DNA methylation and expression analyses reveal epialleles for the foliar disease resistance genes in peanut (Arachis hypogaea L.).

OBJECTIVE: Low DNA sequence polymorphism despite enormous phenotypic variations in peanut indicates the possible role of epigenetic variations. An attempt was made to analyze genome-wide DNA methylation pattern and its influence on gene expression across 11 diverse genotypes of peanut. RESULTS: The genotypes were subjected to bisulfite sequencing after 21 days of sowing (DAS). CHG regions showed the highest (30,537,376) DNA methylation followed by CpG (30,356,066) and CHH (15,993,361) across 11 genotypes. The B sub-genome exhibited higher DNA methylation sites (46,294,063) than the A sub-genome (30,415,166). Overall, the DNA methylation was more frequent in inter-genic regions than in the genic regions. The genes showing altered methylation and expression between the parent (TMV 2) and its EMS-derived mutant (TMV 2-NLM) were identified. Foliar disease resistant genotypes showed significant differential DNA methylation at 766 sites corresponding to 25 genes. Of them, two genes (Arahy.1XYC2X on chromosome 01 and Arahy.00Z2SH on chromosome 17) coding for senescence-associated protein showed differential expression with resistant genotypes recording higher fragments per kilobase of transcript per million mapped reads (FPKM) at their epialleles. Overall, the study indicated the variation in the DNA methylation pattern among the diverse genotypes of peanut and its influence of gene expression.

Does cognitive function influence alaryngeal speech rehabilitation?

BACKGROUND: We sought to define the cognitive domains that influence valved speech rehabilitation. METHODS: Sixteen laryngectomees with primary tracheoesophageal punctures were randomly recruited from one head and neck unit. They were assessed by a consultant neuropsychologist and a speech therapist. Speech therapy time was determined from speech therapy notes. RESULTS: The Digit Symbol Substitution Test, assessing learning speed and processing speed, correlated significantly with speech therapy time in the first (p = .002) and third (p = .014) postoperative years, respectively. Categorical fluency assessment correlated positively with speech therapy time in the first year (p = .009). Learning speed (p = .007) and categorical fluency (p = .041) correlated positively with the fall in speech therapy input between the first and third year after laryngectomy. CONCLUSIONS: Learning speed, processing speed, and categorical fluency strongly influence alaryngeal speech rehabilitation. This study highlights the potential for pre-laryngectomy cognitive assessment to help plan alaryngeal speech rehabilitation. This has significant resource implications.

Contemporary surgical treatment of epistaxis. What is the evidence for sphenopalatine artery ligation?

The optimal surgical management for failed conservative measures in epistaxis remains unclear. Given the growing enthusiasm for endoscopic transnasal sphenopalatine artery ligation, it is prudent and timely to evaluate the evidence base for this technique. This study aims to analyse the current evidence for transnasal endoscopic sphenopalatine artery ligation by reviewing the literature and also by comparing the results with other approaches to the management of epistaxis. A detailed literature search identified 11 publications relating to endoscopic sphenopalatine artery ligation. The total number of patients in the pooled series was 127, of which 98% had control of epistaxis following surgery. These results compared favourably to the results of most other techniques used in the modern treatment of epistaxis. Nonetheless, the total number of patients in the 11 case series is small. It is therefore recommended that all units using this technique audit their results to see if the high success rates achieved in the literature are reproducible. If this is the case, then endoscopic sphenopalatine artery ligation may indeed be the surgical answer to intractable posterior epistaxis.

A critical audit of the surgical management of intractable epistaxis using sphenopalatine artery ligation/diathermy.

An audit of the local practice of sphenopalatine artery (SPA) ligation/diathermy was undertaken following its introduction to the unit in April 1998. The authors looked to the literature for evidence of what was to be taken as a successful result and were surprised at the lack of published data on its efficacy or lack thereof. Fivehundredsixtythree patients were admitted for inpatient management of epistaxis over a two-year period. Ten of these patients required surgical control of epistaxis and went on to have either sphenopalatine artery ligation or diathermy. One had concomitant anterior ethmoidal artery diathermy. Postoperatively, the patients stayed between one to ten days (mean 3.3 days). The mean follow up in the clinic was 42.7 days. A recurrent bleed rate of 33% was found, which is higher than previously published results. Of these failures one had internal maxillary embolization followed by anterior ethmoidal artery ligation. The other two failures were successfully corrected with anterior ethmoidal ligation. The authors discuss and illustrate the possible reasons for failure.

Novel bioemulsifiers from microorganisms for use in foods.

The main objective of this study was to test the range of microorganisms for production of extracellular, high molecular weight emulsifiers for potential use in foods. A standard emulsification assay developed specifically for assessing food emulsifiers was used to examine 24 extracellular microbial products from bacteria, yeasts and algae. Of the 24 products tested, nine had emulsification ability that was as good as and eight had emulsifying properties that were better than those of the commonly used food emulsifiers gum arabic and carboxymethylcellulose. The eight good producer organisms included the yeasts Candida utilis, Candida valida, Hansenula anomala, Rhodospiridium diobovatum and Rhodotorula graminis, the red alga Porphiridium cruentum, and the bacteria Klebsiella spp. and Acinetobacter calcoaceticus. Of these, C. utilis was selected for further study due to the excellent emulsification properties of its extracellular products and food-grade status of the organism. Crude preparations of the bioemulsifier from C. utilis exhibited low viscosity and had a carbohydrate content of over 80%. Preliminary trials showed that the bioemulsifier from this organism had potential for use in salad cream.

Effect of interferon-gamma on the expression of transforming growth factor-beta by human corneal fibroblasts: role in corneal immunoseclusion.

Human corneal fibroblasts (HCF) inhibit T cell alloresponse in mixed leukocyte response-human corneal fibroblast coculture. The inhibition is contact independent, insensitive to indomethacin, and is enhanced by pretreatment of HCF with interferon-gamma (IFN-gamma). To investigate cytokine-dependent mechanisms of inhibition of T cell alloresponse by HCF, the capacity of cultured HCF to produce transforming growth factor-beta (TGF-beta) and the modulatory role of IFN-gamma on their TGF-beta production were investigated by radioreceptor binding inhibition assay (RRA) and the standard mink cell bioassay (BIA). The net total TGF-beta concentration of 4 day culture supernatants from IFN-gamma-treated HCF, measured by RRA, was 11.5 ng/ml. The net total bioactive TGF-beta concentrations of 4 day culture supernatants from HCF, before and after treatment with IFN-gamma, measured by BIA, were 2.0 and 4.8 ng/ml, respectively. These findings indicate that HCF produce TGF-beta and increase their TGF-beta output under the influence of the proinflammatory cytokine IFN-gamma. Media-borne TGF-beta binding proteins appeared to be primarily responsible for the discrepancy between the TGF-beta values measured by RRA and BIA. Active exclusion of TGF-beta binding proteins from intraocular fluids may have an important role in the maintenance of TGF-beta-dependent ocular immune privilege. Corneal fibroblasts may utilize TGF-beta-dependent mechanisms to maintain the immunosecluded environment of the cornea and to preserve the homeostasis of corneal optical competency. Interferon-gamma may enhance corneal immunoseclusion by upregulating the TGF-beta output of the corneal fibroblasts.

Interferon-gamma induces high-affinity transforming growth factor-beta receptor expression on human corneal fibroblasts.

The effect of interferon-gamma (IFN-gamma) on the expression of transforming growth factor-beta (TGF-beta) receptors on cultured human corneal stromal fibroblasts was examined. Scatchard analysis of specific saturable TGF-beta 1 binding data indicated that corneal fibroblasts expressed TGF-beta receptors with an average association constant of 6 x 10 M-1, before and after IFN-gamma treatment. An additional population of higher affinity TGF-beta receptors, with an average association constant of 4 x 10(12) M-1, was demonstrated only on IFN-gamma-treated corneal fibroblasts Interferon-gamma may alter the response of corneal fibroblasts to transforming growth factor-betas by upregulating their higher affinity TGF-beta receptors. The induction of higher affinity TGF-beta receptors by an immune cytokine and an associated autocrine elevation of TGF-beta output by the corneal fibroblasts may be a transient compensatory mechanism that maintains the homeostasis of corneal optical competency through enhancement of corneal immunoseclusion.

A soluble product of human corneal fibroblasts inhibits lymphocyte activation. Enhancement by interferon-gamma.

Corneal stromal fibroblasts expressed HLA-DP, -DQ and -DR Class II MHC antigens in response to interferon-gamma, but did not induce proliferative responses by allogeneic peripheral blood mononuclear cells in vitro when used as stimulator cells in a mixed leukocyte-type reaction. Furthermore, corneal stromal fibroblasts inhibited mixed leukocyte reactions between peripheral blood mononuclear cells of allogeneic donors, even when the corneal stromal fibroblasts were separated from the peripheral blood mononuclear cells by a 0.4 micron pore membrane. Pretreatment of the corneal stromal fibroblasts with interferon-gamma increased the inhibitory activity. Both [3H]thymidine incorporation and interleukin-2 production were inhibited, and the inhibition appeared to be mediated by a soluble factor whose production required protein synthesis. The inhibitory activity was not abolished by including 1-10 micrograms ml-1 indomethacin in the culture media. No inhibition was observed in the proliferation dose-response curves of responder peripheral blood mononuclear cells that had been cultured with corneal stromal fibroblasts for 3 days, prior to culture with allogeneic stimulator peripheral blood mononuclear cells. Thus, the ability of corneal stromal fibroblasts to interfere with alloimmune responses in vitro was dependent upon the continued presence of the fibroblasts and their continued production of a soluble inhibitory factor or factors. Inhibitors of allogeneic reactions that are produced by corneal stromal fibroblasts stimulated by immune cytokines (e.g. interferon-gamma) may play a role in prolonging corneal allograft survival.

Histamine and prostacyclin. Primary and secondary release in allergic conjunctivitis.

The relationship between the release of histamine, a major mast cell mediator of conjunctival type I reactions, and the production of a prostanoid, prostacyclin (prostaglandin I2, PGI2), was examined in a guinea pig model of allergic conjunctivitis. Guinea pigs were sensitized topically and challenged by repeated conjunctival instillation of fluoresceinyl ovalbumin. Histamine and 6-keto-PGF1 alpha, the stable product of the spontaneous degradation of PGI2, were measured in tears by radioimmunoassays. Clinical type I reactions and tear histamine appeared by 8 days and increased up to 22 days during the initial sensitization, with notable variations between animals. The kinetics of histamine and 6-keto-PGF1 alpha release in tears were examined over a 24-hr period after the antigen challenge. Histamine release was maximal during the first 10 min and returned to baseline values by 1 hr in all instances. The 6-keto-PGF1 alpha release also peaked during the first 10 min but continued for an extended period. The ratio of tear 6-keto-PGF1 alpha to histamine increased more than 16-fold over the 2 hr after antigen challenge. Late-phase reactions with second peaks of histamine or 6-keto-PGF1 alpha in the tears were observed in two different guinea pigs 4-8 hr after antigen challenge. Histamine applied to the eyes of naive guinea pigs also induced the release of 6-keto-PGF1 alpha in tears. Histamine appeared to act as a primary mediator, stimulating the secondary production and release of PGI2 by constitutive (eg, vascular) and possibly infiltrating inflammatory cells during an allergic conjunctival reaction.

Experimental ocular onchocerciasis: local and systemic antibody and cell-mediated immune responses.

Hartley guinea pigs injected subconjunctivally with Onchocerca lienalis (OL) microfilariae (Mf) develop punctate corneal opacities resembling the punctate keratitis of human onchocerciasis. Antibody production and antigen-induced proliferative responses were studied in conjunctival-associated lymphoid tissues (CALT), spleens (SL) and peripheral blood lymphocytes (PBL) from experimentally infected guinea pigs. Cultured single cell suspensions of CALT, SL and PBL were assayed for IgG1, IgG2, IgA and IgE antibody production. IgG1, IgG2, and IgA Onchocerca-specific antibodies were found in culture supernatants of CALT, SL and PBL. When initiated 10 days after a challenge injection of OL, CALT cultures produced antibody levels equal to or less than those produced by the corresponding SL cultures. When initiated 66 days after the last injection of Mf, CALT cultures produced significantly more antibody than the corresponding SL cultures. Blastogenic responses to OL Mf antigen were observed in peripheral and splenic lymphocytes of OL-infected guinea pigs. Animals given subconjunctival injections of Mf followed by treatment with a microfilaricide had greater responses to OL antigen than those given Mf alone, while responses to phytomitogens were similar in drug-treated and non-treated animals. The CALT was locally immunologically responsive against the subconjunctivally injected OL Mf, with the capacity for localized memory responses. The local immunologic responses to conjunctival Onchocerca microfilariae may play a significant role in the immunopathological reactions of ocular onchocerciasis.

Class II alloantigen induced on corneal endothelium. Role in corneal allograft rejection.

The expression of Class II major histocompatibility complex (MHC) antigens on corneal cells can be increased in vitro by (gamma-interferon) and in vivo in inflammatory reactions. The expression of Class II MHC by corneal endothelium of New Zealand White (NZW) rabbits during the rejection of corneal allografts was demonstrated by immunoperoxidase staining. Class II MHC expression by corneal endothelial cells may facilitate rejection of corneal allografts.

Massive follicular lymphoid hyperplasia in experimental allergic conjunctivitis. Local antibody production.

Acute and recurrent allergic conjunctival reactions were induced in guinea pigs by repeated conjunctival applications of fluoresceinyl ovalbumin (FL-OA) for up to 30 months. Early type I conjunctival reactions developed 11 to 25 days after the initial conjunctival exposure to FL-OA. Continuous topical challenges during a six- to 30-month period caused a variety of reactions, including papillary changes and massive hyperplasia of the conjunctival-associated lymphoid tissues. Hyperplasia of lymphoid tissues was induced during a shorter period (two to five months) with a mixture of FL-OA and phorbol ester. Culture fluid from hyperplastic conjunctival lymphoid tissue showed a ratio of IgG1/IgG2 antibody production of up to 15. A low level of recurrence of type I reactivity, after an initial desensitization phenomenon due to a loss of reactive mast cells, correlated with prominent follicular hyperplasia of the conjunctival-associated lymphoid tissue.

Activation of Mg-ATPase of skeletal-muscle myosin by bovine retinal pigment epithelial actin.

The morphology and function of actin in cultured bovine retinal pigment epithelial (RPE) cells were studied. Filamentous actin was identified with a fluorescent mushroom toxin, nitrobenzoxadiazole (NBD)-phallacidin, specific for actin. Dark-field microscopy of cultured RPE cells revealed numerous pigment granules; fluorescent microscopy identified scattered lipofuscin granules. One-dimensional SDS polyacrylamide gel electrophoresis of urea-soluble proteins extracted from RPE cells showed a 46,000-dalton protein band which comigrated with authentic muscle actin. Densitometric scanning showed that this protein band comprised 7.6% of the total urea-soluble proteins. An actin-activated skeletal-muscle myosin Mg-ATPase assay, using skeletal-muscle heavy meromyosin as enzyme and [gamma-32P]-ATP as substrate, demonstrated functional actin in RPE cell extracts after DEAE-cellulose anion exchange chromatography. The actin-containing protein fractions were eluted at ionic strengths between 0.19 and 0.36 M KCl. The activation of myosin ATPase by actin in RPE cells provides a molecular basis for the phagocytic activity which is important in maintaining the integrity of retinal photoreceptor cells.

Experimental ocular onchocerciasis in cynomolgus monkeys. II. Chorioretinitis elicited by intravitreal Onchocerca lienalis microfilariae.

Chorioretinitis due to onchocerciasis is a major cause of blindness, and the pathogenesis is poorly understood. We have developed an experimental model for onchocercal chorioretinitis using cynomolgus monkeys (Macaca fascicularis). Two normal monkeys and two monkeys which had received prior sensitization with subcutaneous injections of live Onchocerca lienalis microfilariae were given intravitreal injections of either 0, 10, 50 or 500 live microfilariae. Posterior segment changes included disc edema, venous engorgement, retinal vasculitis, intraretinal hemorrhage, and progressive retinal pigment epithelial (RPE) disturbances. Histopathological findings included perivascular infiltrates with eosinophils, eosinophilic choroiditis, and RPE hypertrophy, hyperplasia and loss of pigment. Microfilariae in the retina had no surrounding inflammation but were found adjacent to areas of RPE alterations. Overall the inflammatory reaction in the two unsensitized monkeys was more severe than that seen in the sensitized monkeys. The retinal appearance of the monkeys resembled that found in human onchocerciasis, and this model appears to be a promising one for future investigations.

Prostacyclin is the major prostaglandin synthesized by bovine retinal capillary pericytes in culture.

Prostaglandin synthesis by bovine retinal pericytes was investigated using high pressure liquid chromatography to separate and identify 3H-labeled prostaglandins released from 3H-arachidonic acid labeled pericyte monolayers. A dominant peak activity corresponding to 6-keto-PGF1 alpha was observed. This peak was eliminated when monolayers were pretreated with cyclooxygenase inhibitors and was augmented when monolayers were stimulated by the calcium ionophore A23187. Suspensions of pericytes and the cell-free media of monolayers incubated with arachidonic acid inhibited adenosine diphosphate-, collagen-, and arachidonic acid-stimulated platelet aggregation in a bioassay for prostacyclin-like activity. This inhibitory activity was unstable at room temperature. Cultures of 7.5 to 10 x 10(5) pericytes (7th passage near-confluence) released nanogram quantities of 6-keto-PGF1 alpha as measured by radioimmunoassay. These results are evidence that prostacyclin is the main prostaglandin synthesized by bovine retinal capillary pericytes in culture. Pericytes may influence the microcirculation via their production and release of this potent vasoactive arachidonic acid metabolite.

Regulation of uptake of inositol by glucose in cultured retinal pigment epithelial cells.

Long term and acute effects of glucose on myo-inositol (MI) uptake were studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. RPE cells were grown under low (5 mM) or high (20, 40, or 50 mM) glucose levels in the growth medium for up to 18 days. The concentrative capacity of confluent RPE cells to accululate [3H]MI (10 microM) was reduced up to 41% as the glucose concentration in the growth medium increased. When the growth medium glucose was switched from 5 to 40 mM, or vice versa, the capacity of cells to accumulate MI was reversed. Treatment of cells grown in 40 or 50 mM glucose with 0.1 mM Sorbinil (an aldose reductase inhibitor) minimally reversed the ability of cells to accumulate MI. RPE cells, grown in 5 mM glucose, were incubated with 10-60 mM D-glucose or its nonmetabolizable analogues (acute effect). Kinetics of MI uptake inhibition by alpha-methyl glucose according to Dixon plots were characteristic of competitive inhibition (Ki = 28 mM). MI uptake was strongly inhibited by phlorizin. The ability of RPE cells to bind 5 microM [3H]phlorizin also was reduced by increased glucose levels in the growth medium. These studies indicated that MI and glucose shared the same transporter system. Glucose in the incubation medium directly interfered with MI binding to the transporter. High glucose feeding of the cells also down-regulated the transporter density. The uptake and function of solutes such as MI in tissues that operate on the glucose carrier system may be severely impaired in diabetes.

Experimental allergic conjunctivitis: production of different isotypes of antibody by conjunctival-associated lymphoid tissue in culture.

Long-term (greater than 2 years) topical, conjunctival application of fluoresceinyl ovalbumin (FL-OA) induced allergic conjunctivitis-like lesions and hyperplasia of conjunctival-associated lymphoid tissue (CALT) in guinea pigs. Single-cell suspensions of CALT and spleen were prepared by collagenase digestion and cultured with or without FL-OA or lipopolysaccharide; the culture supernatants were assayed for IgG, IgA, IgM, and IgE antibody. Absolute values (ng Ab protein/ml) of anti-FL-OA IgG subclasses (IgG1 and IgG2) were measured using purified preparations of IgG1 and IgG2 anti-FL-OA antibody standards in an enzyme-linked immunosorbent assay. Immunohistochemical studies were performed using frozen sectioned CALT tissues as well as cultured single cells. IgG1, IgG2, IgA, IgM, but not IgE, anti-FL-OA antibodies were detected in the culture supernatants of both CALT and spleen. IgG- and IgA-secreting plasma cells were demonstrated in immunoperoxidase-stained CALT and single-cell cultures. The ratio of IgG1 to IgG2 isotypes produced by CALT in vitro was significantly higher than that produced by spleen and also that found in serum. These findings indicated that a site-specific regulation of antibody isotypes may exist within the hyperplastic CALT induced by the long-term topical exposure to FL-OA.

Effect of human corneal fibroblasts on lymphocyte proliferation in vitro.

Cocultivation of human corneal fibroblasts (CFB) with allogeneic peripheral blood mononuclear cells (PBL) induced a minimal lymphocyte proliferative response, even when the fibroblasts had been pre-treated with interferon-gamma (IFN-gamma) and were HLA-DR positive. IFN-gamma-treated CFB did not express detectable HLA-DQ alloantigens. Cocultivation of CFB with PBL in the presence of Concanavalin A (Con A) or Phytohemagglutinin inhibited mitogen-induced lymphocyte proliferation by 40-90% relative to PBL cultured without CFB. Induction of HLA-DR expression on the CFB did not alter their inhibitory properties. Addition of indomethacin to cultures reversed the effect of CFB on Con A responses. However, no difference between proliferative responses to HLA-DR positive or negative CFB was seen in the presence of indomethacin. This weak response to induced Class II alloantigens on CFB suggests that induction of Class II alloantigens on corneal stroma alone may be insufficient to sensitize a recipient for Class II alloantigen-driven corneal graft rejection.