Carbohydrate binding specificity of the B-cell maturation mitogen from Artocarpus integrifolia seeds

Artocarpin, a mannose-specific lectin, is a homotetrameric protein (M(r) 65,000) devoid of covalently attached carbohydrates and consists of four isolectins with pI in the range 5-6.5. Investigations of its carbohydrate binding specificity reveal that among monosaccharides, mannose is preferred over glucose. Among mannooligosaccharides, mannotriose (Man alpha 1-3[Man alpha 1-6]Man) and mannopentaose are the strongest ligands followed by Man alpha 1-3Man. Extension of these ligands by GlcNAc at the reducing ends of mannooligosaccharides tested remarkably improves their inhibitory potencies, while substitution of both the alpha 1-3 and alpha 1-6 mannosyl residues of mannotriose and the core pentasaccharide of N-linked glycans (Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc) by GlcNAc or N-acetyllactosamine in beta 1-2 linkage diminishes their inhibitory potencies. Sialylated oligosaccharides are non-inhibitory. Moreover, the substitution of either alpha 1-3 or alpha 1-6 linked mannosyl residues of M5Gn or both by mannose in alpha 1-2 linkage leads to a considerable reduction of their inhibitory power. Addition of a xylose residue in beta 1-2 linkage to the core pentasaccharide improves the inhibitory activity. Considering the fact that artocarpin has the strongest affinity for the xylose containing hepasaccharide from horseradish peroxidase, which differs significantly from all the mannose/glucose-specific lectins, it should prove a useful tool for the isolation and characterization of glycoproteins displaying such structure.

Cooperative Regulation of NOTCH1 Protein-Phosphatidylinositol 3-Kinase (PI3K) Signaling by NOD1, NOD2, and TLR2 Receptors Renders Enhanced Refractoriness to Transforming Growth Factor-beta (TGF-beta)- or Cytotoxic T-lymphocyte Antigen 4 (CTLA-4)-mediated Impairment of Human Dendritic Cell Maturation

Dendritic cells (DCs) as sentinels of the immune system are important for eliciting both primary and secondary immune responses to a plethora of microbial pathogens. Cooperative stimulation of a complex set of pattern-recognition receptors, including TLR2 and nucleotide-binding oligomerization domain (NOD)-like receptors on DCs, acts as a rate-limiting factor in determining the initiation and mounting of the robust immune response. It underscores the need for ``decoding'' these multiple receptor interactions. In this study, we demonstrate that TLR2 and NOD receptors cooperatively regulate functional maturation of human DCs. Intriguingly, synergistic stimulation of TLR2 and NOD receptors renders enhanced refractoriness to TGF-beta- or CTLA-4-mediated impairment of human DC maturation. Signaling perturbation data suggest that NOTCH1-PI3K signaling dynamics assume critical importance in TLR2- and NOD receptor-mediated surmounting of CTLA-4- and TGF-beta -suppressed maturation of human DCs. Interestingly, the NOTCH1-PI3K signaling axis holds the capacity to regulate DC functions by virtue of PKC delta-MAPK-dependent activation of NF-kappa B. This study provides mechanistic and functional insights into TLR2-and NOD receptor-mediated regulation of DC functions and unravels NOTCH1-PI3K as a signaling cohort for TLR2 and NOD receptors. These findings serve in building a conceptual foundation for the design of improved strategies for adjuvants and immunotherapies against infectious diseases.

Bactericidal/permeability increasing protein: A multifaceted protein with functions beyond LPS neutralization

Bactericidal permeability increasing protein (BPI), a 55-60kDa protein, first reported in 1975, has gone a long way as a protein with multifunctional roles. Its classical role in neutralizing endotoxin (LPS) raised high hopes among septic shock patients. Today, BPI is not just a LPS-neutralizing protein, but a protein with diverse functions. These functions can be as varied as inhibition of endothelial cell growth and inhibition of dendritic cell maturation, or as an anti-angiogenic, chemoattractant or opsonization agent. Though the literature available is extremely limited, it is fascinating to look into how BPI is gaining major importance as a signalling molecule. In this review, we briefly summarize the recent research focused on the multiple roles of BPI and its use as a therapeutic.

CINCINNATA in Antirrhinum majus directly modulates genes involved in cytokinin and auxin signaling

Mutations in the CINCINNATA (CIN) gene in Antirrhinum majus and its orthologs in Arabidopsis result in crinkly leaves as a result of excess growth towards the leaf margin. CIN homologs code for TCP (TEOSINTE-BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTOR 1 AND 2) transcription factors and are expressed in a broad zone in a growing leaf distal to the proliferation zone where they accelerate cell maturation. Although a few TCP targets are known, the functional basis of CIN-mediated leaf morphogenesis remains unclear. We compared the global transcription profiles of wild-type and the cin mutant of A. majus to identify the targets of CIN. We cloned and studied the direct targets using RNA in situ hybridization, DNA-protein interaction, chromatin immunoprecipitation and reporter gene analysis. Many of the genes involved in the auxin and cytokinin signaling pathways showed altered expression in the cin mutant. Further, we showed that CIN binds to genomic regions and directly promotes the transcription of a cytokinin receptor homolog HISTIDINE KINASE 4 (AmHK4) and an IAA3/SHY2 (INDOLE-3-ACETIC ACID INDUCIBLE 3/SHORT HYPOCOTYL 2) homolog in A. majus. Our results suggest that CIN limits excess cell proliferation and maintains the flatness of the leaf surface by directly modulating the hormone pathways involved in patterning cell proliferation and differentiation during leaf growth. 10.1111/(ISSN)1469-8137

PE_PGRS Antigens of Mycobacterium tuberculosis Induce Maturation and Activation of Human Dendritic Cells

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world's population. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). DCs are sentinels of the immune system and are important for eliciting both primary and secondary immune responses to pathogens. In this context, to understand the molecular pathogenesis of tuberculosis and host response to mycobacteria and to conceive prospective vaccine candidates, it is important to understand how cell wall Ags of M.tuberculosis and, in particular, the proline-glutamic acid-polymorphicguanine-cytosine-rich sequence (PE_PGRS) family of proteins modulate DC maturation and function. In this study, we demonstrate that two cell wall-associated/secretory PE_PGRS proteins, PE_PGRS 17 (Rv0978c) and PE_PGRS 11 (Rv0754), recognize TLR2, induce maturation and activation of human DCs, and enhance the ability of DCs to stimulate CD4(+) T cells. We further found that PE_PGRS protein-mediated activation of DCs involves participation of ERK1/2, p38 MAPK, and NF-kappa B signaling pathways. Priming of human DCs with IFN-gamma further augmented PE_PGRS 17 or PE_PGRS 11 Ag-induced DC maturation and secretion of key proinflammatory cytokines. Our results suggest that by activating DCs, PE_PGRS proteins, important mycobacterial cell wall Ags, could potentially contribute in the initiation of innate immune responses during tuberculosis infection and hence regulate the clinical course of tuberculosis. The Journal of Immunology, 2010, 184: 3495-3504.

PE_PGRS Antigens of Mycobacterium tuberculosis Induce Maturation and Activation of Human Dendritic Cells

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world's population. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). DCs are sentinels of the immune system and are important for eliciting both primary and secondary immune responses to pathogens. In this context, to understand the molecular pathogenesis of tuberculosismand host response to mycobacteria and to conceive prospective vaccine candidates, it is important to understand how cell wall Ags of M. tuberculosis and, in particular, the proline-glutamic acid-polymorphic guanine-cytosine-rich sequence (PE_PGRS) family of proteins modulate DC maturation and function. In this study, we demonstrate that two cell wall-associated/secretory PE_PGRS proteins, PE_PGRS 17 (Rv0978c) and PE_PGRS 11 (Rv0754), recognize TLR2, induce maturation and activation of human DCs, and enhance the ability of DCs to stimulate CD4(+) T cells. We further found that PE_PGRS protein-mediated activation of DCs involves participation of ERK1/2, p38 MAPK, and NF-kappa B signaling pathways. Priming of human DCs with IFN-gamma further augmented PE_PGRS 17 or PE_PGRS 11 Ag-induced DC maturation and secretion of key proinflammatory cytokines. Our results suggest that by activating DCs, PE_PGRS proteins, important mycobacterial cell wall Ags, could potentially contribute in the initiation of innate immune responses during tuberculosis infection and hence regulate the clinical course of tuberculosis. The Journal of Immunology, 2010, 184: 3495-3504.

Maturation of a transient outward potassium current in mouse fetal hypothalamic neurons in culture

The whole-cell voltage clamp technique was used to record potassium currents in mouse fetal hypothalamic neurons developing in culture medium from days 1 to 17. The neurons were derived from fetuses of IOPS/OF1 mice on the 14th day of gestation. The mature neurons (>six days in culture) showed both a transient potassium current and a non-inactivating delayed rectifier potassium current. These were identified pharmacologically by using the potassium channel blockers tetraethyl ammonium chloride and 4-aminopyridine, and on the basis of their kinetics and voltage sensitivities. The delayed rectifier potassium current had a threshold of −20 mV, a slow time-course of activation, and was sustained during the voltage pulse. The 4-aminopyridine-sensitive current was transient, and was activated from a holding potential more negative (−80 mV) than that required for evoking the delayed rectifier potassium current (−40 mV). The delayed rectifier potassium current was detectable from day 1 onwards, while the transient potassium current showed a distinct developmental trend. The time-constant of inactivation became faster with age in culture. The half steady-state inactivation potential showed a shift towards less negative membrane potentials with age, and the relationship was best described by a logarithmic regression equation.The developmental trend of the transient potassium current may relate functionally to the progressive morphological changes, and the appearance of synaptic connections during ontogenesis.

Src Homology 3-interacting Domain of Rv1917c of Mycobacterium tuberculosis Induces Selective Maturation of Human Dendritic Cells by Regulating PI3K-MAPK-NF-kappa B Signaling and Drives Th2 Immune Responses

Mycobacterium tuberculosis, an etiological agent of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Pathogenic mycobacteria survive in the host by subverting host innate immunity. Dendritic cells (DCs) are professional antigen-presenting cells that are vital for eliciting immune responses to infectious agents, including pathogenic mycobacteria. DCs orchestrate distinct Th responses based on the signals they receive. In this perspective, deciphering the interactions of the proline-glutamic acid/proline-proline-glutamic acid (PE/PPE) family of proteins of M. tuberculosis with DCs assumes significant pathophysiological attributes. In this study, we demonstrate that Rv1917c (PPE34), a representative member of the proline-proline-glutamic-major polymorphic tandem repeat family, interacts with TLR2 and triggers functional maturation of human DCs. Signaling perturbations implicated a critical role for integrated cross-talk among PI3K-MAPK and NF-kappa B signaling cascades in Rv1917c-induced maturation of DCs. However, this maturation of DCs was associated with a secretion of high amounts of anti-inflammatory cytokine IL-10, whereas Th1-polarizing cytokine IL-12 was not induced. Consistent with these results, Rv1917c-matured DCs favored secretion of IL-4, IL-5, and IL-10 from CD4(+) T cells and contributed to Th2-skewed cytokine balance ex vivo in healthy individuals and in patients with pulmonary tuberculosis. Interestingly, the Rv1917c-skewed Th2 immune response involved induced expression of cyclooxygenase-2 (COX-2) in DCs. Taken together, these results indicate that Rv1917c facilitates a shift in the ensuing immunity toward the Th2 phenotype and could aid in immune evasion by mycobacteria.

Hyper-activation of the TCP4 transcription factor in Arabidopsis thaliana accelerates multiple aspects of plant maturation

Plant organs are initiated as primordial outgrowths, and require controlled cell division and differentiation to achieve their final size and shape. Superimposed on this is another developmental program that orchestrates the switch from vegetative to reproductive to senescence stages in the life cycle. These require sequential function of heterochronic regulators. Little is known regarding the coordination between organ and organismal growth in plants. The TCP gene family encodes transcription factors that control diverse developmental traits, and a subgroup of class II TCP genes regulate leaf morphogenesis. Absence of these genes results in large, crinkly leaves due to excess division, mainly at margins. It has been suggested that these class II TCPs modulate the spatio-temporal control of differentiation in a growing leaf, rather than regulating cell proliferation per se. However, the link between class II TCP action and cell growth has not been established. As loss-of-function mutants of individual TCP genes in Arabidopsis are not very informative due to gene redundancy, we generated a transgenic line that expressed a hyper-activated form of TCP4 in its endogenous expression domain. This resulted in premature onset of maturation and decreased cell proliferation, leading to much smaller leaves, with cup-shaped lamina in extreme cases. Further, the transgenic line initiated leaves faster than wild-type and underwent precocious reproductive maturation due to a shortened adult vegetative phase. Early senescence and severe fertility defects were also observed. Thus, hyper-activation of TCP4 revealed its role in determining the timing of crucial developmental events, both at the organ and organism level.

Cyclic Cationic Peptides Containing Sugar Amino Acids Selectively Distinguishes and Inhibits Maturation of Pre-miRNAs of the Same Family

The discovery of microRNAs (miRNAs) has added a new dimension to the gene regulatory networks, making aberrantly expressed miRNAs as therapeutically important targets. Small molecules that can selectively target and modulate miRNA levels can thus serve as lead structures. Cationic cyclic peptides containing sugar amino acids represent a new class of small molecules that can target miRNA selectively. Upon treatment of these small molecules in breast cancer cell line, we profiled 96 therapeutically important miRNAs associated with cancer and observed that these peptides can selectively target paralogous miRNAs of the same seed family. This selective inhibition is of prime significance in cases when miRNAs of the same family have tissue-specific expression and perform different functions. During these conditions, targeting an entire miRNA family could lead to undesired adverse effects. The selective targeting is attributable to the difference in the three-dimensional structures of precursor miRNAs. Hence, the core structure of these peptides can be used as a scaffold for designing more potent inhibitors of miRNA maturation and hence function.

Mycobacterium tuberculosis WhiB3 Responds to Vacuolar pH-induced Changes in Mycothiol Redox Potential to Modulate Phagosomal Maturation and Virulence.

The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (E-MSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in E-MSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic E-MSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, Mtb Delta whiB3 displayed intramacrophage survival defect, which can be rescued by pharmacological inhibition of phagosomal acidification. Last, Mtb Delta whiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis.

Heme biosynthesis by the malarial parasite - Import of delta-aminolevulinate dehydrase from the host red cell

The mouse and human malarial parasites, Plasmodium berghei and Plasmodium falciparum, respectively, synthesize heme de novo following the standard pathway observed in animals despite the availability of large amounts of heme, derived from red cell hemoglobin, which is stored as hemozoin pigment, The enzymes, delta-aminolevulinate dehydrase (ALAD), coproporphyrinogen oxidase, and ferrochelatase are present at strikingly high levels in the P, berghei infected mouse red cell in vivo, The isolated parasite has low levels of ALAD and the data clearly indicate it to be of red cell origin. The purified enzyme preparations from the uninfected red cell and the parasite are identical in kinetic properties, subunit molecular weight, cross-reaction with antibodies to the human enzyme, and N-terminal amino acid sequence. Immunogold electron microscopy of the infected culture indicates that the enzyme is present inside the parasite and, therefore, is not a contaminant, The parasite derives functional ALAD from the host and the enzyme binds specifically to isolated parasite membrane in vitro, suggestive of the involvement of a receptor in its translocation into the parasite, While, ALAD, coproporphyrinogen oxidase, and ferrochelatase from the parasite and the uninfected red cell supernatant have identical subunit molecular weights on SDS-polyacrylamide gel electrophoresis and show immunological cross-reaction with antibodies to the human enzymes, as revealed by Western analysis, the first enzyme of the pathway, namely, delta-aminolevulinate synthase (ALAS) in the parasite, unlike that of the red cell host, does not cross-react with antibodies to the human enzyme, However, ALAS enzyme activity in the parasite is higher than that of the infected red cell supernatant. We therefore conclude that the parasite, while making its own ALAS, imports ALAD and perhaps most of the other enzymes of the pathway from the host to synthesize heme de novo, and this would enable it to segregate this heme from the heme derived from red cell hemoglobin degradation, ALAS of the parasite and the receptor(s) involved in the translocation of the host enzymes into the parasite would be unique drug targets.

Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order +35 > +1 > -3 > -8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60-72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in 'glowing silkworms'. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.

Cell Dynamics of Model Proteins

We design rapidly folding sequences by assigning the strongest couplings to the contacts present in a target native state in a two dimensional model of heteropolymers. The pathways to folding and their dependence on the temperature are illustrated via a mapping of the dynamics into motion within the space of the maximally compact cells.

Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [P-32] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by Western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [P-32] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC alpha and epsilon, the major isotypes of PKC in BPAECs, by TPA (100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [P-32] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.