Rich Box Research Articles

IgG4-related disease is characterised by the overexpression of immunomodulatory proteins.

Immunoglobulin 4-related disease (IgG4-RD) is a multisystem disease, characterised by tumefactive lesions and a swift response to immunosuppressive therapy. Although elevated serum and tissue IgG4 are characteristic, T cells appear to be the primary driver of this immunologically mediated disease. The overarching goal was to examine the role of immunomodulatory cells in IgG4-RD. Biopsies from patients with IgG4-RD (n = 39) and mimics of this disease (n = 78) were evaluated for IgG4, IgG, CD8, programmed cell death ligand 1 (PD-L1) and a subset (n = 18) evaluated for CD4, purine rich box 1 (PU.1), forkhead box protein 3 (FoxP3), PD-L1, programmed cell death 1 (PD-1), indoleamine 2,3-dioxygenase 1 (IDO1) and lymphocyte-activation gene 3 (LAG3). Data pertaining to demographics and laboratory findings at baseline evaluation was extracted from electronic medical records. When compared to mimics, IgG4-RD showed increased numbers of PD-L1- (P = 0.0001), PD-1- (P = 0.001), IDO1- (P = 0.03), LAG3- (P = 0.04) and FoxP3- (P = 0.04)-positive immune cells. The PD-L1-positive cells were enriched within aggregates of CD4 and CD8-positive T cells. Thirty-one of 39 (80%) IgG4-RD cases showed greater than five PD-L1-positive cells per high-power field (HPF), while four of 78 (5%) mimics of this disease exceeded this cut-point. In IgG4-RD, PD-L1-positive macrophages correlated with PD-1- (P = 0.002), LAG3- (P = 0.001) and IDO1-positive cells (P = 0.001); a-positive correlation was also noted between IgG4/IgG ratio and PD-L1-, PD-1- and IDO1-positive cells. IgG4-RD shows expansion of mechanisms that maintain peripheral tolerance. The spatial and temporal relationship between T cells and the PD-L1-PD-1 axis and the up-regulation of multiple immunomodulatory proteins suggests that these immunoregulatory mechanisms play a significant role in IgG4-RD.

PU.1 Mimic Synthetic Peptides Selectively Bind with GATA-1 and Allow c-Jun PU.1 Binding to Enhance Myelopoiesis.

BackgroundHematopoietic stem cells' commitment to myelopoiesis builds immunity to prevent infection. This process is controlled through transcription factor, especially Purine rich box 1 (PU.1) protein, which plays a central role in regulating myelopoiesis. The β3/β4 region of PU.1 accommodates a coactivator transcription factor, c-Jun, to activate myelopoiesis. However, an erythroid transcription factor, GATA-1, competes with c-Jun for the β3/β4 region, abolishing myelopoiesis and promoting erythropoiesis. This competitive regulation decides the hematopoietic stem cells’ commitment towards either erythroid or myeloid lineage.MethodsTherefore, this study investigated the in vitro and in vivo effect of novel synthetic PU.1 β3/β4 mimic peptide analogs and peptide-loaded hydrophilic poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles.ResultsThe designed peptides significantly increase the expression of corresponding myeloid markers, specifically CD33 in vitro. However, the in vivo delivery of peptide-loaded PLGA nanoparticles, which have sustained release effect of peptides, increases 10.8% of granulocytes as compared to control.ConclusionThe observations showed that the fabricated nanoparticles protected the loaded peptides from the harsh intracellular environment for a longer duration without causing any toxicity. These findings highlight the possibility to use these peptides and peptide-loaded nanoparticles to increase hematopoietic stem cell commitment to myeloid cells in case of opportunistic infection.

Short communication. Single nucleotide polymorphisms in the ovine CSN1S2 gene for alphaS2–casein

This work describes eighteen SNPs (9 transitions and 9 transversions) found in the sequence spanning from exon 7 to 11 of the CSN1S2 ovine gene, thirteen located at the introns 7 (5 SNPs), 8 (2 SNPs), 9 (1 SNP), and 10 (5 SNPs), and five at the exons 10 (4 SNPs) and 11 (1 SNP). The allele frequencies of the SNPs have been obtained from seventeen animals of Merino breed. Among intronic polymorphisms, the two adjacent SNPs g.[435C>A; 436T>G] are located at the pyrimidine rich box near the 3' splice acceptor site of intron 10, which could potentially affect mRNA processing. Analysis of the deduced amino acid sequences from exons 10 and 11 showed that polymorphisms ex10-g.153G>T and ex10-g. 155C>T, which affect the same codon, and ex11-g. 504A>G, are non-synonymous substitutions producing the amino acid changes Asp75 to Tyr75 and Ile105 to Val105, respectively, giving rise to the A and B alleles previously found by direct sequencing of the mature protein. For these SNPs only six of the eight possible combinations were detected, GCA being the most frequent haplotype (0.323), corresponding to Asp75 – Ile105 in the mature protein. The new polymorphisms of the ovine CSN1S2 found in this paper are potential molecular markers that could be used to investigate its association with production traits.

Cellular nucleic acid binding protein binds G‐rich single‐stranded nucleic acids and may function as a nucleic acid chaperone

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression.

Components involved in RNA–protein interaction at the 3′ untranslated region of rbcL mRNA of Populus deltoides

RNA–protein interactions occurring at the 3′ untranslated region (3′ UTR) of the mRNA of rbcL gene from Populus deltoides have been examined. The 3′ UTR of rbcL gene in P. deltoides is highly AT rich and contains invert repeat (IR) sequences capable of forming two stem-loops. Gel mobility shift assays followed by SDS–PAGE analysis revealed that the 3′ IR-RNA region interacts with a set of proteins having molecular weights between 66 and 26 kDa. The protein complex protected an approximately 82-nucleotide segment of 3′ UTR of rbcL mRNA when treated with RNase T1. An internal competition assay using variable lengths of the rbcL 3′ UTR indicated that the RNaseT1 protected segment included both stem-loops and a few sequences downstream including a conserved AU rich box.

Collagen alpha1(I) gene contains an element responsive to tumor necrosis factor-alpha located in the 5' untranslated region of its first exon.

The aims of the present study were to identify the cis-acting element through which tumor necrosis factor-alpha (TNFalpha) inhibits collagen alpha1(I) gene transcription and the trans-acting factors involved in this effect in cultured hepatic stellate cells. Deletion analysis of the collagen alpha1(I) promoter demonstrated that TNFalpha inhibited gene expression through an element located between -59 and + 116 bp relative to the transcription start site. DNase I protection assays revealed a footprint between +68 and +86 bp of the collagen first exon, the intensity of which decreased when the DNA probe was incubated with nuclear protein from TNFalpha-treated hepatic stellate cells. This footprint contained a G+C-rich box. Transfection experiments demonstrated that mutations in this G+C-rich element abrogated the inhibitory effect of TNFalpha on the collagen alpha1(I) promoter. Gel retardation experiments using a radiolabeled oligonucleotide containing sequences of this region confirmed that TNFalpha treatment decreased the formation of two complexes between nuclear proteins and DNA. These complexes were efficiently blocked with an oligonucleotide containing an Spl-binding site and were supershifted with specific Spl and Sp3 antibodies. These results suggest that TNFalpha inhibits collagen alpha1(I) gene expression by decreasing the binding of Spl to a G+C-rich box in the 5' untranslated region of its first exon.

Growth Hormone Promotion of Tubulin Polymerization Stabilizes the Microtubule Network and Protects Against Colchicine-Induced Apoptosis

We have investigated the effect of GH on microtubular physiology in Chinese hamster ovary (CHO) cells stably transfected with the complementary DNA for the rat GH receptor (CHO-GHR1–638). We show here that after 30 min of human GH (hGH) treatment of CHO-GHR1–638 cells, there was a significant increase in the level of polymerization of all four tubulin isoforms (α-, β-,γ -, and tyrosinated α-tubulin) compared with the serum-deprived state. However, this transient increase in the levels of polymerized tubulin after hGH treatment was particularly pronounced for β- and tyr α-tubulin. For α- and γ-tubulin, the hGH-induced increase in polymerization state lasted to approximately 3 h and then declined by 7 h, whereas for β- and tyr α-tubulin there was a decrease in the polymerization state at 1–2 h after hGH treatment compared with the level at 30 min (but still greater than the serum-deprived state) followed by a second but lesser wave of increased polymerization lasting to 7 h. The changes in the polymerization state of the tubulins were not accompanied by comparative changes in the level of total cellular tubulin. The proline rich box 1 region of the GH receptor was required for hGH to stimulate tubulin polymerization indicative that this event is JAK dependent. Increased tubulin polymerization still occurred in response to hGH in a receptor truncation lacking the carboxyl terminal half of the intracellular domain of the GH receptor indicative that hGH induced changes in intracellular calcium concentration is not required for tubulin polymerization. Prior treatment of CHO-GHR1–638 cells with hGH retarded colchicine induced microtubule depolymerization and also prevented colchicine induced apoptotic cell death. The integrity of the microtubule network was not required for GH-induced STAT5 mediated transcription as treatment of cells with colchicine, vincristine, or vinblastine did not alter the fold stimulation of the STAT5 mediated transcriptional response to GH. Thus one consequence of cellular treatment with GH is alteration in microtubule physiology.

Growth hormone promotion of tubulin polymerization stabilizes the microtubule network and protects against colchicine-induced apoptosis.

We have investigated the effect of GH on microtubular physiology in Chinese hamster ovary (CHO) cells stably transfected with the complementary DNA for the rat GH receptor (CHO-GHR(1-638)). We show here that after 30 min of human GH (hGH) treatment of CHO-GHR(1-638) cells, there was a significant increase in the level of polymerization of all four tubulin isoforms (alpha-, beta-, gamma-, and tyrosinated alpha-tubulin) compared with the serum-deprived state. However, this transient increase in the levels of polymerized tubulin after hGH treatment was particularly pronounced for beta- and tyr alpha-tubulin. For alpha- and gamma-tubulin, the hGH-induced increase in polymerization state lasted to approximately 3 h and then declined by 7 h, whereas for beta- and tyr alpha-tubulin there was a decrease in the polymerization state at 1-2 h after hGH treatment compared with the level at 30 min (but still greater than the serum-deprived state) followed by a second but lesser wave of increased polymerization lasting to 7 h. The changes in the polymerization state of the tubulins were not accompanied by comparative changes in the level of total cellular tubulin. The proline rich box 1 region of the GH receptor was required for hGH to stimulate tubulin polymerization indicative that this event is JAK dependent. Increased tubulin polymerization still occurred in response to hGH in a receptor truncation lacking the carboxyl terminal half of the intracellular domain of the GH receptor indicative that hGH induced changes in intracellular calcium concentration is not required for tubulin polymerization. Prior treatment of CHO-GHR(1-638) cells with hGH retarded colchicine induced microtubule depolymerization and also prevented colchicine induced apoptotic cell death. The integrity of the microtubule network was not required for GH-induced STAT5 mediated transcription as treatment of cells with colchicine, vincristine, or vinblastine did not alter the fold stimulation of the STAT5 mediated transcriptional response to GH. Thus one consequence of cellular treatment with GH is alteration in microtubule physiology.

TGF-β-signaling with small molecule FKBP12 antagonists that bind myristoylated FKBP12-TGF-β type I receptor fusion proteins

Background: Growth arrest in many cell types is triggered by transforming growth factor beta (TGF-β), which signals through two TGF-β receptors (type I, TGF-βRI, and type II, TGF-βRII). In the signaling pathway, TGF-β binds to the extracellular domain of TGF-βRII, which can then transphosphorylate TGF-βRI in its glycine/serine (GS)-rich box. Activated TGF-βRI phosphorylates two downstream effectors, Smad2 and Smad3, leading to their translocation into the nucleus. Cell growth is arrested and plasminogen activator inhibitor 1 (PAI-1) is upregulated. We investigated the role of the immunophilin FKBP12, which can bind to the GS box of TGF-βRI, in TGF-β signaling. Results: Overexpression of myristoylated TGF-βRI and TGF-βRII cytoplasmic tails caused constitutive nuclear translocation of a green-fluorescent-protein-Smad2 construct in COS-1 cells, and constitutive activation of a PAI-1 reporter plasmid in mink lung cells. Fusing FKBP12 to TGF-βRI resulted in repression of autosignaling that could be alleviated by FK506M or rapamycin (two small molecules that can bind to FKBP12). Mutation of the FKBP12-binding site in the FKBP12-TGF-βRI fusion protein restored constitutive signaling. An acidic mutation in the FKBP12-TGFβRI protein allowed FKBP12 antagonists to activate signaling in the absence of TGF-βRII. Further mutations in the TGF-βRI FKBP12-binding site resulted in TGF-β signaling that was independent of both TGF-βRII and FKBP12 antagonists. Conclusions: Fusing FKBP12 to TGF-βRI results in a novel receptor that is activated by small molecule FKBP12 antagonists. These results suggest that FKBP12 binding to TGF-βRI is inhibitory and that FKBP12 plays a role in inhibiting TGF-β superfamily signals.

Characterisation of the rat tissue-type plasminogen activator gene promoter -- identification of a TAAT-containing promoter element.

Tissue-type plasminogen activator (tPA) activates plasminogen to the active protease plasmin and is implicated in many biological processes that require extracellular proteolysis. In rat ovarian cells, gonadotropins induce the tPA gene by a cAMP-dependent pathway and this induction correlates with the time of follicular rupture. We have previously identified several promoter elements within the first 621 bp of the rat tPA promoter that are important for constitutive and cAMP-induced expression of the gene, including a cAMP responsive element (CRE), a nuclear factor 1 (NF1) element, a SP1-binding site and a G+C-rich box. In this report we have extended our study by analysing promoter constructs, ranging in size from 7.7 kb to 135 bp fused to the luciferase reporter gene. Transient transfection analysis of rat granulosa cells and human 293 cells, reveal that the proximal 268 bp of the promoter is enough to confer high basal and cAMP-induced expression of the gene. At position -162 to -172, between the previously identified CRE and NF1 sites, a novel TAAT-containing promoter element was identified. Mutational inactivation of the TAAT motif indicates that this element is important for both constitutive and cAMP-induced expression of the gene, and for the binding of a presumably novel nuclear factor that we have termed tPA promoter factor-1 (tPF-1).