Multiphoton ANS fluorescence microscopy as an in vivo sensor for protein misfolding stress

The inability of cells to maintain protein folding homeostasis is implicated in the development of neurodegenerative diseases, malignant transformation, and aging. We find that multiphoton fluorescence imaging of 1-anilinonaphthalene-8-sulfonate (ANS) can be used to assess cellular responses to protein misfolding stresses. ANS is relatively nontoxic and enters live cells and cells or tissues fixed in formalin. In an animal model of Alzheimer’s disease, ANS fluorescence imaging of brain tissue sections reveals the binding of ANS to fibrillar deposits of amyloid peptide (Aβ) in amyloid plaques and in cerebrovascular amyloid. ANS imaging also highlights non-amyloid deposits of glial fibrillary acidic protein in brain tumors. Cultured cells under normal growth conditions possess a number of ANS-binding structures. High levels of ANS fluorescence are associated with the endoplasmic reticulum (ER), Golgi, and lysosomes—regions of protein folding and degradation. Nuclei are virtually devoid of ANS binding sites. Additional ANS binding is triggered by hyperthermia, thermal lesioning, proteasome inhibition, and induction of ER stress. We also use multiphoton imaging of ANS binding to follow the in vivo recovery of cells from protein-damaging insults over time. We find that ANS fluorescence tracks with the binding of the molecular chaperone Hsp70 in compartments where Hsp70 is present. ANS highlights the sensitivity of specific cellular targets, including the nucleus and particularly the nucleolus, to thermal stress and proteasome inhibition. Multiphoton imaging of ANS binding should be a useful probe for monitoring protein misfolding stress in cells.     

Determination of community structure through deconvolution of PLFA-FAME signature of mixed population

Phospholipid fatty acids (PLFAs) as biomarkers are well established in the literature. A general method based on least square approximation (LSA) was developed for the estimation of community structure from the PLFA signature of a mixed population where biomarker PLFA signatures of the component species were known. Fatty acid methyl ester (FAME) standards were used as species analogs and mixture of the standards as representative of the mixed population. The PLFA/FAME signatures were analyzed by gas chromatographic separation, followed by detection in flame ionization detector (GC-FID). The PLFAs in the signature were quantified as relative weight percent of the total PLFA. The PLFA signatures were analyzed by the models to predict community structure of the mixture. The LSA model results were compared with the existing "functional group" approach. Both successfully predicted community structure of mixed population containing completely unrelated species with uncommon PLFAs. For slightest intersection in PLFA signatures of component species, the LSA model produced better results. This was mainly due to inability of the "functional group" approach to distinguish the relative amounts of the common PLFA coming from more than one species. The performance of the LSA model was influenced by errors in the chromatographic analyses. Suppression (or enhancement) of a component's PLFA signature in chromatographic analysis of the mixture, led to underestimation (or overestimation) of the component's proportion in the mixture by the model. In mixtures of closely related species with common PLFAs, the errors in the common components were adjusted across the species by the model.     

The type III secretion system needle,tip, and translocon

Many Gram‐negative bacteria pathogenic to plants and animals deploy the type III secretion system (T3SS) to inject virulence factors into their hosts. All bacteria that rely on the T3SS to cause infectious diseases in humans have developed antibiotic resistance. The T3SS is an attractive target for developing new antibiotics because it is essential in virulence, and part of its structural component is exposed on the bacterial surface. The structural component of the T3SS is the needle apparatus, which is assembled from over 20 different proteins and consists of a base, an extracellular needle, a tip, and a translocon. This review summarizes the current knowledge on the structure and assembly of the needle, tip, and translocon.     

Identification of mammalian protein quality control factors by high-throughput cellular imaging

Protein Quality Control (PQC) pathways are essential to maintain the equilibrium between protein folding and the clearance of misfolded proteins. In order to discover novel human PQC factors, we developed a high-content, high-throughput cell-based assay to assess PQC activity. The assay is based on a fluorescently tagged, temperature sensitive PQC substrate and measures its degradation relative to a temperature insensitive internal control. In a targeted screen of 1591 siRNA genes involved in the Ubiquitin-Proteasome System (UPS) we identified 25 of the 33 genes encoding for 26S proteasome subunits and discovered several novel PQC factors. An unbiased genome-wide siRNA screen revealed the protein translation machinery, and in particular the EIF3 translation initiation complex, as a novel key modulator of misfolded protein stability. These results represent a comprehensive unbiased survey of human PQC components and establish an experimental tool for the discovery of genes that are required for the degradation of misfolded proteins under conditions of proteotoxic stress.     

Noncancer risk assessment of arsenic safe and unsafe water uses: Bayesian estimation on cohort study

Cancer and noncancer risk of arsenic exposure depends on arsenic intake through drinking water and diets. The present study evaluated the probability of noncancer effects of arsenic exposure from drinking water and diets in a cohort of 82 participants in arsenic-endemic rural areas, considering arsenic-safe and arsenic-unsafe water uses for three consecutive years. The risk assessment included the collection of last 24 hours' diet replica and urine of the participants followed by total arsenic analysis of the same. Toxic dose emerging from exposure duration is a nonlinear variable. So, Bayesian estimation of the data for noncancer risk assessment of the variable arsenic consumption was performed. In spite of using arsenic-safe water, we observed arsenic consumption and release. Participants with skin lesions had more arsenic in urine than participants without skin lesions. Future risk for participants without skin lesions was twice due to less arsenic release in urine. For the first time, Bayesian simulation was used to assess noncancer risk on a cohort for a consecutive three-year study. A significant finding was the higher assessed noncancer risk of the participants without skin lesions than the participants with skin lesions.     

Synthesis and SAR of 2-aryl-3-aminomethylquinolines as agonists of the bile acid receptor TGR5

Optimization of a screening hit from uHTS led to the discovery of TGR5 agonist 32, which was shown to have activity in a rodent model for diabetes.     

Modulation of adipose tissue inflammation by bioactive food compounds

Adipose tissue has an important endocrine function in the regulation of whole-body metabolism. Obesity leads to a chronic low-grade inflammation of the adipose tissue, which disrupts this endocrine function and results in metabolic derangements, such as type-2 diabetes. Dietary bioactive compounds, such as polyphenols and certain fatty acids, are known to suppress both systemic and adipose tissue inflammation and have the potential to improve these obesity-associated metabolic disorders. Mechanistically, polyphenolic compounds including non-flavonoids, such as curcumin and resveratrol, and flavonoids, such as catechins (tea-polyphenols), quercetin and isoflavones, suppress nuclear factor-κB (NF-κB) and mitogen-activated protein (MAP) kinases (MAPK) pathways while activating the 5′ adenosine monophosphate-activated protein kinase (AMPK) pathway in adipose tissue. Dietary polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), conjugated linoleic acid (CLA) and monounsaturated fatty acids (MUFA), such as oleic acid, also impart anti-inflammatory effects through several mechanisms. These include activation of AMPK and peroxisome proliferator-activated receptor gamma (PPAR-γ), as well as suppression of toll-like receptors (TLRs) and NF-κB pathway. This review discusses the major molecular mechanisms of dietary polyphenols and fatty acids, alone or in combination, which are responsible for adipose tissue-associated anti-inflammatory effects.     

Dominant-negative mutants of platelet-derived growth factor revert the transformed phenotype of human astrocytoma cells.

Malignant astrocytoma is the most common primary human brain tumor. Most astrocytomas express a combination of platelet-derived growth factor (PDGF) and PDGF receptor which could close an autocrine loop. It is not known whether these autocrine loops contribute to the transformed phenotype of astrocytoma cells or are incidental to that phenotype. Here we show that dominant-negative mutants of the PDGF ligand break the autocrine loop and revert the phenotype of BALB/c 3T3 cells transformed by the PDGF-A or PDGF-B (c-sis) gene. Then, we show that these mutants are selective in that they do not alter the phenotype of 3T3 cells transformed by an activated Ha-ras or v-src gene or by simian virus 40. Finally, we show that these mutants revert the transformed phenotype of two independent human astrocytoma cell lines. They have no effect on the growth of human medulloblastoma, bladder carcinoma, or colon carcinoma cell lines. These observations are consistent with the view that PDGF autocrine loops contribute to the transformed phenotype of at least some human astrocytomas.     

Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides

Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV) peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05⁺ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05⁺ NK cells, including three immunodominant CD8⁺ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05⁺ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002⁺ CD4⁺ lymphocytes co-cultured with Mamu-KIR3DL05⁺ NK cells than with Mamu-KIR3DL05^- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses.     

The use of C14-labelled substrate in histochemical demonstration of different forms of phosphorylase

Active and total phosphorylase activity, using labelled C¹⁴-glucose-1-phosphate as the substrate, is demonstrated by histoautoradiographic method. This method can demonstrate the polysaccharide synthesizedin vitro by phosphorylase without intervention from the unlabelled pre-existing glycogen. C¹⁴-glucose can not replace C¹⁴-glucose-1-phosphate as substrate. The distribution of phosphorylase in tissue sections, except in cases of very low activity, is similar to that obtained by customary dilute Lugol's iodine staining method. The relative difference of intensity between active and total phosphorylase, as revealed by iodine staining, is also reflected by histoautoradiographic method. Histoautoradiographic method has several advantages over the iodine staining method. This method is more sensitive for demonstration of very low phosphorylase activity which may escape detection by iodine staining. Branching enzyme activity, especially when it favors synthesis of glycogen type of polysaccharide instead of amylopectin type, can be better detected by this method. Active phosphorylase substrate medium can be used to demonstrate this activity in plant tissues, where the presence of pre-existing starch often prohibits the use of iodine staining method. Stripping film method for autoradiography is recommended for the study of this enzyme activity.