Focus: Allergic Diseases and Type II Immunity: Allergies and Adaptations: A Perspective on the Need for Culturally Responsive Care to Medically Indicated Dietary Restrictions

In medicine, we tend to think of food as being equivalent to nutrition, and food allergies are understood primarily as a biomedical process. In this piece, I explore how my experience with food allergies intersects with my cultural identity as a second-generation Indian-American. I also offer insights from my experiences in medical training and practice and reflect on the responsibility of health providers to understand the social and cultural context of food allergies.     

Cytokinin and explant types influence in vitro plant regeneration of Leopard Orchid (<Emphasis Type="Italic">Ansellia africana</Emphasis> Lindl.)

The effects of explant and cytokinin types on in vitro plant regeneration of Ansellia africana were investigated. The exogenous addition of cytokinins is not required for the proliferation of new protocorms from Trimmed protocorm cluster (TPC) explants. To the contrary, nodal and shoot-tip explants produced a single shoot in response to the addition of cytokinins. Overall plant growth in terms of shoot length, leaf number, frequency of root organogenesis, root length, and fresh weight/plant were significantly higher in media containing meta-Topolin Riboside (mTR) in both nodal and shoot-tip explants. Thidiazuron (TDZ) and 6-benzyladenine (BA) induced stunted and hypertrophied shoots at their highest level (15 μM). In addition root differentiation and root growth were significantly lower on P668 media with TDZ and BA. Zeatin was capable of inducing a significantly higher root organogenesis frequency and root length in TPC explants as compared to other cytokinins. However, TPC explants produced a significantly greater number of longer shoots (>3 cm) on P668 media with mTR. Hyperhydric shoots were produced from TPC explants. The occurrence of hyperhydricity is discussed with respect to the culture vessel used in this study.     

Synthesis of 2-substituted 17β-hydroxy/17-methylene estratrienes and their in vitro cytotoxicity in human cancer cell cultures

Synthesis of various types of 2-(alkylaminomethyl) and 2-(aroyl) 17β-estradiol analogs are reported. The synthesis of similar types of 2-substituted 17-methylene estratriene analogs was also achieved. Synthesis of chalcone derivatives of 17β-estradiol and 17-methylene estratriene were also realized. All these 2-substituted estratrienes were tested for their antiproliferative activity by using four different cell lines from colon, lung, glioma and breast cancers. Among the various 2-substituted estratrienes, the compounds 10d, 14a-h and 17e were found to have in vitro antiproliferative activity comparable to that of parent analogs 1-4. Comparison of the SAR pattern of these 2-susbtituted estratriene derivatives confirmed that relatively, 17-methylene estratrienes are more active than that of 17β-estradiol analogs.     

Effect of lecithin with vitamin-B complex and tocopheryl acetate on long-term effect of ethanol induced immunomodulatory activities

The alcoholic liver disease usually causes overall immunological alterations which might be attributed to hepatic disease, to ethanol action, and/or to malnourishment. In the present study, efficacy of lecithin with vitamin-B complex to treat ethanol induced immunomodulatory activity was compared with the effect of lecithin alone and tocopheryl acetate (vitamin E). Ethanol (1.6 g/kg body wt/day for 12 weeks) exposure increased thiobarbituric acid reactive substance (TBARS) level, while decreased superoxide dismutase (SOD) activity and reduced glutathione (GSH) content in whole blood hemolysate of 8-10 week-old male BALB/c mice (weighing 20-30 g). The activities of transaminase (AST and ALT) enzymes, interleukin (IL)-10 and gamma interferon (IFN-gamma) elevated, while IL-2 and IL-4 reduced in mice serum due to ethanol exposure. These suggested that oxidative stress and immunomodulatory activities were interdependent and associated with ethanol induced liver damage. Lecithin treatment significantly reduced AST (32.44%), ALT (32.09%), IL-10 (25.63%) activities and TBARS content (12.76%) compared to ethanol treated group. However, lecithin with vitamin-B complex treatment, significantly reduced AST (62.83%); ALT (61.96%); IL-10 (35.88%); IFN-gamma (22.55%) activities and TBARS content (31.58%), while significantly elevated GSH content (36.49%) and SOD activity (61.21%). Tocopheryl acetate treatment significantly reduced AST (62.83%); ALT (61.54%); IL-10 (36.35%): IFN-gamma (23.28%) activities and TBARS content (35.84%). while significantly elevated GSH content (28.76%) and SOD activity (62.42%) compared to ethanol treated group. These findings persuasively argued that lecithin with vitamin-B complex was a new promising therapeutic approach in controlling ethanol induced immunomodulatory activities involving liver damage processes. Prevention of oxidative stress with correction of nutritional deficiency caused alteration in the ethanol-induced immunomodulatory activities and associated liver diseases.     

A minimal element in 5'UTR of insulin mRNA mediates its translational regulation by glucose

Glucose induced translation of insulin in pancreatic beta cells is mediated by the 5'UTR of insulin mRNA. We determined the minimal sequence/structure in the 5'UTR of rat insulin gene1 for this regulation. We show that specific factors in the pancreatic islets bind to the 5'UTR of the insulin mRNA upon glucose stimulation. We identified a minimal 29-nucleotide element in the 5'UTR that is sufficient to form the complex, and confer glucose mediated translation activation. Conserved residues in the predicted stem loop region of the un-translated region (UTR) seem to be important for the complex formation and the translation regulation.     

Influence of oxidatively modified LDL on monocyte-macrophage differentiation

Cellular cytoskeletal remodeling reflects alterations in local biochemical and mechanical changes in terms of stress that manifests relocation of signaling molecules within and across the cell. Although stretching due to load and chemical changes by high homocysteine (HHcy) causes cytoskeletal re-arrangement, the synergism between stretch and HHcy is unclear. We investigated the contribution of HHcy in cyclic stretch-induced focal adhesion (FA) protein redistribution leading to cytoskeletal re-arrangement in mouse aortic endothelial cells (MAEC). MAEC were subjected to cyclic stretch (CS) and HHcy alone or in combination. The redistribution of FA protein, and small GTPases were determined by Confocal microscopy and Western blot techniques in membrane and cytosolic compartments. We found that each treatment induces focal adhesion kinase (FAK) phosphorylation and cytoskeletal actin polymerization. In addition, CS activates and membrane translocates small GTPases RhoA with minimal effect on Rac1, whereas HHcy alone is ineffective in both GTPases translocation. However, the combined effect of CS and HHcy activates and membrane translocates both GTPases. Free radical scavenger NAC (N-Acetyl-Cysteine) inhibits CS and HHcy-mediated FAK phosphorylation and actin stress fiber formation. Interestingly, CS also activates and membrane translocates another FA protein, paxillin in HHcy condition. Cytochalasin D, an actin polymerization blocker and PI3-kinase inhibitor Wortmannin inhibited FAK phosphorylation and membrane translocation of paxillin suggesting the involvement of PI3K pathway. Together our results suggest that CS- and HHcy-induced oxidative stress synergistically contribute to small GTPase membrane translocation and focal adhesion protein redistribution leading to endothelial remodeling.     

Backbone structure of the amantadine-blocked trans-membrane domain M2 proton channel from Influenza A virus

Amantadine is known to block the M2 proton channel of the Influenza A virus. Here, we present a structure of the M2 trans-membrane domain blocked with amantadine, built using orientational constraints obtained from solid-state NMR polarization-inversion-spin-exchange-at-the-magic-angle experiments. The data indicates a kink in the monomer between two helical fragments having 20 degrees and 31 degrees tilt angles with respect to the membrane normal. This monomer structure is then used to construct a plausible model of the tetrameric amantadine-blocked M2 trans-membrane channel. The influence of amantadine binding through comparative cross polarization magic-angle spinning spectra was also observed. In addition, spectra are shown of the amantadine-resistant mutant, S31N, in the presence and absence of amantadine.     

A Dual‐Purpose Bromocoumarin Tag Enables Deep Profiling of the Cellular Cysteinome

Chemical proteomics enables comprehensive profiling of small molecules in complex proteomes. A critical component to understand the interactome of a small molecule is the precise location on a protein where the interaction takes place. Several approaches have been developed that take advantage of bio‐orthogonal chemistry and subsequent enrichment steps to isolate peptides modified by small molecules. These methods rely on target identification at the level of mass spectrometry making it difficult to interpret an experiment when modified peptides are not identified. Herein, an approach in which fluorescence‐triggered two‐dimensional chromatography enables the isolation of small molecule‐conjugated peptides prior to mass spectrometry analysis is described. In this study, a bromocoumarin moiety has been utilized that fluoresces and generates a distinct isotopic signature to locate and identify modified peptides. Profiling of a cellular cysteinome with the use of a bromocoumarin tag demonstrates that two‐dimensional fluorescence‐based chromatography separation can enable the identification of proteins containing reactive cysteine residues. Moreover, the method facilitates the interrogation of low abundance proteins with greater depth and sensitivity than a previously reported isotope‐targeted approach. Lastly, this workflow enables the identification of small‐molecule modified peptides from a protein‐of‐interest.     

Mutations in CKAP2L,the Human Homolog of the Mouse Radmis Gene,Cause Filippi Syndrome

Filippi syndrome is a rare, presumably autosomal-recessive disorder characterized by microcephaly, pre- and postnatal growth failure, syndactyly, and distinctive facial features, including a broad nasal bridge and underdeveloped alae nasi. Some affected individuals have intellectual disability, seizures, undescended testicles in males, and teeth and hair abnormalities. We performed homozygosity mapping and whole-exome sequencing in a Sardinian family with two affected children and identified a homozygous frameshift mutation, c.571dupA (p.Ile191Asnfs^∗6), in CKAP2L, encoding the protein cytoskeleton-associated protein 2-like (CKAP2L). The function of this protein was unknown until it was rediscovered in mice as Radmis (radial fiber and mitotic spindle) and shown to play a pivotal role in cell division of neural progenitors. Sanger sequencing of CKAP2L in a further eight unrelated individuals with clinical features consistent with Filippi syndrome revealed biallelic mutations in four subjects. In contrast to wild-type lymphoblastoid cell lines (LCLs), dividing LCLs established from the individuals homozygous for the c.571dupA mutation did not show CKAP2L at the spindle poles. Furthermore, in cells from the affected individuals, we observed an increase in the number of disorganized spindle microtubules owing to multipolar configurations and defects in chromosome segregation. The observed cellular phenotypes are in keeping with data from in vitro and in vivo knockdown studies performed in human cells and mice, respectively. Our findings show that loss-of-function mutations in CKAP2L are a major cause of Filippi syndrome.     

Interferon-mediated ISG15 conjugation restricts dengue virus 2 replication

ISGylation, an ubiquitin-like post-translational modification by ISG15, has been reported to participate in the interferon (IFN)-mediated antiviral response. In this study, we analyzed the functional role of ISGylation in dengue virus 2 (DENV-2) replication. Overexpression of ISG15 was found to significantly suppress the amount of extracellular infectious virus released, while intracellular viral RNA was unaffected. This effect was not observed with a conjugation-defective ISG15 mutant. In addition, extracellular virus infectivity was decreased by ISG15 overexpression. To further clarify the role of ISGylation in the anti-DENV-2 response, we depleted endogenous ISG15 by RNA interference and analyzed the virus production in the absence or presence of type-I IFN. Results showed a significant reduction in extracellular DENV-2 RNA levels for cells treated with IFN, and that these DENV-2 RNA levels could be partially restored by the ISG15 knockdown. Among various DENV-2 proteins, NS3 and NS5 were subjected to the ISGylation. These results demonstrate that IFN-inducible ISGylation suppresses DENV-2 particle release, and that ISG15 is one of the mediators of IFN-induced inhibition of DENV-2 replication. ISG15 therefore functions as a host antiviral factor against DENV-2 infection.