MHC Class I Phenotype and Function of Human β2-Microglobulin Transgenic Murine Lymphocytes

Lymphoid cells from β₂-microglobulin (β₂m) knockout mice transgenic for human (h) β₂m (C57BL/10 mβ₂m^?/hβ₂m⁺) were compared with normal mice for their binding to exogenously added hβ₂m, binding to a H-2D^b peptide and for functional activity in a one-way allogenic MLC. Based on data from cellular binding studies, Scatchard analyses and flow cytometry, it is concluded that exogenous hβ₂m does not bind to hybrid MHC class I (MHC-I) molecules composed of mouse heavy chain/hβ₂m molecules expressed on lymphocytes of transgenic mice. Immunoprecipitation and SDS-PAGE analysis of metabolically labelled normal C57BL/6 lymph node cells showed binding of exogenous hβ₂m to MHC-I, in particular, to the H-2D^b molecule through an exchange with endogenous mouse β₂m. In contrast to normal H-2D^b molecules, hybrid H-2D^b expressed on the surface of transgenic lymphocytes binds radiolabelled peptide in the absence of exogenous added hβ₂m suggesting that a stable fraction of hybrid H-2D^b molecules is empty or contain peptides with very low affinity. In a one-way allogenic mixed lymphocyte culture, transgenic splenocytes were found to be far less stimulatory than normal splenocytes. In contrast, transgenic alloreactive cytotoxic T lymphocytes developed earlier in MLC than their non-transgenic counterparts. These data indicate that the hybrid mouse heavy chain/hβ₂m complex alters the alloantigenic repertoire and influences important aspects of T-cell activation.     

Structural Implications of β‐Cardiac Myosin Heavy Chain Mutations in Human Disease

Over 500 disease‐causing point mutations have been found in the human β‐cardiac myosin heavy chain, many quite recently with modern sequencing techniques. This review shows that clusters of these mutations occur at critical points in the sequence and investigates whether the many studies on these mutants reveal information about the function of this protein. Anat Rec, 297:1670–1680, 2014. © 2014 Wiley Periodicals, Inc.     

Effect of Hypoxia on Endothelial Nitric Oxide Synthase,NO Production,Intracellular Survival Signaling (p‐ERK1/2 and p‐AKT) and Apoptosis in Human Term Trophoblast

Citation Park M‐H, Galan HL, Arroyo JA. Effect of hypoxia on endothelial nitric oxide synthase, NO production, intracellular survival signaling (p‐ERK1/2 and p‐AKT) and apoptosis in human term trophoblast. Am J Reprod Immunol 2011; 65: 407–414 Problem Hypoxia is commonly associated with complicated pregnancies such as intrauterine growth restriction. We evaluated the effects of hypoxia on phospho (p)‐eNOS, p‐ERK, p‐AKT and apoptosis in human trophoblast. Method of study Isolated trophoblast were cultured in 21% oxygen or 2% oxygen for 24, 48 and 72 hr. p‐eNOS, p‐ERK and p‐AKT protein were assessed by Western blot and apoptosis by TUNEL assay. NOx was determined in the culture media. Results Compared to controls, hypoxia‐exposed CT showed the following: (1) decreased eNOS at 48 and 72 hr, (2) increased p‐eNOS at 48 hr, (3) no differences in total NOx production, (4) increased p‐ERK at 24, 48 and 72 hr, (5) increased p‐AKT at 24 hr (P < 0.05) and (6) increased apoptosis at 48 hr. Conclusion Hypoxia increases activation of p‐ERK and induces apoptosis of cultured trophoblast. Hypoxia decreases overall total eNOS but increases p‐eNOS, which may allow for NO production to be maintained in trophoblast cells.     

2‐(Pro‐1‐ynyl)‐5‐(5,6‐dihydroxypenta‐1,3‐diynyl) Thiophene Induces Apoptosis Through Reactive Oxygen Species‐Mediated JNK Activation in Human Colon Cancer SW620 Cells

2‐(Pro‐1‐ynyl)?5‐(5,6‐dihydroxypenta‐1,3‐diynyl) thiophene (PYDDT) is a naturally occurring thiophene isolated from the roots of Echinops grijsii, a Chinese herbal medicine used to treat colon cancer, breast cancer, and lung cancer. There are many reports on the clinical use of Echinops grijsii alone or in combination with other herbs to treat malignant tumors. We previously reported that the expression and activity of phase II enzymes including GSTs and NQO1 could be induced through the activation of Keap1‐Nrf2 pathway by the treatment of PYDDT. In this study, we reported the anticancer effect and mechanism of PYDDT against human colon cancer SW620 cells. Our results demonstrate that treatment of SW620 cells with PYDDT leads to induction of mitochondrial‐mediated apoptosis, which is characterized by the cleavage of PARP, activation of caspase 9 and caspase 3, release of cytochrome c from mitochondria, loss of mitochondrial membrane potential, down‐regulation of Bcl‐2, and mitochondrial translocation of Bax. The PYDDT treatment caused the production of reactive oxygen species (ROS), and the activation of JNK but not p38 mitogen‐activated protein kinases and ERK1/2. Specific JNK inhibitor SP600125 prevented the PYDDT‐induced down‐regulation of Bcl‐2, mitochondrial translocation of Bax, activation of caspase 3, and apoptosis of SW620 cells. Moreover, PYDDT‐induced apoptosis as well as activation of JNK was abrogated by the pretreatment with antioxidant N‐acetylcysteine. Taken together, these findings suggest that PYDDT induces apoptosis in SW620 cells through a ROS/JNK‐mediated mitochondrial pathway. Anat Rec, 298:376–385, 2015. © 2014 Wiley Periodicals, Inc.     

The Interaction between Beta 2-Microglobulin (ßm) and Purified Class-I Major Histocompatibility (MHC) Antigen

The function of MHC class-I molecules is to sample peptides from the intracellular environment and present them to CD8⁺ cytotoxic T lymphocytes. To understand the molecular details of the assembly (and disassembly) of peptide-ß₂m-class-I complexes a biochemical peptidc-class-I binding assay has been generated recently and this paper reports on a similar assay for the interaction between ß₂m and class I. As a model system human ß₂m binding to mouse class I was used. The assay is strictly biochemical using purified reagents which interact in solution and complex formation is determined by size separation. It is specific and highly sensitive. The observed affinity of the interaction, K_D, is close to 0.4 nw. The rate of association at 37 C is very fasi (the k_a is around 5 × 10⁴/M/s) whereas the dissociation is slow (the k_d is around 8 × 10⁻⁶/s); the ratio of dissociation to association yields a calculated K_D close to the observed value. At 37° C almost all of the purified class I participates in binding of the exogenously offered ß₂m showing that a considerable exchange of the endogenous ß₂m occurs. Finally, it was demonstrated that exogenous ß₂m enhances binding to MHC class-I of short perfectly-matching peplides as well as longer peptides.     

Poly(2‐isopropyl‐2‐oxazoline) Bearing a Spiropyran Side‐Chain: Synthesis and Evaluation of Photo and Temperature‐Responsive Properties

Poly(2‐isopropyl‐2‐oxazoline)s with varied loading of the spiropyran side‐chain are synthesized, and their photochemical and thermosensitive properties are characterized in aqueous solution. The absorption spectrum of the sample aqueous solution (0.1 wt%) indicates that 7.8 mol% of the pendant groups are in the zwitterionic merocyanine form. The introduction of spiropyran groups causes a marked decrease in the cloud point of the aqueous solution even when the pendant groups contain the zwitterionic merocyanine form. The cloud point of the aqueous solution of the samples in the spiropyran form is slightly (less than 1 °C) lower than that bearing the merocyanine group. Moreover, the photoinduced phase transition is demonstrated at an optimum temperature for the spiropyran‐bearing poly(2‐isopropyl‐2‐oxazoline). This material can be made applicable to a light‐driven drug releasing system by introducing a hydrophobic group to form micelles and increasing the photoinduced change of the cloud point.     

On the Heterologous Interaction between β2-Microglobulin and the Heavy Chain of Rat Major Histocompatibility Complex Class 1 Antigens

The heterologous interaction between β₂-microglobulin (β₂m) and rat major histocompatibility complex (MHC) (RT1) antigens was measured in a two-step binding assay consisting of binding of radiolabelled β₂-m to RT1 antigens and immunoprecipitation of β₂m-RT1 antigen complexes with RT1 antisera. The effects of varying the concentrations of the three reactants involved were studied. The molecular events taking place in the two steps were analysed by gel chromatography. The β₂m-RT1 antigen complex had the apparent size of albumin and reacted completely with specific alloantisera. RT1 antigens prepared from Wistar/Furth (RT1^u) and Brown Norway (RT1ⁿ), respectively, both effectively bound heterologous β₂m. The times for association and dissociation, respectively, at 37°C, were of the same order, but dissociation was slightly slower. Association was markedly temperature-dependent and was considerably slower at low temperatures. All these processes were slower for RT1^u than for RT1^u antigens. The association constant for the interaction between RT1^u antigens and ¹²⁵I-human β₂m was estimated by Scatchard analysis to be about 10⁹ M^-1. Contribution to the heterologous interaction by products from various rat MHC subloci (A, B, and C) was investigated by the introduction of sublocus-specific antisera in step 2. The reaction apparently involved neither class 2 antigens (sublocus B) nor the presumed rat Qa homologue (sublocus C). Classical class 1 antigens (suhlocus A) clearly contributed to the binding. However, a monoclonal antibody against products from rat MHC class 1 genes only precipitated less than half of the RTI antigen-complexed β₂m. Thus, at least two RT1^u class 1 alloantigen molecules seem to participate in the reaction. This, in turn, indicates that the rat genome may contain multiple class 1 genes, an is the case for most other mammals investigated.     

Chain Dynamics of Poly(oxyethylene) in Nanotubes of α‐Cyclodextrin by Solid‐State 2H NMR

Summary: Solid‐state ²H NMR spectroscopy was used to examine the chain dynamics of perdeuterated poly(oxyethylene) (d‐POE) inside α‐cyclodextrin (α‐CD) nanotubes. The nanotubes were prepared by aqueous self‐assembly of α‐CD onto either low‐molecular‐weight (1.5 kg/mol) or high‐molecular‐weight (25.8 kg/mol) monodisperse d‐POE. At a given temperature, POE chain segments exhibit faster dynamics when included inside the CD nanotubes as compared to the bulk. Even at 150 K, when no large‐angle dynamics are detected in bulk POE, evidence for chain motions in the nanotube‐confined POE is observed. The ²H line shapes representing this motion were modeled by a discrete 3‐site jump using a ln‐Gaussian distribution of correlation times. An activation energy of 15 ± 1 kJ/mol was determined and the motion envisioned as gauche defects propagating along the primarily trans chains included within CD nanotubes. As the nanotube length decreased, the jump angle became much less defined and more isotropic motions were observed for POE segments at elevated temperatures (>270 K).

     

Synthesis of New Hydrogels by Copolymerization of Poly(2‐methyl‐2‐oxazoline) Bis(macromonomers) and N‐Vinylpyrrolidone

New non‐ionic hydrogels were synthesized by radical homopolymerization of vinyl end‐functionalized poly(2‐methyl‐2‐oxazoline) bis(macromonomers), or by radical copolymerization of these bis(macromonomers) with N‐vinyl‐2‐pyrrolidone (NVP). The poly(2‐methyl‐2‐oxazoline) bis(macromonomers) were synthesized through “living” cationic ring‐opening polymerization of 2‐methyl‐2‐oxazoline (MeOXA), using, simultaneously, the known “initiating” and “end‐capping” method for synthesis of macromonomers. Chloromethyl styrene was used as initiator and N‐(4‐vinylbenzyl)‐piperazine was used as the terminating agent. Well defined poly(2‐methyl‐2‐oxazoline) bis(macromonomers) were obtained with P_n = 4, 11, and 17. The hydrogel structures were characterized by high‐resolution magic angle spinning NMR technique and their solvent absorption capacity was tested by swelling experiments in different solvents. The bis(macromonomers) were characterized by NMR spectroscopy and gel permeation chromatography.

Schematic of polymerization     

Amorphous‐Smectic Glassy Main‐Chain LCPs, 1. Poly(ether esters) Derived from Hydroxybibenzoic Acid and (R,S)‐ and (R)‐2‐Methylpropane‐1,3‐diol

The synthesis and the characterization of main‐chain liquid‐crystalline poly(ether esters), derived from hydroxybibenzoic acid and (R,S)‐ and (R)‐2‐methylpropane‐1,3‐diol, are reported. These polymers show an interesting thermal behavior. They develop mesophases with a slow rate of formation, allowing the easy quenching of the melt into: a) the glassy amorphous state, b) the glassy liquid‐crystalline state, or c) a mixture of both, depending on the thermal treatment. The extent of the transformation and the symmetry of the different phases have been determined by means of calorimetric and X‐ray diffraction methods. Dielectric spectroscopy results provide additional evidence for the detection of distinct glass transitions. The results show that the racemic polymer forms a low‐ordered SmC_alt mesophase, while a more ordered phase is obtained in the case of the enantiomerically pure polymer. The comparison of the properties of the different states evidences the special behavior and properties of the glass transition (T_g) in these polymers. Emphasis is paid to the location of the T_g of the liquid‐crystalline state in comparison to the T_g of the amorphous state. It is found that the glass transition of the SmC_alt glass in R,S‐PBO3 (the poly(ether ester) derived from hydroxybibenzoic acid and (R,S)‐2‐methylpropane‐1,3‐diol) appears at lower temperatures than the glass transition of the amorphous state. However, in R‐PBO3 (the poly(ether ester) derived from hydroxybibenzoic acid and (R)‐2‐methylpropane‐1,3‐diol), where the more ordered phase is present, the glass transition follows the classical tendency of semicrystalline polymers.

     

Studies on the Synthesis and Conductivity of a Novel Reactive Ladder‐Like Poly(β‐cyanoethylsilsesquioxane) and Poly[(β‐cyanoethylsilsesquioxane)‐co‐(β‐methylsilsesquioxane)]

Two novel reactive poly(β‐cyanoethylsilsesquioxane) ( CN‐T ) and poly[(β‐cyanoethylsilsesquioxane)‐co‐(β‐methylsilsesquioxane)] ( CN‐Me‐T ) have been synthesized successfully for the first time via stepwise coupling polymerization (SCP). A variety of characterization methods including FTIR, ¹H NMR, ²⁹Si NMR, X‐ray diffraction (XRD), differential scanning calorimetry (DSC) and vapor pressure osmometry (VPO) were combined to demonstrate that the structures of the title polymers possess ordered ladder‐like structures. As expected, the ionic conductivity of these polymers mixed homogeneously with lithium perchlorate reached 10^?6 S · cm^?1 at room temperature and obviously increased with the raise of temperature.

     

Adenovirus‐mediated Interferon‐γ Gene Therapy Induced Human Pancreatic Carcinoma Capan‐2 Cell Apoptosis In Vitro and In Vivo

Pancreatic cancer is one of the most lethal human malignancies with a very low 5‐year survival rate, which highlights urgent needs for more effective therapeutic strategies. In this study, we examined the potential therapeutic effects of an adenovirus encoding human interferon gamma (Ad‐IFNγ) on pancreatic carcinoma cells Capan‐2 in vitro and in vivo. The results indicated that Ad‐IFNγ could significantly inhibit tumor cell growth via inducing cell apoptosis. After infection, IFNγ expressed durably and stably in xenografts, predominantly in tumor tissue, while much less in blood and liver. Thus, adenovirus‐mediated intratumoral injection of human IFNγ gene could be an effective gene therapeutic system for the treatment of pancreatic carcinoma. Anat Rec, 296:604–610, 2013. © 2013 Wiley Periodicals, Inc.     

Thermal,Mechanical, and Surface Properties of Poly(2‐N‐alkyl‐2‐oxazoline)s

Thermal, mechanical, and surface properties of a library of poly(2‐oxazoline)s are investigated. These polymers are suitable to study structure/property relationships as their cationic ROP and the relative facile monomer synthesis allow for control over the molecular structure. The number of carbon atoms in the linear side‐chain is systematically varied from methyl to nonyl. Relations between chemical structures, thermal transitions, surface energies, and elastic moduli are discussed. It is shown that the mechanical and thermal properties of the polymers depend on the presence of a crystalline phase in the material. The amorphous polymers reveal a decrease in the reduced moduli along with a decrease in their respective glass transition temperature with increasing length of the side‐chain.

     

Tailoring Optical Properties of π‐Conjugated Statistical Co‐Oligomers Composed of 3‐Pentylthiophene and 3‐[(E)‐2‐(1‐Naphthyl)­vinyl]thiophene through the Monomer Ratio in the Main Chain

Synthesis and characterization of π‐conjugated homo‐ and co‐oligomers based on thiophene substituted in position 3 by pentyl (3PT) and naphthylvinyl (NVT) side groups are presented. The resultant co‐oligomers have statistical structure and the proportion of monomers can be fully controlled by the composition of the reaction mixture. The approach here offers a facile way to synthesis of materials with tunable optoelectrical properties that is an alternative for complicated organometallic synthesis involving water and oxygen sensitive Sn, Pd, or Mg compounds. The UV–vis absorption and fluorescence emission spectra depend on and can be easily tuned by the [3PT]:[NVT] monomer molar ratio. Combination of these two monomers has a strong synergistic effect on the color of the fluorescence emission that varies in a broad range, from orange to blue color. X‐ray diffraction, calorimetric, and materials display electrical properties characteristic for organic semiconductors.

     

Lipopolysaccharide (LPS)‐induced Interferon (IFN)‐gamma Production by Decidual Mononuclear Cells (DMNC) is Interleukin (IL)‐2 and IL‐12 Dependent

Citation Negishi M, Izumi Y, Aleemuzzaman S, Inaba N, Hayakawa S. Lipopolysaccharide (LPS)‐induced interferon (IFN)‐gamma production by decidual mononuclear cells (DMNC) is interleukin (IL)‐2 and IL‐12 dependent. Am J Reprod Immunol 2011; 65: 20–27 Problem Th1‐shifted immune response is believed to be harmful for successful pregnancy because of activation of maternal cytotoxic T lymphocytes and natural killer cells. However, its effects on Toll‐like receptor (TLR)‐mediated innate immune response are so far unknown and this study has been undertaken to address the issue. Method of study Decidual tissues were obtained from 16 pregnant women undergoing elective termination during the first trimester pregnancy for socioeconomic reasons. Decidual Mononuclear Cells (DMNC) were stimulated with suboptimal doses of IL‐2 and IL‐12 with/without LPS, considered to be a TLR4 ligand, for 48 hr. Productions of IFN‐γ and tumor necrosis factor (TNF)‐α in culture supernatant were measured with ELISA. Results (i) IFN‐γ production was induced with LPS alone which was strongly up‐regulated in the presence of IL‐2 and IL‐12. (ii) TNF‐α was also induced by LPS but was not affected by the presence of IL‐2 and IL‐12. Conclusion IL‐2 and IL‐12 up‐regulated the production of IFN‐γ in DMNC through increasing their susceptibility to LPS. TNF‐α production is independent of such a mechanism.     

Poly(ε‐caprolactone) and Poly(ω‐pentadecalactone)‐Based Networks with Two‐Way Shape‐Memory Effect through [2+2] Cycloaddition Reactions

In this work, the synthesis and characterization of triple‐shape and two‐way shape‐memory effect of novel poly(ester‐urethane)s (PURs) made of a poly(ε‐caprolactone) (PCL) and poly(ω‐pentadecalactone) (PPDL) segments and N,N‐bis(2‐hydroxyethyl)cinnamide (BHECA) monomer by reactive extrusion (REx) is reported. PCL and PPDL are chosen as semicrystalline segments because of their inherent ability to undergo tensile elongation upon cooling, as prerequisite for the two‐way shape‐memory effect. BHECA is used as the “cross‐linker” due to its ability to participate in reversible [2+2] cycloaddition reaction and to mainly maintain the crystalline features of semicrystalline precursors within these PURs. This novel simple strategy is considered extremely versatile and adaptive because of the possibility to vary crystallizable segments and coupling agents, thus paving the way to the design of a multitude of triple (or more) shape‐memory polymers with two‐way behavior. Feasibility with PURs containing PCL, PPDL, and BHECA is demonstrated by adjusting the shape‐memory behavior (one and two‐way effects), and studying the structure–property relationships of the resulting PURs by DSC, DMTA, and 2D wide angle X‐ray scattering analyses while varying the weight composition of the two semicrystalline segments.     

Synthesis and properties of fluorinated polyimides, 2. Derived from novel 2,6‐bis(3′‐trifluoromethyl‐p‐aminobiphenyl ether)pyridine and 2,5‐bis(3′‐trifluoromethyl‐p‐aminobiphenyl ether)thiophene

Novel diamine monomers, 2,6‐bis(3′‐trifluoromethyl‐p‐aminobiphenyl ether)pyridine (TABP) and 2,5‐bis(3′‐trifluoromethyl‐p‐aminobiphenyl ether)thiophene (TABT) have been synthesized, which lead to a number of novel fluorinated polyimides by thermal imidization routes when reacted with different commercially available dianhydrides like pyromellitic dianhydride (PMDA), benzophenone tetracarboxylic acid dianhydride (BTDA) or 2,2‐bis(3,4‐dicarboxyphenyl) hexafluoropropane (6FDA). The polyimides ABTP/6FDA and ABTT/6FDA are soluble in several organic solvents such as N,N‐dimethylformamide (DMF), N,N‐dimethylacetamide (DMAc) and tetrahydrofuran (THF). The polyimide ABTP/PMDA is only soluble in N‐methylpyrollidone (NMP) whereas ABTT/PMDA is insoluble in all solvents. The polyimides ABTP/6FDA (2c) and ABTT/6FDA (3c) exhibited number‐average molecular weight 32 940 (polydispersity index (PDI), 1.97) and 24 760 (PDI, 1.87) respectively. These polyimide films have low water absorption rate 0.3–0.9% and low dielectric constant 2.92–3.29 at 1 MHz. These polyimides showed very high thermal stability even up to 532 °C for 5% weight loss in synthetic air and glass transition temperature up to 287 °C (by DSC) in nitrogen. All polyimides formed tough transparent films, with tensile strength up to 115 MPa, a modulus of elasticity up to 2.91 GPa and elongation at break up to 12% depending upon the exact repeating unit structure.

TGA thermograms of the thiophene ring poly(ether imide)s.     

Physically and Chemically Cross‐Linked Poly{[(maleic anhydride)‐alt‐styrene]‐co‐(2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid)}/Poly(ethylene glycol) Proton‐Exchange Membranes

Novel proton exchange membranes were solvent‐cast from DMF solutions of the terpolymers poly[(MA‐alt‐S)‐co‐AMPS], containing hydrophobic phenyl and reactive hydrophilic carboxylic and organo‐sulfonic acid fragments with different compositions, and PEGs with different molecular weights and amounts. These membranes were formed as a result of physical (via H‐bonding) and chemical (via PEG) cross‐linking. The structures of membranes were confirmed by FT‐IR and ¹H‐ and ¹³C NMR spectroscopy. Mechanical and thermal properties, swellability, and proton conductivity of these membranes were significantly affected both by the chemical composition of the terpolymers (mainly the AMPS content) and also the cross‐linker (PEG) molecular weight and content in the final form of the membranes. It was concluded that the membranes prepared by using the terpolymer with an AMPS content of 36.84 mol‐% and PEG with a molecular weight of 1 450 and with an initial PEG content of 30 wt.‐% are the most suitable ones for fuel cell applications.

     

Homo and Block Copolymers of Poly(β‐benzyl‐L‐aspartate)s and Poly(γ‐benzyl‐L‐glutamate)s of Different Architectures

Homopolypeptides of linear and star‐like architectures were prepared by polymerizing benzylic‐protected L ‐glutamic acid and L ‐aspartic acid N‐carboxyanhydrides (Glu NCA, Asp NCA) in DMF. The polymerization rate of the Glu NCA is faster than that of Asp NCA. Using a simple monoamino initiator, its hydrochloride, di‐, tri‐, and tetraamino functional initiators, homopolypeptides with well‐defined structures and molar masses were obtained. The molar‐mass averages of the poly(γ‐benzyl‐L ‐glutamate)s lie very close to calculated values, according to the initial [M]:[I] ratios, while those of the linear poly(β‐benzyl‐L ‐aspartate)s were lower than the predicted ones. PBAs had somewhat broader molar‐mass distributions than PBGs.