Cloning and characterization of a mitochondrial glyoxalase II from Brassica juncea that is upregulated by NaCl, Zn, and ABA

A cDNA (1061 bp) Bj glyII was cloned from a mannitol induced library of Brassica juncea. It encoded a protein of 335 amino acids with a molecular weight of 36.52 kDa. The deduced amino acid sequence of the clone showed 92% and 56% identity with Pennisetum and rice glyoxalase II, respectively, and 30% identity was observed with the human glyoxalase II. Search for the identical residues revealed the presence of highly conserved THHHXDH domain which is involved in zinc binding. p-NN and pSORT analysis of this sequence revealed a N-terminal mitochondrial target peptide. The cDNA was cloned in pMAL and a fusion protein with MBP (78 kDa) was expressed in Escherichia coli. The recombinant protein was purified approximately sixfold by affinity purification on amylose column and showed its pH optima at 7.0. The K(m) was determined to be 120 microM using S-d-lactoylglutathione as substrate. The expression of Bj glyII under various abiotic stress conditions showed that it is upregulated by salinity, heavy metal stress, and ABA.     

Inhibition of chlorophyll biosynthesis by mercury in excised etiolated maize leaf segments during greening: effect of 2-oxoglutarate

Mercury (0.01-1.0 mM) inhibited chlorophyll formation in greening maize leaf segments. However, supplementing incubation medium with 2-oxoglutarate, maintained substantially higher level of chlorophyll in absence of metal after an initial period of 8 hr. On preincubation of leaf segments with HgCl2, per cent inhibition of chlorophyll synthesis by metal was same in the presence and absence of 2-oxoglutarate. Supply of 2-oxoglutarate (0.1-10.0 mM) exerted concentration dependent effect on chlorophyll formation in absence or presence of metal. Increase in delta-amino levulinic acid dehydratase as well as NADH-glutamate synthase activity and decrease in NADH-glutamate dehydrogenase activity by 2-oxoglutarate in the presence of Hg suggested that glutamate for delta-amino levulinic acid synthesis could be made available from NH4+ assimilation via., glutamine synthetase/glutamate synthase pathway during mercury toxicity.     

Amelioration of radiation-induced fibrosis: inhibition of transforming growth factor-beta signaling by halofuginone

Radiation-induced fibrosis is an untoward effect of high dose therapeutic and inadvertent exposure to ionizing radiation. Transforming growth factor-beta (TGF-beta) has been proposed to be critical in tissue repair mechanisms resulting from radiation injury. Previously, we showed that interruption of TGF-beta signaling by deletion of Smad3 results in resistance to radiation-induced injury. In the current study, a small molecular weight molecule, halofuginone (100 nm), is demonstrated by reporter assays to inhibit the TGF-beta signaling pathway, by Northern blotting to elevate inhibitory Smad7 expression within 15 min, and by Western blotting to inhibit formation of phospho-Smad2 and phospho-Smad3 and to decrease cytosolic and membrane TGF-beta type II receptor (TbetaRII). Attenuation of TbetaRII levels was noted as early as 1 h and down-regulation persisted for 24 h. Halofuginone blocked TGF-beta-induced delocalization of tight junction ZO-1, a marker of epidermal mesenchymal transition, in NMuMg mammary epithelial cells and suggest halofuginone may have in vivo anti-fibrogenesis characteristics. After documenting the in vitro cellular effects, halofuginone (intraperitoneum injection of 1, 2.5, or 5 microg/mouse/day) efficacy was assessed using ionizing radiation-induced (single dose, 35 or 45 Gy) hind leg contraction in C3H/Hen mice. Halofuginone treatment alone exerted no toxicity but significantly lessened radiation-induced fibrosis. The effectiveness of radiation treatment (2 gray/day for 5 days) of squamous cell carcinoma (SCC) tumors grown in C3H/Hen was not affected by halofuginone. The results detail the molecular effects of halofuginone on the TGF-beta signal pathway and show that halofuginone may lessen radiation-induced fibrosis in humans.     

Eukaryotic initiation factor 2-associated glycoprotein, p67, shows differential effects on the activity of certain kinases during serum-starved conditions

Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 is the major regulatory step in the initiation of protein synthesis in mammals. P67, a cellular glycoprotein, protects phosphorylation of eIF2alpha from kinases. P67 has five conserved amino acid residues at the D251, D262, H331, E364, and E459 positions. To determine the roles of these conserved amino acid residues in eIF2alpha phosphorylation during serum-starved conditions, we constitutively expressed D251A, D262A, H331A, E364A, and E459A mutants in rat tumor hepatoma cells. We find that the point mutants D251A, H331A, and E364A lower the levels of eIF2alpha phosphorylation. These low levels of phosphorylation decrease when serum-starved cells are grown in medium containing serum. To understand the mechanism of action of the p67 mutants in eIF2alpha phosphorylation during serum-starvation, we performed detailed biochemical analyses with the D251A mutant. We find that neither the O-GlcNAc modification on the D251A mutant nor the binding of D251A mutant with eIF2gamma has significant effects on eIF2alpha phosphorylation during serum-starved conditions. However, the D251A mutant inhibits p67's activity to suppress the activity of ERK1/2. Our data suggest that both p67 and the D251A mutant bind to ERK1, thus strengthening the idea that p67 regulates the activity of ERK1. During serum-starvation conditions, both PKR and PERK are phosphorylated and the D251A mutant shows increased stability of PERK as well as a slight decrease in its activity. Altogether, our data provide evidence to suggest that p67 modulates the expression and activity of certain eIF2alpha-specific kinases.     

Determinants of antileukemia effects of allogeneic NK cells

In HLA-nonidentical bone marrow transplantation, we studied the characteristics of donor NK cells, recipient leukemia cells, and the cytokine environment that predict the antileukemia effects of allogeneic NK cells. We found that the risk of relapse in pediatric patients with hematologic malignancies was best predicted by a model taking into consideration the presence of inhibitory killer cell Ig-like receptors (KIRs) on the donor's NK cells and the absence of corresponding KIR ligand in the recipient's HLA repertoire (a receptor-ligand model). The risk of relapse was prognosticated less precisely by the Perugia donor-recipient KIR ligand-ligand mismatch model or by a natural cytotoxicity model. In contrast to the ligand-ligand model, we found that the new receptor-ligand model was accurate when analysis was applied to patients with lymphoid malignancy. These findings corroborate our observations that the recipient's KIR repertoire, which was derived from highly purified, HLA-disparate CD34+ cells, resumed a donor-specific pattern within 3 mo of transplantation, but did not correlate evidently with the donor or recipient ligand repertoire. In an in vitro assay and an in vivo mouse model, human NK cell cytotoxicity toward human leukemia cells with 11q23 chromosomal rearrangement increased with the number of receptor-ligand mismatch pairs or prestimulation with IL-12 and IL-18. These findings provide new insights into the determinants of antileukemia effects of allogeneic NK cells and therapeutic strategies.     

Monomeric recombinant TCR ligand reduces relapse rate and severity of experimental autoimmune encephalomyelitis in SJL/J mice through cytokine switch

Our previous studies demonstrated that oligomeric recombinant TCR ligands (RTL) can treat clinical signs of experimental autoimmune encephalomyelitis (EAE) and induce long-term T cell tolerance against encephalitogenic peptides. In the current study, we produced a monomeric I-A(s)/PLP 139-151 peptide construct (RTL401) suitable for use in SJL/J mice that develop relapsing disease after injection of PLP 139-151 peptide in CFA. RTL401 given i.v. or s.c. but not empty RTL400 or free PLP 139-151 peptide prevented relapses and significantly reduced clinical severity of EAE induced by PLP 139-151 peptide in SJL/J or (C57BL/6 x SJL)F(1) mice, but did not inhibit EAE induced by PLP 178-191 or MBP 84-104 peptides in SJL/J mice, or MOG 35-55 peptide in (C57BL/6 x SJL/J)F(1) mice. RTL treatment of EAE caused stable or enhanced T cell proliferation and secretion of IL-10 in the periphery, but reduced secretion of inflammatory cytokines and chemokines. In CNS, there was a modest reduction of inflammatory cells, reduced expression of very late activation Ag-4, lymphocyte function-associated Ag-1, and inflammatory cytokines, chemokines, and chemokine receptors, but enhanced expression of Th2-related factors, IL-10, TGF-beta3, and CCR3. These results suggest that monomeric RTL therapy induces a cytokine switch that curbs the encephalitogenic potential of PLP 139-151-specific T cells without fully preventing their entry into CNS, wherein they reduce the severity of inflammation. This mechanism differs from that observed using oligomeric RTL therapy in other EAE models. These results strongly support the clinical application of this novel class of peptide/MHC class II constructs in patients with multiple sclerosis who have focused T cell responses to known encephalitogenic myelin peptides.     

Molecular docking of potential inhibitors with the mTOR protein

The mTOR (mammalian or mechanistic Target of Rapamycin) is linked with oral cancer. Therefore, it is of interest to study the molecular docking-based binding of paclitaxel (a FDA approved drug for oral cancer) and its analogues with mTOR. Hence, we report the binding features of 10-Deacetyltaxol, 7-Epi-10-deacetyltaxol, 7-Epi-Taxol and 6alpha-Hydroxypaclitaxel with mTOR for further consideration.     

Comparative genomics of emerging human ehrlichiosis agents

Anaplasma (formerly Ehrlichia) phagocytophilum, Ehrlichia chaffeensis, and Neorickettsia (formerly Ehrlichia) sennetsu are intracellular vector-borne pathogens that cause human ehrlichiosis, an emerging infectious disease. We present the complete genome sequences of these organisms along with comparisons to other organisms in the Rickettsiales order. Ehrlichia spp. and Anaplasma spp. display a unique large expansion of immunodominant outer membrane proteins facilitating antigenic variation. All Rickettsiales have a diminished ability to synthesize amino acids compared to their closest free-living relatives. Unlike members of the Rickettsiaceae family, these pathogenic Anaplasmataceae are capable of making all major vitamins, cofactors, and nucleotides, which could confer a beneficial role in the invertebrate vector or the vertebrate host. Further analysis identified proteins potentially involved in vacuole confinement of the Anaplasmataceae, a life cycle involving a hematophagous vector, vertebrate pathogenesis, human pathogenesis, and lack of transovarial transmission. These discoveries provide significant insights into the biology of these obligate intracellular pathogens.     

Probing the Role of CXC Motif in Chemokine CXCL8 for High Affinity Binding and Activation of CXCR1 and CXCR2 Receptors

All chemokines share a common structural scaffold that mediate a remarkable variety of functions from immune surveillance to organogenesis. Chemokines are classified as CXC or CC on the basis of conserved cysteines, and the two subclasses bind distinct sets of GPCR class of receptors and also have markedly different quaternary structures, suggesting that the CXC/CC motif plays a prominent role in both structure and function. For both classes, receptor activation involves interactions between chemokine N-loop and receptor N-domain residues (Site-I), and between chemokine N-terminal and receptor extracellular/transmembrane residues (Site-II). We engineered a CC variant (labeled as CC-CXCL8) of the chemokine CXCL8 by deleting residue X (CXC → CC), and found its structure is essentially similar to WT. In stark contrast, CC-CXCL8 bound poorly to its cognate receptors CXCR1 and CXCR2 (K_i > 1 μm). Further, CC-CXCL8 failed to mobilize Ca²⁺ in CXCR2-expressing HL-60 cells or recruit neutrophils in a mouse lung model. However, most interestingly, CC-CXCL8 mobilizes Ca²⁺ in neutrophils and in CXCR1-expressing HL-60 cells. Compared with the WT, CC-CXCL8 binds CXCR1 N-domain with only ∼5-fold lower affinity indicating that the weak binding to intact CXCR1 must be due to its weak binding at Site-II. Nevertheless, this level of binding is sufficient for receptor activation indicating that affinity and activity are separable functions. We propose that the CXC motif functions as a conformational switch that couples Site-I and Site-II interactions for both receptors, and that this coupling is critical for high affinity binding but differentially regulates activation.     

Importance of macrophage cholesterol content on the flux of cholesterol mass

Net flux of cholesterol represents the difference between efflux and influx and can result in net cell-cholesterol accumulation, net cell-cholesterol depletion, or no change in cellular cholesterol content. We measured radiolabeled cell-cholesterol efflux and cell-cholesterol mass using cholesterol-normal and -enriched J774 and elicited mouse peritoneal macrophage cells. Net cell-cholesterol effluxes were observed when cholesterol-enriched J774 cells were incubated with 3.5% apolipoprotein (apo) B depleted human serum, HDL₃, and apo A-I. Net cell-cholesterol influxes were observed when cholesterol-normal J774 cells were incubated with the same acceptors except apo A-I. When incubated with 2.5% individual sera, cholesterol mass efflux in free cholesterol (FC)-enriched J774 cells correlated with the HDL-cholesterol (HDL-C) concentrations (r² = 0.4; P=0.003), whereas cholesterol mass influx in cholesterol-normal J774 cells correlated with the LDL cholesterol (LDL-C) concentrations (r² = 0.6; P<0.0001) of the individual sera. A positive correlation was observed between measurements of [³H]cholesterol efflux and reductions in cholesterol mass (r² = 0.4; P=0.001) in FC-enriched J774 cells. In conclusion, isotopic efflux measurements from cholesterol-normal or cholesterol-enriched cells provide an accurate measurement of relative ability of an acceptor to remove labeled cholesterol under a specific set of experimental conditions, i.e., efflux potential. Moreover, isotopic efflux measurements can reflect changes in cellular cholesterol mass if the donor cells are enriched with cholesterol.