Advanced glycation endproduct modified basement membrane attenuates endothelin-1 induced [Ca2+]i signalling and contraction in retinal microvascular pericytes

PURPOSE: To assess the effects of advanced glycation endproduct (AGE) modification of vascular basement membrane (BM) on endothelin-1 (Et-1) induced intracellular [Ca2+] ([Ca2+]i) homeostasis and contraction in retinal microvascular pericytes (RMP). METHODS: RMPs were isolated from bovine retinal capillaries and propagated on AGE modified BM extract (AGE-BM) or non-modified native BM. Cytosolic Ca2+ was estimated using fura-2 microfluorimetry and cellular contraction determined by measurement of planimetric cell surface area. ETA receptor mRNA and protein expression was assessed by real time RT-PCR and western blotting, respectively. RESULTS: Exogenous endothelin-1 (Et-1) evoked rises in [Ca2+]i and contraction in RMPs were found to be mediated entirely through ETA receptor (ETAR) activation. Both peak and plateau phases of the Et-1 induced [Ca2+]i response and contraction were impaired in RMPs propagated on AGE modified BM. ETAR mRNA expression remained unchanged in RMPs exposed to native or AGE-BM, but protein expression for ETAR (66 kDa) was lower in the AGE exposed cells. CONCLUSIONS: These results suggest that substrate derived AGE crosslinks can influence RMP physiology by mechanisms which include disruption of ETA receptor signalling. AGE modification of vascular BMs may contribute to the retinal hemodynamic abnormalities observed during diabetes.

Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica

A cysteine proteinase released in vitro by Fasciola hepatica was purified to homogeneity by Sephacryl S-200 gel filtration chromatography followed by QAE-Sephadex chromatography. The purified enzyme resolves as a single band with an apparent molecular size of 27 kDa on reducing SDS-polyacrylamide gel electrophoresis; however, under non-reducing conditions it migrates as multiple bands, each with enzymatic activity, in the apparent molecular size range 60-90 kDa. The sequence of the first 20 N-terminal amino acids of the enzyme shows considerable homology with cathepsin L-like proteinases. Immunolocalisation studies revealed that the cathepsin L-like proteinase is concentrated within vesicles in the gut epithelial cells of liver fluke.

Fasciola hepatica in the rat:immune responses associated with the development of resistance to infection

F. hepatica infections were established in rats and immune responses were monitored during primary and challenge infections. Antibody levels peaked at 3 weeks post-primary infection and at 6 days post-challenge infection. No significant correlation was found between antibody titre and number of flukes recovered at autopsy. Immunoblotting revealed a limited number of immunogenic polypeptides. When antibodies from these reactive bands were eluted and tested by IFA they all gave identical binding patterns: on juvenile fluke sections tegumental syncytium, tegumental cells and gut cells were labelled, while on adult sections the same antibodies labelled gut cells, reproductive tissue, excretory ducts and flame cells. This suggested that these tissues shared a common epitope or range of epitopes. A pronounced eosinophilia was observed throughout the infection period studied and infected liver sections showed massive cellular infiltration. Histochemical and immunocytochemical investigation of infected liver revealed the presence of large numbers of eosinophils, neutrophils, lymphocytes and phagocytes. The implications of these findings, to an understanding of concomitant immunity in the rat are discussed.

Localisation of actin in the liver fluke, Fasciola hepatica

Adult and 3-week-old juvenile Fasciola hepatica were examined for the presence of the cytoskeletal protein actin. Techniques of direct fluorescence using fluorescein isothiocyanate (FITC)-phalloidin and of indirect immunofluorescence using a monoclonal anti-actin antibody (MAA) demonstrated actin in the testes, sub-tegumental and gut musculature, tegumental cell bodies and tegumental spines. In contrast, polyclonal anti-actin antibody (PAA) revealed immunostaining only in the vitellaria. Effective removal of the tegument with 1% (w/v) sodium dodecyl sulphate (SDS) was confirmed by scanning electron microscopy (SEM), and this enabled immunoblotting of whole fluke and tegumental fractions with and without spines. Whole fluke fractions produced three bands corresponding to molecules exhibiting relative molecular weights of 43, 28 and 15 kDa, respectively, whereas the tegumental fraction with spines revealed a single band corresponding to 15 kDa in size. The fraction without spines displayed no bands. The present study localised actin in a number of different tissue types within the liver fluke. Using MAA, three forms of actin have been identified in the whole fluke and a single one in the tegumental spines.

Fasciola hepatica:localization and partial characterization of tubulin

The localisation and distribution of the cytoskeletal protein tubulin in the adult liver fluke Fasciola hepatica have been determined by an indirect immunofluorescence technique using a monoclonal antibody raised against beta-tubulin. Tubulin was demonstrated in the tegumental syncytium and in the tegumental cell bodies and their cytoplasmic connections with the surface syncytium. Immunostaining was also evident in the nerve fibres innervating sensory receptors in the tegument, in the nerve plexus innervating the sub-tegumental musculature and in the cytoplasmic extensions of the nurse cells within the vitelline follicle. Immunoblotting of whole fluke fractions produced a single band corresponding to a molecule of approximately 54 kDa in size. This figure corresponds with previous data obtained on tubulin from other helminth and eukaryotic sources.

Detection and partial characterization of human B cell colony stimulating activity in synovial fluids of patients with rheumatoid arthritis

The joint fluids of 37 patients with rheumatoid arthritis, eight patients with traumatic injuries to their joints, two patients with Reiter's syndrome and three patients with psoriatic arthritis were tested for the presence of B cell colony stimulating activity (B cell CSA). B cell CSA was found in all of the joint fluids from the patients with rheumatoid arthritis but in none of the joint fluids from patients with traumatic injuries to their joints or in the joint fluids from the patients with Reiter's syndrome. A trace of B cell CSA was found in the joint fluid of one of the three patients with psoriatic arthritis. There was a positive correlation (r = 0.796) between the amount of rheumatoid factor present in the joint fluids and the titre of B cell CSA. This correlation was highly significant (P less than 0.001). The B cell CSA was localized to component(s) with molecular weight ranges 115-129 kD and 64-72 kD and an isoelectric point of 6.8. Its activity was sensitive to reduction with 2-mercaptoethanol and to the oxidising action of potassium periodate.

Biochemical and physiochemical characteristics of a pre-B cell stimulating factor produced by the plasmacytoma cell line LICR LON HMY2

We have characterized the pre-B cell colony stimulating activity (pre-B cell CSA) from LICR LON HMY2 conditioned medium (CM) by a variety of biochemical techniques. Pre-B cell CSA was found to be associated with a heat stable glycoprotein which has an isoelectric point of 8.3 and a mol. wt, as determined by polyacrylamide gel electrophoresis, of 28-32 kD. The relationship of this activity to previously described factors acting on cells of the B cell lineage is discussed.

Roles of multiple flagellins in flagellar formation and flagellar growth post bdelloplast lysis in Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus cells have a single polar flagellum whose helical pitch and diameter characteristically change near the midpoint, resulting in a tapered wave. There are six flagellin genes in the genome: fliC1 to fliC6. Accordingly, the flagellar filament is composed of several similar flagellin species. We have used knockout mutants of each gene and analyzed the mutational effects on the filament length and on the composition and localization of each flagellin species in the filament by electron microscopy and one- and two-dimensional polyacrylamide gel electrophoresis. The location and amounts of flagellins in a filament were determined to be as follows: a small amount of FliC3 at the proximal end, followed by a large amount of FliC5, a large amount of FliC1, a small amount of FliC2 in this order, and a large amount of FliC6 at the distal end. FliC4 was present at a low level, but the location was not determined. Filament lengths of newly born progeny cells increased during prolonged incubation in nutrient-deficient buffer. The newly formed part of the elongated filament was composed of mainly FliC6. Reverse transcription PCR analysis of flagellar gene expression over 5 days in buffer showed that fliC gene expression tailed off over 5 days in the wild-type cells, but in the fliC5 mutant, expression of the fliC2, fliC4, and fliC6 genes was elevated on day 5, suggesting that they may be expressed to compensate for the absence of a major component, FliC5.

Studying N-linked glycosylation of receptor tyrosine kinases

Metabolic alterations have been identified as a frequent event in cancer. This is often associated with increased flux through glycolysis, and also a secondary pathway to glycolysis, hexosamine biosynthetic pathway (HBP). HBP provides substrate for N-linked glycosylation, which occurs in the endoplasmic reticulum and the Golgi apparatus. N-linked glycosylation supports protein folding and correct sorting of proteins to plasma membrane and secretion. This process generates complex glycoforms, which can be recognized by other proteins and glycosylation of receptor tyrosine kinases (RTK) can also regulate their plasma-membrane retention time. Of special interest for experimental biologists, plants produce proteins, termed lectins, which bind with high specificity to glyco-conjugates. For the purposes of molecular biology, plant lectins can be conjugated to different moieties, such as agarose beads, which enable precipitation of specifically glycosylated proteins. In this chapter, we describe in detail how to perform pull-down experiments with commercially available lectins to identify changes in the glycosylation of RTKs.

Transient receptor potential (TRP) channel expression in pulpal fibroblasts

Introduction: Transient receptor potential (TRP) channels are widely, but not uniformly, distributed in tissues. To date the dominant focus of attention has been on TRP expression and functionality in neurons. However, their expression and activation in selected non-neuronal cells suggest TRPs have a potential role in coordinating cross-talk during the inflammatory process. Fibroblasts comprise the major cell type in the dental pulp and play an important role in pulpal inflammation. Objectives: The aim of this study was to investigate the expression and functionality of the TRP channels TRPA1, TRPM8, TRPV4 and TRPV1 in human dental pulp fibroblasts. Methods: Dental pulp fibroblasts were derived by explant culture of pulps removed from extracted healthy teeth. Fibroblasts were cultured in DMEM supplemented with 10% FCS, 100U/ml penicillin and 100µg/ml streptomycin. Protein expression of TRP channels was investigated by SDS- polyacrylamide gel electrophoresis and Western blotting of cell lysates from fibroblast cells in culture. TRPA1, TRPM8, TRPV4 and TRPV1 expression was determined by specific antibodies, detected using appropriate anti-species antibodies and chemiluminescence. Functionality of TRP channels was determined by Ca2+ microfluorimetry. Cells were grown on cover slips and incubated with Fura 2AM prior to stimulation with icilin (TRPA1 agonist), menthol (TRPM8 agonist), 4 alpha-phorbol 12,13-didecanoate (4alphaPDD) (TRPV4 agonist) or capsaicin (TRPV1 agonist). Emitted fluorescence (F340/F380) was used to determine intracellular [Ca2+] levels. Results: Fibroblast expression of TRPA1, TRPM8, TRPV4 and TRPV1 was confirmed at the protein level by Western blotting. Increased intracellular [Ca2+] levels in response to icillin, methanol, 4alphaPDD and capsacin, indicated functional expression of TRPA1, TRPM8, TRPV4 and TRPV respectively. Conclusions: The presence and functionality of TRP channels on dental pulp fibroblasts suggests a potential role for these cells in the pulpal neurogenic inflammatory response. (Supported by a research grant from the Royal College of Surgeons of Edinburgh).

Functional expression of thermo-sensitive TRP channels in human odontoblasts

Introduction: Accumulating evidence supports a role for odontoblasts in initiating tooth pain, however direct ionic mechanisms underlying dentine nociceptive function remain unclear. The transient receptor potential (TRP) ion channels are directly related to cellular mechanisms of nociception and thermo-sensitive function but their expression by human odontoblasts remains to be determined. Objectives: To investigate the expression and functionality of the thermo-sensitive TRP channels TRPV1, TRPV4, TRPM8 and TRPA1 in human odontoblasts. Methods: Human odontoblasts were derived from dental pulp of immature permanent third molars by explant method. Cell lysates of odontoblasts were subject to SDS- polyacrylamide gel electrophoresis and proteins were blotted onto nitrocellulose membranes. Blots were probed with primary antibodies to TRPA1, TRPM8, TRPV4 and TRPV1. Detection of bound primary antibodies was achieved using appropriate anti-species antibody conjugates and chemiluminescent substrates. Functionality of the channels was determined with Ca2+ microfluorimetry, where cells grown in cover slips and incubated with Fura 2AM prior to stimulation with capsaicin (TRPV1 agonist), 4 alpha-phorbol 12,13-didecanoate (4áPDD) (TRPV4 agonist), icilin (TRPA1 agonist) and menthol (TRPM8 agonist). Emitted fluorescence was measured and the fluorescence ratio (R) was calculated as F340/F380 to determine the level of [Ca2+]i. Results: Western blotting confirmed the molecular localisation of thermo-sensitive TRP channels in human odontoblasts. Functionality assays revealed increase in [Ca2+]i in response to capsacin, icillin, methanol and 4áPDD indicating functional expression of TRPV1, TRPA1, TRPM8 and TRPV4 respectively. Conclusions: Functional expression of thermo-sensitive TRP channels in human odontoblasts may indicate a crucial role for odontoblasts in thermally induced dental pain. (Supported by a Research Grant from the Royal College of Surgeons of Edinburgh)

Heteroduplex analysis of VDJ amplified segments from rearranged IgH genes for clonality assessments in B-cell non-Hodgkin's lymphoma. A comparison between different strategies.

BACKGROUND AND OBJECTIVE: The main difficulty of PCR-based clonality studies for B-cell lymphoproliferative disorders (B-LPD) is discrimination between monoclonal and polyclonal PCR products, especially when there is a high background of polyclonal B cells in the tumor sample. Actually, PCR-based methods for clonality assessment require additional analysis of the PCR products in order to discern between monoclonal and polyclonal samples. Heteroduplex analysis represents an attractive approach since it is easy to perform and avoids the use of radioactive substrates or expensive equipment. DESIGN AND METHODS: We studied the sensitivity and specificity of heteroduplex PCR analysis for monoclonal detection in samples from 90 B-cell non Hodgkin's lymphoma (B-NHL) patients and in 28 individuals without neoplastic B-cell disorders (negative controls). Furthermore, in 42 B-NHL and in the same 28 negative controls, we compared heteroduplex analysis vs the classical PCR technique. We also compared ethidium bromide (EtBr) vs. silver nitrate (AgNO(3)) staining as well as agarose vs. polyacrylamide gel electrophoresis (PAGE). RESULTS: Using two pair consensus primers sited at VH (FR3 and FR2) and at JH, 91% of B-NHL samples displayed monoclonal products after heteroduplex PCR analysis using PAGE and AgNO(3) staining. Moreover, no polyclonal sample showed a monoclonal PCR product. By contrast, false positive results were obtained when using agarose (5/28) and PAGE without heteroduplex analysis: 2/28 and 8/28 with EtBr and AgNO(3) staining, respectively. In addition, false negative results only appeared with EtBr staining: 13/42 in agarose, 4/42 in PAGE without heteroduplex analysis and 7/42 in PAGE after heteroduplex analysis. INTERPRETATION AND CONCLUSIONS: We conclude that AgNO(3) stained PAGE after heteroduplex analysis is the most suitable strategy for detecting monoclonal rearrangements in B-NHL samples because it does not produce false-positive results and the risk of false-negative results is very low.

Stability of 5-aminolevulinic acid in novel non-aqueous gel and patch-type systems intended for topical application.

Aminolevulinic acid (ALA) stability within topical formulations intended for photodynamic therapy (PDT) is poor due to dimerisation to pyrazine-2,5-dipropionic acid (PY). Most strategies to improve stability use low pH vehicles, which can cause cutaneous irritancy. To overcome this problem, a novel approach is investigated that uses a non-aqueous vehicle to retard proton-induced charge separation across the 4-carbonyl group on ALA and lessen nucleophilic attack that leads to condensation dimerisation. Bioadhesive anhydrous vehicles based on methylvinylether-maleic anhydride copolymer patches and poly(ethyleneglycol) or glycerol thickened poly(acrylic acid) gels were formulated. ALA stability fell below pharmaceutically acceptable levels after 6 months, with bioadhesive patches stored at 5°C demonstrating the best stability by maintaining 86.2% of their original loading. Glycerol-based gels maintained 40.2% in similar conditions. However, ALA loss did not correspond to expected increases in PY, indicating the presence of another degradative process that prevented dimerisation. Nuclear magnetic resonance (NMR) analysis was inconclusive in respect of the mechanism observed in the patch system, but showed clearly that an esterification reaction involving ALA and both glycerol and poly(ethyleneglycol) was occurring. This was especially marked in the glycerol gels, where only 2.21% of the total expected PY was detected after 204 days at 5°C. Non-specific esterase hydrolysis demonstrated that ALA was recoverable from the gel systems, further supporting esterified binding within the gel matrices. It is conceivable that skin esterases could duplicate this finding upon topical application of the gel and convert these derivatives back to ALA in situ, provided skin penetration is not affected adversely.

A metal-organic gel used as a template for a porous organic polymer

A polymeric metal-organic gel is described, which acts as a template in the preparation of macroporous polymethylmethacrylate, and can be easily removed post polymerisation.