Electrophoretic Analysis of Human Parotid Salivary Proteins with Application to the Study of Rheumatoid Arthritis and Sjogren's Syndrome

Human parotid saliva contains many proteins with diverse functions. In the course of a number of diseases, especially where the normal function of the salivary gland is affected, changes may occur in the levels of certain of these. Therefore the analysis of some of these proteins may be of diagnostic significance. This study has focused on the development and refinement of electrophoretic and of protein detection techniques in order to allow the fractionation of proteins in small volumes of human saliva with the minimum of sample preparation. In order to give an example of their possible diagnostic significance, the electrophoretic techniques which were developed were applied to the fractionation and partial characterisation of the anionic salivary proteins associated with connective tissue disorders such as rheumatoid arthritis and Sjogren's Syndrome. The saliva of patients with rheumatoid arthritis and Sjogren's Syndrome contains additional anionic proteins, which are either present in very low levels or below detection limits in the saliva of normal healthy individuals. Research into the identity of these proteins has been largely hindered by the relatively high electrolyte and low protein content of human parotid saliva, making it necessary to desalt and concentrate the saliva samples prior to carrier ampholyte-based isoelectric focusing. Desalting requires relatively large volumes (preferably > 2ml) of saliva, which may be difficult or even impossible to obtain from diseased glands. Also, one-dimensional isoelectric focusing cannot separate these anionic proteins from the acidic proline-rich proteins of human saliva, as both groups of proteins have overlapping isoelectric points. In this study, a hybrid carrier ampholyte-immobilised pH gradient isoelectric focusing technique was developed to analyse human salivary proteins. Immobilised pH gradients (IPG's) of 3 pH ranges were prepared: broad-range (pH 4-9) IPG was used for the general study of human salivary proteins; while 2 narrow, acidic range IPG's (pH 2.8-4.5 and pH 3.5-5.0) were used to analyse proteins of low isoelectric points, such as the anionic salivary proteins associated with rheumatoid arthritis and Sjogren's Syndrome. This method allowed the difficulties involved when conventional carrier ampholyte-based isoelectric focusing is used to be circumvented, thus making it possible to fractionate the proteins in small volumes (approximately 50ul) of human saliva without prior treatment except for centrifugation. Parotid salivary proteins were also analysed by onedimensional SDS-PAGE. SDS-PAGE gels were subjected to im-muno- and lectin affinity-blotting in order to characterise or identify some of the protein bands. Proline-rich proteins were recognised by their characteristic pink-staining with the dye Coommassie Brilliant Blue R250, and some of the bands which were revealed have been correlated with proline-rich proteins which have been isolated and partially characterised by other research groups. SDS-PAGE failed to reveal any obvious differences between the band patterns of normal subjects and those of patients with rheumatoid arthritis or Sjogren's Syndrome. Two-dimensional gel electrophoresis was also carried out using hybrid carrier ampholyte-immobilised pH gradient polyacrylamide gels in the first dimension and thin-layer SDS-polyacrylamide gradient gels in the second. By means of a combination of staining and electroblotting of the two-dimensional gels onto nitrocellulose followed by probing with specific antisera, a two-dimensional map of human parotid salivary proteins, in which most of the major components have been identified, has been obtained. These techniques were applied to the investigation of the nature of the anionic salivary proteins associated with rheumatoid arthritis and Sjogren's Syndrome. Two-dimensional electrophoresis with pH 3.5-5.0 IPG's in the first dimension followed by silver staining revealed these proteins to be heterogeneous (pI's approximately 3.65-4.75) and of a single relative molecular weight (approximately 32,000). In normal healthy controls these silver stained components were less heterogeneous (pI's approximately 3.65-4.25). Incubation with neuraminidase showed that their heterogeneity was largely due to differing contents of sialic acid in their carbohydrate side-chains. In order to attempt to identify these proteins, the one-dimensional IPG and two-dimensional gels were electroblotted and the blots were probed with a variety of antisera. The proteins appeared to be immunoreactive with antisera to human tissue kallikrein, a protein the level of which has often been reported to be elevated in the saliva of patients with Sjogren's Syndrome. (Abstract shortened by ProQuest.)

Penentuan Hasil Tindakbalas Antara Asid Kromotropik Dan Formaldehid

Identiti tindakbalas antara asid kromotropik dan formaldehid berlebihan tanpa asid sulfuric pekat dibuktikan sebagai dimer di mana dua molekul asid kromotropik diikat melalui dua titisan olefinik, berasaskan kepada spectrum N.M.R Proton, mikroanalisisuntuk C,H, N dan S, dan pentitratan Karl Fischer

Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

<p>Abstract</p> <p>Background</p> <p>Nasopharyngeal carcinoma (NPC) is an epithelial malignancy, which commonly occurs in Southern China, Taiwan, North Africa and Southeast Asia. Nasopharyngeal carcinoma is strongly associated with Epstein-Barr virus infection. The p53 tumour suppressor protein is rarely mutated in NPC suggesting that the inactivation of p53 pathway in NPC could be due to the presence of EBV proteins. The aim of this work was to determine the effects of EBV proteins namely LMP1 and LMP2A on the expression levels of p53 protein.</p> <p>Findings</p> <p>In this work we found that LMP1, but not LMP2A, decreased p53 protein levels. Overexpression of LMP1 resulted in increased ubiquitination of p53 suggesting that the decreased p53 protein levels by LMP1 was due to increased degradation of the protein. The reduction of p53 protein levels was independent of the PI3K-Akt pathway.</p> <p>Conclusions</p> <p>LMP1, but not LMP2A, reduced p53 protein levels through the increase in the polyubiquitination of p53 protein and was independent of the PI3K-Akt pathway.</p

Investigation of twenty selected medicinal plants from Malaysia for anti-Chikungunya virus activity

Chikungunya virus is a reemerging arbovirus transmitted mainly by Aedes mosquitoes. As there are no specific treatments available, Chikungunya virus infection is a significant public health problem. This study investigated 120 extracts from selected medicinal plants for anti-Chikungunya virus activity. The plant materials were subjected to sequential solvent extraction to obtain six different extracts for each plant. The cytotoxicity and antiviral activity of each extract were examined using African monkey kidney epithelial (Vero) cells. The ethanol, methanol and chloroform extracts of Tradescantia spathacea (Commelinaceae) leaves showed the strongest cytopathic effect inhibition on Vero cells, resulting in cell viabilities of 92.6% &plusmn; 1.0% (512 &mu;g/ml), 91.5% &plusmn; 1.7% (512 &mu;g/ml) and 88.8% &plusmn; 2.4% (80 &mu;g/ml) respectively. However, quantitative RT-PCR analysis revealed that the chloroform extract of Rhapis excelsa (Arecaceae) leaves resulted in the highest percentage of reduction of viral load (98.1%), followed by the ethyl acetate extract of Vernonia amygdalina (Compositae) leaves (95.5%). The corresponding 50% effective concentrations (EC50) and selectivity indices for these two extracts were 29.9 &plusmn; 0.9 and 32.4 &plusmn; 1.3 &mu;g/ml, and 5.4 and 5.1 respectively. Rhapis excelsa and Vernonia amygdalina could be sources of anti-Chikungunya virus agents. [Int Microbiol 19(3):175-182 (2016)]Keywords: Chikungunya virus &middot; antivirals &middot; cytotoxicity &middot; sequential extraction &middot; medicinal plant

Interactions between Plant Extracts and Cell Viability Indicators during Cytotoxicity Testing: Implications for Ethnopharmacological Studies

Purpose: To compare the cytotoxicity of six medicinal plants (Acmella ciliata, Amaranthus tricolor, Coriandrum sativum, Glebionis coronaria, Kyllinga brevifolia and Tradescantia zebrina) using 3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake (NRU) assays.Methods: Hexane, chloroform, ethyl acetate, ethanol, methanol and water extracts were obtained for each plant by sequential solvent extraction. Cytotoxicity was evaluated in triplicate, from 640 to 5 μg/mL, two-fold, serially on monkey kidney epithelial (Vero) cells.Results: The hexane, chloroform and ethyl acetate extracts of the six plants were more toxic to the Vero cells compared to the ethanol, methanol and water extracts. Thirty one percent (11/36) and 75 % (27/36) of the extracts showed significant cytotoxicity (p &lt; 0.05) in MTT and NRU assays, respectively. The 78, 52 and 7 % cytotoxicity levels detected in 27 extracts using the MTT assay were significantly (p &lt; 0.05) underestimated at 640, 320 and 160 μg/mL, respectively, using NRU assay. Nine extracts from five plants exhibited significantly lower (p &lt; 0.05) 50 % cytotoxic concentration (CC50) when NRU assay was employed compared to MTT assay. At 640 μg/mL, 10 of the 21 extracts were also found to react chemically with MTT, causing a 2.0 – 29.1-fold increase in the absorbance value (550 nm) compared to control.Conclusion: The plant extracts of A. ciliata, A. tricolor, C. sativum, G. coronaria, K. brevifolia and T. zebrina show concentration- and extraction method-dependent cytotoxicity using MTT and NRU assays. NRU assay appears to be more sensitive and reliable than MTT assay for cell viability evaluation of the plant extracts.Keywords: Acmella ciliata, Amaranthus tricolor, Coriandrum sativum, Glebionis coronaria, Kyllinga brevifolia and Tradescantia zebrina, Extraction, Medicinal plant, Neutral red uptake assay, Vero cel

Age-related changes relevant to health in women: design, recruitment, and retention strategies for the Longitudinal Assessment of Women (LAW) Study

Objectives: The primary aim was to assess the age-related changes that occur in older women. This paper describes the study rationale and methods, recruitment, and retention strategies. Methods: The Longitudinal Assessment of Women (LAW) Study was a longitudinal, observational, and multidisciplinary evaluation of a population-based cohort of urban-living women, aged between 40 and 80 years at recruitment and randomly invited from a district in Brisbane (a city in Australia) via the electoral roll. Five hundred eleven women were recruited and stratified into four age groups (40-49, 50-59, 60-69, 70-79 years) and were assessed on three or four occasions each year, using interviews and diagnostic instruments (echocardiography, applination tonometry, dual-energy x-ray absorptiometry [DEXA]) Retention strategies included flexibility, accessibility, personalized attention, and feedback. Results: From a sample frame of 1598 names, there were 1082 respondents, of whom 511 (47%) were successfully recruited from those eligible to participate. Recruitment was quickest for the oldest age group, 70-79 years, and slowest for the age group 40-49 years; all age groups achieved their required quota. A scheduling program was developed to minimize the number of visits and maximize the use of allocated time. The largest dropout was seen in year 1 of the study, with very few thereafter. Of the 9 deaths, cancer was the cause in 7. The retention rate after 5 years was 95.5%. Conclusions: The design of the present study, with careful attention to coordination and a personal approach, facilitated the completion of a 5-year study, enabling a collection of a set of wide-ranging data from almost all the women recruited. The information thus collected will form the basis of cross-linking analysis of the risk factors associated with health problems in aging women

Pathogenic role of exosomes in Epstein-Barr Virus (EBV)-associated cancers

Exosomes are 40- to 100-nm membrane-bound small vesicles that carry a great variety of cellular cargoes including proteins, DNA, messenger RNAs (mRNAs), and microRNAs (miRNAs). These nanovesicles are detected in various biological fluids such as serum, urine, saliva, and seminal fluids. Exosomes serve as key mediators in intercellular communication by facilitating the transfer and exchange of cellular components from cells to cells. They contain various pathogenic factors whereby their adverse effects have been implicated in multiple viral infections and cancers. Interestingly, accumulating evidences showed that exosomes derived from tumour viruses or oncoviruses, exacerbate virus-associated cancers by remodelling the tumour microenvironment. In this review, we summarize the contributing factors of Epstein-Barr virus (EBV) products-containing exosomes in viral pathogenesis and their potential implications in EBV-driven malignancies. Understanding the biological role of these exosomes in the disease would undoubtedly boost the development of a more comprehensive strategy to combat EBV-associated cancers and to better predict the therapeutic outcomes. Furthermore, we also highlight the potentials and challenges of EBV products-containing exosomes being employed as diagnostic markers and therapeutic targets for EBV-related cancers. Since these aspects are rather underexplored, we attempt to underline interesting areas that warrant further investigations in the future

Audit Fees: To Disclose or Not to Disclose?

The following article by Associate Professor Khoo Teng Aun and Associate Professor Hwang Soo Chiat argues that the disclosure of audit fees can provide a more level playing field in Singapore, and would also be consistent with the other major capital markets in the world. The article was first published in the Business Times on 29 June 2010.</p

Exosomes in Human Immunodeficiency Virus Type I Pathogenesis: Threat or Opportunity?

Nanometre-sized vesicles, also known as exosomes, are derived from endosomes of diverse cell types and present in multiple biological fluids. Depending on their cellular origins, the membrane-bound exosomes packed a variety of functional proteins and RNA species. These microvesicles are secreted into the extracellular space to facilitate intercellular communication. Collective findings demonstrated that exosomes fromHIV-infected subjects sharemany commonalities withHuman ImmunodeficiencyVirus Type I (HIV-1) particles in terms of proteomics and lipid profiles. These observations postulated that HIV-resembled exosomes may contribute to HIV pathogenesis. Interestingly, recent reports illustrated that exosomes from body fluids could inhibit HIV infection, which then bring up a new paradigm for HIV/AIDS therapy. Accumulative findings suggested that the cellular origin of exosomes may define their effects towards HIV-1. This review summarizes the two distinctive roles of exosomes in regulating HIV pathogenesis. We also highlighted several additional factors that govern the exosomal functions. Deeper understanding on how exosomes promote or abate HIV infection can significantly contribute to the development of new and potent antiviral therapeutic strategy and vaccine designs