Systematic analysis of N-linked sugar chains from whole tissue employing partial automation

A partially automated technique for the isolation and characterization of N-linked sugar chains from glycoproteins of crude tissue samples is established. The N-linked sugar chains from the acetone-extracted tissues are made free by a process of hydrazinolysis and subsequently N-acetylated by GlycoPrep 1000 (Oxford Glycosystems). These free sugar chains are further converted to pyridylamino derivatives by GlycoTag (Takara). Characterization of these sugar chains is achieved by a combination of HPLC columns using a highly sensitive fluorescence detector at femtomole levels. Tissue sample can be successfully pyridylaminated and analyzed to give highly reproducible results with consistent yield, requiring fewer purification steps, minimum skills, and less time. Moreover, fixed tissues can also be analyzed employing this technique, giving a similar sugar chain pattern compared to normal tissue samples. Using this method we show that the pattern of N-linked sugar chains present in human sera or in one small region of brain is strikingly similar among the different individuals. However, the absence of a highlighted peak in one of the samples suggests this method can be extrapolated to identify changes, if any, associated with disorders such as inflammation or cancer. Furthermore, this two-dimensional display of sugar chains would discover the function-specific molecules as we see in proteins.     

A protocol for shoot multiplication from foliar meristem of Vanda spathulata (L.) Spreng

Leaf explants collected from flowering plants of Vanda spathulata were cultured in Mitra medium with combinations of 6-benzyladenine (BA; 13.2-88.8 microM) and indole-3-acetic acid (IAA; 0.0 -85.6 microM). Combination of BA (66.6 microM) and IAA (28.5 microM) induced maximum shoots (17.33) from foliar meristems (leaf base). BA individually did not induce caulogenesis in leaf explants. For optimized multiplication, BA:IAA (2:1 microM) was essential at 22.2- 88.8 microM of BA. Re-cultured leaf explants produced lesser number of shoots compared to original explants and were nearly equal at combinations of 22.2-44.4 microM of BA and 5.7-28.5 microM of IAA. Rooting of shoots (> 95%) occurred in medium containing banana pulp (75 gl(-1)) and IAA (5.7 microM) within 3-9 weeks. Plantlets with 2-5 roots of 2-5 cm length established easily in community pots at 80-90% rates without hardening.     

Interaction of microtubules with active principles of Xanthium strumarium

Indigenous variety of Xanthium strumarium (X. strumarium) was screened for its antimitotic activity using the microtubule-tubulin system isolated from mammalian tissue. A preliminary phytochemical screening of the whole extracts of the plant was carried out followed by partial purification of the whole extract of X.strumarium. The separated fractions obtained were identified and used for in vitro polymerization studies. The whole as well as partially separated chemical constituents of X. strumarium showed effective inhibition of tubulin polymerization. The results thus suggest that X. strumarium may possess antimitotic components.     

Transbilayer movement of dipalmitoylphosphatidylcholine in proteoliposomes reconstituted from detergent extracts of endoplasmic reticulum. Kinetics of transbilayer transport mediated by a single flippase and identification of protein fractions enriched in flippase activity

Phospholipid translocation (flip-flop) across membrane bilayers is typically assessed via assays utilizing partially water-soluble phospholipid analogs as transport reporters. These assays have been used in previous work to show that phospholipid translocation in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum is facilitated by specific membrane proteins (flippases). To extend these studies to natural phospholipids while providing a framework to guide the purification of a flippase, we now describe an assay to measure the transbilayer translocation of dipalmitoylphosphatidylcholine, a membrane-embedded phospholipid, in proteoliposomes generated from detergent-solubilized rat liver endoplasmic reticulum. Translocation was assayed using phospholipase A(2) under conditions where the vesicles were determined to be intact. Phospholipase A(2) rapidly hydrolyzed phospholipids in the outer leaflet of liposomes and proteoliposomes with a half-time of approximately 0.1 min. However, for flippase-containing proteoliposomes, the initial rapid hydrolysis phase was followed by a slower phase reflecting flippase-mediated translocation of phospholipids from the inner to the outer leaflet. The amplitude of the slow phase was decreased in trypsin-treated proteoliposomes. The kinetic characteristics of the slow phase were used to assess the rate of transbilayer equilibration of phospholipids. For 250-nm diameter vesicles containing a single flippase, the half-time was 3.3 min. Proportionate reductions in equilibration half-time were observed for preparations with a higher average number of flippases/vesicle. Preliminary purification steps indicated that flippase activity could be enriched approximately 15-fold by sequential adsorption of the detergent extract onto anion and cation exchange resins.     

Humor modulates the mesolimbic reward centers

Humor plays an essential role in many facets of human life including psychological, social, and somatic functioning. Recently, neuroimaging has been applied to this critical human attribute, shedding light on the affective, cognitive, and motor networks involved in humor processing. To date, however, researchers have failed to demonstrate the subcortical correlates of the most fundamental feature of humor-reward. In an effort to elucidate the neurobiological substrate that subserves the reward components of humor, we undertook a high-field (3 Tesla) event-related functional MRI study. Here we demonstrate that humor modulates activity in several cortical regions, and we present new evidence that humor engages a network of subcortical regions including the nucleus accumbens, a key component of the mesolimbic dopaminergic reward system. Further, the degree of humor intensity was positively correlated with BOLD signal intensity in these regions. Together, these findings offer new insight into the neural basis of salutary aspects of humor.     

Development of a universal radioactive DNA methyltransferase inhibition test for high-throughput screening and mechanistic studies

DNA methylation is an important epigenetic mark in eukaryotes, and aberrant pattern of this modification is involved in numerous diseases such as cancers. Interestingly, DNA methylation is reversible and thus is considered a promising therapeutic target. Therefore, there is a need for identifying new small inhibitors of C5 DNA methyltransferases (DNMTs). Despite the development of numerous in vitro DNMT assays, there is a lack of reliable tests suitable for high-throughput screening, which can also give insights into inhibitor mechanisms of action. We developed a new test based on scintillation proximity assay meeting these requirements. After optimizing our assay on human DNMT1 and calibrating it with two known inhibitors, we carried out S-Adenosyl-l-Methionine and DNA competition studies on three inhibitors and were able to determine each mechanism of action. Finally, we showed that our test was applicable to 3 other methyltransferases sources: human DNMT3A, bacterial M.SssI and cellular extracts as well.     

Two novel IL-1 family members, IL-1 delta and IL-1 epsilon, function as an antagonist and agonist of NF-kappa B activation through the orphan IL-1 receptor-related protein 2

IL-1 is of utmost importance in the host response to immunological challenges. We identified and functionally characterized two novel IL-1 ligands termed IL-1delta and IL-1epsilon. Northern blot analyses show that these IL-1s are highly abundant in embryonic tissue and tissues containing epithelial cells (i.e., skin, lung, and stomach). In extension, quantitative real-time PCR revealed that of human skin-derived cells, only keratinocytes but not fibroblasts, endothelial cells, or melanocytes express IL-1delta and epsilon. Levels of keratinocyte IL-1delta are approximately 10-fold higher than those of IL-1epsilon. In vitro stimulation of keratinocytes with IL-1beta/TNF-alpha significantly up-regulates the expression of IL-1epsilon mRNA, and to a lesser extent of IL-1delta mRNA. In NF-kappaB-luciferase reporter assays, we demonstrated that IL-1delta and epsilon proteins do not initiate a functional response via classical IL-1R pairs, which confer responsiveness to IL-1alpha and beta or IL-18. However, IL-1epsilon activates NF-kappaB through the orphan IL-1R-related protein 2 (IL-1Rrp2), whereas IL-1delta, which shows striking homology to IL-1 receptor antagonist, specifically and potently inhibits this IL-1epsilon response. In lesional psoriasis skin, characterized by chronic cutaneous inflammation, the mRNA expression of both IL-1 ligands as well as IL-1Rrp2 are increased relative to normal healthy skin. In total, IL-1delta and epsilon and IL-1Rrp2 may constitute an independent signaling system, analogous to IL-1alphabeta/receptor agonist and IL-1R1, that is present in epithelial barriers of our body and takes part in local inflammatory responses.     

Treatment Satisfaction and Well-Being in Patients with Myopic Choroidal Neovascularization Treated with Ranibizumab in the REPAIR Study

The Ranibizumab for the Treatment of Choroidal Neovascularisation (CNV) Secondary to Pathological Myopia (PM): an Individualized Regimen (REPAIR) trial was a prospective study exploring the efficacy and safety of intravitreal ranibizumab 0.5 mg using an individualized treatment regimen over 12 months. The current study investigated the impact of treatment with ranibizumab as needed (pro re nata [PRN]) on individuals with myopic choroidal neovascularization (mCNV) in the REPAIR study, using patient-reported outcome measures (PROMs) for treatment satisfaction and well-being. This study included 65 adults with mCNV and a best-corrected visual acuity (BCVA) letter score of 24–78 in the study eye. Patients completed the Macular Disease Treatment Satisfaction Questionnaire (MacTSQ) at months 1, 6 and 12, and the 12-item Well-Being Questionnaire (W-BQ12) at baseline and months 1, 6 and 12. Subgroup analyses investigated the relationship between PROM scores and treatment in the better- or worse-seeing eye (BSE/WSE), number of injections received, baseline BCVA, BCVA improvement and age. Pearson correlations between change in BCVA, MacTSQ scores and W-BQ12 scores were calculated. The main outcome measures were treatment satisfaction measured with the MacTSQ (score 0–72) and well-being measured with the W-BQ12 (score 0–36). Treatment satisfaction significantly increased over the study period (p = 0.0001). Mean MacTSQ scores increased by 9.7 and 10.0 in patients treated in their WSE and BSE, respectively. Treatment satisfaction was highest in individuals receiving only one injection at month 1; however, by month 12, scores were similar across injection subgroups. Patients aged 68 years or older had the highest MacTSQ scores. Well-being scores also significantly increased over the study period (p = 0.03). Mean W-BQ12 scores increased by 1.7 in patients treated in their WSE and by 2.1 in patients treated in their BSE. Individuals aged 40 years or younger had the greatest increases in general well-being. Patients who experienced stable or improved BCVA at month 12 had greater increases in W-BQ12 scores than those who experienced a decrease. Correlations between BCVA, MacTSQ scores and W-BQ12 scores were largely non-significant. In conclusion, treatment satisfaction and well-being increased during treatment with ranibizumab PRN. Although directly comparable data are limited for the MacTSQ and W-BQ12 in mCNV, these results complement PROM outcomes reported in related studies.     

Trace Element Studies on Tinospora cordifolia (Menispermaceae), Ocimum sanctum (Lamiaceae), Moringa oleifera (Moringaceae), and Phyllanthus niruri (Euphorbiaceae) Using PIXE

Traditionally, Tinospora cordifolia (Willd.) Hook. F. & Thomson (Menispermaceae), Ocimum sanctum L. (Lamiaceae), Moringa oleifera Lam. (Moringaceae), and Phyllanthus niruri L. (Euphorbiaceae) are some of the commonly used medicinal plants in India for curing ailments ranging from common cold, skin diseases, and dental infections to major disorders like diabetes, hypertension, jaundice, rheumatism, etc. To understand and correlate their medicinal use, trace element studies on the aqueous extract of these medicinal plants have been carried out using particle-induced X-ray emission technique. A 2-MeV proton beam was used to identify and characterize major and minor elements namely Cl, K, Ca, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, Br, and Sr in them. Results have revealed that these elements are present in varying concentrations in the selected plants. Notable results include very high concentrations of Cl, K, and Ca in all the leaf samples, appreciable levels of Mn in all plants, high Zn content in T. cordifolia, and the aqueous extract of Moringa leaves compared to others and relative higher concentrations of Cr in all the plants.