Tetanus Toxin Enhances Protein Kinase C Activity Translocation and Increases Polyphosphoinositide Hydrolysis in Rat Cerebral Cortex Preparations

Abstract: Tetanus toxin (TeTx) has been recently demonstrated to be a Zn²⁺-dependent endopeptidase that cleaves synaptobrevin, a protein in part responsible for neurotransmitter release. Nevertheless, certain aspects of TeTx action, for example, the causal relationship between TeTx and protein kinase C (PKC; EC 2.7.1.37) activity cannot be explained by this cleavage alone. In the present study, primary neurons from fetal rat brain, synaptosomes, and whole slices have been used to examine this issue. Low doses of TeTx (≤ 10^?8M) caused PKC activity translocation in a manner similar to that produced by 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA (≤ 10^?7M) caused sustained PKC activity translocation, whereas TeTx produced translocation followed by relocation, depending on the dose and time of exposure. Immunoidentification with a monoclonal antibody recognizing both α and β isoforms revealed that TeTx induced moderate losses of PKC in the cytosolic fraction, without a comparable increase in the particulate fraction. Although moderate losses of activity were also noticed in the cytosolic fraction, the inconsistency with respect to activity translocation may be explained by translocation of additional PKC isoforms that are not identified by the antibody. Comparable levels of water-soluble inositol phosphate-labeled intermediates were obtained after treatment of cerebral cells and/or cortical brain slices with TeTx. Significant increases of 19 and 114% in the water-soluble myo-[2-³H]inositol-labeled inositol phosphate metabolites were found in cerebral cell culture and brain slices, respectively, after treatment with 10^?8M TeTx. TeTx (10^?8M) increased to the same degree the water-soluble inositol phosphate levels as did serotonin (10^?5M) or carbachol (10^?6M). It is suggested that part of the signaling cascade of TeTx consists of a component involving inositol phospholipid hydrolysis, which is associated with PKC activity translocation.     

Identifying novel regulators of shoot meristem development

New organs are initiated throughout the life span of higher plants. This process occurs at the shoot meristem, which is initiated during embryogenesis and is later responsible for generating the above-ground portion of the plant. The shoot meristem can be thought of as having two zones, a central zone containing meristematic cells in an undifferentiated state, and a surrounding peripheral zone where cells enter a specific developmental pathway toward a differentiated state. Recent advances have revealed several genes that specifically regulate meristem development inArabidopsis. However, extensive mutagenesis by several labs have identified only a handful, of loci that appear to specifically regulate shoot meristem development. We have undertaken an enhancer/suppressor mutagenesis of an existing meristem mutant (clv1) and have identified novel regulators of meristem development. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Validation of computational fluid dynamics methodology used for human upper airway flow simulations

An anatomically accurate human upper airway model was constructed from multiple magnetic resonance imaging axial scans. This model was used to conduct detailed Computational Fluid Dynamics (CFD) simulations during expiration, to investigate the fluid flow in the airway regions where obstruction could occur. An identical physical model of the same airway was built using stereo lithography. Pressure and velocity measurements were conducted in the physical model. Both simulations and experiments were performed at a peak expiratory flow rate of 200 L/min. Several different numerical approaches within the FLUENT commercial software framework were used in the simulations; unsteady Large Eddy Simulation (LES), steady Reynolds-Averaged Navier-Stokes (RANS) with two-equation turbulence models (i.e. k?ε, standard k?ω, and k?ω Shear Stress Transport (SST)) and with one-equation Spalart–Allmaras model. The CFD predictions of the average wall static pressures at different locations along the airway wall were favorably compared with the experimental data. Among all the approaches, standard k?ω turbulence model resulted in the best agreement with the static pressure measurements, with an average error of ～20% over all ports. The highest positive pressures were observed in the retroglossal regions below the epiglottis, while the lowest negative pressures were recorded in the retropalatal region. The latter is a result of the airflow acceleration in the narrow retropalatal region. The largest pressure drop was observed at the tip of the soft palate. This location has the smallest cross section of the airway. The good agreement between the computations and the experimental results suggest that CFD simulations can be used to accurately compute aerodynamic flow characteristics of the upper airway.     

Involvement of lectin pathway activation in the complement killing of Giardia intestinalis

Giardia intestinalis (syn. G. lamblia, G. duodenalis) is a flagellated unicellular eukaryotic microorganism that commonly causes diarrheal disease throughout the world. In humans, the clinical effects of Giardia infection range from the asymptomatic carrier state to a severe malabsorption syndrome possibly due to different virulence of the Giardia strain, the number of cysts ingested, the age of the host, and the state of the host immune system at the time of infection.The question about how G. intestinalis is controlled by the organism remains unanswered. Here, we investigated the role of the complement system and in particular, the lectin pathway during Giardia infections. We present the first evidence that G. intestinalis activate the complement lectin pathway and in doing so participate in eradication of the parasite. We detected rapid binding of mannan-binding lectin, H-ficolin and L-ficolin to the surface of G. intestinalis trophozoites and normal human serum depleted of these molecules failed to kill the parasites. Our finding provides insight into the role of lectin pathway in the control of G. intestinalis and about the nature of surface components of parasite.     

Human plasma membrane-derived vesicles inhibit the phagocytosis of apoptotic cells - Possible role in SLE

Plasma membrane-derived vesicles (PMVs) also known as microparticles, are small membrane-bound vesicles released from the cell membrane via blebbing and shedding. PMVs have been linked with various physiological functions as well as pathological conditions such as inflammation, autoimmune disease and cardiovascular disease. PMVs are characterised by the expression of phosphatidylserine (PS) on the plasma membrane. PS, also expressed on apoptotic cells (ACs) enables macrophages to phagocytose ACs. As it is widely known that PMV production is increased during apoptosis, we were able to show that PMVs could compete dose dependently with ACs for the PS receptor on macrophages, so reducing phagocytosis of ACs. In a clinical setting this may result in secondary necrosis and further pathological conditions. In SLE in which there are raised PMV levels, there is an anti-phospholipid-mediated increase in PMV release, which can be abrogated by depletion of IgG. Our work provides an insight into how PMVs may play a role in the aetiology of autoimmune disease, in particular SLE.     

Human plasma membrane-derived vesicles halt proliferation and induce differentiation of THP-1 acute monocytic leukemia cells

Plasma membrane-derived vesicles (PMVs) are small intact vesicles released from the cell surface that play a role in intercellular communication. We have examined the role of PMVs in the terminal differentiation of monocytes. The myeloid-differentiating agents all-trans retinoic acid/PMA and histamine, the inflammatory mediator that inhibits promonocyte proliferation, induced an intracellular Ca(2+)-mediated PMV (as opposed to exosome) release from THP-1 promonocytes. These PMVs cause THP-1 cells to enter G(0)-G(1) cell cycle arrest and induce terminal monocyte-to-macrophage differentiation. Use of the TGF-β receptor antagonist SB-431542 and anti-TGF-β1 Ab showed that this was due to TGF-β1 carried on PMVs. Although TGF-β1 levels have been shown to increase in cell culture supernatants during macrophage differentiation and dendritic cell maturation, the presence of TGF-β1 in PMVs is yet to be reported. In this study, to our knowledge we show for the first time that TGF-β1 is carried on the surface of PMVs, and we confirm the presence within PMVs of certain leaderless proteins, with reported roles in myeloid cell differentiation. Our in vitro findings support a model in which TGF-β1-bearing PMVs, released from promonocytic leukemia cells (THP-1) or primary peripheral blood monocytes on exposure to sublytic complement or after treatment with a differentiation therapy agent, such as all-trans retinoic acid, significantly reduce proliferation of THP-1 cells. Such PMVs also induce the terminal differentiation of primary peripheral blood monocytes as well as THP-1 monocytes.     

NGF blocks polyunsaturated fatty acids biosynthesis in n-3 fatty acid-supplemented PC12 cells

Regulation of polyunsaturated fatty acid (PUFA) biosynthesis in proliferating and NGF-differentiated PC12 pheochromocytoma cells deficient in n-3 docosahexaenoic acid (DHA 22:6n-3) was studied. A dose- and time-dependent increase in eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3) and DHA in phosphatidylethanolamine (PtdEtn) and phosphatidylserine (PtdSer) glycerophospholipids (GPL) via the elongation/desaturation pathway following alpha-linolenic acid (ALA, 18:3n-3) supplements was observed. That was accompanied by a marked reduction of eicosatrienoic acid (Mead acid 20:3n-9), an index of PUFA deficiency. EPA supplements were equally effective converted to 22:5n-3 and 22:6n-3. On the other hand, supplements of linoleic acid (LNA, 18:2n-6) were not effectively converted into higher n-6 PUFA intermediates nor did they impair elongation/desaturation of ALA. Co-supplements of DHA along with ALA did not interfere with 20:5n-3 biosynthesis but reduced further elongation to 22-hydrocarbon PUFA intermediates. A marked decrease in the newly synthesized 22:5n-3 and 22:6n-3 following ALA or EPA supplements was observed after nerve growth factor (NGF)-induced differentiation. NGF also inhibited the last step in 22:5n-6 formation from LNA. These results emphasize the importance of overcoming n-3 PUFA deficiency and raise the possibility that growth factor regulation of the last step in PUFA biosynthesis may constitute an important feature of neuronal phenotype acquisition.     

Three-dimensional folding and functional organization principles of the Drosophila genome

Chromosomes are the physical realization of genetic information and thus form the basis for its readout and propagation. Here we present a high-resolution chromosomal contact map derived from a modified genome-wide chromosome conformation capture approach applied to Drosophila embryonic nuclei. The data show that the entire genome is linearly partitioned into well-demarcated physical domains that overlap extensively with active and repressive epigenetic marks. Chromosomal contacts are hierarchically organized between domains. Global modeling of contact density and clustering of domains show that inactive domains are condensed and confined to their chromosomal territories, whereas active domains reach out of the territory to form remote intra- and interchromosomal contacts. Moreover, we systematically identify specific long-range intrachromosomal contacts between Polycomb-repressed domains. Together, these observations allow for quantitative prediction of the Drosophila chromosomal contact map, laying the foundation for detailed studies of chromosome structure and function in a genetically tractable system.