Multiple lipid interactions of the Sendai virus fusogenic protein

The membrane topology of the envelope of Sendai virus was investigated using various radioactive photoactivable hydrophobic reagents: 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and the two phospholipid analogues, 1-palmitoyl-2-(2-azido-4-nitro)benzoyl-sn -glycero-3- phospho[3H]choline and 1-myristoyl-2,12-amino-(4-N-3-nitro-1-azidophenyl)dodecanoyl-sn-glycero- 3-phospho[14C]choline. The hemagglutinin-neuraminidase glycoprotein and the fusogenic (F) glycoprotein were labeled by all three probes, confirming that these proteins are integral components of the viral envelope. The labeled F glycoprotein, composed of the two subunits F1 and F2, was cleaved in situ with trypsin to yield two fragments, F32 (32 kDa) and F19 (19 kDa). F2 was not labeled by any of the probes, suggesting an external location; whereas F19 was labeled by all probes and hence contains the portion of the F glycoprotein which traverses the viral envelope. Fragment F32 reacted both with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and with 1-palmitoyl-2-(2-azido-4-nitro)benzoyl-sn-glycero-3-phospho[3H]choline, but not with 1-myristoyl-2,12-amino-(4-N-3-nitro-1-azidophenyl)dodecanoyl-sn-glycero- 3- phospho[14C]choline. This result opens the possibility that the F glycoprotein is formed by a loop-like structure having multiple interactions with viral lipids.     

Histo-morphological analysis of rice callus cultures reveals differential regeneration response with varying media combinations

Genetic transformation of most indica rice (Oryza sativa) cultivars is hampered by poor in vitro culture performance and low regeneration potential. Histological study of primary calli can provide substantial information on their regeneration potential and can be used for early grading of calli expected to develop plantlets on regeneration media. The study was aimed to undertake histological analysis of primary calli derived from mature seeds of five indica rice cultivars viz. KSK-133, KS-282, Shaheen Basmati, Super Basmati, and DilRosh in order to assess their regeneration potential on different media combinations supplemented with various hormone concentrations (N6 + 2 mg/L 2,4-Dichlorophenoxyacetic acid; N6 + 2 mg/L 2–4 D + 2 mg/L Benzylaminopurine and MS + 2 mg/L 2,4-D). Calli with regeneration capability were subjected to histological assays by examining toulidine blue stained 5–8 μm thin sections for the presence of meristematic zones exhibiting embryogenic callus features. Based on our observations, formation of embryoids or embryoid-like structures was pronounced in KSK-133 and KS-282 calli. However, DilRosh, Super Basmati and Shaheen Basmati did not show these characteristic features. Three-week-old calli of all rice cultivars were transferred into regeneration medium (MS + 2 mg/L BAP + 1 mg/L Naphthaleneacetic acid). KSK-133 and KS-282 showed the highest regeneration potential (81% and 76%, respectively). These data were supported by histological observations where characteristic embryogenic units (EU) were noticed in these genotypes. These meristematic regions displayed high mitotic activity and stained relatively dark. The embryogenic calli cells were found heavily cytoplasmic with prominent nuclei and were located on the callus surface or inside surrounded by parenchymal cells.

     

Programmed cell death induced by high levels of cytokinin in Arabidopsis cultured cells is mediated by the cytokinin receptor CRE1/AHK4

High levels of cytokinins (CKs) induce programmed cell death (PCD) both in animals and plant cells. High levels of the CK benzylaminopurine (BA) induce PCD in cultured cells of Arabidopsis thaliana by accelerating a senescence process characterized by DNA laddering and expression of a specific senescence marker. In this report, the question has been addressed whether members of the small family of Arabidopsis CK receptors (AHK2, AHK3, CRE1/AHK4) are required for BA-induced PCD. In this respect, suspension cell cultures were produced from selected receptor mutants. Cell growth and proliferation of all receptor mutant and wild-type cell cultures were similar, showing that the CK receptors are not required for these processes in cultured cells. The analysis of CK metabolites instead revealed differences between wild-type and receptor mutant lines, and indicated that all three receptors are redundantly involved in the regulation of the steady-state levels of isopentenyladenine- and trans-zeatin-type CKs. By contrast, the levels of cis-zeatin-type CKs were controlled mainly by AHK2 and AHK3. To study the role of CK receptors in the BA-induced PCD pathway, cultured cells were analysed for their behaviour in the presence of high levels of BA. The results show that CRE1/AHK4, the strongest expressed CK receptor gene of this family in cultured cells, is required for PCD, thus linking this process to the known CK signalling pathway.     

Fenofibrate activates the biochemical pathways and the de novo expression of genes related to lipid handling and uncoupling protein-3 functions in liver of normal rats

Fibrates (anti-hyperlipidemic agents) enhance the mRNA expression of uncoupling protein 2 (UCP2) in the liver and that of uncoupling protein 3 (UCP3) in skeletal muscle in standard-diet-fed rats and induce a de novo expression of UCP3 (mRNA and protein) in the liver of high-fat-fed rats. Here, we report that in the liver of normal rats, fenofibrate induces a de novo expression of UCP3 and a 6-fold increase in UCP2 mRNA, whereas UCP2 protein was not detectable. Indeed, we evidenced an ORF in UCP2 exon 2 potentially able to inhibit the expression of the protein. Fenofibrate increases the expression and activity of hepatic enzymes and cofactors involved in lipid handling and UCP3 activity and, as is the case for UCP3, induces other muscle-specific genes (e.g., Carnitine palmitoyl transferase 1b and Ubiquinone biosynthesis protein COQ7 homolog). In addition, we demonstrated that in mitochondria from fenofibrate-treated rats a palmitoyl-carnitine-induced GDP-sensitive uncoupling takes place, involving UCP3 rather than other uncouplers (i.e., UCP2 and Adenine Nucleotide Translocase). Thus, the liver of fenofibrate-treated standard-diet- fed rat is a useful model for investigations of the biochemical functions of UCP3 and allowed us to demonstrate that fenofibrate programs a gene-expression pattern able to modulate lipid handling and UCP3 activation.     

Receptor-Dependent and -Independent Axonal Retrograde Transport of Poliovirus in Motor Neurons

Poliovirus (PV), when injected intramuscularly into the calf, is incorporated into the sciatic nerve and causes an initial paralysis of the inoculated limb in transgenic (Tg) mice carrying the human PV receptor (hPVR/CD155) gene. We have previously demonstrated that a fast retrograde axonal transport process is required for PV dissemination through the sciatic nerves of hPVR-Tg mice and that intramuscularly inoculated PV causes paralytic disease in an hPVR-dependent manner. Here we showed that hPVR-independent axonal transport of PV was observed in hPVR-Tg and non-Tg mice, indicating that several different pathways for PV axonal transport exist in these mice. Using primary motor neurons (MNs) isolated from these mice or rats, we demonstrated that the axonal transport of PV requires several kinetically different motor machineries and that fast transport relies on a system involving cytoplasmic dynein. Unexpectedly, the hPVR-independent axonal transport of PV was not observed in cultured MNs. Thus, PV transport machineries in cultured MNs and in vivo differ in their hPVR requirements. These results suggest that the axonal trafficking of PV is carried out by several distinct pathways and that MNs in culture and in the sciatic nerve in situ are intrinsically different in the uptake and axonal transport of PV.In humans, paralytic poliomyelitis results from the invasion of the central nervous system by circulating poliovirus (PV), probably via the blood-brain barrier. This conclusion is supported by the finding that circulating PV after intravenous inoculation in mice appears to cross the blood-brain barrier at a high rate in a human PV receptor (hPVR/CD155)-independent manner (44). After the virus enters the central nervous system, it replicates in neurons, especially in motor neurons (MNs), inducing the cell death that causes paralytic poliomyelitis. Along with this route of dissemination, a neuron-specific pathway has been reported in humans (31), monkeys (18), and PV-sensitive transgenic (Tg) mice carrying the hPVR gene (34, 37). This neuron-specific pathway appears to be important in causing “provocation poliomyelitis,” which is triggered by injuries after PV ingestion (11). Using differentiated PC12 cells and a PV-sensitive Tg mouse line, we have shown that intramuscularly inoculated PV is taken up by endocytosis at synapses.hPVR is a member of the immunoglobulin (Ig) superfamily, with three linked extracellular Ig-like domains, followed by a membrane-spanning domain and a cytoplasmic domain. Two membrane-bound forms (α and δ) and two secreted forms (β and γ) of hPVR derived by alternative splicing are likely to be expressed in human cells (23). Membrane-bound hPVRs are considered to play important roles in the early steps of infection, such as the binding of the virus to the cell surface, its entry into the cell, and the uncoating of the virus. The N-terminal Ig-like domain harbors the sites for PV binding, and anti-hPVR monoclonal antibodies (MAbs) directed against this region block PV infection (9, 24, 39).hPVR has the ability to alter the conformation of PV from the 160S intact infectious particle to a 135S particle from which the viral capsid protein VP4 is missing (2, 29). PV-related materials recovered from the sciatic nerves of PV-sensitive Tg mice after intramuscular inoculation with PV were mainly composed of intact 160S virions. The amount of 160S particles recovered was greatly reduced by coinjection with MAb p286, which specifically recognizes hPVR (34). Thus, most of the intramuscularly inoculated PV is incorporated into the sciatic nerves of PV-sensitive Tg mice as intact particles in an hPVR-dependent manner. This surprising finding might be due to either of two alternative, yet not mutually exclusive, possibilities: (i) a small number of PVRs bound per virion does not result in a conformational change in the viral capsid with a loss of VP4, but it is sufficient to induce endocytosis of the virus on the cell surface, or (ii) a cellular inhibitor(s) of PV uncoating may exist in the endocytic pathway responsible for PV uptake and transport in Tg mice (34).This mouse strain also allowed us to demonstrate that PV inoculated into the calf was incorporated into the sciatic nerve and retrogradely transported through the axons as intact virion particles. Furthermore, PV dissemination via the neural pathway has been found to rely on a fast retrograde axonal transport system and was inhibited by MAb p286 (34). Moreover, the efficient direct interaction of the hPVR cytoplasmic domain with Tctex-1, a light chain of cytoplasmic dynein (21), has been suggested to play an important role in retrograde transport, together with microtubule integrity (33). Cytoplasmic dynein, a minus-end-directed microtubule-based motor complex (13, 14, 17, 43), is implicated in the transport of early and late endosomes, lysosomes, synaptic vesicles, and endoplasmic reticulum along microtubules (1, 8, 13, 14, 17, 43). Notwithstanding the recent progress in the understanding of PV trafficking, the molecular determinants of the axonal transport of PV in MNs have not yet been elucidated.Despite the importance of axonal retrograde transport in health and disease, the direct visualization of retrograde transport and its quantitative analysis have been hampered by the lack of a reliable assay for living MNs. Such an assay was established in MNs by using a nontoxic fluorescent fragment of tetanus toxin (TeNT H_C), which binds to MNs and is retrogradely transported (28). Here, we applied this assay to the visualization of PV in living MNs.We employed hPVR-Tg and non-Tg mice, together with cultured MNs isolated from these mice, to clarify the mechanisms of axonal retrograde transport of PV. Experiments involving cultured MNs showed that the entry and axonal transport of PV are strictly hPVR dependent. However, hPVR-independent axonal transport of PV can be observed in non-Tg as well as in hPVR-Tg mice, suggesting that multiple axonal transport routes for PV are present in vivo.     

Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses

The clostridial neurotoxins responsible for tetanus and botulism are proteins consisting of three domains endowed with different functions: neurospecific binding, membrane translocation and proteolysis for specific components of the neuroexocytosis apparatus. Tetanus neurotoxin (TeNT) binds to the presynaptic membrane of the neuromuscular junction, is internalized and transported retroaxonally to the spinal cord. The spastic paralysis induced by the toxin is due to the blockade of neurotransmitter release from spinal inhibitory interneurons. In contrast, the seven serotypes of botulinum neurotoxins (BoNTs) act at the periphery by inducing a flaccid paralysis due to the inhibition of acetylcholine release at the neuromuscular junction. TeNT and BoNT serotypes B, D, F and G cleave specifically at single but different peptide bonds, of the vesicle associated membrane protein (VAMP) synaptobrevin, a membrane protein of small synaptic vesicles (SSVs). BoNT types A, C and E cleave SNAP-25 at different sites located within the carboxyl-terminus, while BoNT type C additionally cleaves syntaxin. The remarkable specificity of BoNTs is exploited in the treatment of human diseases characterized by a hyperfunction of cholinergic terminals.     

An NSF function distinct from ATPase-dependent SNARE disassembly is essential for Golgi membrane fusion

The precise biochemical role of N-ethylmaleimide-sensitive factor (NSF) in membrane fusion mediated by SNARE proteins is unclear. To provide further insight into the function of NSF, we have introduced a mutation into mammalian NSF that, in Drosophila dNSF-1, leads to temperature-sensitive neuroparalysis. This mutation is like the comatose mutation and renders the mammalian NSF temperature sensitive for fusion of postmitotic Golgi vesicles and tubules into intact cisternae. Unexpectedly, at the temperature that is permissive for membrane fusion, this mutant NSF binds to, but cannot disassemble, SNARE complexes and exhibits almost no ATPase activity. A well-charaterized NSF mutant containing an inactivating point mutation in the catalytic site of its ATPase domain is equally active in the Golgi-reassembly assay. These data indicate that the need for NSF during postmitotic Golgi membrane fusion may be distinct from its ATPase-dependent ability to break up SNARE pairs.     

Functional recycling of C2 domains throughout evolution: a comparative study of synaptotagmin, protein kinase C and phospholipase C by sequence, structural and modelling approaches

The C2 domain is one of the most frequent and widely distributed calcium-binding motifs. Its structure comprises an eight-stranded beta-sandwich with two structural types as if the result of a circular permutation. Combining sequence, structural and modelling information, we have explored, at different levels of granularity, the functional characteristics of several families of C2 domains. At the coarsest level, the similarity correlates with key structural determinants of the C2 domain fold and, at the finest level, with the domain architecture of the proteins containing them, highlighting the functional diversity between the various sub-families. The functional diversity appears as different conserved surface patches throughout this common fold. In some cases, these patches are related to substrate-binding sites whereas in others they correspond to interfaces of presumably permanent interaction between other domains within the same polypeptide chain. For those related to substrate-binding sites, the predictions overlap with biochemical data in addition to providing some novel observations. For those acting as protein-protein interfaces, our modelling analysis suggests that slight variations between families are a result of not only complementary adaptations in the interfaces involved but also different domain architecture. In the light of the sequence and structural genomic projects, the work presented here shows that modelling approaches along with careful sub-typing of protein families will be a powerful combination for a broader coverage in proteomics.