Differential sensitivity of oleosins to proteolysis during oil body mobilization in sunflower seedlings

Until now, there has been no conclusive demonstration of any in vivo oleosin degradation at the early stages of oil body mobilization. The present work on sunflower (Helianthus annuus L.) has demonstrated limited oleosin degradation during seed germination. Seedling cotyledon homogenization in Tris-urea buffer, followed by SDS-PAGE, revealed three oleosins (16, 17.5 and 20 kDa). Incubation of oil bodies with total soluble protein from 4-day-old seedlings resulted in oleosin degradation. In vitro and in vivo degradation of the 17.5-kDa oleosin was faster than the other two, indicating its greater susceptibility to proteolysis. Oleosin degradation by the total soluble protein resulted in a transient 14.5-kDa polypeptide, followed by an 11-kDa protease-protected fragment, which appeared post-germinatively and accumulated corresponding to increased rate of lipid mobilization. A 65-kDa protease, active at pH 7.5-9.5, was zymographically detected in the total soluble protein. Its activity increased along with in vivo accumulation of the protease-protected fragment during seed germination and accompanying lipid mobilization. Protease-treated oil bodies were more susceptible to maize lipase action. Differential proteolytic sensitivity of different oleosins in the oil body membranes could be a determinant of oil body longevity during seed germination.     

Analytical validation of quantitative immunohistochemical assays of tumor infiltrating lymphocyte biomarkers

Tumor infiltrating lymphocytes (TIL), especially T-cells, have both prognostic and therapeutic applications. The presence of CD8+ effector T-cells and the ratio of CD8+ cells to FOXP3+ regulatory T-cells have been used as biomarkers of disease prognosis to predict response to various immunotherapies. Blocking the interaction between inhibitory receptors on T-cells and their ligands with therapeutic antibodies including atezolizumab, nivolumab, pembrolizumab and tremelimumab increases the immune response against cancer cells and has shown significant improvement in clinical benefits and survival in several different tumor types. The improved clinical outcome is presumed to be associated with a higher tumor infiltration; therefore, it is thought that more accurate methods for measuring the amount of TIL could assist prognosis and predict treatment response. We have developed and validated quantitative immunohistochemistry (IHC) assays for CD3, CD8 and FOXP3 for immunophenotyping T-lymphocytes in tumor tissue. Various types of formalin fixed, paraffin embedded (FFPE) tumor tissues were immunolabeled with anti-CD3, anti-CD8 and anti-FOXP3 antibodies using an IHC autostainer. The tumor area of stained tissues, including the invasive margin of the tumor, was scored by a pathologist (visual scoring) and by computer-based quantitative image analysis. Two image analysis scores were obtained for the staining of each biomarker: the percent positive cells in the tumor area and positive cells/mm² tumor area. Comparison of visual vs. image analysis scoring methods using regression analysis showed high correlation and indicated that quantitative image analysis can be used to score the number of positive cells in IHC stained slides. To demonstrate that the IHC assays produce consistent results in normal daily testing, we evaluated the specificity, sensitivity and reproducibility of the IHC assays using both visual and image analysis scoring methods. We found that CD3, CD8 and FOXP3 IHC assays met the fit-for-purpose analytical acceptance validation criteria and that they can be used to support clinical studies.     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Antidyslipidemic activity of furano-flavonoids isolated from Indigofera tinctoria

Flavonoids appear to play a major role in reducing the risk of cardiovascular diseases by decreasing the blood lipid levels. In continuation of our drug discovery program on antidyslipidemic agents we have isolated three furano-flavones 1-3 and a rare flavonol glycoside 4 from the aerial parts of Indigofera tinctoria. Our results disclose that the treatment with diastereomeric flavonoid mixture 1 and 2 (80:20) significantly decreased the plasma triglycerides (TG) by 60%, total cholesterol (TC) 19%, glycerol (Gly) 13%, and free fatty acid (FFA) 25% accompanied with increase in high density lipoproteins-cholesterol (HDL-C) by 8% and HDL-C/TC ratio 36% in high fat diet (HFD) fed dyslipidemic hamsters at the dose of 50 mg/kg body weight. The flavonoid 3 has exhibited moderate antidyslipidemic activity.     

Discovery of curcumin inspired sulfonamide derivatives as a new class of carbonic anhydrase isoforms I,II, IX,and XII inhibitors

A series of curcumin inspired sulfonamide derivatives was prepared from various chalcones and 4-sulfamoyl benzaldehyde via Claisen–Schmidt condensation. All new compounds were assayed as inhibitors of four human isoforms of the metalloenzyme carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I, II, IX and XII. Interesting inhibitory activities were observed against all these isoforms. hCA I, an isoform involved in several eye diseases was inhibited moderately with K_Is in the range of 191.8–904.2?nM, hCA II, an antiglaucoma drug target was highly inhibited by the new sulfonamides, with K_Is in the range of 0.75–8.8?nM. hCA IX, a tumor-associated isoform involved in cancer progression and metastatic spread was potently inhibited by the new sulfonamides, with K_Is in the range of 2.3–87.3?nM, whereas hCA XII, and antiglaucoma and anticancer drug target, was inhibited with K_Is in the range of 6.1–71.8?nM. It is noteworthy that one of the new compounds, 5d, was found to be almost 9 times more selective against hCA II (K_I =?0.89?nM) over hCA IX and hCA XII, whereas 5e was 3 and 70 times more selective against hCA II (K_I =?0.75?nM) over hCA IX and hCA XII, respectively.     

Artificial viral envelopes containing recombinant human immunodeficiency virus (HIV) gp160.

An artificial viral envelope was constructed, resembling the human immunodeficiency virus (HIV) envelope with respect to ultrastructure, size, phospholipid profile and lipid:cholesterol ratio. Recombinant HIV surface protein gp160 was anchored in the outer surface of the envelope membrane using a double detergent dialysis. The envelopes remained physically stable for several months. Immunolabeling with anti-gp160/41 monoclonal antibody revealed surface insertion and availability of gp160 for binding. Cell fusion and cytosolic transfer of the encapsulated fluorescent marker FITC-dextran was demonstrated. Flow cytometry indicated more efficient transfer of the fluorescent marker to cells which were approximately 60% CD4+ (REX-1B), relative to cells which were only approximately 18% CD4+ (KG-1). However, plain lipid envelopes without gp160 fused very efficiently with both cell types, indicating their potential usefulness as "fusogenic liposomes". Complete artificial viral envelopes may serve as subunit vaccines, and receptor-targeted delivery systems for drugs, toxins and genetic constructs.     

Syntheses and biological evaluation of 3-substituted amino-1-aryl-6-hydroxy-hex-2-ene-1-ones as antioxidant and hypolipidemic agents

A new series of compounds belonging to 3-substituted amino-1-aryl-6-hydroxy-hex-2-ene-1-ones (4-12a-e) have been synthesized and evaluated for antioxidant and hypolipidemic activities. Amongst all the synthesized compounds, seven compounds, namely 5b, 5d, 6e, 8a, 8b, 10b and 11a, exhibit better antioxidant activity than probucol. Two compounds, 5d and 10b, have been evaluated in detail for antioxidant and hypolipidemic activities and show comparable activity profile to that of probucol and guggulipid. From the present study it may be postulated that the mechanism of action of these compounds could be through activation of lecithin cholesterol acyltransferase (LCAT), liver lipolytic activity, increased faecal bile acid secretion and inhibition of hepatic cholesterol biosynthesis.     

Synexin and GTP increase surfactant secretion in permeabilized alveolar type II cells

We have previously suggested that synexin (annexin VII), a Ca(2+)-dependent phospholipid binding protein, may have a role in surfactant secretion, since it promotes membrane fusion between isolated lamellar bodies (the surfactant-containing organelles) and plasma membranes. In this study, we investigated whether exogenous synexin can augment surfactant phosphatidylcholine (PC) secretion in synexin-deficient lung epithelial type II cells. Isolated rat type II cells were cultured for 20-22 h with [(3)H]choline to label cellular PC. The cells were then treated with beta-escin, which forms pores in the cell membrane and releases cytoplasmic proteins including synexin. These cells, however, retained lamellar bodies. The permeabilized type II cells were evaluated for PC secretion during a 30-min incubation. Compared with PC secretion under basal conditions, the presence of Ca(2+) (up to 10 microM) did not increase PC secretion. In the presence of 1 microM Ca(2+), synexin increased PC secretion in a concentration-dependent manner, which reached a maximum at approximately 5 microg/ml synexin. The secretagogue effect of synexin was abolished when synexin was inactivated by heat treatment (30 min at 65 degrees C) or by treatment with synexin antibodies. GTP or its nonhydrolyzable analog beta:gamma-imidoguanosine-5'-triphosphate also increased PC secretion in permeabilized type II cells. The PC secretion was further increased in an additive manner when a maximally effective concentration of synexin was added in the presence of 1 mM GTP, suggesting that GTP acts by a synexin-independent mechanism to increase membrane fusion. Thus our results support a direct role for synexin in surfactant secretion. Our study also suggests that membrane fusion during surfactant secretion may be mediated by two independent mechanisms.     

Markers of small cell lung cancer

Lung cancer is the number one cause of cancer death; however, no specific serum biomarker is available till date for detection of early lung cancer. Despite good initial response to chemotherapy, small-cell lung cancer (SCLC) has a poor prognosis. Therefore, it is important to identify molecular markers that might influence survival and may serve as potential therapeutic targets. The review aims to summarize the current knowledge of serum biomarkers in SCLC to improve diagnostic efficiency in the detection of tumor progression in lung cancer. The current knowledge on the known serum cytokines and tumor biomarkers of SCLC is emphasized. Recent findings in the search for novel diagnostic and therapeutic molecular markers using the emerging genomic technology for detecting lung cancer are also described. It is believed that implementing these new research techniques will facilitate and improve early detection, prognostication and better treatment of SCLC.