Tyrosine-Specific Chemical Modification with <i>in Situ</i> Hemin-Activated Luminol Derivatives

Tyrosine-specific chemical modification was achieved using <i>in situ</i> hemin-activated luminol derivatives. Tyrosine residues in peptide and protein were modified effectively with N-methylated luminol derivatives under oxidative conditions in the presence of hemin and H₂O₂. Both single and double modifications of the tyrosine residue occurred in the reaction of angiotensin II with N-methylated luminol derivative <b>9</b>. Tyrosine-specific chemical modification of the model protein bovine serum albumin (BSA) revealed that the surface-exposed tyrosine residues were selectively modified with <b>9</b>. We succeeded in the functionalization of several proteins using azide-conjugated compound <b>18</b> using alkyne-conjugated probes by copper­(I)-catalyzed azide–alkyne cycloaddition (CuAAC) or dibenzocyclooctyne (DBCO)-mediated copper-free click chemistry. This tyrosine-specific modification was orthogonal to conventional lysine modification by <i>N</i>-hydroxysuccinimide (NHS) ester, and dual functionalization by fluorescence modification of tyrosine residues and PEG modification of lysine residues was achieved without affecting the modification efficiency

Chemically Programmed Antibodies As HIV‑1 Attachment Inhibitors

Herein, we describe the design and application of two small-molecule anti-HIV compounds for the creation of chemically programmed antibodies. <i>N</i>-Acyl-β-lactam derivatives of two previously described molecules BMS-378806 and BMS-488043 that inhibit the interaction between HIV-1 gp120 and T-cells were synthesized and used to program the binding activity of aldolase antibody 38C2. Discovery of a successful linkage site to BMS-488043 allowed for the synthesis of chemically programmed antibodies with affinity for HIV-1 gp120 and potent HIV-1 neutralization activity. Derivation of a successful conjugation strategy for this family of HIV-1 entry inhibitors enables its application in chemically programmed antibodies and vaccines and may facilitate the development of novel bispecific antibodies and topical microbicides

Bortezomib treatment enhances T cell apoptosis by inhibiting NF-κB activation.

<p>(<b>A</b>) The percentages of Annexin-V/7-AAD⁺ CD4⁺ and CD8⁺ T cells in mesenteric lymph nodes 7 days after the administration of DSS. Bar graphs indicate mean (± SEM) percentages of mesenteric lymph node Annexin-V/7-AAD⁺ CD4⁺ and CD8⁺ T cells following bortezomib or control treatment in one representative experiment with 4 mice per group. (<b>B</b>) IκBα mRNA expression in mesenteric lymph nodes CD4⁺ and CD8⁺ T cells. Transcript levels were quantified by real-time PCR analysis and were normalized with an internal control. Values represent means (± SEM) from ≥4 mice of each group. A, B) Significant differences between sample means are indicated; *<i>P</i><0.05; **<i>P</i><0.01. Similar results were obtained in at least two independent experiments.</p

Bortezomib treatment reduces IFN-γ production by CD4⁺ and CD8⁺ T cells in mesenteric lymph nodes during DSS-induced colitis.

<p>IFN-γ production by mesenteric lymph node CD4⁺ (<b>A</b>) or CD8⁺ (<b>B</b>) T cells 7 days following DSS administration as determined by intracellular cytokine staining with flow cytometry analysis. Bar graphs indicate mean (± SEM) percentages and numbers of draining lymph node IFN-γ-producing CD4⁺ or CD8⁺ T cells following bortezomib or control treatment in one representative experiment with three mice per group. Significant differences between PBS-treated mice versus other group are indicated; *<i>P</i><0.05, **<i>P</i><0.01. Similar results were obtained in at least two independent experiments.</p

Bortezomib treatment affects cytokine production in DSS-induced colits.

<p>Cytokine mRNA expression in the colon (<b>A</b>) and mesenteric lymph nodes (<b>B</b>) in mice treated with bortezomib or control during DSS-induced colitis. Colon tissue and mesenteric lymph nodes were collected from control- or bortezomib-treated mice 7 days following DSS administration. Transcript levels were quantified by real-time PCR analysis and were normalized with an internal control. Values represent means (± SEM) from ≥4 mice of each group. Significant differences between sample means are indicated; *<i>P</i><0.05, **<i>P</i><0.01. Results represent one of two independent experiments producing similar results.</p

Bortezomib suppressed the severity of DSS-induced colitis.

<p>Mice ingested either DSS solution or normal drinking water. Mice were treated with 200 µl of bortezomib (0.75 mg/kg) or PBS (control) intravenously twice weekly, starting 2 days prior to DSS administration. The severity of intestinal injury was evaluated by quantitatively measuring body weight (<b>A</b>) and DAI scores (<b>B</b>). DAI scores were based on weight loss, stool consistency, and bleeding. Values represent means (±SEM) from ≥4 mice per group. Significant differences between sample means are indicated: *<i>P</i><0.05; **<i>P</i><0.01. Similar results were obtained in at least two independent experiments.</p

Profile of infiltrating cells in the colon and mesenteric lymph nodes during DSS-induced colitis in mice treated with bortezomib or control.

<p>(<b>A</b>) The numbers of neutrophils, CD4⁺ T cells, CD8⁺ T cells, B220⁺ B cells, and F4/80⁺ macrophages per one field of view (×200) in the colon were counted. (<b>B</b>) Bortezomib affects CD4⁺ and CD8⁺ T cell numbers in mesenteric lymph nodes during DSS-induced colitis. Bar graphs indicate CD4⁺ T cells, CD8⁺ T cells, and B220⁺ cells in mesenteric lymph nodes 7 days after induction of colitis. A, B) Values represent means (± SEM) from ≥4 mice per group. Significant differences between sample means are indicated: *<i>P</i><0.05. Similar results were obtained in at least two independent experiments.</p

Deregulated JNK signaling enhances apoptosis during hyperthermia

c-Jun N-terminal kinases (JNKs) comprise a subfamily of mitogen-activated protein kinases (MAPKs). The JNK group is known to be activated by a variety of stimuli. However, the molecular mechanism underlying heat-induced JNK activation is largely unknown. The aim of this study was to clarify how JNK activity is stimulated by heat. The expression levels of various MAPK members in HeLa cells, with or without hyperthermia treatment, were evaluated via western blotting. The kinase activity of MAPK members was assessed through in vitro kinase assays. Cell death was assessed in the absence or presence of siRNAs targeting MAPK-related members. Hyperthermia decreased the levels of MAP3Ks, such as ASK1 and MLK3 which are JNK kinase kinase members, but not those of the downstream MAP2K/SEK1 and MAPK/JNK. Despite the reduced or transient phosphorylation of ASK1, MLK3, or SEK1, downstream JNK was phosphorylated in a temperature-dependent manner. In vitro kinase assays demonstrated that heat did not directly stimulate SEK1 or JNK. However, the expression levels of DUSP16, a JNK phosphatase, were decreased upon hyperthermia treatment. DUSP16 knockdown enhanced the heat-induced activation of ASK1–SEK1–JNK pathway and apoptosis. JNK was activated in a temperature-dependent manner despite reduced or transient phosphorylation of the upstream MAP3K and MAP2K. Hyperthermia-induced degradation of DUSP16 may induce activation of the ASK1–SEK1–JNK pathway and subsequent apoptosis.</p

Upregulation of hepatic nuclear receptors in extremely preterm ovine fetuses undergoing artificial placenta therapy

Extremely preterm infants have low Nuclear Receptor (NR) expression in their developing hepatobiliary systems, as they rely on the placenta and maternal liver for compensation. NRs play a crucial role in detoxification and the elimination of both endogenous and xenobiotic substances by regulating key genes encoding specific proteins. In this study, we utilized an Artificial Placenta Therapy (APT) platform to examine the liver tissue expression of NRs of extremely preterm ovine fetuses. This fetal model, resembling a “knockout placenta,” lacks placental and maternal support, while maintaining a healthy extrauterine survival. Six ovine fetuses at 95 ± 1 d gestational age (GA; term = ∼150 d)/∼600 g delivery weight were maintained on an APT platform for a period of 120 h (APT Group). Six age-matched, in utero control fetuses were delivered at 99–100 d GA (Control Group). Fetal liver tissue samples and blood samples were collected at delivery from both groups and assessed mRNA expression of NRs and target transporters involved in the hepatobiliary transport system using quantitative PCR. Data were tested for group differences with ANOVA (p  mRNA expression of NRs was identified in both the placenta and the extremely preterm ovine fetal liver. The expression of HNF4α, LRH1, LXR, ESR1, PXR, CAR, and PPARα/γ were significantly elevated in the liver of the APT Group compared to the Control Group. Moreover, target transporters NTCP, OATP1B3, BSEP, and MRP4 were upregulated, whereas MRP2 and MRP3 were unchanged. Although there was no evidence of liver necrosis or apoptotic changes histologically, there was an impact in the fetal liver of the ATP group at the tissue level with a significant increase in TNFα mRNA, a cytokine involved in liver inflammation, and blood elevation of transaminases. A number of NRs in the fetal liver were significantly upregulated after loss of placental-maternal support. However, the expression of target transporter genes appeared to be insufficient to compensate role of the placenta and maternal liver and avoid fetal liver damage, potentially due to insufficient excretion of organic anions.</p

Additional file 2: Figure S2. of Fli1-haploinsufficient dermal fibroblasts promote skin-localized transdifferentiation of Th2-like regulatory T cells

Signal intensity of IL-33. Signal intensity of IL-33 was analyzed and summarized. To quantify signal intensity of IL-33, color images were converted to grayscale, and then the brightness was measured in five different randomly selected fibroblasts and epidermal areas per specimen. WT Wild-type mice; Basal Under physiological condition; BLM Bleomycin-treated. (PDF 57 kb