Glycosylation of dodecyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and dodecyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside as saccharide primers in cells

Syntheses of oligosaccharides expressed on cells are indispensable for the improvement of the functional analyses of the oligosaccharides and their applications. We are developing saccharide primers for synthesizing oligosaccharides using living cells. In this study, dodecyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (GlcNAc-C12) and dodecyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside (LacNAc-C12) were examined for their abilities to prime the syntheses of neolacto-series oligosaccharides in HL60 cells. When GlcNAc-C12 was incubated with HL60 cells in serum-free medium for 2 days, 14 kinds of glycosylated products were collected from the culture medium. They were separated by high-performance liquid chromatography. The sequences of the products were determined to be neolacto-series oligosaccharides including Lewis(X), sialyl Lewis(X), polylactosamine, and sialylpolylactosamine by mass spectrometry. GlcNAc-C12 was also glycosylated by B16 cells and gave sialyllactosamine. Furthermore, LacNAc-C12 gave similar glycosylated products to GlcNAc-C12.     

p53 regulates the proliferation, differentiation and spontaneous transformation of mesenchymal stem cells

Mesenchymal stem cells (MSC) have been extensively studied and gained wide popularity due to their therapeutic potential. Spontaneous transformation of MSC, from both human and murine origin, has been reported in many studies. MSC transformation depends on the culture conditions, the origin of the cells and the time on culture; however, the precise biological characteristics involved in this process have not been fully defined yet. In this study, we investigated the role of p53 in the biology and transformation of murine bone marrow (BM)-derived MSC. We demonstrate that the MSC derived from p53KO mice showed an augmented proliferation rate, a shorter doubling time and also morphologic and phenotypic changes, as compared to MSC derived from wild-type animals. Furthermore, the MSC devoid of p53 had an increased number of cells able to generate colonies. In addition, not only proliferation but also MSC differentiation is controlled by p53 since its absence modifies the speed of the process. Moreover, genomic instability, changes in the expression of c-myc and anchorage independent growth were also observed in p53KO MSC. In addition, the absence of p53 implicates the spontaneous transformation of MSC in long-term cultures. Our results reveal that p53 plays a central role in the biology of MSC.     

Monosialylated biantennary N-glycoforms containing GalNAc-GlcNAc antennae predominate when human EPO is expressed in goat milk

Recently, our group reported the expression of recombinant human erythropoietin in goat milk (rhEPO-milk) as well as in the mammary epithelial cell line GMGE (EPO-GMGE) by cell culture using the adenoviral transduction system. N-Glycosylation characterization of rhEPO-milk by Normal-Phase HPLC profiling of the fluorophore, 4-aminobenzoic acid-labeled enzymatically released N-glycan pool from rhEPO-goat milk, combined with MALDI, ESI-MS and LC/MS, revealed that low branched, core-fucosylated, N-glycans predominate. The labeled N-glycans were separated into neutral and charged fractions by anion exchange chromatography and the charged N-glycans were found to be mostly α2,6-monosialylated with Neu5Ac or Neu5Gc in a ratio of 1:1. Unlike the N-glycans from rhEPO produced in CHO cells, where the glycans are multiantennary highly sialylated, core-fucosylated oligosaccahrides, or even in the goat mammary gland epithelial cell line cultured in vitro in which multiantennary, core- and outer-arm fucosylated, monosialylated N-glycans are the most abundant species, a large proportion of the N-glycans from rhEPO-milk were monosialylated, biantennary, antennae mostly terminating with the more unusual GalNAc-GlcNAc motive and without outer-arm fucosylation. These findings, emphasizing the difference in the N-glycan repertoire between the rhEPO-milk and EPO-GMGE, are consistent with the principle that glycosylation is cell-type dependent and that the cell environment is crucial as well.     

In vitro culture of embryonic cells from the shrimp, Penaeus chinensis

Studies were carried out in our laboratory to develop cell cultures from embryonic tissue of Penaeus chinensis. Good yields of dissociated, uncontaminated, viable cell suspensions were obtained by physical disruption of harvested embryonic tissues. Primary cultures in the form of proliferating foci of cells were readily initiated using MPS medium (osmolality: 2.4%) with 20% heat-inactivated fetal bovine serum. Insulin-like growth factor-II (IGF-II) and basic fibroblast growth factor (bFGF) at different concentrations were also added into the culture medium. Passage of primary cultures resulted in rapid proliferation and good adherence of lymphocytes in the presence of IGF-II at 80 ng/ml and bFGF at 20 ng/ml. Cells maintained in subculture for up to 10 passages still had good cellular morphology and division rates.     

Flow cytometric determination of zoospore DNA content and population DNA distribution in cultured Pfiesteria spp. (Pyrrhophyta)

The relative cellular DNA content from 23 different clonal cultures of Pfiesteria spp. zoospores was determined using a DNA fluorochrome and flow cytometry. Significant differences between Pfiesteria piscicida and P. shumwayae were detected, both in mean zoospore DNA content and population cell cycle DNA distribution. Intraspecific differences in DNA content were found between clonal zoospore cultures established from different geographical regions. Long-term cultures (years) of P. piscicida were available for testing, and a negative correlation was observed between zoospore DNA content and time in culture. Zoospore cell cycle-related DNA distributions were also markedly different between the two species in these clonal cultures. In most cultures tested, P. piscicida zoospores exhibited bimodal DNA flow histograms with G1-S-G2+M distributions, typical of eukaryotic asynchronously cycling cells. In contrast, cultures of P. shumwayae zoospores exhibited one DNA peak distribution, indicative of synchronized cells. The data are consistent with the hypothesis that P. shumwayae zoospores are interphasic cells, and mitosis in zoospore cultures of this species predominantly occurs as benthic or adherent non-motile division cysts. Light microscopy observations of the nuclear condition of electrostatically sorted zoospores of each Pfiesteria species also support this hypothesis. If highly conserved, this disparity in modes of vegetative reproduction would ramify the population dynamics of the two Pfiesteria species.     

Impairment of the activity of the xenobiotic-metabolizing enzymes arylamine N-acetyltransferases 1 and 2 (NAT1/NAT2) by peroxynitrite in mouse skeletal muscle cells

Reactive nitrogen species and their by-products, such as peroxynitrite, modulate many physiological functions of skeletal muscle. Peroxynitrite generation occuring under specific conditions, such as inflammation, may also lead to skeletal muscle dysfunction and pathologies. Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes (XMEs) involved in the detoxification and/or metabolic activation of several drugs and chemicals. In addition to other XMEs, such as gluthatione S-transferases or cytochromes P450, NAT enzymes are expressed in skeletal muscle. We show here that functional NAT1 and NAT2 isoforms are expressed in mouse myotubes and that peroxynitrite may impair their activity in these cells. We show that this inactivation is likely due to the irreversible modification of NATs catalytic cysteine residue in vivo. Our results suggest that peroxynitrite-dependent inactivation of muscle XMEs such as NATs may contribute to muscle dysfunction by impairing the biotransformation activity of this key cellular defense enzyme system.     

Sporulating bacteria prefers predation to cannibalism in mixed cultures

Predatory behavior, a property associated with ecosystems, is not commonly observed in microorganisms. However, cannibalistic tendencies have been observed in microorganisms under stress. For example, pure culture of Bacillus subtilis exhibits cannibalism under nutrient limitation. It has been proposed that a fraction of cells in the population produce Spo0A, a regulatory protein that is responsible for delaying sporulation. Cells containing spo0A would produce a killing factor by activating skf operon and an associated pump to export the factor. Cells that do not contain spo0A in the population are lysed. However in addition to the competition among the cells of B. subtilis, these cells also compete with other organisms for the limited nutrients. In this work, we report the cannibalistic behavior of B. subtilis in presence of Escherichia coli under severe nutritional limitation. We demonstrate that B. subtilis lyses cells of E. coli using an antibacterial factor under the regulation of Spo0A. Our experiments also suggest that B. subtilis prefers predation of E. coli to cannibalism in mixed cultures. B. subtilis also demonstrated predation in mixed cultures with other soil microorganisms, such as, Xanthomonas campestris, Pseudomonas aeruginosa and Acinetobactor lwoffi. This may offer B. subtilis a niche to survive in an environment with limited nutrients and under competition from other microorganisms.     

The transglutaminase activating metalloprotease inhibitor from Streptomyces mobaraensis is a glutamine and lysine donor substrate of the intrinsic transglutaminase

Transglutaminase (TGase) from Streptomyces mobaraensis is an extra-cellular enzyme that cross-links proteins to high molecular weight aggregates. Screening for intrinsic substrates now revealed the dual Streptomyces subtilisin inhibitor-like inhibitor Streptomyces subtilisin and transglutaminase activating metalloprotease (TAMEP) inhibitor (SSTI), equally directed against subtilisin and the TGase activating metalloprotease TAMEP, is both a glutamine and a lysine donor protein. Reactivity of glutamines is lost during culture, most likely by TGase mediated deamidation, and, accordingly, cross-linking only occurred if SSTI from early cultures was used. Interestingly, release of buried endo-glutamines by the lipoamino acid N-lauroylsarcosine could restore SSTI reactivity. Formation of lipoamino acids by Streptomycetes suggests such compounds could also modulate in vivo TGase mediated SSTI cross-linking.     

N-Methyltransferase involved in gramine biosynthesis in barley: cloning and characterization

The indole alkaloid gramine occurs in leaves of certain barley (Hordeum vulgare L.) cultivars but not in others. A gene sequence in barley that earlier was characterized as a jasmonate-induced O-methyltransferase (MT) (EC 2.1.1.6, GenBank accession U54767) was here found to be absent in some barley cultivars and breeding lines that all lacked gramine. The cDNA was cloned and expressed in Escherichia coli and the recombinant protein purified. The purified recombinant protein methylated two substrates in the pathway to gramine: 3-aminomethylindole (AMI) and N-methyl-3-aminomethylindole (MAMI) at a high rate, with Km-values of 77 microM and 184 microM, respectively. In contrast, the protein did not exhibit any detectable methylation with the earlier suggested substrate for O-methylation, caffeic acid. A number of cultivars and breeding lines of barley were analyzed for presence of the U54767 gene sequence and MT protein and the enzyme activity in vitro with MAMI or caffeic acid as substrates. The results showed a clear relationship between the presence of the MT gene, the MT protein and N-methyltransferase activity, and confirmed the identification of the gene as coding for an N-methyltransferase (NMT, EC 2.1.1) and being involved in gramine biosynthesis.     

No overall hyposialylation in hereditary inclusion body myopathy myoblasts carrying the homozygous M712T GNE mutation

Hereditary inclusion body myopathy (HIBM) is a unique group of neuromuscular disorders characterized by adult-onset, slowly progressive distal and proximal muscle weakness, which is caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme in the biosynthetic pathway of sialic acid. In order to investigate the consequences of the mutated GNE enzyme in muscle cells, we have established cell cultures from muscle biopsies carrying either kinase or epimerase mutations. While all myoblasts carrying a mutated GNE gene show a reduction in their epimerase activity, only the cells derived from the patient carrying a homozygous epimerase mutation present also a significant reduction in the overall membrane bound sialic acid. These results indicate that although mutations in each of the two GNE domains result in an impaired enzymatic activity and the same HIBM phenotype, they do not equally affect the overall sialylation of muscle cells. This lack of correlation suggests that the pathological mechanism of the disease may not be linked solely to the well-characterized sialic acid pathway.     

Responses of the coastal bacterial community to viral infection of the algae Phaeocystis globosa

The release of organic material upon algal cell lyses has a key role in structuring bacterial communities and affects the cycling of biolimiting elements in the marine environment. Here we show that already before cell lysis the leakage or excretion of organic matter by infected yet intact algal cells shaped North Sea bacterial community composition and enhanced bacterial substrate assimilation. Infected algal cultures of Phaeocystis globosa grown in coastal North Sea water contained gamma- and alphaproteobacterial phylotypes that were distinct from those in the non-infected control cultures 5 h after infection. The gammaproteobacterial population at this time mainly consisted of Alteromonas sp. cells that were attached to the infected but still intact host cells. Nano-scale secondary-ion mass spectrometry (nanoSIMS) showed ∼20% transfer of organic matter derived from the infected ¹³C- and ¹⁵N-labelled P. globosa cells to Alteromonas sp. cells. Subsequent, viral lysis of P. globosa resulted in the formation of aggregates that were densely colonised by bacteria. Aggregate dissolution was observed after 2 days, which we attribute to bacteriophage-induced lysis of the attached bacteria. Isotope mass spectrometry analysis showed that 40% of the particulate ¹³C-organic carbon from the infected P. globosa culture was remineralized to dissolved inorganic carbon after 7 days. These findings reveal a novel role of viruses in the leakage or excretion of algal biomass upon infection, which provides an additional ecological niche for specific bacterial populations and potentially redirects carbon availability.     

Expression of a fungal cyanamide hydratase in transgenic soybean detoxifies cyanamide in tissue culture and in planta to provide cyanamide resistance

Embryogenic tissue cultures of soybean were transformed by particle bombardment with a vector pCHZ-II that carries the coding sequence for cyanamide hydratase (Cah), an enzyme that converts toxic cyanamide to urea, from the soil fungus Myrothecium verrucaria. The Cah gene was driven by the constitutive Arabidopsis thaliana actin-2 promoter and terminated with its cognate terminator. This vector also carries the hygromycin phosphotransferase gene (hpt) driven by the potato (Solanum tuberosum) ubiquitin-3 promoter. Twelve individual lines of transgenic plants that were obtained under hygromycin selection expressed Cah mRNA and exhibited resistance to hygromycin in leaf tissue culture, while the untransformed tissues were sensitive. Cah enzyme activity was present in extracts of transformed leaves and embryogenic tissue cultures when measured by a colorimetric assay and the presence of the Cah protein was confirmed by enzyme-linked immunosorbent assay (ELISA). Cah expression detoxified cyanamide in leaf callus and embryogenic cultures as well as in whole plants as shown by cyanamide resistance. The Cah-expressing plants grew and set seeds normally indicating that the Cah enzyme activity did not affect soybean plant metabolism. We also describe a test whereby callus was formed on cultured leaf tissue in the presence of hygromycin or cyanamide only if the hpt or Cah gene was expressed, respectively. This test is a convenient and cost-effective way to follow the marker gene in the primary regenerated plants and subsequent generations, which is particularly reliable for the hpt gene expression using hygromycin.     

Improved tolerance to bile salts of aggregated Bifidobacterium longum produced during continuous culture with immobilized cells

The effect of cell immobilization and continuous culture was studied on selected physiological and technological characteristics of Bifidobacterium longum NCC2705 cultivated for 20 days in a two stage continuous fermentation system. Continuous immobilized cell (IC) cultures with and without glucose limitation exhibited formation of macroscopic cell aggregates after 12 and 9 days, respectively. Auto-aggregation resulted in underestimation of viable cell counts by plate counts by more than 2 log units CFU/ml compared with qPCR method. Modifications of cell membrane composition might partially explain aggregate formation in IC cultures. Decreases in the ratio of unsaturated to saturated fatty acid content from 1.74 to 0.58 might also contribute to the enhanced tolerance of IC cells to porcine bile salts and aminoglycosidic antibiotics compared with free cells from batch cultures.The enhanced resistance against bile salts in combination with auto-aggregation may confer an advantage to probiotic bacteria produced by IC technology.     

Truncated hemoglobin o of Mycobacterium tuberculosis: the oligomeric state change and the interaction with membrane components

Being an obligate aerobe, the Mycobacterium tuberculosis cells would have to evolve a mechanism to collect and deliver the hardly available O(2) to survive in granulomas and to maintain the low level of respiration during latency. The M. tuberculosis truncated hemoglobin o (trHbO), when heterologously expressed in Escherichia coli cells, was found to significantly enhance the cellular respiration and cell growth. This study was undertaken in an attempt to understand the molecular details for trHbO to promote the cellular respiration, focusing on the ways through which trHbO is recruited to the cell membrane and O(2) molecules are delivered. Our data demonstrate that the trHbO protein is able to promote the growth of E. coli cells in a fashion that depends on the presence of the respiratory chain terminal oxidase cytochrome o complex (or Cyo complex). The trHbO protein appears to interact with the Cyo B subunit of the Cyo complex directly, likely in a dynamic manner. The trHbO is also able to bind membrane lipids in a non-specific way, during the process electrostatic and hydrophobic interactions both likely exist. Besides, binding with membrane induces the dissociation of trHbO from dimers to monomers. In light of these observations, a hypothesis was made to explain how trHbO might serve as an O(2) collector and/or reservoir for M. tuberculosis cells under O(2)-limiting or lacking conditions.     

Interaction of bHLH-PAS proteins involved in juvenile hormone reception in Drosophila

The Methoprene-tolerant (Met) bHLH-PAS gene is involved in juvenile hormone (JH) action in Drosophila melanogaster as a likely component of a JH receptor. We expressed Met in Drosophila S2 cells and explored for MET partners using pull-down assays. MET-MET interaction was found to occur. The germ-cell expressed (gce) gene is another D. melanogaster bHLH-PAS gene with high homology to Met, and GCE formed heterodimers with MET. In the presence of JH or either of two JH agonists, MET-MET and MET-GCE formation was drastically reduced. Interaction between GCE and MET having N- or C-terminus truncations, bHLH or PAS-A domain deletions, or a point mutation in the PAS-B domain failed to occur. However, JH-dependent interaction occurred between GCE and MET having point mutations in bHLH or PAS-A. During development, changes in JH titer may alter partner binding by MET and result in different gene expression patterns.     

A streamlined method for the isolation and quantitation of nanomole levels of exported polyamines in cell culture media

A number of years ago, our laboratory published a method for the isolation of small amounts of polyamines from cell culture media using the ion-exchange resin Bio-Rex 70. We have used this technique extensively to study the export of putrescine and cadaverine from cultured mammalian cells. Unfortunately, this method was highly inefficient in isolating the polyamines spermidine and spermine and was incapable of recovering the acetylated polyamine N(1)-acetylspermidine. In response to these shortcomings, we modified our previous protocol to quantitatively isolate the polyamines N(1)-acetylspermidine, putrescine, cadaverine, N(1)-acetylspermine, spermidine, and spermine. The new method, which is much faster to perform and more efficient than the one previously described, employs the use of disposable minicolumns and a single resin washing step using a weak solution of sodium carbonate at pH 9.3. This new protocol also eliminates the column elution step in favor of directly derivatizing the polyamines with dansyl chloride on the ion-exchange resin. High-performance liquid chromatography analysis of the dansylated polyamines isolated by this procedure showed that 75% of N(1)-acetylspermidine and nearly 100% of the other polyamines present in nanomolar levels were recovered from small amounts of cell culture medium. This new protocol is a valuable new tool for the study of the intracellular/extracellular dynamics of polyamine pools in cultured cells. [A detailed laboratory protocol for this procedure (containing all of the information in this paper but in a condensed form) can be requested by e-mailing the authors.]     

Stereoselectivity of the demethylation of nicotine piperidine homologues by Nicotiana plumbaginifolia cell suspension cultures

The metabolism of (R,S)-N-methylanabasine and (R,S)-N-methylanatabine has been studied in a cell suspension culture of Nicotiana plumbaginifolia. Both substrates are effectively demethylated, anabasine or anatabine, respectively, accumulating in the medium. Similarly, there is strong stereoselectivity for the (R)-isomers of both substrates. The kinetics of metabolism of (R,S)-N-methylanabasine differ significantly from those of nicotine in that no further degradation of the initial demethylation product occurs. (R,S)-N-Methylanatabine, however, shows kinetics closer to those of nicotine, with loss of alkaloid from the system. Further more, (R,S)-N-methylanabasine does not diminish (S)-nicotine demethylation, indicating a lack of competition. However, the metabolism of (S)-nicotine is affected by the presence of (R,S)-N-methylanabasine. Hence, the demethylation of the piperidine homologues of nicotine is seen to be similar but not identical to that of the pyridine analogues. The implications of these different metabolic profiles in relation to the demethylation activity are discussed.     

Monitoring stem cell proliferation and differentiation in primary midgut cell cultures from Heliothis virescens larvae using flow cytometry

In the midgut of Heliothis virescens larvae, proliferation and differentiation of stem cell populations allow for midgut growth and regeneration. Basic epithelial regenerative function can be assessed in vitro by purifying these two cell type populations, yet efficient high throughput methods to monitor midgut stem cell proliferation and differentiation are not available. We describe a flow cytometry method to differentiate stem from mature midgut cells and use it to monitor proliferation, differentiation and death in primary midgut stem cell cultures from H. virescens larvae. Our method is based on differential light scattering and vital stain fluorescence properties to distinguish between stem and mature midgut cells. Using this method, we monitored proliferation and differentiation of H. virescens midgut cells cultured in the presence of fetal bovine serum (FBS) or AlbuMAX II. Supplementation with FBS resulted in increased stem cell differentiation after 5 days of culture, while AlbuMAX II-supplemented medium promoted stem cell proliferation. These data demonstrate utility of our flow cytometry method for studying stem cell-based epithelial regeneration, and indicate that AlbuMAX II-supplemented medium may be used to maintain pluripotency in primary midgut stem cell cultures.     

WAXS and 13C NMR study of Gluconoacetobacter xylinus cellulose in composites with tamarind xyloglucan

- Model composites, produced using cellulose from stationary cultures of the bacterium Gluconoacetobacter xylinus and tamarind xyloglucan, were examined by wide-angle X-ray scattering (WAXS) and CP/MAS solid-state (13)C NMR spectroscopy. The dominant crystallite allomorph of cellulose produced in culture media with or without xyloglucan was cellulose I(alpha) (triclinic). The presence of xyloglucan in the culture medium reduced the cross-section dimensions of the cellulose crystallites, but did not affect the crystallite allomorph. However, when the composites were refluxed in buffer, the proportion of cellulose I(beta) allomorph increased relative to that of cellulose I(alpha). In contrast, cellulose I(alpha) remained the dominant form when cellulose, produced in the absence of xyloglucan, was then heated in the buffer. Hence the presence of xyloglucan has a profound effect on the formation of the cellulose crystallites by G. xylinus.