Isolation and characterization of Methanoplanus endosymbiosus sp. nov., an endosymbiont of the marine sapropelic ciliate Metopus contortus quennerstedt

Epifluorescence microscopy revealed the presence of a methanogenic bacterium as an endosymbiont in the sapropelic marine ciliate Metopus contortus. The in situ methanogenic activity of the symbiont could be demonstrated. The isolated endosymbiont was an irregular, disc-shaped bacterium with a diameter of 1.6–3.4 m. It had a generation time of 7 or 12 hours on growth on H₂/CO₂ or formate, respectively. The temperature range for growth was between 16 and 36°C with an optimum at 32°C. The optimal pH range for growth was 6.8 to 7.3. Salts, with an optimum concentration of 0.25 M, and tungsten were required for growth. The mol% G+C was 38.7%. The cell envelope consisted of proteins and a glycoprotein with an apparent molecular weight of 110,000. Morphology, antigenic relationship and the G+C content established the isolate MC1 as a new species of the genus Methanoplanus, and the name Methanoplanus endosymbiosus is proposed.Abbreviations G+C Guanine+cytosine - SDS sodium dodecylsulfate - PIPES piperazine-N,N-bis (2-ethane) sulfonic acid     

Betaine as a Growth Factor for Pediococcus soyae

Besides P-factor, Pediococcus soyae requires betaine (glycine-betaine) as a specific growth promotant. The maximal growth is obtained with the supplementation of both betaine and P-factor to the synthetic medium, while betaine only gives the half-maximal growth. When the organism is cultured in 18% salted media, the addition of both betaine and P-factor is essential for the occurrence of growth. Thus, betaine is requisite for Ped. soyae as a growth promoting factor and also as a factor bestowing the osmotelerant ability.     

Carnitine as a Growth Factor for Pediococcus soyae

Pediococcus soyae, the soy sauce lactic acid bacteria, requires two kinds of factors for growth as well as for salt-tolerance. These two factors are: glycine-betaine and an unknown substance tentatively named P-factor. The present paper reports that carnitine (vitamin Bt) is substitutable for glycine-betaine for its growth promoting effect on the organism. This is the first microorganism requiring carnitine as a specific growth factor.     

Succinate as a Growth Factor for Bacteroides melaninogenicus

Rumen strains of the obligate anaerobe Bacteroides melaninogenicus normally require medium supplemented with both heme and vitamin K. Sodium succinate was found to be an additional growth factor in that this compound can replace the requirement for heme in the presence of vitamin K, allowing good growth of the organism, and succinate can also partially replace the requirement for vitamin K in the presence of heme. The addition of succinate to a medium supplemented with both vitamin K and heme increases the growth rate of the culture. This ability to stimulate growth was specific for succinate, and cells grown without heme but with vitamin K and succinate were insensitive to cyanide. These experiments demonstrate a central role for succinate in the metabolism of B. melaninogenicus.     

Nerve Growth Factor as a Mitogen for a Pancreatic Carcinoid Cell Line

Abstract: Carcinoid tumors are a group of neuroendocrine neoplasms distributed widely throughout the body but most commonly occurring in the gut. These tumors retain many characteristics of their neural crest origin, including secretion of neuroactive peptides and responsiveness to neurotrophic substances. Nerve growth factor (NGF), a neurotrophic protein involved in maintenance and differentiation of peripheral sympathetic and sensory neurons, regulates growth of several neural tumor cells by inducing a differentiated phenotype and subsequent inhibition of cell growth rate. We examined the actions of NGF in a functioning human pancreatic carcinoid cell line (termed BON). NGF has no effect on the cytoarchitecture or constitutive secretion of bioamines in this carcinoid cell line. NGF, however, stimulates the in vitro cellular proliferation of BON cells. BON cells possess mRNA for the NGF receptors (p75^LNGFR and p140^trkA) and membrane-associated tyrosine kinase activity is increased in response to NGF. Both the mitogenic activity of NGF, as well as the receptor-linked tyrosine kinase activity, can be abrogated in BON cells by the trkA inhibitor K-252a and specific anti-NGF antibody. Our studies demonstrate that NGF is a mitogen for this carcinoid cell line without effect on cellular phenotype or cytoarchitecture. NGF may play a role in the development and progression of human carcinoid tumors.     

Identification of Chlorogenic Acid as a Resistance Factor for Thrips in Chrysanthemum

Western flower thrips (Frankliniella occidentalis) has become a key insect pest of agricultural and horticultural crops worldwide. Little is known about host plant resistance to thrips. In this study, we investigated thrips resistance in chrysanthemum (Dendranthema grandiflora). We identified thrips-resistant chrysanthemums applying bioassays. Subsequently, nuclear magnetic resonance (NMR)-based metabolomics was applied to compare the metabolome of thrips-resistant and -susceptible chrysanthemums. NMR facilitates wide-range coverage of the metabolome. We show that thrips-resistant and -susceptible chrysanthemums can be discriminated on basis of their metabolomic profiles. Thrips-resistant chrysanthemums contained higher amounts of the phenylpropanoids chlorogenic acid and feruloyl quinic acid. Both phenylpropanoids are known for their inhibitory effect on herbivores as well as pathogens. Thus, chlorogenic and feruloyl quinic acid are the compounds of choice to improve host plants resistance to thrips in ornamentals and crops. The effect of chlorogenic acid on thrips was further studied in bioassays with artificial diets. These experiments confirmed the negative effects on thrips. Our results prove NMR to be an important tool to identify different metabolites involved in herbivore resistance. It constitutes a significant advance in the study of plant-insect relationships, providing key information on the implementation of herbivore resistance breeding strategies in plants.     

Genome sequence of the mud-dwelling archaeon Methanoplanus limicola type strain (DSM 2279T), reclassification of Methanoplanus petrolearius as Methanolacinia petrolearia and emended descriptions of the genera Methanoplanus and Methanolacinia

Methanoplanus limicola Wildgruber et al. 1984 is a mesophilic methanogen that was isolated from a swamp composed of drilling waste near Naples, Italy, shortly after the Archaea were recognized as a separate domain of life. Methanoplanus is the type genus in the family Methanoplanaceae, a taxon that felt into disuse since modern 16S rRNA gene sequences-based taxonomy was established. Methanoplanus is now placed within the Methanomicrobiaceae, a family that is so far poorly characterized at the genome level. The only other type strain of the genus with a sequenced genome, Methanoplanus petrolearius SEBR 4847^T, turned out to be misclassified and required reclassification to Methanolacinia. Both, Methanoplanus and Methanolacinia, needed taxonomic emendations due to a significant deviation of the G+C content of their genomes from previously published (pre-genome-sequence era) values. Until now genome sequences were published for only four of the 33 species with validly published names in the Methanomicrobiaceae. Here we describe the features of M. limicola, together with the improved-high-quality draft genome sequence and annotation of the type strain, M3^T. The 3,200,946 bp long chromosome (permanent draft sequence) with its 3,064 protein-coding and 65 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

The Role of Thiamine as a Factor for the Growth of Brochothrix thermosphacta

Thiamine enhances growth of the meat spoilage organism, Brochothrix thermosphacta. It fulfils some, but not all, of the yeast-extract requirement of this organism. Oxythiamine acts as a growth inhibitor at high concentrations and the inhibition is readily reversed by thiamine. The possible roles of thiamine in the metabolism of Br. thermosphacta are discussed.     

Identification of a Sam68 Ribonucleoprotein Complex Regulated by Epidermal Growth Factor

Sam68, Src associated in mitosis of 68 kDa, is a known RNA-binding protein and a signaling adaptor protein for tyrosine kinases. However, the proteins associated with Sam68 and the existence of a Sam68 complex, its mass, and regulation are, however, unknown. Herein we identify a large Sam68 complex with a mass >1 MDa in HeLa cells that is composed of ∼40 proteins using an immunoprecipitation followed by a mass spectrometry approach. Many of the proteins identified are RNA-binding proteins and are known components of a previously identified structure termed the spreading initiation center. The large Sam68 complex is a ribonucleoprotein complex, as treatment with RNases caused a shift in the molecular mass of the complex to 200–450 kDa. Moreover, treatment of HeLa cells with phorbol 12-myristate 13-acetate or epidermal growth factor induced the disassociation of Sam68 from the large complex and the appearance of Sam68 within the smaller complex. Actually, in certain cell lines such as breast cancer cell lines MCF-7 and BT-20, Sam68 exists in equilibrium between a large and a small complex. The appearance of the small Sam68 complex in cells correlates with the ability of Sam68 to promote the alternative splicing of CD44 and cell migration. Our findings show that Sam68 exists in equilibrium in transformed cells between two complexes and that extracellular signals, such as epidermal growth factor stimulation, promote alternative splicing by modulating the composition of the Sam68 complex.     

Identification of Pigment Epithelium-Derived Factor as an Adipocyte-Derived Inflammatory Factor

Obesity is a major risk factor for insulin resistance, type 2 diabetes mellitus and cardiovascular disease. The pathophysiology of obesity is associated with chronic low-grade inflammation. Adipose tissue in obesity is significantly infiltrated by macrophages that secrete cytokines. The mechanisms of interaction between macrophages and adipocytes, leading to macrophage activation and increased cytokine release, remain to be elucidated. We reasoned that an adipocyte-derived factor might stimulate activation of macrophages. We have identified pigment epithelium-derived factor (PEDF) as a mediator of inflammation that is secreted by adipocytes and mediates macrophage activation. Recombinant PEDF activates macrophages to release tumor necrosis factor (TNF) and interleukin-1 (IL-1). The PEDF receptor adipose triglyceride lipase (ATGL) is required for PEDF-mediated macrophage activation. Selective inhibition of ATGL on macrophages attenuates PEDF-induced TNF production, and PEDF enhances the phosphorylation of p38 and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases. PEDF administration to rats results in increased serum TNF levels, and insulin resistance. Together, these findings suggest that PEDF secreted by adipocytes contributes to the onset and maintenance of chronic inflammation in obesity, and may be a therapeutic target in ameliorating insulin resistance.     

Identification of TRAPPC8 as a Host Factor Required for Human Papillomavirus Cell Entry

Human papillomavirus (HPV) is a non-enveloped virus composed of a circular DNA genome and two capsid proteins, L1 and L2. Multiple interactions between its capsid proteins and host cellular proteins are required for infectious HPV entry, including cell attachment and internalization, intracellular trafficking and viral genome transfer into the nucleus. Using two variants of HPV type 51, the Ma and Nu strains, we have previously reported that MaL2 is required for efficient pseudovirus (PsV) transduction. However, the cellular factors that confer this L2 dependency have not yet been identified. Here we report that the transport protein particle complex subunit 8 (TRAPPC8) specifically interacts with MaL2. TRAPPC8 knockdown in HeLa cells yielded reduced levels of reporter gene expression when inoculated with HPV51Ma, HPV16, and HPV31 PsVs. TRAPPC8 knockdown in HaCaT cells also showed reduced susceptibility to infection with authentic HPV31 virions, indicating that TRAPPC8 plays a crucial role in native HPV infection. Immunofluorescence microscopy revealed that the central region of TRAPPC8 was exposed on the cell surface and colocalized with inoculated PsVs. The entry of Ma, Nu, and L2-lacking PsVs into cells was equally impaired in TRAPPC8 knockdown HeLa cells, suggesting that TRAPPC8-dependent endocytosis plays an important role in HPV entry that is independent of L2 interaction. Finally, expression of GFP-fused L2 that can also interact with TRAPPC8 induced dispersal of the Golgi stack structure in HeLa cells, a phenotype also observed by TRAPPC8 knockdown. These results suggest that during viral intracellular trafficking, binding of L2 to TRAPPC8 inhibits its function resulting in Golgi destabilization, a process that may assist HPV genome escape from the trans-Golgi network.     

Identification of Glycosyltransferase 8 Family Members as Xylosyltransferases Acting on O-Glucosylated Notch Epidermal Growth Factor Repeats

The epidermal growth factor repeats of the Notch receptor are extensively glycosylated with three different O-glycans. O-Fucosylation and elongation by the glycosyltransferase Fringe have been well studied and shown to be essential for proper Notch signaling. In contrast, biosynthesis of O-glucose and O-N-acetylglucosamine is less well understood. Recently, the isolation of the Drosophila mutant rumi has shown that absence of O-glucose impairs Notch function. O-Glucose is further extended by two contiguous α1,3-linked xylose residues. We have identified two enzymes of the human glycosyltransferase 8 family, now named GXYLT1 and GXYLT2 (glucoside xylosyltransferase), as UDP-d-xylose:β-d-glucoside α1,3-d-xylosyltransferases adding the first xylose. The enzymes are specific for β-glucose-terminating acceptors and UDP-xylose as donor substrate. Generation of the α1,3-linkage was confirmed by nuclear magnetic resonance. Activity on a natural acceptor could be shown by in vitro xylosylation of a Notch fragment expressed in a UDP-xylose-deficient cell line and in vivo by co-expression of the enzymes and the Notch fragment in insect cells followed by mass spectrometric analysis of peptide fragments.     

Production of Skatole and para-Cresol by a Rumen Lactobacillus sp

The objective of this study was to examine the substrate specificity of several ruminal strains of a Lactobacillus sp. which previously was shown to produce skatole (3-methylindole) by the decarboxylation of indoleacetic acid. A total of 13 compounds were tested for decarboxylase activity. The Lactobacillus strains produced p-cresol (4-methylphenol) by the decarboxylation of p-hydroxyphenylacetic acid, but did not produce either o-cresol or m-cresol from the corresponding hydroxyphenylacetic acid isomers. These strains also decarboxylated 5-hydroxyindoleacetic acid to 5-hydroxyskatole and 3,4-dihydroxyphenylacetic acid to methylcatechol. Skatole and p-cresol were produced in a 0.5:1 ratio, when indoleacetic acid and p-hydroxyphenylacetic acid were combined in equimolar concentrations. Competition studies with indoleacetic acid and p-hydroxyphenylacetic acid suggested that two different decarboxylating enzymes are involved in the production of skatole and p-cresol by these strains. This is the first demonstration of both skatole production and p-cresol production by a single bacterium.