Mechanosensory circuits coordinate two opposing motor actions in Drosophila feeding.

Mechanoreception detects physical forces in the senses of hearing, touch, and proprioception. Here, we show that labellar mechanoreception wires two motor circuits to facilitate and terminate Drosophila feeding. Using patch-clamp recordings, we identified mechanosensory neurons (MSNs) in taste pegs of the inner labella and taste bristles of the outer labella, both of which rely on the same mechanoreceptor, NOMPC (no mechanoreceptor potential C), to transduce mechanical deflection. Connecting with distinct brain motor circuits, bristle MSNs drive labellar spread to facilitate feeding and peg MSNs elicit proboscis retraction to terminate feeding. Bitter sense modulates these two mechanosensory circuits in opposing manners, preventing labellar spread by bristle MSNs and promoting proboscis retraction by peg MSNs. Together, these labeled-line circuits enable labellar peg and bristle MSNs to use the same mechanoreceptors to direct opposing feeding actions and differentially integrate gustatory information in shaping feeding decisions.

The Role of Biotin in Bacterial Physiology and Virulence: a Novel Antibiotic Target for Mycobacterium tuberculosis.

Biotin is an essential cofactor for enzymes present in key metabolic pathways such as fatty acid biosynthesis, replenishment of the tricarboxylic acid cycle, and amino acid metabolism. Biotin is synthesized de novo in microorganisms, plants, and fungi, but this metabolic activity is absent in mammals, making biotin biosynthesis an attractive target for antibiotic discovery. In particular, biotin biosynthesis plays important metabolic roles as the sole source of biotin in all stages of the Mycobacterium tuberculosis life cycle due to the lack of a transporter for scavenging exogenous biotin. Biotin is intimately associated with lipid synthesis where the products form key components of the mycobacterial cell membrane that are critical for bacterial survival and pathogenesis. In this review we discuss the central role of biotin in bacterial physiology and highlight studies that demonstrate the importance of its biosynthesis for virulence. The structural biology of the known biotin synthetic enzymes is described alongside studies using structure-guided design, phenotypic screening, and fragment-based approaches to drug discovery as routes to new antituberculosis agents.

In HepG2 cells, coexisting carnitine deficiency masks important indicators of marginal biotin deficiency.

BACKGROUND: A large number of birth defects are related to nutrient deficiencies; concern that biotin deficiency is teratogenic in humans is reasonable. Surprisingly, studies indicate that increased urinary 3-hydroxyisovalerylcarnitine (3HIAc), a previously validated marker of biotin deficiency, is not a valid biomarker in pregnancy. OBJECTIVE: In this study we hypothesized that coexisting carnitine deficiency can prevent the increase in 3HIAc due to biotin deficiency. METHODS: We used a 2-factor nutrient depletion design to induce isolated and combined biotin and carnitine deficiency in HepG2 cells and then repleted cells with carnitine. To elucidate the metabolic pathogenesis, we quantitated intracellular and extracellular free carnitine, acylcarnitines, and acylcarnitine ratios using liquid chromatography-tandem mass spectrometry. RESULTS: Relative to biotin-sufficient, carnitine-sufficient cells, intracellular acetylcarnitine increased by 90%, propionylcarnitine more than doubled, and 3HIAc increased by >10-fold in biotin-deficient, carnitine-sufficient (BDCS) cells, consistent with a defensive mechanism in which biotin-deficient cells transesterify the acyl-coenzyme A (acyl-CoA) substrates of the biotin-dependent carboxylases to the related acylcarnitines. Likewise, in BDCS cells, the ratio of acetylcarnitine to malonylcarnitine and the ratio of propionylcarnitine to methylmalonylcarnitine both more than tripled, and the ratio of 3HIAc to 3-methylglutarylcarnitine (MGc) increased by >10-fold. In biotin-deficient, carnitine-deficient (BDCD) cells, the 3 substrate-derived acylcarnitines changed little, but the substrate:product ratios were masked to a lesser extent. Moreover, carnitine repletion unmasked biotin deficiency in BDCD cells as shown by increases in acetylcarnitine, propionylcarnitine, and 3HIAc (each increased by >50-fold). Likewise, ratios of acetylcarnitine:malonylcarnitine, propionylcarnitine:methylmalonylcarnitine, and 3HIAc:MGc all increased by >8-fold. CONCLUSIONS: Our findings provide strong evidence that coexisting carnitine deficiency masks some indicators of biotin deficiency and support the potential importance of the ratios of acylcarnitines arising from the acyl-CoA substrates and products for biotin-dependent carboxylases in detecting the biotin deficiency that is masked by coexisting carnitine deficiency.

Biotin: biochemical, physiological and clinical aspects.

Significant progress has been made in our understanding of the biochemical, physiological and nutritional aspects of the water-soluble vitamin biotin (vitamin H). It is well know now that biotin plays important roles in a variety of critical metabolic reactions in the cell, and thus, is essential for normal human health, growth and development. This is underscored by the serious clinical abnormalities that occur in conditions of biotin deficiency, which include, among other things, growth retardation, neurological disorders, and dermatological abnormalities (reviewed in 1). Studies in animals have also shown that biotin deficiency during pregnancy leads to embryonic growth retardation, congenital malformation and death (Watanabe 1983; Cooper and Brown 1958; Mock et al. 2003; Zempleni and Mock 2000). The aim of this chapter is to provide coverage of current knowledge of the biochemical, physiological, and clinical aspects of biotin nutrition. Many sections of this chapter have been the subject of excellent recent reviews by others (Wolf 2001; McMahon 2002; Mock 2004; Rodriguez-Melendez and Zempleni 2003; Said 2004; Said et al. 2000; Said and Seetheram 2006), and thus, for more information the reader is advised to consider these additional sources.

Biotin increases glucokinase expression via soluble guanylate cyclase/protein kinase G, adenosine triphosphate production and autocrine action of insulin in pancreatic rat islets.

Besides its role as a carboxylase prosthetic group, biotin has important effects on gene expression. However, the molecular mechanisms through which biotin exerts these effects are largely unknown. We previously found that biotin increases pancreatic glucokinase expression. We have now explored the mechanisms underlying this effect. Pancreatic islets from Wistar rats were treated with biotin, in the presence or absence of different types of inhibitors. Glucokinase mRNA and 18s rRNA abundance were determined by real-time PCR. Adenosine triphosphate (ATP) content was analyzed by fluorometry. Biotin treatment increased glucokinase mRNA abundance approximately one fold after 2 h; the effect was sustained up to 24 h. Inhibition of soluble guanylate cyclase or protein kinase G (PKG) signalling suppressed biotin-induced glucokinase expression. The cascade of events downstream of PKG in biotin-mediated gene transcription is not known. We found that inhibition of insulin secretion with diazoxide or nifedipine prevented biotin-stimulated glucokinase mRNA increase. Biotin treatment increased islet ATP content (control: 4.68+/-0.28; biotin treated: 6.62+/-0.26 pmol/islet) at 30 min. Inhibition of PKG activity suppressed the effects of biotin on ATP content. Insulin antibodies or inhibitors of phosphoinositol-3-kinase/Akt insulin signalling pathway prevented biotin-induced glucokinase expression. The nucleotide 8-Br-cGMP mimicked the biotin effects. We propose that the induction of pancreatic glucokinase mRNA by biotin involves guanylate cyclase and PKG activation, which leads to an increase in ATP content. This induces insulin secretion via ATP-sensitive potassium channels. Autocrine insulin, in turn, activates phosphoinositol-3-kinase/Akt signalling. Our results offer new insights into the pathways that participate in biotin-mediated gene expression.

Cocultures of rat sensorimotor cortex and spinal cord slices to investigate corticospinal tract sprouting.

STUDY DESIGN: Experimental study of corticospinal axonal sprouting in an organotypic slice culture model. OBJECTIVE: To develop an in vitro model that simplifies the study of various factors regulating neuronal regeneration. SUMMARY OF BACKGROUND DATA: Spinal cord injury leads to permanent neurologic damage, mainly due to the inability of the adult central nervous system to regenerate. Much attention has been focused on promoting axonal regeneration and sprouting, either by exogenous administration of various neurotrophic factors or by the antagonization of factors inhibiting regeneration. METHODS: An in vitro system that allows coculture of slices from rat sensorimotor cortex and spinal cord (p4) was established. Two groups of cultures were investigated: In the first group, intact spinal cord slices were cultured adjacent to sensorimotor cortex slices, while in the second group the spinal cord slices were sagittally cut into halves, with the sectioned interface placed directly adjacent to the sensorimotor cortex, to prevent the spinal white matter from interference. Each group was further divided into 2 subgroups: The neurotrophin-3 (NT-3) group, where the culture medium contained 50 ng/mL NT-3 and the control group treated with normal culture medium. Sensorimotor cortex pyramidal neurons were anterogradely labeled with Mini-Ruby, a 10 kD biotinylated dextran amine. RESULTS: Cocultures of cortical and spinal cord tissue were propagated in vitro, and axonal sprouting occurred. The group of cocultures treated with NT-3 showed an improved cortical cytoarchitecture, and sprouting axons were more frequently observed. In NT-3-treated cocultures where spinal cord gray matter was directly opposed to cortical slices sprouting axons entered the adjacent spinal cord tissue. This phenomenon was not observed if spinal cord pia mater and white matter were opposed to the cortical slices, or if NT-3 was absent. CONCLUSION: Our data suggest that the absence of repellent factors such as white matter and the presence of neurotrophic factors promote axonal sprouting. Cocultures of sensorimotor cortex and spinal cord slices combined with anterograde axonal labeling could provide a valuable in vitro model for the simplified screening of factors influencing corticospinal tract regeneration.

Impaired biotinidase activity disrupts holocarboxylase synthetase expression in late onset multiple carboxylase deficiency.

Biotinidase catalyzes the hydrolysis of the vitamin biotin from proteolytically degraded biotin-dependent carboxylases. This key reaction makes the biotin available for reutilization in the biotinylation of newly synthesized apocarboxylases. This latter reaction is catalyzed by holocarboxylase synthetase (HCS) via synthesis of 5'-biotinyl-AMP (B-AMP) from biotin and ATP, followed by transfer of the biotin to a specific lysine residue of the apocarboxylase substrate. In addition to carboxylase activation, B-AMP is also a key regulatory molecule in the transcription of genes encoding apocarboxylases and HCS itself. In humans, genetic deficiency of HCS or biotinidase results in the life-threatening disorder biotin-responsive multiple carboxylase deficiency, characterized by a reduction in the activities of all biotin-dependent carboxylases. Although the clinical manifestations of both disorders are similar, they differ in some unique neurological characteristics whose origin is not fully understood. In this study, we show that biotinidase deficiency not only reduces net carboxylase biotinylation, but it also impairs the expression of carboxylases and HCS by interfering with the B-AMP-dependent mechanism of transcription control. We propose that biotinidase-deficient patients may develop a secondary HCS deficiency disrupting the altruistic tissue-specific biotin allocation mechanism that protects brain metabolism during biotin starvation.

ANG II and calmodulin/CaMKII regulate surface expression and functional activity of NBCe1 via separate means.

We recently reported that ANG II inhibits NBCe1 current and surface expression in Xenopus laevis oocytes (Perry C, Blaine J, Le H, and Grichtchenko II. Am J Physiol Renal Physiol 290: F417-F427, 2006). Here, we investigated mechanisms of ANG II-induced changes in NBCe1 surface expression. We showed that the PKC inhibitor GF109203X blocks and EGTA reduces surface cotransporter loss in ANG II-treated oocytes, suggesting roles for PKC and Ca(2+). Using the endosomal marker FM 4-64 and enhanced green fluorescent protein (EGFP)-tagged NBCe1, we showed that ANG II stimulates endocytosis of NBCe1. To eliminate the possibility that ANG II inhibits NBCe1 recycling, we demonstrated that the recycling inhibitor monensin decreases surface expression, accumulates NBCe1-EGFP in endosomes, and inhibits NBCe1 current. Monensin and ANG II applied together produce greater inhibition of NBCe1 current than either did alone. This additive effect of monensin and ANG II suggests that ANG II stimulates internalization of NBCe1. We used the calmodulin (CaM) antagonist W13, which controls recycling by blocking the exit of the endocytosed cargo from early endosomes, to determine the role of CaM in NBCe1 trafficking. We demonstrated that W13 decreases surface expression of NBCe1, accumulates NBCe1-EGFP in endosomal-like formations, and inhibits NBCe1 current. W13 and ANG II applied together produce greater inhibition of NBCe1 current than either does alone, while W13 and monensin applied together do not. The additive effect of ANG II and W13 and lack of additive effect of monensin and W13 suggest that CaM is not involved in ANG II stimulation of internalization but controls recycling of endocytosed NBCe1. The CaM-activated enzyme CaM kinase II (CaMKII) applied with ANG II also gives an additive inhibitory effect, suggesting a role for CaMKII in NBCe1 recycling.

Roles of biotin in growing ovarian follicles and embryonic development in domestic fowl.

It is well known that biotin deficiency causes morphological anomalies in hatchlings of fowl. An abundance of biotin in the yolk, therefore, is greatly required for maintaining reproductive functions. Although there is growing evidence for the molecular significance of the vitamin in the processes of the reproductive system in mammals, little is known about its use and roles in fowl's yolk. This review focuses on studies on the roles of biotin in the ovarian follicles and embryos of domestic fowl. Recent studies from our laboratory showed an analysis of the mechanism of biotin supply from the maternal body to follicles, or the yolk using the eggs of domestic fowl. In growing ovarian follicles, biotin was incorporated into the follicles, particularly in a large amount immediately before ovulation. Biotin incorporated from the early stage to the growth stage of ovarian follicles was stored in the protein-binding form and became free biotin again immediately before ovulation. This may be because the biotin necessary for the maintenance of embryonic growth and development should be used immediately after fertilization. In embryos, free biotin in the yolk increased shortly after fertilization. A large amount of free biotin was incorporated into the embryo at the age of 3-4 d. Biotin supply to the embryo differed among embryonic growth stages and organs, suggesting involvement in the formation of each tissue and organ. Thus, a large amount of biotin was utilized for embryonic growth, suggesting its important role in normal embryonic growth. These findings show that biotin is an essential nutrient and may play a major role in the normal morphogenesis of embryos in domestic fowl.

Epigenetic regulation of chromatin structure and gene function by biotin.

Covalent modifications of histones are a crucial component of epigenetic events that regulate chromatin structures and gene function. Evidence exists that distinct lysine residues in histones are modified by covalent attachment of the vitamin biotin, catalyzed by biotinidase and holocarboxylase synthetase. Biotinylation of histones appears to be conserved across species. The following biotinylation sites were identified using both MS and enzymatic biotinylation of synthetic peptides: K9, K13, K125, K127, and K129 in histone H2A; K4, K9, and K18 in histone H3; and K8 and K12 in histone H4. Evidence was provided that biotinylated histone H4 is enriched in pericentromeric heterochromatin, and that biotinylation of histone H4 participates in gene silencing, mitotic condensation of chromatin, and the cellular response to DNA damage. Biotinylation of histones is a reversible process and depends on the exogenous biotin supply, but the identities of histone debiotinylases remain uncertain. We propose that some effects of biotin deficiency can be attributed to abnormal chromatin structures.

Phospholipid biotinylation of polydimethylsiloxane (PDMS) for protein immobilization.

Polydimethylsiloxane (PDMS) surfaces can be functionalized with biotin groups by adding biotinylated phospholipids to the PDMS prepolymer before curing. The addition of beta-D-dodecyl-N-maltoside (DDM) in the solution blocks non-specific protein binding on these functionalized PDMS surfaces. We characterize the surface by measuring fluorescently labeled streptavidin binding. Single molecule tracking shows that the phospholipids are not covalently linked to PDMS polymer chains, but the surface functionalization is not removed by washing. We demonstrate the immobilization of biotinylated antibodies and lectins through biotin-avidin interactions.

The expression of genes encoding ribosomal subunits and eukaryotic translation initiation factor 5A depends on biotin and bisnorbiotin in HepG2 cells.

Biotin affects gene expression at both the transcriptional and the posttranscriptional level; biotin metabolites might have biotin-like activities with regard to gene expression. Here, human hepatocarcinoma (HepG2) cells were used (i) to identify clusters of biotin-dependent genes, (ii) to determine whether the naturally occurring metabolite bisnorbiotin affects gene expression and (iii) to determine whether biotin and bisnorbiotin affect the expression of genes coding for ribosomal subunits and translation initiation factors. HepG2 cells were cultured in media containing deficient (0.025 nmol/L), physiological (0.25 nmol/L, control) and pharmacological (10 nmol/L) concentrations of biotin; a fourth treatment group consisted of cells cultured in biotin-deficient medium (0.025 nmol/L) supplemented with bisnorbiotin (0.225 nmol/L). Gene expression was quantified by using DNA microarrays and reverse transcriptase polymerase chain reaction. The expression of 1803 genes depended on biotin concentrations in culture media; the expression of 618 genes depended on bisnorbiotin. Biotin deficiency was associated with increased expression of a gene cluster encoding ribosomal subunits and eukaryotic translation initiation factor 5A; this effect was reversed by supplementation with biotin and bisnorbiotin. Additional prominent clusters of (bisnor)biotin-dependent genes included DNA-, RNA-, and nucleotide-binding proteins, consistent with a role for biotin in cell signaling and gene expression. Collectively, these data suggest that bisnorbiotin has biotin-like activities regarding gene expression, and that clusters of (bisnor)biotin-dependent genes include genes that play roles in translational activity.

Efecto de la biotina sobre la expresión genética y el metabolismo / Effect of biotin upon gene expression and metabolism

During the last few decades, an increasing number of vitamin-mediated effects has been discovered at the level of gene expression in addition to their well-known roles as substrates and cofactors; the best recognized examples are the lipophilic vitamins A and D. Although little is known about water-soluble vitamins as genetic modulators, there are increasing examples of their effect on gene expression. Biotin is a hydro soluble vitamin that acts as a prosthetic group of carboxylases. Besides its role as carboxylase cofactor, biotin affects several systemic functions such as development, immunity and metabolism. In recent years, significant progress has been made in the identification of genes that are affected by biotin at the transcriptional and post-transcriptional levels as well as in the elucidation of mechanisms that mediate the effects of biotin on the gene expression. These studies bring new insights into biotin mediated gene expression and will lead to a better under-standing of biotin roles in the metabolism and in systemic functions.

En décadas recientes, diversas investigaciones han demostrado que las vitaminas afectan la expresión genética. Los casos mejor estudiados son los de las vitaminas A y D. Existe menos información para las vitaminas hidrosolubles sobre su efecto en la expresión de los genes, sin embargo, se sabe que éstas también los modifican. La biotina es una vitamina hidrosoluble que actúa como grupo prostético de las carboxilasas. Además de su función como cofactor de enzimas, participa en el desarrollo embrionario, en la proliferación celular, en funciones inmunológicas y en el metabolismo. Ha habido un notable avance en la identificación de genes cuya expresión está regulada por la biotina. Asimismo, se han investigado los mecanismos moleculares a través de los cuales la biotina efectúa estas acciones. Estos estudios brindan nuevas claves para entender el papel de la biotina en la expresión genética, en el metabolismo, y en otras funciones biológicas de esta vitamina.

Uptake, localization, and noncarboxylase roles of biotin.

Evidence is emerging that biotin participates in processes other than classical carboxylation reactions. Specifically, novel roles for biotin in cell signaling, gene expression, and chromatin structure have been identified in recent years. Human cells accumulate biotin by using both the sodium-dependent multivitamin transporter and monocarboxylate transporter 1. These transporters and other biotin-binding proteins partition biotin to compartments involved in biotin signaling: cytoplasm, mitochondria, and nuclei. The activity of cell signals such as biotinyl-AMP, Sp1 and Sp3, nuclear factor (NF)-kappaB, and receptor tyrosine kinases depends on biotin supply. Consistent with a role for biotin and its catabolites in modulating these cell signals, greater than 2000 biotin-dependent genes have been identified in various human tissues. Many biotin-dependent gene products play roles in signal transduction and localize to the cell nucleus, consistent with a role for biotin in cell signaling. Posttranscriptional events related to ribosomal activity and protein folding may further contribute to effects of biotin on gene expression. Finally, research has shown that biotinidase and holocarboxylase synthetase mediate covalent binding of biotin to histones (DNA-binding proteins), affecting chromatin structure; at least seven biotinylation sites have been identified in human histones. Biotinylation of histones appears to play a role in cell proliferation, gene silencing, and the cellular response to DNA repair. Roles for biotin in cell signaling and chromatin structure are consistent with the notion that biotin has a unique significance in cell biology.

Biotin deficiency stimulates survival pathways in human lymphoma cells exposed to antineoplastic drugs.

Cells may respond to nutrient deficiency or death signals with nuclear translocation of the transcription factor nuclear factor kappaB (NF-kappaB), which activates transcription of anti-apoptotic genes. Here we tested the hypothesis that biotin deficiency stimulates NF-kappaB-dependent survival pathways in human lymphoma cells, enhancing resistance to antineoplastic agents. Lymphoma (Jurkat) cells were cultured in biotin-deficient (0.025 nmol/L) and biotin-supplemented (10 nmol/L) media. If cells were treated with antineoplastic agents (taxol, doxorubicin or vinblastine), nuclear translocation of two NF-kappaB proteins (p50 and p65) was >25% greater in biotin-deficient compared with biotin-supplemented cells. The transcriptional activities of the following NF-kappaB-dependent reporter genes were 16-59% greater in biotin-deficient compared with biotin-supplemented cells treated with various antineoplastic agents: (1) reporter expression driven by a TATA box and five NF-kappaB repeats and (2) reporter expression driven by the regulatory region of the anti-apoptotic Bfl-1 gene. Collectively, these findings are consistent with activation of survival pathways in biotin-deficient lymphoma cells. Finally, cells were treated with antineoplastic agents for 48 h and cell survival was monitored at timed intervals. Biotin deficiency was associated with enhanced survival of cells treated with doxorubicin and vinblastine, but did not affect survival of cells treated with taxol. Collectively, these observations suggest that biotin deficiency may enhance resistance of cancer cells to antineoplastic agents.

[Effect of biotin upon gene expression and metabolism]. / Efecto de la biotina sobre la expresión genética y el metabolismo.

During the last few decades, an increasing number of vitamin-mediated effects has been discovered at the level of gene expression in addition to their well-known roles as substrates and cofactors; the best recognized examples are the lipophilic vitamins A and D. Although little is known about water-soluble vitamins as genetic modulators, there are increasing examples of their effect on gene expression. Biotin is a hydro soluble vitamin that acts as a prosthetic group of carboxylases. Besides its role as carboxylase cofactor, biotin affects several systemic functions such as development, immunity and metabolism. In recent years, significant progress has been made in the identification of genes that are affected by biotin at the transcriptional and post-transcriptional levels as well as in the elucidation of mechanisms that mediate the effects of biotin on the gene expression. These studies bring new insights into biotin mediated gene expression and will lead to a better under-standing of biotin roles in the metabolism and in systemic functions.

Biotin deficiency decreases life span and fertility but increases stress resistance in Drosophila melanogaster.

Biotin deficiency is associated with fetal malformations and activation of cell survival pathways in mammals. In this study we determined whether biotin status affects life span, stress resistance, and fertility in the fruit fly Drosophila melanogaster. Male and female flies of the Canton-S strain had free access to diets containing 6.0 (control), 4.8, 2.5, or 0 pmol biotin/100 mg. Biotin concentrations in diets correlated with activities of biotin-dependent propionyl-CoA carboxylase and biotin concentrations in fly homogenates, but not with biotinylation of histones (DNA-binding proteins). Propionyl-CoA carboxylase activities and biotin concentrations were lower in males than in females fed diets low in biotin. The life span of biotin-deficient males and females was up to 30% shorter compared to biotin-sufficient controls. Exposure to oxidative stress reversed the effects of biotin status on survival in male flies: survival times increased by 40% in biotin-deficient males compared to biotin-sufficient controls. Biotin status did not affect survival of females exposed to oxidative stress. Exposure of flies to cold, heat, and oxidative stress was associated with mobilization of biotin from yet unknown sources. Biotin deficiency decreased fertility of flies. When biotin-deficient males and females were mated, the hatching rate (larvae hatched per egg) decreased by about 28% compared to biotin-sufficient controls. These findings are consistent with the hypothesis that biotin affects life span, stress resistance, and fertility in fruit flies.