Search

The protocorm is a structure that is formed upon germination of an orchid seed. It lacks cotyledons and is ovoid in shape. The protocorm-like body (PLB), on the other hand, is a protocorm-like organ induced from somatic tissues. PLBs have been widely used for orchid micropropagation. Because of its unique structure and its application in the orchid industry, PLB development has drawn considerable interest from orchid and developmental biologists. Our previous genome-wide comparative transcriptome study demonstrated that protocorms and PLBs share similar molecular signatures and suggested that SHOOT MERISTEMLESS (STM)-dependent organogenesis is important for PLB development. Here, we show that overexpression of Phalaenopsis aphrodite STM (PaSTM) greatly enhances PLB regeneration from vegetative tissue-based explants of Phalaenopsis orchids, confirming its regulatory role in PLB development. Expression of PaSTM restored shoot meristem function of the Arabidopsis (Arabidopsis thaliana) stm-2 mutant. Moreover, we identified class S11 MYB transcription factors (TFs) as targets downstream of PaSTM. A cis-acting element, TTGACT, identified in the promoters of S11 MYB TFs was found to be important for PaSTM binding and activation. Overexpression of PaSTM or its downstream targets, PaMYB13, PaMYB14, and PaMYB17, enhanced de novo shoot regeneration in Arabidopsis, indicating the active role of the PaSTM-S11 PaMYB module in organogenesis. In summary, our data demonstrate that PaSTM is important for PLB development. The STM-S11 MYB regulatory module is evolutionarily conserved and may regulate shoot or shoot-related organ development in plants.

Some inner C-cyanation and C-alkylation complexes have been described. Reactions of 2-N-substituted N-confused tetraphenylporphyrin (2-NCH2YNCTPPH) with Co(SCN)2 afford C-alkylation (14) and C-cyanation (15–17) complexes. The crystal structures for the inner C-alkylation product of thiocyanato(2-aza-2-[p-methylbenzyl]-5,10,15,20-tetraphenyl-21-benzyl-21-carbaporphyrinato-N,N′,N″)cobalt(II)·0.5 octane solvate [Co(2-NCH2-p-CH3C6H4-21-CH2C6H5NCTPP)(NCS)·0.5C8H18; 14·0.5C8H18] and the inner C-cyanation products of thiocyanato(2-aza-2-ethoxycarbonylmethyl-5,10,15,20-tetraphenyl-21-cyano-21-carbaporphyrinato-N,N′,N″)cobalt(II) [Co(2-NCH2COOCH2CH3-21-CN NCTPP) (NCS); 15], thiocyanato(2-aza-2-methyl-5,10,15,20-tetraphenyl-21-cyano-21-carbaporphyrinato-N,N′,N″)cobalt(II)[Co(2-NCH3-21-CNNCTPP) (NCS); 16], and thiocyanato(2-aza-2-benzyl-5,10,15,20-tetraphenyl-21-cyano-21-carbaporphyrinato-N,N′,N″)cobalt(II) [Co(2-NCH2C6H5-21-CNNCTPP) (NCS); 17] have been reported. The geometry around Co2+ ion in these four complexes is a distorted tetrahedron with a C2v symmetry. Two possible mechanisms were proposed to explain the formation of inner C-alkylation (14·0.5C8H18) versus C-cyanation (15–17) products in these complexes with an axial *NCS− ligand.

Six four-coordinated complexes of the chemical formulae [Hg(2-N CH2COOCH2CH3-21-H-NCTPP)X] with X = Cl (5), Br (6), I (7), [Hg(2-NCH3-21-H-NCTPP)Cl] (4) and [Hg(2-NCH2COOCH2C6H5-21-H-NCTPP)X] with X = Cl (8), I (9) are synthesized and structurally determined. The bond path for the weak η1-H(17)–C(17)⋯Hg agostic interactions between the Hg center and H(17) in complexes 4–9 was a through-space interaction from Hg to agostic carbon [C(17)] followed by a through-bond interaction from C(17) to an agostic proton [H(17)]. The magnitude of J[Hg–H(17)] [or the agostic upfield shift Δδago of the C(17)] for these complexes increases as the halide ligand varies from iodide to chloride, ranging from 33.2 Hz (or 14.3 ppm) for I− to 36 Hz (or 15.8 ppm) for Br− and 36.9 Hz (or 16.0 ppm) for Cl−. The plot of J[Hg–H(17)] for the agostic proton H(17) versus |Δδago| for the agostic carbon atom C(17) in compounds 3–9 was linearly expressed as J[Hg–H(17)] = 2.29 |Δδago| + 0.13.

The crystal structures of 2-aza-2-[p-(isopropyl)benzyl]-5,10,15,20-tetraphenyl-21-carbaporphyrinato-N,N′,N″)palladium(II) [Pd (2-NCH2-p-C6H4-isoC3H7NCTPP); 3], (2-azo-2-[p-(isopropyl)]-5,10,15,20-tetraphenyl-21-carbaporphyrinato-N,N′,N″)platinum(II) [Pt(2-NCH2-p-C6H4-isoC3H7NCTPP); 4], iodo(2-azo-2-[p-(isopropyl)benzyl]-5,10,15,20-tetraphenyl-21-hydrogen-21-carbaporphyrinato-N,N′,N″)mercury(II) toluene solvate [Hg(2-NCH2-p-C6H4-isoC3H7-21-H-NCTPP)I⋅C6H5CH3; 5⋅C6H5CH3], (2-azo-2-[p-methylbenzyl]-5,10,15,20-tetraphenyl-21-carbaporphyrinato-N,N′,N″)nickel(II) [Ni(2-NCH2-p-C6H4-CH3NCTPP); 6] and iodo(2-azo-2-[p-methylbenzyl]-5,10,15,20-tetraphenyl-21-hydrogen-21-carbaporphyrinato-N,N′,N″)mercury(II) [Hg(2-NCH2-p-C6H4-CH3-21-H-NCTPP)I; 7] were determined. The coordination sphere around the Pd2+ (or Pt2+, Ni2+) ion in 3 (or 4, 6) is distorted square planar (DSP) whereas for Hg2+ in 5 (or 7), it is a distorted tetrahedral complex with the T-based pyramid of the C2v symmetry. The coupling of H(17) in 5 (7) to 199Hg with J [199Hg–H(17)] = 34.8 Hz (5) and J [199Hg–H(17)] = 34.2 Hz (7), supported the assignment of agostic η2–C–H(17)⋯Hg interactions in these two mercury complexes.

Six four-coordinated complexes of the chemical formulae [Hg(2-N CH2COOCH2CH3-21-H-NCTPP)X] with X = Cl (5), Br (6), I (7), [Hg(2-NCH3-21-H-NCTPP)Cl] (4) and [Hg(2-NCH2COOCH2C6H5-21-H-NCTPP)X] with X = Cl (8), I (9) are synthesized and structurally determined. The bond path for the weak η1-H(17)-C(17)Hg agostic interactions between the Hg center and H(17) in complexes 4-9 was a through-space interaction from Hg to agostic carbon [C(17)] followed by a through-bond interaction from C(17) to an agostic proton [H(17)]. The magnitude of J[Hg-H(17)] [or the agostic upfield shift Δδago of the C(17)] for these complexes increases as the halide ligand varies from iodide to chloride, ranging from 33.2 Hz (or 14.3 ppm) for I- to 36 Hz (or 15.8 ppm) for Br- and 36.9 Hz (or 16.0 ppm) for Cl-. The plot of J[Hg-H(17)] for the agostic proton H(17) versus |Δδago| for the agostic carbon atom C(17) in compounds 3-9 was linearly expressed as J[Hg-H(17)] = 2.29 |Δδago| + 0.13.

Orchidaceae is one of the most abundant and diverse families in the plant kingdom and its unique developmental patterns have drawn the attention of many evolutionary biologists. Particular areas of interest have included the co-evolution of pollinators and distinct floral structures, and symbiotic relationships with mycorrhizal flora. However, comprehensive studies to decipher the molecular basis of growth and development in orchids remain scarce. Cell proliferation governed by cell-cycle regulation is fundamental to growth and development of the plant body. We took advantage of recently released transcriptome information to systematically isolate and annotate the core cell-cycle regulators in the moth orchid Phalaenopsis aphrodite. Our data verified that Phalaenopsis cyclin-dependent kinase A (CDKA) is an evolutionarily conserved CDK. Expression profiling studies suggested that core cell-cycle genes functioning during the G1/S, S, and G2/M stages were preferentially enriched in the meristematic tissues that have high proliferation activity. In addition, subcellular localization and pairwise interaction analyses of various combinations of CDKs and cyclins, and of E2 promoter-binding factors and dimerization partners confirmed interactions of the functional units. Furthermore, our data showed that expression of the core cell-cycle genes was coordinately regulated during pollination-induced reproductive development. The data obtained establish a fundamental framework for study of the cell-cycle machinery in Phalaenopsis orchids. Electronic supplementary material The online version of this article (doi:10.1007/s11103-013-0128-y) contains supplementary material, which is available to authorized users.

Background and Purpose— Low-dose lipopolysaccharide (LPS) preconditioning provides neonatal rats long-term neuroprotection against hypoxic ischemia (HI). Upregulating endothelial nitric oxide synthase (eNOS) protects against cerebral ischemia; however, whether eNOS is required for LPS preconditioning-induced protection in neonatal rats is unknown. We hypothesized that Akt activation, which upregulates eNOS in neurons and endothelial cells, is required for LPS preconditioning-induced tolerance against HI in the neonatal brain. Methods— Six-day-old rat pups were intraperitoneally injected with LPS (0.05 mg/kg) or normal saline 24 hours before HI. Immunoblotting and immunohistochemistry were used to determine the phospho-Akt (pAkt Ser473), phospho-eNOS (peNOS Ser1177), and eNOS levels and immunofluorescence to determine the cellular distribution of eNOS and pAkt Ser473. Pharmacological and genetic approaches were used to regulate Akt and eNOS, and the weight loss of cerebral hemispheres on postnatal Day 21 was used to assess outcomes. Results— eNOS, peNOS (Ser1177), and pAkt (Ser473) levels were significantly higher in LPS- than in normal saline-treated rats 24 hours postinjection. LPS-induced eNOS was expressed primarily in neurons and vascular endothelial cells. N-omega(ω)-nitro- l -arginine and antisense oligodeoxynucleotide treatment significantly reduced eNOS expression in neurons and endothelial cells and inhibited LPS-induced protection against HI in rat pups. l -arginine and adenovirus eNOS transfection upregulated eNOS and protected the rat pups against HI. Wortmannin treatment before LPS preconditioning significantly reduced eNOS expression in neurons and endothelial cells, which inhibited LPS-induced protection against HI. Conclusions— Akt-mediated eNOS upregulation in neurons and vascular endothelial cells is required for LPS-induced tolerance against HI in the neonatal rat brain.

Enterovirus 71 (EV71) causes hand, foot, and mouth disease and severe neurological disorders in children. Human scavenger receptor class B member 2 (hSCARB2) and P-selectin glycoprotein ligand-1 (PSGL-1) are identified as receptors for EV71. The underling mechanism of PSGL-1-mediated EV71 entry remains unclear. The endocytosis required for EV71 entry were investigated in Jurkat T and mouse L929 cells constitutively expressing human PSGL-1 (PSGL-1-L929) or human rhabdomyosarcoma (RD) cells displaying high SCARB2 but no PSGL-1 by treatment of specific inhibitors or siRNA. We found that disruption of clathrin-dependent endocytosis prevented EV71 infection in RD cells, while there was no influence in Jurkat T and PSGL-1-L929 cells. Disturbing caveolar endocytosis by specific inhibitor or caveolin-1 siRNA in Jurkat T and PSGL-1-L929 cells significantly blocked EV71 infection, whereas it had no effect on EV71 infection in RD cells. Confocal immunofluorescence demonstrated caveola, and EV71 was directly colocalized. pH-dependent endosomal acidification and intact membrane cholesterol were important for EV71 infection, as judged by the pretreatment of inhibitors that abrogated the infection. A receptor-dominated endocytosis of EV71 infection was observed: PSGL-1 initiates caveola-dependent endocytosis and hSCARB2 activates clathrin-dependent endocytosis.

Although several factors participating in enterovirus 71 (EV71) entry and replication had been reported, the precise mechanisms associated with these events are far from clear. In the present study, we showed that heat shock protein 90 (HSP90) is a key element associated with EV71 entry and replication in a human rhabdomyosarcoma of RD cells. Inhibition of HSP90 by pretreating host cells with HSP90β siRNA or blocking HSP90 with a HSP90-specific antibody or geldanamycin (GA), a specific inhibitor of HSP90, as well as recombinant HSP90β resulted in inhibiting viral entry and subsequent viral replication. Co-immunprecipitation of EV71 with recombinant HSP90β and colocalization of EV71-HSP90 in the cells demonstrated that HSP90 was physically associated with EV71 particles. HSP90 seems to mediate EV71 replication by preventing proteosomal degradation of the newly synthesized capsid proteins, but does not facilitate viral gene expression at transcriptional level. This was evident by post-treatment of host cells with GA, which did not affect the expression of viral transcripts but accelerated the degradation of viral capsid proteins and interfered with the formation of assembled virions. In vivo studies were carried out using human SCARB2-transgenic mice to evaluate the protection conferred by HSP90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), an analog of geldanamycin, that elicited similar activity but with less toxicity. The results showed that the administration of 17-AAG twice conferred the resistance to hSCARB2 mice challenged with C2, C4, and B4 genotypes of EV71. Our data supports HSP90 plays an important role in EV71 infection. Targeting of HSP90 with clinically available drugs might provide a feasible therapeutic approach to treat EV71 infection.

Enterovirus (EV) 71 infection is known to cause hand-foot-and-mouth disease (HFMD) and in severe cases, induces neurological disorders culminating in fatality. An outbreak of EV71 in South East Asia in 1997 affected over 120,000 people and caused neurological disorders in a few individuals. The control of EV71 infection through public health interventions remains minimal and treatments are only symptomatic. Recently, human scavenger receptor class B, member 2 (SCARB2) has been reported to be a cellular receptor of EV71. We expressed human SCARB2 gene in NIH3T3 cells (3T3-SCARB2) to study the mechanisms of EV71 entry and infection. We demonstrated that human SCARB2 serves as a cellular receptor for EV71 entry. Disruption of expression of SCARB2 using siRNAs can interfere EV71 infection and subsequent inhibit the expression of viral capsid proteins in RD and 3T3-SCARB2 but not Vero cells. SiRNAs specific to clathrin or dynamin or chemical inhibitor of clathrin-mediated endocytosis were all capable of interfering with the entry of EV71 into 3T3-SCARB2 cells. On the other hand, caveolin specific siRNA or inhibitors of caveolae-mediated endocytosis had no effect, confirming that only clathrin-mediated pathway was involved in EV71 infection. Endocytosis of EV71 was also found to be pH-dependent requiring endosomal acidification and also required intact membrane cholesterol. In summary, the mechanism of EV71 entry through SCARB2 as the receptor for attachment, and its cellular entry is through a clathrin-mediated and pH-dependent endocytic pathway. This study on the receptor and endocytic mechanisms of EV71 infection is useful for the development of effective medications and prophylactic treatment against the enterovirus.

Identification of HLA-restricted CD8+ T cell epitopes is important to study RSV-induced immunity and illness. We algorithmically analyzed the sequence of the fusion protein (F) of respiratory syncytial virus (RSV) and generated synthetic peptides that can potentially bind to HLA-A*0201. Four out of the twenty-five 9-mer peptides tested: peptides 3 (F33–41), 13 (F214–222), 14 (F273–281), and 23 (F559–567), were found to bind to HLA-A*0201 with moderate to high affinity and were capable of inducing IFN-γ and IL-2 secretion in lymphocytes from HLA-A*0201 transgenic (HLA-Tg) mice pre-immunized with RSV or recombinant adenovirus expressing RSV F. HLA-Tg mice were immunized with these four peptides and were found to induce both Th1 and CD8+ T cell responses in in vitro secondary recall. Effector responses induced by these peptides were observed to confer differential protection against live RSV challenge. These peptides also caused better recovery of body weight loss induced by RSV. A significant reduction of lung viral load was observed in mice immunized with peptide 23, which appeared to enhance the levels of inflammatory chemokines (CCL17, CCL22, and IL-18) but did not increase eosinophil infiltration in the lungs. Whereas, significant reduction of infiltrated eosinophils induced by RSV infection was found in mice pre-immunized with peptide 13. Our results suggest that HLA-A2-restricted epitopes of RSV F protein could be useful for the development of epitope-based RSV vaccine.

Hypoxic ischemia (HI) in newborns causes long-term neurologic abnormalities. Systemic lipopolysaccharide (LPS) is neuroprotective in neonatal rats when injected 24 h before HI. However, the effect on HI-induced neuroinflammation and the long-term outcome of LPS preconditioning in neonatal rats have not been examined. In a rat-pup HI model, compared with normal saline (NS), 0.3 mg/kg of LPS injected 24 h before HI greatly increased microglial cell and macrophage activation and up-regulated TNF-alpha and inducible NOS expression 12-h postinjection and resulted in high mortality during HI. In contrast, 0.05 mg/kg of LPS elicited very little microglia and macrophage activation and TNF-alpha and inducible NOS expression and resulted in low mortality. Given 24 h before HI, low-dose (0.05 mg/kg) LPS greatly reduced microglia and macrophage activation, TNF-alpha expression, and reactive oxygen species production 24-h post-HI compared with NS-treated rats. Rats in the low-dose LPS group also showed significantly better learning and memory and less brain damage in adulthood. Learning and memory performance among the LPS-HI, LPS, and NS groups was not significantly different. We conclude that low-dose LPS preconditioning in neonatal rats greatly reduces HI-induced neuroinflammation and provides long-term neuroprotection against behavioral and pathologic abnormalities.

Enterovirus 71 (EV71) and coxsackievirus (CVA) are the most common causative factors for hand, foot, and mouth disease (HFMD) and neurological disorders in children. Lack of a reliable animal model is an issue in investigating EV71-induced disease manifestation in humans, and the current clinical therapies are symptomatic. We generated a novel EV71-infectious model with hSCARB2-transgenic mice expressing the discovered receptor human SCARB2 (hSCARB2). The challenge of hSCARB2-transgenic mice with clinical isolates of EV71 and CVA16 resulted in HFMD-like and neurological syndromes caused by E59 (B4) and N2838 (B5) strains, and lethal paralysis caused by 5746 (C2), N3340 (C4), and CVA16. EV71 viral loads were evident in the tissues and CNS accompanied the upregulated pro-inflammatory mediators (CXCL10, CCL3, TNF-α, and IL-6), correlating to recruitment of the infiltrated T lymphocytes that result in severe diseases. Transgenic mice pre-immunized with live E59 or the FI-E59 vaccine was able to resist the subsequent lethal challenge with EV71. These results indicate that hSCARB2-transgenic mice are a useful model for assessing anti-EV71 medications and for studying the pathogenesis induced by EV71.