perfused livers, whereas staurosporin (1 mumol/L) showed only a modest inhibition of the uptake of the cation. It is inferred that the effect of protein kinase C modulators on hepatobiliary transport of organic cations occurs at the level of carrier-mediated transport in the canalicular membrane. Because bile flow was only slightly affected by these agents, effects on biliary excretion rate of the cation are unlikely to be caused by changes in bile flow. With regard to the cyclic AMP second-messenger system, neither glucagon (concentration range of 10(-9) to 10(-6) mol/L), a receptor-mediated activator of adenylate cyclase, nor forskolin (100 mumol/L), a direct activator of adenylate cyclase and dibutyryl cyclic AMP (100 mumol/L), affected the biliary excretion rate and the hepatic uptake rate of the cation in these preparations. In conclusion, cell-to-bile transport of the organic cation tri-n-butylmethylammonium at the canalicular level is directly or indirectly regulated by protein kinase C. Neither the protein kinase C nor the cyclic AMP second-messenger systems seem to be involved in the hepatic uptake process of the cation.

Bacterial growth at the extremes of temperature has remained a fascinating aspect in the study of membrane function and structure. The stability of the integral membrane proteins of thermophiles make them particularly amenable to study. Respiratory enzymes of thermophiles appear to be functionally similar to the mesophilic enzymes but differ in their thermostability and unusual high turnover rates. Energy coupling at extreme temperatures seems inefficient as suggested by the high maintenance coefficients and the high permeability of the cell membrane to protons. Nevertheless, membranes maintain their structure at these extremes through changes in fatty acid acyl chain composition. Archaebacteria synthesize novel membrane-spanning lipids with unique physical characteristics. Thermophiles have adapted to life at extreme temperatures by using sodium ions rather than protons as coupling ions in solute transport. Genetic and biochemical studies of these systems now reveal fundamental principles of such adaptations. The recent development of reconstitution techniques using membrane-spanning lipids allows a rigorous biochemical characterization of membrane proteins of extreme thermophiles in their natural environment.

The adaptation of bacteria to the vigorous environmental changes they undergo is crucial to their survival. They achieve this adaptation partly via intricate regulation of the transcription of their genes. In this study, we infer the transcriptional network of the Gram-positive model organism, Bacillus subtilis. We use a data integration workflow, exploiting both motif and expression data, towards the generation of condition-dependent transcriptional modules. In building the motif data, we rely on both known and predicted information. Known motifs were derived from DBTBS, while predicted motifs were generated by a de novo motif detection method that utilizes comparative genomics. The expression data consists of a compendium of microarrays across different platforms. Our results indicate that a considerable part of the B. subtilis network is yet undiscovered; we could predict 417 new regulatory interactions for known regulators and 453 interactions for yet uncharacterized regulators. The regulators in our network showed a preference for regulating modules in certain environmental conditions. Also, substantial condition-dependent intra-operonic regulation seems to take place. Global regulators seem to require functional flexibility to attain their roles by acting as both activators and repressors.

BACKGROUND: D-ribose, a five carbon sugar, can be produced via mutant strains with impairment in the non-oxidative part of the pentose phosphate pathway (PPP). D-ribose is an important intermediate in the pharmaceutical industry. This work focuses on the effect of various substrates on D-ribose production with a transketolase deficient strain of Bacillus pumilus IFO13322. RESULT: D-ribose titer is found to increase with a concomitant decrease in the yield (on a g product g−1 substrate basis) with increasing glucose concentration. Likewise, higher concentration of ammonium sulfate led to lower product yield while glucose and corn steep liquor concentrations were kept unaltered. Among the complex nitrogen substrates, cas amino acids showed better product yield than corn steep liquor. When tested for supplements of defined amino acids, media supplemented with tyrosine, phenylalanine, tryptophan and histidine gave better yields and higher biomass growth. This is possibly due to the fact that mutant strain is an aromatic amino acids auxotroph owing to impairment in aromatic amino acid biosynthesis. Further, higher phosphate concentrations in the media led to greater biomass formation and lower D-ribose yield. CONCLUSIONS: At higher glucose or ammonium sulfate concentrations, a greater proportion of the carbon was channeled toward byproducts such as acetate, and acetoin and biomass formation. Thus, the relative rates of PPP and in turn the D-ribose yield were dependent on not only the carbon substrate concentration but also the nitrogen and phosphorus substrates. Copyright © 2008 Society of Chemical Industry

Clostridium fervidus is a thermophilic, anaerobic bacterium which uses solely Na+ as a coupling ion for energy transduction. Important features of the primary Na+ pump (ATPase) that generates the sodium motive force are presented. The advantage of using a sodium rather than a proton motive force at high temperatures becomes apparent from the effect of temperature on H+ and Na+ permeation in liposomes.

The cytoplasmic membrane of bacteria and archaea determine to a large extent the composition of the cytoplasm. Since the ion and in particular the proton and/or the sodium ion electrochemical gradients across the membranes are crucial for the bioenergetic conditions of these microorganisms, strategies are needed to restrict the permeation of these ions across their cytoplasmic membrane. The proton and sodium permeabilities of all biological membranes increase with the temperature. Psychrophilic and mesophilic bacteria, and mesophilic, (hyper)thermophilic and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains low and constant (homeo-proton permeability). Thermophilic bacteria, however, have more difficulties to restrict the proton permeation across their membrane at high temperatures and these organisms have to rely on the less permeable sodium ions for maintaining a high sodium-motive force for driving their energy requiring membrane-bound processes. Transport of solutes across the bacterial and archaeal membrane is mainly catalyzed by primary ATP driven transport systems or by proton or sodium motive force driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary ATP-driven uptake systems for their carbon and energy sources. Several high-affinity ABC transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments.

The radiolabelled amino acid analogue L-3-[I-125]iodo-alpha-methyl-tyrosine (IMT) is under evaluation in brain tumours, where it reflects amino acid transport activity, but is also taken up in many other tumour types. This study investigated the uptake mechanism of IMT in tumour cells not derived from brain tumours, in comparison with the native amino acid C-14-tyrosine (Tyr) from which IMT is derived. Human GLC4 small-cell lung cancer cells in log-phase were incubated with IMT and Tyr. Tracer uptake was determined in various buffers, incubation periods, concentrations of specific amino acid transport blockers, pH and temperature. IMT uptake was very fast, reaching a plateau within 5 min, while Tyr kept on accumulating for > 60 min. Based on steady-state experiments, > 90% of IMT uptake could be attributed to amino acid transport activity. The L transport system was the most important, both fur IMT and Tyr. IMT uptake into GLC4 tumour cells is almost completely the result of amino acid transport activity (especially the L system) and is very similar to Tyr uptake. Therefore, also outside the brain, IMT is a metabolic tracer that may reflect the increased amino acid transport that is characteristic for malignant tumours. ((C) 2001 Lippincott Williams & Wilkins).

The radiolabeled amino acid L-3.[I-123]-iodo-alpha-methyl-tyrosine (IMT) is a new tumor tracer that accumulates in many tumors and is suitable for single photon emission computed tomography (SPECT) imaging. Using IMT SPECT, we studied 32 patients with a soft-tissue tumor suspected to be a soft-tissue sarcoma to determine whether: (a) tumors can be visualized; (b) benign and malignant lesions can be distinguished; and (c) IMT uptake is related to tumor grade and proliferation. Whole-body imaging was performed 15 min after administration of 300 MBq IMT, biopsy, or resection 1-2 weeks later, IMT uptake was quantified using a region-of-interest method resulting in tumor:background (T:B) ratios. These were compared with tumor grade, mitotic index, tumor cellularity, vascularity, and the Ki-67 proliferation index. Eleven patients had a benign tumor, and 21 patients had a soft-tissue sarcoma, Six benign tumors demonstrated minor IMT uptake, and five lipomas had no uptake. All malignant tumors had high uptake and were clearly visualized. T:B ratios in malignant tumors (3.83 +/- 1.16) were higher (P

The V-type Na+-ATPase of the thermophilic, anaerobic bacterium Caloramator fervidus was purified to homogeneity. The subunit compositions of the catalytic V-1 and membrane-embedded V-0 parts were determined and the structure of the enzyme complex was studied by electron microscopy. The V-1 headpiece consists of seven subunits present in one to three copies, and the V-0 part of two subunits in a ratio of 5:2. An analysis of over 7500 single particle images obtained by electron microscopy of the purified V1V0 enzyme complex revealed that the stalk region, connecting the V-1 and V-0 parts, contains two peripheral stalks in addition to a central stalk. One of the two is connected to the V-0 part, while the other is connected to the first via a bar-like structure that is positioned just above V-0, parallel with the plane of the membrane. Ln projection, this bar seems to contact the central stalk. The data show that the stator structure that prevents rotation of the static part of V-0 relative to V-1 in the rotary catalysis mechanism of energy coupling in ATPases/ATPsynthases is more complex than previously thought. (C) 2000 Academic Press.

Transport of acidic amino acids in Bacillus subtilis is an electrogenic process in which L-glutamate or L-aspartate is symported with at least two protons. This is shown by studies of transport in membrane vesicles in which a proton motive force is generated by oxidation of ascorbate-phenazine methosulfate or by artificial ion gradients. An inwards-directed sodium gradient had no (stimulatory) effect on proton motive force-driven L-glutamate uptake. The transporter is specific for L-glutamate and L-aspartate. L-Glutamate transport is inhibited by beta-hydroxyaspartate and cysteic acid but not by alpha-methyl-glutamate. The gene encoding the L-glutamate transport protein of B. subtilis (gltPBsu) was cloned by complementation of Escherichia coli JC5412 for growth on glutamate as the sole source of carbon, energy, and nitrogen, and its nucleotide sequence was determined. Putative promoter, terminator, and ribosome binding site sequences were found in the flanking regions. UUG is most likely the start codon. gltPBsu encodes a polypeptide of 414 amino acid residues and is homologous to several proteins that transport glutamate and/or structurally related compounds such as aspartate, fumarate, malate, and succinate. Both sodium- and proton-coupled transporters belong to this family of dicarboxylate transporters. Hydropathy profiling and multiple alignment of the family of carboxylate transporters suggest that each of the proteins spans the cytoplasmic membrane 12 times with both the amino and carboxy termini on the inside.

Clostridium fervidus is a thermophilic, anaerobic bacterium which uses solely Na+ as a coupling ion for energy transduction. Important features of the primary Na+ pump (ATPase) that generates the sodium motive force are presented. The advantage of using a sodium rather than a proton motive force at high temperatures becomes apparent from the effect of temperature on H+ and Na+ permeation in liposomes.

A purified protein fraction of Bacillus thuringiensis subsp. aizawai (HD-133)-activated toxin was subjected to amino acid modification by phenylglyoxal as an arginine-directed reagent. The effect of phenylglyoxylation was assayed on the K+-dependent accumulation of leucine into brush border membrane vesicles from Bombyx mori larval midgut. Phenylglyoxal inactivates the toxin in a dose- and time-dependent manner. Loss of inhibitory effect of the toxin followed a pseudo-first-order kinetics. Observed rates of inactivation were linearly dependent upon the square of phenylglyoxal concentration and a second-order constant of 4.17 M−2 min−1 was calculated. These results indicate the involvement of highly reactive arginine residues in the formation of specific toxin domains implicated in the interaction with membrane receptor(s).

To investigate whether hepatobiliary transport of organic cations is under regulatory control, we studied transport of tri-n-butylmethylammonium in the isolated perfused rat liver and in isolated rat hepatocytes. Transport was investigated in the presence of modulators of the protein kinase C and the cyclic AMP second-messenger system. In the isolated perfused rat liver, it was observed that compounds modulating protein kinase C activity clearly affected the biliary excretion process of the cation tri-n-butylmethylammonium. Phorbol 12-myristate 13-acetate, a compound that directly stimulates protein kinase C, elevated the biliary excretion rate of tri-n-butylmethylammonium in a concentration-dependent manner, reaching a twofold increase at 60 nmol/L of the phorbol ester. The inactive derivative 4alpha-phorbol 12, 13-didecanoate (60 nmol/L) did not show any effect. Vasopressin (48 nmol/L), a receptor-mediated activator of protein kinase C, stimulated the excretion rate of the cation by about 50%. Staurosporin (1 mumol/L), an inhibitor of protein kinase C, clearly decreased the biliary excretion rate of the cation and also blocked its stimulation by phorbol 12-myristate 13-acetate. Neither phorbol 12-myristate 13-acetate nor vasopressin (at concentrations ranging from 10(-9) to 10(-6) mol/L) affected the initial uptake velocity of tri-n-butylmethylammonium in isolated hepatocytes and isolated perfused livers, whereas staurosporin (1 mumol/L) showed only a modest inhibition of the uptake of the cation. It is inferred that the effect of protein kinase C modulators on hepatobiliary transport of organic cations occurs at the level of carrier-mediated transport in the canalicular membrane. Because bile flow was only slightly affected by these agents, effects on biliary excretion rate of the cation are unlikely to be caused by changes in bile flow. With regard to the cyclic AMP second-messenger system, neither glucagon (concentration range of 10(-9) to 10(-6) mol/L), a receptor-mediated activator of adenylate cyclase, nor forskolin (100 mumol/L), a direct activator of adenylate cyclase and dibutyryl cyclic AMP (100 mumol/L), affected the biliary excretion rate and the hepatic uptake rate of the cation in these preparations. In conclusion, cell-to-bile transport of the organic cation tri-n-butylmethylammonium at the canalicular level is directly or indirectly regulated by protein kinase C. Neither the protein kinase C nor the cyclic AMP second-messenger systems seem to be involved in the hepatic uptake process of the cation.

The thermophilic, peptidolytic, anaerobic bacterium Clostridium fervidus is unable to generate a pH gradient in the range of 5.5-8.0, which limits growth of the organism to a narrow pH range (6.3-7.7). A significant membrane potential (delta psi approximately -60 mV) and chemical gradient of Na+ (-Z delta pNa approximately -60 mV) are formed in the presence of metabolizable substrates. Energy-dependent Na+ efflux is inhibited by the Na+/H+ ionophore monensin but is stimulated by uncouplers, suggesting that the Na+ gradient is formed by a primary pumping mechanism rather than by secondary Na+/H+ antiport. This primary sodium pump was found to be an ATPase that has been characterized in inside-out membrane vesicles and in proteoliposomes in which solubilized ATPase was reconstituted. The enzyme is stimulated by Na+, resistant to vanadate, and sensitive to nitrate, which is indicative of an F/V-type Na(+)-ATPase. In the proteoliposomes Na+ accumulation depends on the presence of ATP, is inhibited by the ATPase inhibitor nitrate, and is completely prevented by the ionophore monensin but is stimulated by protonophores and valinomycin. These and previous observations, which indicated that secondary amino acid transport uses solely Na+ as coupling ion, demonstrate that energy transduction at the membrane in C. fervidus is exclusively dependent on a Na+ cycle.

The genes encoding the Na+/H+/L-glutamate symport proteins of the thermophilic organisms Bacillus stearothermophilus (gltT(Bs)) and Bacillus caldotenax (gltT(Bc)) were cloned by complementation of Escherichia coli JC5412 for growth on glutamate as sole source of carbon, energy and nitrogen. The nucleotide sequences of the gltT(Bs) and gltT(Bc), genes were determined. In both cases the translated sequences corresponded with proteins of 421 amino acid residues (96.7% amino acid identity between GltT(Bs) and GltT(Bc)). Putative promoter, terminator and ribosome-binding-site sequences were found in the flanking regions. These expression signals were functional in E coli. The hydropathy profiles indicate that the proteins are hydrophobic and could form 12 membrane-spanning regions. The Na+/H+ CoUpled L-glutamate symport proteins GltT(Bs) and GltT(Bc) are homologous to the strictly H+ coupled L-glutamate transport protein of E. coli K-12 (overall 57.2% identity). Functional expression of glutamate transport activity was demonstrated by uptake of glutamate in whole cells and membrane vesicles. In accordance with previous observations (de Vrij et al., 1989; Heyne et al., 1991), glutamate uptake was driven by the electrochemical gradients of sodium ions and protons.

The applicability of protein synthesis rate (PSR) determination with L-[1-C-11]tyrosine (C-11-TYR) and PET was assessed in patients suspected of a primary or recurrent brain tumor. Methods: Simultaneous to intravenous injection of 265 MBq of C-11-TYR, dynamic PET acquisition was started and continued for 50 min. Arterial samples were taken and analyzed for C-11-TYR and metabolites. Based on this data, a model was proposed and the corresponding PSR calculated. Results: Plasma metabolites were (CO2)-C-11, C-11-proteins and C-11-L-DOPA, constituting more than 50% of total plasma radioactivity at 40 min postinjection. Plasma (CO2)-C-11 reached a plateau of around 25% of total plasma radioactivity at 20 min postinjection. Plasma C-11-protein was not detected before 10 min postinjection, but increased exponentially afterwards to 20% at 40 min postinjection. Plasma C-11-L-DOPA was the only acid-soluble radioactive metabolite detected and was less than 8% at 40 min postinjection. Using a five-compartment model, it was shown that while the net PSR was dependent on the recycling of amino acids from protein, the amino acid incorporation was not, which was thus used for subsequent analysis. It was found that our curve-fitting results were unreliable due to the exchange of C-11-TYR between plasma and erythrocytes whereas the graphical Patlak-Gjedde analysis is hardly influenced by this transport phenomenon. The average amino acid incorporation rate thus calculated was 0.7 nmole/ml . min for nontumor tissue with a tumor versus nontumor average ratio of 1.7. Conclusion: The assessment of the PSR with TYR-PET is valuable and relatively simple to implement clinically.