Lotus corniculatus nodulation specificity is changed by the presence of a soybean lectin gene

Plant lectins have been implicated as playing an important role in mediating recognition and specificity in the Rhizobium-legume nitrogen-fixing symbiosis. To test this hypothesis, we introduced the soybean lectin gene Le1 either behind its own promoter or behind the cauliflower mosaic virus 35S promoter into Lotus corniculatus, which is nodulated by R. loti. We found that nodulelike outgrowths developed on transgenic L. corniculatus plant roots in response to Bradyrhizobium japonicum, which nodulates soybean and not Lotus spp. Soybean lectin was properly targeted to L. corniculatus root hairs, and although infection threads formed, they aborted in epidermal or hypodermal cells. Mutation of the lectin sugar binding site abolished infection thread formation and nodulation. Incubation of bradyrhizobia in the nodulation (nod) gene-inducing flavonoid genistein increased the number of nodulelike outgrowths on transgenic L. corniculatus roots. Studies of bacterial mutants, however, suggest that a component of the exopolysaccharide surface of B. japonicum, rather than Nod factor, is required for extension of host range to the transgenic L. corniculatus plants.     

Decay of Normal and 5-Fluorouracil-Substituted Messenger Ribonucleic Acid of Alkaline Phosphatase in Escherichia coli

The decay of alkaline phosphatase messenger ribonucleic acid (mRNA) in Escherichia coli was studied in cells sensitized to actinomycin D by ethylenediaminetetra-acetate treatment. It was found that in the wild-type strain (K-10) as well as in a nonsense mutant (S26C200), which is phenotypically reversible by treatment with 5-fluorouracil, mRNA decays with a half-life of 2.0 to 2.5 min. Similarly, in a 3-min pulse of labeled 5-fluorouracil, 70% of the incorporated analogue is contained in the labile RNA fraction decomposing with a half-life of 1.8 min.     

Immunocytolocalization of two protection-inducing antigens of Trichinella spiralis during its enteral phase in immune and non-immune mice

Monoclonal antibodies (mAb) recognizing epitopes on the 48K (beta stichocyte specific) and the 50/55K antigen (alpha stichocyte specific) were used as first ligands for immunocytolocalization on de-paraffinized sections of infected gut tissue of non-immune and immune CFW strain mice. The enteral phase was studied at 6, 14, 23, 30 hr and 7 days after initiation of infection via the oral route, times corresponding in worm development to the first (L1), second (L2), and third (L3) stage larva and adult. No change in the intensity of the immune reaction with either mAb was noted in parasites developing within immune or non-immune mice for any of the time-points studied. The 48K and the 50/55K antigens were present within the stichocytes at 6 hr. Enterocytes adjacent to some worms also stained positive for both epitopes at this time. Throughout worm development, the amount of each antigen within the worm diminished, until almost none was left at 30 hr. At day 7, the 48K antigen was present within a few stichocyte cells, the canalicular tree, and within the lumen of the midgut. The 50/55K antigen at this time point was localized within only a few stichocyte granules and on the lining of the worm's gut. Embryo stages did not possess either the 48K or 50/55K epitopes. A marked increase in cells bearing IgG in the lamina propria was noted in immune mice when compared with their non-immune counterparts.     

Epithelium-dependent extracellular matrix synthesis in transforming growth factor-beta 1-growth-inhibited mouse mammary gland

Exogenous transforming growth factor beta (TGF-beta 1) was shown in earlier studies to reversibly inhibit mouse mammary ductal growth. Using small plastic implants to treat regions of developing mammary glands in situ, we now report that TGF-beta 1 growth inhibition is associated with an ectopic accumulation of type I collagen messenger RNA and protein, as well as the glycosaminoglycan, chondroitin sulfate. Both macromolecules are normal components of the ductal extracellular matrix, which, under the influence of exogenous TGF-beta 1, became unusually concentrated immediately adjacent to the epithelial cells at the tip of the ductal growth points, the end buds. Stimulation of extracellular matrix was confined to aggregations of connective tissue cells around affected end buds and was not present around the TGF-beta 1 implants themselves, indicating that the matrix effect was epithelium dependent. Ectopic matrix synthesis was specific for TGF-beta 1 insofar as it was absent at ducts treated with other growth inhibitors, or at ducts undergoing normal involution in response to endogenous regulatory processes. These findings are consistent with the matrix-stimulating properties of TGF-beta 1 reported for other systems, but differ in their strict dependence upon epithelium. A possible role for endogenous TGF-beta 1 in modulating a mammary epithelium-stroma interaction is suggested.     

Synthesis of linear multimers of OriC and pBR322 derivatives in Escherichia coli K-12: role of recombination and replication functions.

Inactivation of RecBCD nuclease (exonuclease V) and SbcB nuclease (exonuclease I) in Escherichia coli K-12 diverts most of plasmid replication activity from circular monomer production to the synthesis of linear multimers. Linear multimer synthesis has been demonstrated in plasmids of diverse origins and copy numbers, including E. coli minichromosomes. The effect of dnaA, dnaB, recF, and recJ mutations on the rate of linear multimer synthesis in sbcB cells after gam inactivation of RecBCD nuclease was investigated. Results are consistent with the hypothesis that homologous recombination, but not activities at the plasmid origin of replication, is involved in initiation of linear multimer synthesis.     

Acetate causes alcohol hangover headache in rats

Background

The mechanism of veisalgia cephalgia or hangover headache is unknown. Despite a lack of mechanistic studies, there are a number of theories positing congeners, dehydration, or the ethanol metabolite acetaldehyde as causes of hangover headache.

Methods

We used a chronic headache model to examine how pure ethanol produces increased sensitivity for nociceptive behaviors in normally hydrated rats.

Results

Ethanol initially decreased sensitivity to mechanical stimuli on the face (analgesia), followed 4 to 6 hours later by inflammatory pain. Inhibiting alcohol dehydrogenase extended the analgesia whereas inhibiting aldehyde dehydrogenase decreased analgesia. Neither treatment had nociceptive effects. Direct administration of acetate increased nociceptive behaviors suggesting that acetate, not acetaldehyde, accumulation results in hangover-like hypersensitivity in our model. Since adenosine accumulation is a result of acetate formation, we administered an adenosine antagonist that blocked hypersensitivity.

Discussion

Our study shows that acetate contributes to hangover headache. These findings provide insight into the mechanism of hangover headache and the mechanism of headache induction.     

Natural Product Celastrol Destabilizes Tubulin Heterodimer and Facilitates Mitotic Cell Death Triggered by Microtubule-Targeting Anti-Cancer Drugs

Background

Microtubule drugs are effective anti-cancer agents, primarily due to their ability to induce mitotic arrest and subsequent cell death. However, some cancer cells are intrinsically resistant or acquire a resistance. Lack of apoptosis following mitotic arrest is thought to contribute to drug resistance that limits the efficacy of the microtubule-targeting anti-cancer drugs. Genetic or pharmacological agents that selectively facilitate the apoptosis of mitotic arrested cells present opportunities to strengthen the therapeutic efficacy.

Methodology and Principal Findings

We report a natural product Celastrol targets tubulin and facilitates mitotic cell death caused by microtubule drugs. First, in a small molecule screening effort, we identify Celastrol as an inhibitor of neutrophil chemotaxis. Subsequent time-lapse imaging analyses reveal that inhibition of microtubule-mediated cellular processes, including cell migration and mitotic chromosome alignment, is the earliest events affected by Celastrol. Disorganization, not depolymerization, of mitotic spindles appears responsible for mitotic defects. Celastrol directly affects the biochemical properties of tubulin heterodimer in vitro and reduces its protein level in vivo. At the cellular level, Celastrol induces a synergistic apoptosis when combined with conventional microtubule-targeting drugs and manifests an efficacy toward Taxol-resistant cancer cells. Finally, by time-lapse imaging and tracking of microtubule drug-treated cells, we show that Celastrol preferentially induces apoptosis of mitotic arrested cells in a caspase-dependent manner. This selective effect is not due to inhibition of general cell survival pathways or mitotic kinases that have been shown to enhance microtubule drug-induced cell death.

Conclusions and Significance

We provide evidence for new cellular pathways that, when perturbed, selectively induce the apoptosis of mitotic arrested cancer cells, identifying a potential new strategy to enhance the therapeutic efficacy of conventional microtubule-targeting anti-cancer drugs.     

Sustained PU.1 Levels Balance Cell-Cycle Regulators to Prevent Exhaustion of Adult Hematopoietic Stem Cells

1. Download : Download high-res image (312KB)
2. Download : Download full-size image

Highlights? PU.1 prevents hematopoietic stem cell (HSC) exhaustion ? PU.1 is a master regulator of cell-cycle genes in HSCs ? PU.1 binding mediates chromosome looping in HSCs ? Positive autoregulation via a ?14 kb enhancer sustains PU.1 levels in HSCs     

Characterization of the B-Raf interactome in mouse hippocampal neuronal cells

B-Raf links a variety of extracellular stimuli downstream of cell surface receptors, constituting a determining factor in the ability of neurons to activate ERK. A detailed study of the B-Raf interactome is necessary to clarify the intricacy of B-Raf-dependent signal transduction. We used a mouse hippocampal cell line (HT22) that expresses B-Raf at high levels, to identify B-Raf associated proteins under endogenous expression conditions, avoiding artificial interactions from overexpression studies. We used stringent procedures to co-immunoprecipitate proteins that specifically associate with endogenous B-Raf with the help of gel electrophoresis separation and off-line LC-MALDI-MS/MS proteomic analysis. Our stringent protein identification criteria allowed confident identification of B-Raf interacting proteins under non-stimulating conditions. The presence of previously reported B-Raf interactors among the list of proteins identified confirms the quality of proteomic data. We identified tubulin and actin as B-Raf interactors for the first time, among structural and accessory proteins of cell cytoskeleton, molecular chaperones (Hsc70, GRP78), and cellular components involved in aspects of mRNA metabolism and translation. Interactions were validated in HT22 cells and in the neuronal cell line Neuro-2a providing further evidence that the identified proteins are B-Raf interactors, which constitute a basis for understanding MAPK pathway regulation in neurons.     

All pyruvylated galactose in Schizosaccharomyces pombe N-glycans is present in the terminal disaccharide, 4, 6-O-[(R)-(1- carboxyethylidine)]-Galbeta1,3Galalpha1-

The large N-linked oligosaccharides released from Schizosaccharomyces pombe by endo-beta-N-acetylglucosaminidase H were examined to determine how the negatively chargedpyruvylated galactoses present (Gemmill,T.R., and Trimble,R.B., 1996, J. Biol. Chem ., 271, 25945-25949) were attached to the oligosaccharide chains. Binding of biotinylated human serum amyloid P and peanut agglutinin to native and depyruvylated S.pombe glycoproteins, respectively, indicated that the pyruvylated epitope was likely to be in the beta configuration. Examination by high- field 1H NMR of whole glycans and a disaccharide fragment released from them on partial acid hydrolysis showed that the pyruvylated galactose species was in fact beta1,3-linked to a second galactose, and this occurred an average of five to six times on nominal Gal57Man64GlcNAc N- glycans. The pyruvate-2,(4,6)Gal-beta1,3Gal epitope is chemically similar to acetaldehyde-Galbeta1,3Gal groups found on the glycoproteins from Paramyxovirus-infected bovine kidney cells (Prehm, P., Scheid,A. and Choppin,P.W. ,1979, J. Biol. Chem ., 254, 9669-9677). The 1:1 stoichiometry between pyruvate and beta-linked galactose in these S.pombe glycans indicates that either pyruvate addition to terminal beta1,3Gal is highly efficient or that pyruvylated Gal is transferred en bloc to alpha1,2-linked Gal residues in theN-linked chains. In contradiction to many galactomannan-producing fungi, which add substantial amounts of Gal in the furanose form to their glycoproteins, all detectable Gal in the large S.pombe galactomannans is in the pyranose form, as found in higher eukaryotes. The current work shows that the S.pombe outer chain structure is a poly-alpha1,6Man backbone 2- O-substituted with either Gal or the pyruvylated galactobiose and contains little alpha1,2-linked or 2-O-substituted Man. This is in contrast to the S. cerevisiae outer chain, which is poly-alpha1,6Man substituted with alpha1,2-linked Man sidechains (Ballou,C.E. ,1990, Methods Enzymol , 185, 440-470).