Utilization of D-ribitol by Lactobacillus casei BL23 requires a mannose-type phosphotransferase system and three catabolic enzymes.

Lactobacillus casei strains 64H and BL23, but not ATCC 334, are able to ferment D-ribitol (also called D-adonitol). However, a BL23-derived ptsI mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates D-ribitol via a PTS. We identified an 11-kb region in the genome sequence of L. casei strain BL23 (LCABL_29160 to LCABL_29270) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in D-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented D-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a D-ribitol-5-phosphate (D-ribitol-5-P) 2-dehydrogenase, a D-ribulose-5-P 3-epimerase, a D-ribose-5-P isomerase, and a D-xylulose-5-P phosphoketolase. In the first catabolic step, the protein D-ribitol-5-P 2-dehydrogenase uses NAD(+) to oxidize D-ribitol-5-P formed during PTS-catalyzed transport to D-ribulose-5-P, which, in turn, is converted to D-xylulose-5-P by the enzyme D-ribulose-5-P 3-epimerase. Finally, the resulting D-xylulose-5-P is split by D-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate D-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as D-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in L. casei strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation.

Protein phosphorylation and regulation of carbon metabolism in gram-negative versus gram-positive bacteria.

Bacteria impose regulatory mechanisms on metabolic processes to ensure that the needs of the cell are met but not exceeded. Here, we discuss the basic features of a mechanism by which carbohydrate catabolism in Gram-positive bacteria is regulated. Although the physiological consequences of this regulation are the same as in Gram-negative bacteria, the mechanism is entirely different. These regulatory processes evidently evolved late, after the divergence of Gram-negative bacteria, even though the targets of regulation are universal.

Phosphorylation of either crh or HPr mediates binding of CcpA to the bacillus subtilis xyn cre and catabolite repression of the xyn operon.

Carbon catabolite repression (CCR) of several Bacillus subtilis catabolic genes is mediated by ATP-dependent phosphorylation of Ser46 of the histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP): sugar phosphotransferase system. A recently discovered HPr-like protein of B. subtilis, Crh, cannot be phosphorylated by PEP and enzyme I but becomes phosphorylated at Ser46 by the ATP-dependent, metabolite-activated HPr kinase. Genetic data suggested that Crh is also implicated in CCR. We here demonstrate that in a ptsH1 crh1 mutant, in which Ser46 of both HPr and Crh is replaced with an alanyl residue, expression of the beta-xylosidase-encoding xynB gene was completely relieved from CCR. No effect on CCR could be observed in strains carrying the crh1 allele, suggesting that under the experimental conditions P-Ser-HPr can substitute for P-Ser-Crh in CCR. By contrast, a ptsH1 mutant was slightly relieved from CCR of xynB, indicating that P-Ser-Crh can substitute only partly for P-Ser-HPr. Mapping experiments allowed us to identify the xyn promoter and a catabolite responsive element (cre) located 229 bp downstream of the transcription start point. Using DNase I footprinting experiments, we could demonstrate that similar to P-Ser-HPr, P-Ser-Crh stimulates binding of CcpA to the xyn cre. Fructose 1,6-bisphosphate was found to strongly enhance binding of the P-Ser-HPr/CcpA and P-Ser-Crh/CcpA complexes to the xyn cre, but had no effect on binding of CcpA alone.

Cooperative and non-cooperative DNA binding modes of catabolite control protein CcpA from Bacillus megaterium result from sensing two different signals.

Carbon catabolite repression (CCR) of several operons in Bacillus subtilis and Bacillus megaterium is mediated by the cis-acting cre sequence and trans-acting catabolite control protein (CcpA). We describe purification of CcpA from B. megaterium and its interaction with regulatory sequences from the xyl operon. Specific interaction of CcpA with cre as scored by DNase I footprints at concentrations similar to the in vivo situation requires the presence of effectors. We have found two molecular effectors for CcpA activity, which lead to different recognition modes of DNA. The heat-stable phosphotransfer protein HPr from the PTS sugar uptake system triggers non-cooperative binding of CcpA to cre when phosphorylated at Ser46 (HPr-Ser46-P). Glucose 6-phosphate (Glc-6-P) triggers cooperative binding of CcpA to cre and two auxiliary cre* sites, one of which overlaps the -35 box of the xyl promoter. Binding to cre* depends on the presence of the functional cre sequence. A mutation in cre abolishes carbon catabolite repression in vivo and binding of CcpA to cre and cre* in vitro, indicating looping of the intervening DNA. The two triggers are not simultaneously active. The acidity of the buffer determines which of them activates CcpA when both are present in vitro. Glc-6-P is preferred at pH values below 5.4, and HPr-Ser46-P is preferred at neutral pH. The Ccpa dimers present at neutral pH form tetramers and higher oligomers at pH 4.6, explaining cooperativity of binding to DNA. CcpA is the first member of the LacI/GalR family of regulators, for which oligomerization without the leucine zipper at the C terminus is demonstrated.

The 1.9 A resolution structure of phospho-serine 46 HPr from Enterococcus faecalis.

The histidine-containing phosphocarrier protein HPr is a central component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which transfers metabolic carbohydrates across the cell membrane in many bacterial species. In Gram-positive bacteria, phosphorylation of HPr at conserved serine 46 (P-Ser-HPr) plays several regulatory roles within the cell; the major regulatory effect of P-Ser-HPr is its inability to act as a phosphocarrier substrate in the enzyme I reaction of the PTS. In order to investigate the structural nature of HPr regulation by phosphorylation at Ser46, the structure of the P-Ser-HPr from the Gram- positive bacterium Enterococcus faecalis has been determined. X-ray diffraction analysis of P-Ser-HPr crystals provided 10,043 unique reflections, with a 95.1 % completeness of data to 1.9 A resolution. The structure was solved using molecular replacement, with two P-Ser-HPr molecules present in the asymmetric unit. The final R-value and R(Free) are 0.178 and 0.239, respectively. The overall tertiary structure of P-Ser-HPr is that of other HPr structures. However the active site in both P-Ser-HPr molecules was found to be in the "open" conformation. Ala16 of both molecules were observed to be in a state of torsional strain, similar to that seen in the structure of the native HPr from E. faecalis. Regulatory phosphorylation at Ser46 does not induce large structural changes to the HPr molecule. The B-helix was observed to be slightly lengthened as a result of Ser46 phosphorylation. Also, the water mediated Met51-His15 interaction is maintained, again similar to that of the native E. faecalis HPr. The major structural, and thus regulatory, effect of phosphorylation at Ser46 is disruption of the hydrophobic interactions between EI and HPr, in particular the electrostatic repulsion between the phosphoryl group on Ser46 and Glu84 of EI and the prevention of a potential interaction of Met48 with a hydrophobic pocket of EI.

Interaction of phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, with the NADPH-binding site of mammalian catalases.

Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D-glucose cotransporter. In an attempt to purify the Na+/D-glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi-Gel affinity chromatography column to which 3-aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crassa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 microM NADPH from phlorizin columns. Irradiation in the presence of [3H]4-azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 microM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity.

Clinical aspects of obesity in childhood and adolescence.

The level of fatness of a child at which morbidity acutely and/or later in life increases is determined on an acturial basis. Direct measurements of body fat content, e.g. hydrodensitometry, bioimpedance, or DEXA, are useful tools in scientific studies. However, body mass index (BMI) is easy to calculate and is generally accepted now to be used to define obesity in children and adolescents clinically. An increased risk of death from cardiovascular disease in adults has been found in subjects whose BMI had been greater than the 75th percentile as adolescents. Childhood obesity seems to substantially increase the risk of subsequent morbidity whether or not obesity persists into adulthood. The genetic basis of childhood obesity has been elucidated to some extent through the discovery of leptin, the ob gene product, and the increasing knowledge on the role of neuropeptides such as POMC, neuropeptide Y (NPY) and the melanocyte concentrating hormone receptors (for example, MC4R). Environmental/exogenous factors largely contribute to the development of a high degree of body fatness early in life. Twin studies suggest that approximately 50% of the tendency toward obesity is inherited. There are numerous disorders including a number of endocrine disorders (Cushing's syndrome, hypothyroidism, etc.) and genetic syndromes (Prader-Labhard-Willi syndrome, Bardet Biedl syndrome, etc.) that can present with obesity. A simple diagnostic algorithm allows for the differentiation between primary or secondary obesity. Among the most common sequelae of primary childhood obesity are hypertension, dyslipidemia, back pain and psychosocial problems. Therapeutic strategies include psychological and family therapy, lifestyle/behaviour modification and nutrition education. The role of regular exercise and exercise programmes is emphasized. Surgical procedures and drugs used in adult obesity are still not generally recommended in children and adolescents with obesity. As obesity is the most common chronic disorder in industrialized societies, its impact on individual lives as well as on health economics has to be recognized more widely. This review is aimed towards defining the clinical problem of childhood obesity on the basis of current knowledge and towards outlining future research areas in the field of energy homoesostasis and food intake in relation to child health. Finally, one should aim to increase public awareness of the ever increasing health burden and economic dimension of the childhood obesity epidemic that is present around the globe.

Cloning and characterization of the Bacillus subtilis prkA gene encoding a novel serine protein kinase.

We have cloned and sequenced a 3574-bp Bacillus subtilis (Bs) DNA fragment located between the nrdA and citB genes at about 169 degrees on the chromosome. An Escherichia coli strain, LBG1605, carrying a mutated ptsH gene (encoding HPr (His-containing protein) of the bacterial phosphotransferase system (PTS)) and complemented for PTS activity with the ptsH of Staphylococcus carnosus, exhibited reduced mannitol fermentation activity when transformed with a plasmid bearing this 3574-bp Bs fragment. This fragment contained an incomplete and two complete open reading frames (ORFs). The product of the first complete ORF, a protein composed of 235 amino acids (aa) (25038 Da), was found to be responsible for the observed reduced mannitol fermentation. The 3' part of this 705-bp second ORF and the 428-bp incomplete first ORF encode aa sequences exhibiting almost 40% sequence identify. However, the function of these two proteins remains unknown. The third ORF, the 1893-bp prkA gene, encodes a protein (PrkA) of 72889 Da. PrkA possesses the A-motif of nucleotide-binding proteins and exhibits distant homology to eukaryotic protein kinases. Several of the essential aa in the loops known to form the active site of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase appeared to be conserved in PrkA. After expression of prkA and purification of PrkA, we could demonstrate that PrkA can indeed phosphorylate a Bs 60-kDa protein at a Ser residue.

Purification of a putative Na+/D-glucose cotransporter from pig kidney brush border membranes on a phlorizin affinity column.

Phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, was derivatised to 3-aminophlorizin and subsequently coupled to Affi-Gel 15. Affinity chromatography of pig kidney brush border membranes solubilised in Triton X-100 allowed the purification of a 60 kDa protein on this resin. We consider this protein to be the Na+/D-glucose cotransporter, or part of it, for the following reasons: (i) binding of this protein to Affi-Gel 15 specifically requires phlorizin covalently attached to the resin and is lowered when phlorizin is replaced by phloretin; (ii) binding of the 60 kDa protein to a phlorizin affinity column requires the presence of Na+; (iii) polyclonal as well as monoclonal antibodies against the 60 kDa protein inhibit binding of phlorizin to brush border membranes from rabbit and pig kidney.

Metabolite-sensitive, ATP-dependent, protein kinase-catalyzed phosphorylation of HPr, a phosphocarrier protein of the phosphotransferase system in gram-positive bacteria.

In this review article we summarize the recent information available concerning important mechanistic and physiological aspects of the protein kinase-mediated phosphorylation of seryl residue-46 in HPr, a phosphocarrier protein of the phosphoenolpyruvate: sugar phosphotransferase system in Gram-positive bacteria. Emphasis is placed upon the information recently obtained in two laboratories through the use of site-specific mutants of the HPr protein. The results show that (i) in contrast to eukaryotic protein kinases, the HPr(ser) kinase recognizes the tertiary structure of HPr rather than a restricted part of the primary sequence of the protein; (ii) like seryl protein kinases of eukaryotes, the HPr(ser) kinase can phosphorylate a threonyl residue, but not a tyrosyl residue when such a residue replaces the regulatory seryl residue in position-46 of the protein; (iii) the regulatory consequences of seryl phosphorylation are due to the introduction of a negative charge at position-46 in the protein rather than the bulky phosphate group; and (iv) PTS protein-HPr interactions influence the conformation of HPr, thereby retarding or stimulating the rate of kinase-catalyzed seryl-46 phosphorylation. The physiological consequences of HPr(ser) phosphorylation in vivo are still a matter of debate.

Analysis of a ptsH homologue from Streptomyces coelicolor A3(2).

A ptsH homologue of Streptomyces coelicolor A3(2) was identified in the emerging genome sequence, cloned in Escherichia coli and the S. coelicolor HPr over-produced and purified. The protein was phosphorylated in vitro in a phosphoenolpyruvate (PEP)-dependent manner by purified enzyme I (EI) from Bacillus subtilis, and much less efficiently in an ATP-dependent manner by purified HPr kinase, also from B. subtilis. There was no indication of ATP-dependent phosphorylation of the purified protein by cell extracts of either S. coelicolor or Streptomyces lividans. Deletion of the ptsH homologue from the S. coelicolor and S. lividans chromosomes had no effect on growth when fructose was supplied as sole carbon source, and in S. coelicolor it had no effect on glucose repression of agarase and galactokinase synthesis, suggesting that the HPr encoded by this gene does not play an essential role in fructose transport nor a general role in carbon catabolite repression.

Photoaffinity labeling of plasma membrane proteins involved in the transport of loop diuretics into hepatocytes.

To identify proteins involved in the hepatocellular uptake of loop diuretics, [3H]bumetanide was photoactivated by light flash in the presence of either intact isolated rat hepatocytes, rat liver basolateral plasma membranes or integral membrane proteins extracted from the basolateral plasma membranes. Proteins of 52-54, 48, 33, 27, 25 and 23 kDa in sodium dodecyl sulfate (SDS) gel electrophoresis were radiolabeled on intact hepatocytes. On liver basolateral plasma membranes a 50-52 kDa protein was the most intensely labeled protein. After separation into integral and associated membrane proteins by extraction with Triton X-114, radioactive labeling was only found in integral membrane proteins with a molecular weight of 50-52 kDa. Photoactivated bumetanide irreversibly inhibited the hepatocellular uptake of cholate, taurocholate but not of serine. Binding proteins for photoactivated bumetanide were absent on AS 30-D ascites hepatoma cells. Labeling of all proteins was sodium dependent in intact hepatocytes but was sodium independent in plasma membranes. Labeling was prevented by non-labeled bumetanide and by the loop diuretics piretanide and furosemide. Labeling protection was further achieved with organic anions such as bromosulfophthalein, rifampicin, probenecid and by the bile acids taurocholate, deoxycholate and dehydrocholate. The radiolabeled proteins did not belong to the bumetanide-sensitive NaCl/KCl co-transport system which apparently does not occur in intact isolated rat hepatocytes.

Leptin, the ob gene product, in female health and disease.

Leptin is a recently discovered hormone which is involved in the regulation of body weight. It provides a molecular basis for the lipostatic theory of the regulation of energy balance. White adipose tissue is the main site of leptin synthesis and there is some evidence of ob gene expression in brown fat. Leptin seems to play a key role in the control of body fat stores by coordinated regulation of feeding behaviour, metabolic rate, autonomic nervous system regulation and body energy balance in rodents, primates and humans. Apart from the function of leptin in the central nervous system on the regulation of energy balance, it may well be one of the hormonal factors that signal the body's readiness for sexual maturation and reproduction to the brain. During late pregnancy and at birth when maternal fat stores have been developed leptin levels are high. Leptin could then be a messenger molecule signaling the adequacy of the fat stores for reproduction and maintenance of pregnancy. At later stages of gestation leptin could signal the expansion of fat stores in order to prepare the expectant mother for the energy requirements of full term gestation, labour and lactation. This overview focuses on those topics of leptin research which are of particular interest in reproductive medicine and gynecology.

Body fat mass, leptin and puberty.

Leptin, the ob gene product, provides a molecular basis for the lipostatic theory of the regulation of energy balance. Leptin circulates as a monomeric 16 kDa protein in rodent and human plasma and is also bound to leptin binding proteins that may form large high molecular weight complexes. Initial models of leptin action included leptin-deficient ob/ob mice and leptin-insensitive db/db mice. Peripheral or central administration of leptin reduced body weight, adiposity, and food intake in ob/ob mice but not in db/db mice. In ob/ob mice leptin treatment restored fertility. Leptin interacts with many messenger molecules in the brain. For example, leptin suppresses neuropeptide Y (NPY) expression in the arcuate nucleus. Increased NPY activity has an inhibitory effect on the gonadotropin axis and represents a direct mechanism for inhibiting sexual maturation and reproductive function in conditions of food restriction and/or energy expenditure. By modulating the hypothalamo-pituitary-gonadal axis both directly and indirectly, leptin may thus serve as the signal from fat to the brain about the adequacy of fat stores for pubertal development and reproduction. Normal leptin secretion is necessary for normal reproductive function to proceed and leptin may be a signal allowing for the point of initiation of and progression toward puberty.

Leptin as a metabolic regulator during fetal and neonatal life and in childhood and adolescence.

Body weight is regulated by a feedback loop in which peripheral signals report nutritional information to an integratory center in the brain. The cloning of the ob gene is consistent with this concept and suggests that body fat content in adult rodents is regulated by a negative feedback loop centered in the hypothalamus/1-8/. In a recent report, two severely obese children with congenital leptin deficiency due to a homozygous frame-shift mutation involving the deletion of a single guanine nucleotide in codon 133 of the ob gene have been described. This discovery provides the first genetic evidence that leptin is an important regulator of energy balance in humans. However, it has become increasingly clear that apart from leptin's function in the central nervous system and in regulation of energy balance, leptin also acts in the periphery and might be important as a hormone modulating processes in regard to reproduction, glucose metabolism and insulin resistance, as well as growth and development of many tissues and organs either directly or indirectly. This report reviews some of the topics of leptin research that are of particular importance and relevance for pediatric and adolescent medicine and for pediatric endocrinology in particular.

Regulation of carbon metabolism in gram-positive bacteria by protein phosphorylation.

The main function of the bacterial phosphotransferase system is to transport and to phosphorylate mono- and disaccharides as well as sugar alcohols. However, the phosphotransferase system is also involved in regulation of carbon metabolism. In Gram-positive bacteria, it is implicated in carbon catabolite repression and regulation of expression of catabolic genes by controlling either catabolic enzyme activities, transcriptional activators or antiterminators. All these different regulations follow a protein phosphorylation mechanism.

Practical aspects of managing preschool children with type 1 diabetes.

Day-to-day variations in diet and physical exercise, large variations in the glucose response to small changes in insulin doses, and high insulin sensitivity are characteristic of preschool children with diabetes. Hence, difficulties in achieving adequate metabolic control and stable glycaemia in preschool children are common. In addition, hypoglycaemic episodes tend to be frequent and severe in this age group. Problems identifying and treating hypoglycaemia present an additional challenge for the diabetes team and for the family caring for the young child with diabetes. Specific glucose targets are provided for this age group: premeal levels of 6-12 mmoll(-1)(110-220 mg dl(-1)) with bedtime levels above 8 mmoll(-1)(140 mg dl(-1)). It is important to note that children who suffer severe hypoglycaemic events at a young age show evidence of subtle cognitive deficits when tested during adolescence. The question of whether or not the years before pubertal onset contribute less towards the development of diabetes-related microvascular complications than do the years starting with the onset of puberty remains controversial. Twice-daily or multiple insulin injections, dietary adjustments and considerations, home blood-glucose monitoring, family education, support groups and 24-h hotline information facilities can help to achieve good metabolic control without severe hypoglycaemia in the preschool child. In general, good metabolic control without severe hypoglycaemia can be achieved using frequent counselling and a caring team approach.

Stimulation of dihydroxyacetone and glycerol kinase activity in Streptococcus faecalis by phosphoenolpyruvate-dependent phosphorylation catalyzed by enzyme I and HPr of the phosphotransferase system.

Recently we reported the phosphoenolpyruvate (PEP)-dependent phosphorylation of a 55-kilodalton protein of Streptococcus faecalis catalyzed by enzyme I and histidine-containing protein (HPr) of the phosphotransferase system (J. Deutscher, FEMS Microbiol. Lett. 29:237-243, 1985). The purified 55-kilodalton protein was found to exhibit dihydroxyacetone kinase activity. Glycerol was six times more slowly phosphorylated than dihydroxyacetone. The Kms were found to be 0.7 mM for ATP, 0.45 mM for dihydroxyacetone, and 0.9 mM for glycerol. PEP-dependent phosphorylation of dihydroxyacetone kinase stimulated phosphorylation of both substrates about 10-fold. Fructose 1,6-diphosphate at concentrations higher than 2 mM inhibited the activity of phosphorylated and unphosphorylated dihydroxyacetone kinase in a noncompetitive manner. The rate of PEP-dependent phosphorylation of dihydroxyacetone kinase was about 200-fold slower than the phosphorylation rate of III proteins (also called enzyme III or factor III), which so far have been considered the only phosphoryl acceptors of histidyl-phosphorylated HPr. P-Dihydroxyacetone kinase was found to be able to transfer its phosphoryl group in a backward reaction to HPr. Following [32P]PEP-dependent phosphorylation and tryptic digestion of dihydroxyacetone kinase, we isolated a labeled peptide composed of 37 amino acids, as determined by amino acid analysis. The single histidyl residue of this peptide most likely carries the phosphoryl group in phosphorylated dihydroxyacetone kinase.