Circumventing the Crabtree effect: forcing oxidative phosphorylation (OXPHOS) via galactose medium increases sensitivity of HepG2 cells to the purine derivative kinetin riboside.

Small-molecule compound-based therapies have provided new insights into cancer treatment against mitochondrial impairment. N6-furfuryladenosine (kinetin riboside, KR) is a purine derivative and an anticancer agent that selectively affects the molecular pathways crucial for cell growth and apoptosis by interfering with mitochondrial functions and thus might be a potential mitotoxicant. Metabolism of cancer cells is predominantly based on the Crabtree effect that relies on glucose-induced inhibition of cell respiration and thus on oxidative phosphorylation (OXPHOS), which supports the survival of cancer cells in metabolic stress conditions. The simplest way to circumvent this phenomenon is to replace glucose with galactose in the culture environment. Consequently, cells become more sensitive to mitochondrial perturbations caused by mitotoxicants. In the present study, we evaluated several cellular parameters and investigated the effect of KR on mitochondrial functions in HepG2 cells forced to rely mainly on OXPHOS. We showed that KR in the galactose environment is a more potent apoptosis-inducing agent. KR decreases the mitochondrial membrane potential, reduces glutathione level, depletes cellular ATP, and induces reactive oxygen species (ROS) production in the OXPHOS state, leading to the loss of cell viability. Taken together, these results demonstrate that KR directly acts on the mitochondria to limit their function and that the sensitivity of cells is dependent on their ability to cope with energetic stress.

Role of galactose in cellular senescence.

Cellular senescence has been proposed to play critical roles in tumor suppression and organismal aging, but the molecular mechanism of senescence remains incompletely understood. Here we report that a putative lysosomal carbohydrate efflux transporter, Spinster, induces cellular senescence in human primary fibroblasts. Administration of d-galactose synergistically enhanced Spinster-induced senescence and this synergism required the transporter activity of Spinster. Intracellular d-galactose is metabolized to galactose-1-phosphate by galactokinase. Galactokinase-deficient fibroblasts, which accumulate intracellular d-galactose, displayed increased baseline senescence. Senescence of galactokinase-deficient fibroblasts was further enhanced by d-galactose administration and was diminished by restoration of wild-type galactokinase expression. Silencing galactokinase in normal fibroblasts also induced senescence. These results suggest a role for intracellular galactose in the induction of cellular senescence.

A cell size- and cell cycle-aware stochastic model for predicting time-dynamic gene network activity in individual cells.

BACKGROUND: Despite the development of various modeling approaches to predict gene network activity, a time dynamic stochastic model taking into account real-time changes in cell volume and cell cycle stages is still missing. RESULTS: Here we present a stochastic single-cell model that can be applied to any eukaryotic gene network with any number of components. The model tracks changes in cell volume, DNA replication, and cell division, and dynamically adjusts rates of stochastic reactions based on this information. By tracking cell division, the model can maintain cell lineage information, allowing the researcher to trace the descendants of any single cell and therefore study cell lineage effects. To test the predictive power of our model, we applied it to the canonical galactose network of the yeast Saccharomyces cerevisiae. Using a minimal set of free parameters and across several galactose induction conditions, the model effectively captured several details of the experimentally-obtained single-cell network activity levels as well as phenotypic switching rates. CONCLUSION: Our model can readily be customized to model any gene network in any of the commonly used cells types, offering a novel and user-friendly stochastic modeling capability to the systems biology field.

Population diversification in a yeast metabolic program promotes anticipation of environmental shifts.

Delineating the strategies by which cells contend with combinatorial changing environments is crucial for understanding cellular regulatory organization. When presented with two carbon sources, microorganisms first consume the carbon substrate that supports the highest growth rate (e.g., glucose) and then switch to the secondary carbon source (e.g., galactose), a paradigm known as the Monod model. Sequential sugar utilization has been attributed to transcriptional repression of the secondary metabolic pathway, followed by activation of this pathway upon depletion of the preferred carbon source. In this work, we demonstrate that although Saccharomyces cerevisiae cells consume glucose before galactose, the galactose regulatory pathway is activated in a fraction of the cell population hours before glucose is fully consumed. This early activation reduces the time required for the population to transition between the two metabolic programs and provides a fitness advantage that might be crucial in competitive environments.

Galactofuranose in eukaryotes: aspects of biosynthesis and functional impact.

Galactofuranose (Galf) is the five-membered ring form of galactose. It is widely distributed among several branches of the eukaryotic kingdom. This review highlights recent advances in our understanding of the biosynthesis and function of Galf-containing glycoconjugates in fungal Aspergillus spp. and the protozoan trypanosomatid parasites. We give an overview of the biosynthetic pathways leading to the production of glycolipids, glycoproteins and polysaccharides containing Galf in these species and their biological relevance. Remarkably, modification of the cell surface caused by Galf absence often results in morphological abnormalities and an impaired cell wall function in these organisms. Galf-deficient mutants are generally hypersensitive to drugs, exhibit a constitutive osmotic stress phenotype and/or have an attenuated virulence. Since Galf has never been found in mammals and higher plants, Galf-biosynthetic pathways have raised much interest as targets for drug development to combat microbial infections.

Astrocyte activation but not neuronal impairment occurs in the hippocampus of mice after 2 weeks of d-galactose exposure.

AIMS: The objective of this study is to prove that activation of astrocytes precedes neuron cell death in the neurodegenerative process induced by d-galactose (d-gal) exposure. MAIN METHODS: Male adult mice were given intraperitoneal injection of d-gal (200 mg/kg per day) for 2 weeks. The whole brain homogenate and hippocampal sections were then prepared for biochemical analyses, immunohistochemistry and electron microscopy, respectively. KEY FINDINGS: There were no statistically significant differences in brain oxidative and antioxidative parameters between d-gal-treated mice and saline controls. There was also lack of morphological impairment in hippocampal neuronal soma, dendrites and synapses in the model mice. In contrast, hippocampal astrocytes were dramatically activated, and perisynaptic processes of astrocytes were swelling as revealed by ultrastructural analysis. Moreover, d-gal-treated group showed increases in immunostaining levels of glutamate transporter-1 and aquaporin-4 in the hippocampus, which might increase uptake of glutamate from the synaptic cleft into astrocytes. SIGNIFICANCE: These results reveal that astrocytes undergo structural and biochemical changes while no impairment of neuronal elements occurs after 2 weeks of d-gal exposure. Thus, targeting astrocytes may be a promising strategy for the treatment of neurodegenerative diseases at the early stages.

Engineering of GAL1 promoter-driven expression system with artificial transcription factors.

We isolated and characterized artificial transcription factors (ATFs) that functionally activate GAL1 promoter in yeast. These ATFs transformed the yeast galactose-dependent GAL1 promoter system into a galactose-independent one. The ATFs were identified by screening a combinatorial library of zinc finger-containing transcription factors for components that activated the transcription of a reporter gene under the control of a truncated GAL1 promoter from which the GAL4p-binding sites were deleted. We also showed that these ATFs activate transcription from GAL1 promoter by binding directly to specific sequence elements in the promoter.

Lectin/glycan interactions play a role in recognition in a coral/dinoflagellate symbiosis.

Recognition is an important stage in the establishment of highly specific mutualistic associations. Yet, for the majority of symbioses, very few of the mechanisms involved in recognition and specificity are known. In this study, we provide evidence for a recognition mechanism at the onset of symbiosis between larvae of the coral Fungia scutaria and their endosymbiotic dinoflagellate algae. This recognition step occurs during initial cellular contact between the symbiotic partners through a lectin/glycan interaction. We determined that an intact algal cell surface was required for successful infection of F. scutaria larvae. Modification of the algal cell surface by enzymatic digestion with trypsin or N-glycosidase significantly reduced infection success, and implicated algal cell surface glycans in recognition. Using flow cytometry, alpha-mannose/alpha-glucose and alpha-galactose residues were identified as potential recognition ligands on the algal cell surface. Finally, inhibition of these cell surface glycans significantly reduced infection of F. scutaria larvae by the algae. These data provide evidence that the algal cell surface contains glycan ligands, such as alpha-mannose/alpha-glucose and alpha-galactose, which play a role in recognition during initial contact at the onset of symbiosis with F. scutaria larvae.

A reinvestigation of the secondary structure of functionally active vSGLT, the vibrio sodium/galactose cotransporter.

The bacterial Na(+)/galactose cotransporter vSGLT of Vibrio parahaemolyticus is a member of the sodium:solute symporter family (SSS). Previous studies using electron microscopy have shown that vSGLT is a monomeric protein. Computational and experimental topological analyses have consistently indicated that this protein possesses 14 transmembrane alpha-helices. Our previous study using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) to quantitate secondary structure content had indicated, in contrast, an alpha-helical content of only 35%, too little to be consistent with the 14-span model [le Coutre, J., et al. (2002) Biochemistry 41, 8082-6]. ATR-FTIR had also indicated that upon binding of Na(+) and d-galactose, the alpha-helical content increased to 53%. Here we revisit the vSGLT secondary structural distribution using an alternative approach, ultraviolet circular dichroism spectropolarimetry (CD), which is highly accurate in determining the alpha-helical content of a protein in solution. CD spectra were obtained from actively functional, soluble vSGLT and, as an internal check, from a fusion protein of vSGLT and the beta-barrel green fluorescent protein (GFP). Far-UV CD of vSGLT indicates a predominating 85% alpha-helical content, and an absence of beta-strands. Far-UV CD of the vSGLT-GFP fusion corroborates this profile, indicating an equivalent alpha-helical content, and a beta-strand content consistent with the GFP contribution. No detectable substrate-induced macroscopic changes in secondary structure are apparent in the far UV. In the near UV, increases in positive CD intensity occur in a stepwise manner with added substrates, implying changing environments of aromatic amino acid residues. CD thus confirms the current 14-transmembrane span model of vSGLT and reveals distinct substrate-induced conformational changes. The high percentage of alpha-helical structure found requires, when considered in the context of membrane topology, that nearly a third of the total alpha-helical fraction lies in extramembrane domains, which distinguishes this cotransporter from the unrelated lactose and glycerol 3-phosphate transporters.

Genome-wide transcriptional dependence on TAF1 functional domains.

Transcription factor IID (TFIID) plays a central role in regulating the expression of most eukaryotic genes. Of the 14 TBP-associated factor (TAF) subunits that compose TFIID, TAF1 is one of the largest and most functionally diverse. Yeast TAF1 can be divided into four regions including a putative histone acetyltransferase domain and TBP, TAF, and promoter binding domains. Establishing the importance of each region in gene expression through deletion analysis has been hampered by the cellular requirement of TAF1 for viability. To circumvent this limitation we introduced galactose-inducible deletion derivatives of previously defined functional regions of TAF1 into a temperature-sensitive taf1ts2 yeast strain. After galactose induction of the TAF1 mutants and temperature-induced elimination of the resident Taf1ts2 protein, we examined the properties and phenotypes of the mutants, including their impact on genome-wide transcription. Virtually all TAF1-dependent genes, which comprise approximately 90% of the yeast genome, displayed a strong dependence upon all regions of TAF1 that were tested. This finding might reflect the need for each region of TAF1 to stabilize TAF1 against degradation or may indicate that all TAF1-dependent genes require the many activities of TAF1. Paradoxically, deletion of the region of TAF1 that is important for promoter binding interfered with the expression of many genes that are normally TFIID-independent/SAGA (Spt-Ada-Gcn5-acetyltransferase)-dominated, suggesting that this region normally prevents TAF1 (TFIID) from interfering with the expression of SAGA-regulated genes.

A role for fucose alpha(1-2) galactose carbohydrates in neuronal growth.

We report a fucose alpha(1-2) galactose-mediated pathway for the modulation of neuronal growth and morphology. Our studies provide strong evidence for the presence of Fucalpha(1-2)Gal glycoproteins and lectin receptors in hippocampal neurons. Additionally, we show that manipulation of Fucalpha(1-2)Gal-associated proteins using small-molecule and lectin probes induces dramatic changes in neuronal morphology. These findings may provide a novel pathway to stimulate neuronal growth and regeneration.

Chaperone-like functions of high-mannose type and complex-type N-glycans and their molecular basis.

It has recently become apparent that high-mannose type N-glycans directly promote protein folding, whereas complex-type ones play a crucial role in the stabilization of protein functional conformations through hydrophobic interactions with the hydrophobic protein surfaces. Here an attempt was made to understand more deeply the molecular basis of these chaperone-like functions with the aid of information obtained from spacefill models of N-glycans. The promotion of protein folding by high-mannose N-glycans seemed to be based on their unique structure, which includes a hydrophobic region similar to the cyclodextrin cavity. The promotive features of high-mannose N-glycans newly observed under various conditions furnished strong support for the view that both intra- and extramolecular high-mannose N-glycans are directly involved in the promotion of protein folding in the endoplasmic reticulum. Further, it was revealed that the N-acetyllactosamine units in complex-type N-glycans have an amphiphilic structure and greatly contribute to the formation of extensive hydrophobic surfaces and, consequently, to the N-glycan-protein hydrophobic interactions. The processing of high-mannose type N-glycans to complex-type ones seems to be an ingenious device to enable the N-glycans to perform these two chaperone-like functions.

Boundary lubrication by lubricin is mediated by O-linked beta(1-3)Gal-GalNAc oligosaccharides.

Lubrication of mammalian joints is mediated by lubricin, a product of megakaryocyte stimulating factor gene (MSF; GenBank accession #U70136) expression. Lubricin (M(r) approximately 240 kDa) is a mucinous glycoprotein which is 50% (w/w) post-translationally modified with beta(1-3)Gal-GalNAc incompletely capped with NeuAc, and lubricates apposed cartilaginous surfaces in the boundary mode through an unknown mechanism. Both bovine and human lubricin were purified from synovial fluid and digested with recombinant glycosidases. Released oligosaccharides were identified and quantified by fluorophore assisted carbohydrate electrophoresis (FACE). Corresponding digests of human lubricin were also assayed in a friction apparatus oscillating latex rubber against polished glass at a pressure of 0.35 x 10(6) N/m(2) and the coefficient of friction (mu) was measured. Digestion with alpha2,3-neuraminidase decreased lubricating ability by 19.3%. Partial removal of beta(1-3)Gal-GalNAc moieties by endo-alpha-N-acetyl-D-galactosaminidase reduced lubricating ability by 77.2%. Human lubricin digested with combined alpha2,3-neuraminidase and beta1-3,6-galactosidase continued to lubricate at 52.2% of its nominal value. Both bovine and human lubricin released 48.6% and 54.4% of total beta(1-3)Gal-GalNAc sidechains following digestion with endo-alpha-N-acetyl-D-galactosaminidase. Biological boundary lubrication by synovial fluid in vitro is provided primarily by extensive O-linked beta(1-3)Gal-GalNAc.

Glucose and galactose absorption after ingestion of milk containing hydrolysed lactose in calves with diarrhoea.

Ten calves which had contracted acute diarrhoea caused by rotavirus, coronavirus and Cryptosporidium were used to test the hypothesis that feeding lactose-hydrolysed cow's milk instead of unprocessed cow's milk improves sugar absorption in diarrhoeic calves. The animals were rehydrated with an orally administered solution containing electrolytes and glucose. Thereafter the calves received one test meal of whole fresh cow's milk whose lactose had been hydrolysed by added lactase and one test meal of unprocessed cow's milk at an interval of 24 h in a cross-over design trial. In comparison with unprocessed milk, the intake of milk containing hydrolysed lactose resulted in a slight decrease of mean breath hydrogen concentration (P = 0.18), but also a slight decrease of mean blood galactose concentration (P = 0.14). There was no treatment effect on mean plasma glucose concentration. Peak plasma glucose and blood galactose concentration tended to be delayed after the intake of lactose-hydrolysed milk, which implies that gastric emptying was probably delayed. The results show that feeding milk which contains hydrolysed lactose does not significantly improve lactose utilization in calves that are suffering from benign infectious diarrhoea.

Adhesion of human lung mast cells to bronchial epithelium: evidence for a novel carbohydrate-mediated mechanism.

Mast cells contribute to the pathophysiology of asthma through their immunomediator-secretory activity in response to both immunological and nonimmunological stimuli, and infiltrate the bronchial epithelium in this disease. We hypothesized that human lung mast cells (HLMC) localize to the bronchial epithelium via a specific cell-cell adhesion mechanism. We investigated the adhesion of HLMC to primary bronchial epithelial cells and the bronchial epithelial cell line BEAS-2B. HLMC adhered avidly to both primary cultures of bronchial epithelial cells and BEAS-2B cells (mean adhesion 68.4 and 60.1%, respectively) compared with eosinophil adhesion to BEAS-2B (mean adhesion 10.3%). HLMC adhesion did not alter after epithelial activation with cytokines, did not require Ca2+, and was not integrin-mediated. IgE-dependent activation of HLMC produced an approximately 40% inhibition of adhesion. There was significant attenuation of adhesion after incubation of HLMC with pronase, beta-galactosidase, and endo-alpha-N-acetylgalactosaminidase, indicating that HLMC adhere to bronchial epithelial cells via galactose-bearing carbohydrates expressed on a cell-surface peptide(s).

Attachment of Fusobacterium nucleatum PK1594 to mammalian cells and its coaggregation with periodontopathogenic bacteria are mediated by the same galactose-binding adhesin.

It has been shown that Fusobacterium nucleatum PK1594 coaggregates with Prophyromonas gingivalis PK1924 through a galactose-binding adhesin. In the present study, attachment of F. nucleatum PK1594 to a variety of mammalian cells was characterized. F. nucleatum PK1594 attached to all eukaryotic cells tested, including human buccal epithelial cells, gingival and periodontal ligament fibroblasts, HeLa cells and murine lymphocytes, macrophages, and polymorphonuclear leukocytes. These attachments were (i) inhibited by galactose, lactose and N-acetylgalactosamine and (ii) inhibited by monoclonal antibody specific for the galactose-binding adhesin of F. nucleatum PK1594. In addition, a coaggregation-defective mutant of F. nucleatum PK1594 (PK2172), which does not exhibit galactose binding activity, did not attach to the mammalian cells. Coaggregation of F. nucleatum PK1594 with P. gingivalis PK 1924 and Actinobacillus actinomycetemcomitans JP2, but not with other bacteria, showed a similar pattern with sugars, monoclonal antibody, and the adhesin-deficient mutant. The results suggest that the attachment of F. nucleatum PK1594 to mammalian cells and its coaggregation with periodontal pathogens are mediated by the same galactose-binding adhesin.

Sequential steps in synaptic targeting of sensory afferents are mediated by constitutive and developmentally regulated glycosylations of CAMs.

Sensory afferents in the leech are labeled with both constitutive and developmentally regulated glycosylations (markers) of their cell adhesion molecules (CAMs). Their constitutive mannose marker, recognized by Lan3-2 monoclonal antibody (mAb), mediates the formation of their diffuse central arbors. We show that, at the ultrastructural level, these arbors consist of large, loosely organized axons rich with filopodia and synaptic vesicles. Perturbing the mannose-specific adhesion of this first targeting step leads to a gain in cell-cell contact but a loss of filopodia and synaptic vesicles. During the second targeting step, galactose markers divide afferents into different subsets. We focus on the subset labeled by the marker recognized by Laz2-369 mAb. Initially, the galactose marker appears where afferents contact central neurons. Subsequently it spreads proximally and distally, covering the entire afferent surface. Afferents now gain cell-cell contact, with central neurons and self-similar afferents, but lose filopodia and synaptic vesicles. Extant synaptic vesicles prevail where afferents are apposed to central neurons. These neurons develop postsynaptic densities and en passant synapses are forming. Perturbing the galactose-specific adhesion of this second targeting step causes a loss of cell-cell contact but a gain in filopodia and synaptic vesicles, essentially returning afferents to the first targeting step. The transformation of afferent growth, progressing from mannose- to galactose-specific adhesion, is consistent with a change from cell-matrix to cell-cell adhesion. By performing opposing functions in a temporal sequence, constitutive and developmentally regulated glycosylations of CAMs collaborate in the synaptogenesis of afferents and the consolidation of self-similar afferents.

Glutamate and cyclic AMP regulate the expression of galactokinase in Mycobacterium smegmatis.

It was found that Mycobacterium smegmatis is unable to utilize galactose as the sole carbon source because the sugar alone cannot induce galactokinase. However, galactokinase was induced by glutamate alone, and was further stimulated by galactose. Rifampicin completely inhibited the glutamate-mediated expression of galK in both the absence and presence of galactose. Extracellular cAMP stimulated the expression of the enzyme only in the presence of glutamate plus galactose. The galK gene from M. smegmatis, including its upstream promoter region, was cloned in a plasmid in Escherichia coli. The expression of kinase from these clones in E. coli was dependent on cAMP and its receptor protein (CRP). The expression of UDP-galactose 4-epimerase was constitutive. This and other evidence suggests that the galK gene is not linked to galT and galE in the mycobacterial genome. In a glutamate-independent galactose-utilizing mutant (gin-1 mutant) of M. smegmatis, galK was expressed in the absence of both galactose and glutamate, while in the presence of galactose this expression was increased twofold in the absence of glutamate and fourfold in its presence. Extracellularly added cAMP reduced the expression of the enzyme in the presence of galactose plus glutamate nearly to the basal level. It is proposed that in M. smegmatis the galK gene is expressed from two different promoters; the expression from one promoter is dependent on glutamate but not on galactose and cAMP, while that from the other requires all three components. The role of galactose is possibly to derepress the latter promoter.

Galactose-inducible expression systems in Candida maltosa using promoters of newly-isolated GAL1 and GAL10 genes.

The GAL1 and GAL10 gene cluster encoding the enzymes of galactose utilization was isolated from an asporogenic yeast, Candida maltosa. The structure of the gene cluster in which both genes were divergently transcribed from the central promoter region resembled those of some other yeasts. The expression of both genes was strongly induced by galactose and repressed by glucose in the medium. Galactose-inducible expression vectors in C. maltosa were constructed on low- and high-copy number plasmids using the promoter regions of both genes. With these vectors and the beta-galactosidase gene from Kluyveromyces lactis as a reporter, galactose-inducible expression was confirmed. Homologous overexpression of members of the cytochrome P-450 gene family in C. maltosa was also successful by using a high-copy-number vector under the control of these promoters.