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Electroantennogram and oviposition bioassay responses of Culex quinquefasciatus and Culex tarsalis (Diptera: Culicidae) to chemicals in odors from Bermuda grass infusions.

Odors were collected from the headspace above fermented infusions of Bermuda grass, which commonly are used as attractants in traps for gravid mosquitoes. Coupled gas chromatography-electroantennogram detection (GC-EAD) was used to identify 9 compounds (phenol, p-cresol, 4-ethylphenol, indole, 3-methylindole, nonanal, 2-undecanone, 2-tridecanone, naphthalene) from odor extracts that elicited significant antennal responses from antennae of gravid female Culex quinquefasciatus Say and Culex tarsalis Coquillett mosquitoes. Several of these compounds at appropriate concentrations were weakly attractive to gravid female mosquitoes in laboratory bioassays and/or stimulated more oviposition than water controls. In addition, dimethyltrisulfide, a significant component of odor extract which did not elicit strong responses from female mosquito antennae in GC-EAD assays, also appeared to stimulate oviposition at 1 concentration. A reconstituted blend of the 10 compounds resulted in much stronger and more consistent responses than individual compounds. The blend was attractive to gravid Cx. quinquefasciatus and enhanced oviposition over a range of concentrations spanning 3 orders of magnitude. One concentration of the blend also attracted gravid Cx. tarsalis and enhanced oviposition. However, at the highest concentration tested, the blend was repellent to both species. Overall, these studies demonstrated that gravid mosquitoes are attracted to oviposition sites by blends of compounds rather than by individual chemicals, and that the concentration of compounds in the odor is a critical factor in determining whether responses are positive (attractive, stimulatory) or negative (repellent, deterrent).

Animals↗

Isolation and partial characterization of Bermuda grass pollen allergen, BG-60a.

In an earlier study we showed that Bermuda grass (Cynodon dactylon) pollen contains at least 12 IgE-binding proteins that can be analysed by immunoblot technique. One of the active components (BG-60) proved to be a basic protein of glycoprotein nature. It contained about 28% carbohydrate as determined from the dry weight and consisted of four molecules. One of the components was purified from the pollen extract by a combination of ammonium sulphate precipitation, ion-exchange chromatography on carboxymethyl-TSK, gel filtration on Ultrogel AcA 44 and chromatofocusing. Its molecular weight was approximately 60 kD by SDS-PAGE and 34 kD by gel filtration chromatography. The isoelectric point of the antigen was about 9.7. The homogeneity of the antigen BG-60a was assessed by one single arc of immunoprecipitation both in immunodiffusion and crossed immunoelectrophoresis and by one single band after SDS-PAGE. Its allergenicity was demonstrated by direct intradermal skin test on allergic patients and by examining IgE-binding reactivity with allergic patients' serum.

Adolescent↗

A unique human IgE-binding epitope on the Bermuda grass pollen recognized by mouse lambda-type monoclonal antibodies.

A group of six mouse monoclonal antibodies (MoAbs) with the unusual lambda-type light chain were generated by fusion of NS-1 cells with splenic cells derived from BALB/c mice immunized with crude extracts of Bermuda grass pollen (BGP). Four of them were IgG1, one was IgG2b, and one was IgG3. Binding inhibition assay showed that they recognized the same (or very similar) epitope. Using sera from BGP-allergic patients, it was found that the specific binding between the IgE antibodies and the MoAb 26-11-fixed antigen could be blocked by MoAb 26-11 itself and another MoAb 9-13 in a dose-dependent manner. It appears that the epitope recognized by the lambda-type MoAbs is a human IgE-binding antigenic determinant. Further physico-chemical analyses showed that this epitope was stable under heat but sensitive to treatments of sodium periodate and proteinase K. Results from these studies indicate that this unique epitope which leads to the generation of lambda-type MoAbs is part of a glycoprotein.

Animals↗

Analysis of allergenic components of Bermuda grass pollen by monoclonal antibodies.

A panel of 16 monoclonal antibodies (MoAbs) directed against Bermuda grass (Cynodon dactylon) pollen (BGP) were generated for identification and purification of the major allergenic components of the eliciting antigen (Ag). Radioimmunoprecipitation (RIP) analysis revealed that there were at least eight antigenic components with molecular weights (MW) ranging from 12 kilodalton (12 kDa) to 200 kDa. Each of these components has distinct biochemical characteristics based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Among them, Cyn d Bd67K and Cyn d Bd58K were basic proteins, Cyn d Bd35K consisted of at least four isomeric components with isoelectric points ranging from 6.2 to 7.2. The other antigens (Cyn d Bd68K, 48K, 38K, Cyn d Bd200K, Cyn d Bd46K, Cyn d Bd25K and Cyn d Bd12K) were all acidic proteins. The IgE binding capacity of all these antigens was determined with sera from 11 BGP-allergics by using a modified radioallergosorbent test. All but one of the antigens (Cyn d Bd200K) were found to react with human IgE from sera of BGP-allergic patients. Among those human IgE-binding molecules, Cyn d Bd35K reacted with allergic sera most frequently (10 of 11), followed by Cyn d Bd58K (8 of 11) and Cyn d Bd46K (7 of 11) respectively. Our results suggest that Cyn d Bd35K, Cyn d Bd58K, and Cyn d Bd46K are major allergens of BGP, and the MoAbs we obtained should be valuable tools for further purification of these allergens.

Allergens↗

Purification and structural analysis of the novel glycoprotein allergen Cyn d 24, a pathogenesis-related protein PR-1, from Bermuda grass pollen.

Bermuda grass pollen (BGP) contains a very complex mixture of allergens, but only a few have been characterized. One of the allergens, with an apparent molecular mass of 21 kDa, has been shown to bind serum IgE from 29% of patients with BGP allergy. A combination of chromatographic techniques (ion exchange and reverse phase HPLC) was used to purify the 21 kDa allergen. Immunoblotting was performed to investigate its IgE binding and lectin-binding activities, and the Lysyl-C endopeptidase digested peptides were determined by N-terminal sequencing. The cDNA sequence was analyzed by RACE PCR-based cloning. The protein mass and the putative glycan structure were further elucidated using MALDI-TOF mass spectrometry. The purified 21 kDa allergen was designated Cyn d 24 according to the protocol of International Union of Immunological Societies (IUIS). It has a molecular mass of 18,411 Da by MALDI-TOF analysis and a pI of 5.9. The cDNA encoding Cyn d 24 was predicted to produce a 153 amino acid mature protein containing tow conserved sequences seen in the pathogen-related protein family. Carbohydrate analysis showed that the most abundant N-linked glycan is a alpha(3)-fucosylated pauci-mannose (Man3GlcNAc2) structure, without a Xyl beta-(1,2)-linked to the branching beta-Man. Thus, Cyn d 24 is a glycoprotein and the results of the sequence alignment indicate that this novel allergen is a pathogenesis-related protein 1. To the best of our knowledge, this is the first study to identify any grass pollen allergen as a pathogenesis-related protein 1.

Allergens↗

Phylogeny of 16S rRNA, ribulose 1,5-bisphosphate carboxylase/oxygenase, and adenosine 5'-phosphosulfate reductase genes from gamma- and alphaproteobacterial symbionts in gutless marine worms (oligochaeta) from Bermuda and the Bahamas.

Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5'-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes.

Adenosine Phosphosulfate↗

Microbial metal tolerance in bermuda carbonate sediments.

The recovery of aerobic heterotrophic bacteria from Bermuda carbonate sediments on metal-supplemented media varied as much as 44-fold over a 15-cm depth. Distributional relationships with sulfate-reducing bacteria and sediment character indicated that metal tolerance was a function of metal bioavailability.

Journal Article↗

Water balance in embryos of Fundulus heteroclitus and F. bermudae in seawater.

The purpose of this study is to examine the mechanisms of osmoregulation in embryos of Fundulus heteroclitus and F. bermudae. The turnover rate of tritiated water in intact embryos of F. heteroclitus increases from 0.05 to 0.013 h-1 at 4 days to 0.12 +/- 0.030 h-1 at 7 days and to 0.18 +/- 0.060 h-1 at 10 days, corresponding to estimated diffusional water permeabilities of 0.4, 1.0, and 1.6 x 10(-6) cm . s-1, respectively. These values are low when compared to other organisms but not sufficiently low to maintain embryos in water balance. The osmolality of the embryonic extracellular fluids decreases during development from 362 +/- 9.2 mosmol . kg-1 at 4 days to 344 +/- 6.9 mosmol . kg-1 at 10 days. Water loss is balanced by drinking seawater at a rate of 600 pl . mg-1 . h-1 for 7- to 8-day embryos. The embryo possesses a pair of branchial chambers that communicate anteriorly with the pharynx through embryonic gill slits and posteriorly with the perivitelline space by means of pores. In the absence of a mouth it is probably through these pores that seawater is injested.

Animals↗

Oligoclonal analysis of the atopic T cell response to the group 1 allergen of Cynodon dactylon (bermuda grass) pollen: pre- and post-allergen-specific immunotherapy.

BACKGROUND: Bermuda grass pollen (BGP) is an increasingly important seasonal aeroallergen in Australia and other subtropical and temperate regions. BGP shares minimal allergenic cross-reactivity with pollens of rye grass or other Pooideae grasses often used for desensitization regimens in grass pollen allergy. Current allergen immunotherapy is seldom used in asthmatic patients due to IgE-mediated side effects. Since clinically effective immunotherapy is linked with altered allergen-specific T cell response, characterisation of human T cell reactivity to Cyn d 1, the major B cell allergen of BGP, should permit the design of effective and safe immunotherapy for BGP allergy. METHODS: Short-term BGP-specific CD4+ T cell lines were established from peripheral blood of 14 BGP-sensitive patients before and after conventional 50% BGP and 50% 7-grass mix subcutaneous specific allergen immunotherapy (SIT). T cell diversity of antigen specificity and function was assessed by proliferation and cytokine production to BGP, Cyn d 1 and Cyn d 1 peptides. RESULTS: Three highly immunogenic regions of Cyn d 1 were identified in 13/14 patients pre-SIT: Cyn d 1 (109-128), (181-209) and (217-241). The SIT regimen was clinically efficacious. Following SIT, decreased proliferation to BGP, Cyn d 1 and Cyn d 1 peptides was observed with a marked decrease in the IL-5:IFN-gamma ratio. CONCLUSIONS: Cyn d 1 is a major T cell allergen of BGP. Decreased Cyn d 1-specific IL-5 dominant T cell responses were observed in association with clinically effective treatment with the 50% BGP and 50% 7-grass mix. Identified dominant T cell regions of Cyn d 1 should facilitate safer vaccine development for BGP-induced asthma in addition to rhinitis.

Adult↗

An experimental model of allergic asthma in cats sensitized to house dust mite or bermuda grass allergen.

BACKGROUND: Animal models are used to mimic human asthma, however, not all models replicate the major characteristics of the human disease. Spontaneous development of asthma with hallmark features similar to humans has been documented to occur with relative frequency in only one animal species, the cat. We hypothesized that we could develop an experimental model of feline asthma using clinically relevant aeroallergens identified from cases of naturally developing feline asthma, and characterize immunologic, physiologic, and pathologic changes over 1 year. METHODS: House dust mite (HDMA) and Bermuda grass (BGA) allergen were selected by screening 10 privately owned pet cats with spontaneous asthma using a serum allergen-specific IgE ELISA. Parenteral sensitization and aerosol challenges were used to replicate the naturally developing disease in research cats. The asthmatic phenotype was characterized using intradermal skin testing, serum allergen-specific IgE ELISA, serum and bronchoalveolar lavage fluid (BALF) IgG and IgA ELISAs, airway hyperresponsiveness testing, BALF cytology, cytokine profiles using TaqMan PCR, and histopathologic evaluation. RESULTS: Sensitization with HDMA or BGA in cats led to allergen-specific IgE production, allergen-specific serum and BALF IgG and IgA production, airway hyperreactivity, airway eosinophilia, an acute T helper 2 cytokine profile in peripheral blood mononuclear cells and BALF cells, and histologic evidence of airway remodeling. CONCLUSIONS: Using clinically relevant aeroallergens to sensitize and challenge the cat provides an additional animal model to study the immunopathophysiologic mechanisms of allergic asthma. Chronic exposure to allergen in the cat leads to a variety of immunologic, physiologic, and pathologic changes that mimic the features seen in human asthma.

Animals↗

Monoclonal antibody-based ELISA to quantify the major allergen of Cynodon dactylon (Bermuda grass) pollen, Cyn d 1.

BACKGROUND: Pollen of Bermuda grass (Cynodon dactylon) is an important cause of pollinosis in many areas of the world. Most patients show sensitivity to the major allergen Cyn d 1, a glycoprotein composed of a number of isoforms with a molecular mass of 31-32 kDa. The aim of this work was to develop a monoclonal antibody (mAb)-based ELISA to quantify Cyn d 1, and to assess the correlation of the allergen content with the biological activity of C. dactylon pollen extracts. METHODS: After fusion of myeloma cells with spleen cells from a BALB/c mouse immunized with C. dactylon pollen extract, Cyn d 1-specific mAbs secreting hybridomas were selected, and the antibodies characterized. One of them (4.4.1) was used as the capture antibody in an ELISA method for Cyn d 1 quantitation. An anti-Cyn d 1 rabbit serum was used as the second antibody. Cyn d 1 was purified by immunoaffinity chromatography with mAb 4.4.1, characterized, and used as the standard in the assay. RESULTS: The identity, purity and isoallergen composition of affinity-purified Cyn d 1 was confirmed by N-terminal amino acid sequencing, SDS-PAGE, Western blot and 2D electrophoresis. The Cyn d 1 ELISA is highly specific and sensitive, with a detection limit of 0.24 ng/ml and a linear range of 1.1-9.2 ng/ml. An excellent correlation was found when the content of Cyn d 1, measured in 16 different extracts, was compared with the allergenic activity of the same extracts determined by RAST inhibition. CONCLUSIONS: The results prove the usefulness of the Cyn d 1 ELISA for the standardization of C. dactylon-allergen products on the basis of major allergen content.

Allergens↗

Using monoclonal antibodies to characterize a sequential epitope on the group I allergen of Bermuda grass pollen.

BACKGROUND: Cyn d 1, the group I allergen of Bermuda grass pollen, had been purified and characterized. METHODS: A sequential B cell epitope on Cyn d 1 was studied with monoclonal antibodies (MoAbs). Cyn d 1 was cleaved by Achromobacter protease I into fragments, and the resulting peptides were fractionated on reversed-phase columns before being reacted with anti-Cyn d 1 MoAbs in a radioimmunoassay. A Cyn d 1 fragment recognized by its MoAb was selected for Edman degradation. A synthetic peptide was constructed according to the determined sequence. RESULTS: The epitope on Cyn d 1 recognized by MoAb 18-53 was found to be conformation independent, since its activity was not changed after sodium periodate, guanidine or urea treatment. The enzyme-cleaved fragment containing this epitope was determined to be DVDKPPFDGMTACGNEPIF which corresponds to the N-terminal 46-64 residues of Cyn d 1. The presence of this sequence in the epitope recognized by MoAb 18-53 was demonstrated by enzyme immunoassay and further confirmed by inhibition of binding enzyme immunoassay with synthetic peptides. Some cross-reactivity with the N-terminal 45-63 residues of Lol p 1 was also found. CONCLUSIONS: The primary structure of a sequential epitope on Cyn d 1 was determined, and its activity was confirmed with peptides synthesized according to the determined sequence.

Allergens↗

Identification of a Ca2+ binding protein as a new Bermuda grass pollen allergen Cyn d 7: IgE cross-reactivity with oilseed rape pollen allergen Bra r 1.

cDNA clones encoding two isoforms of an allergen from pollen of Bermuda grass (Cynodon dactylon) have been isolated using IgE from allergic patients. Homologous transcripts are present in pollen of 15 other grasses tested. This allergen, tentatively designated as Cyn d 7, contains two calcium binding domains and shows significant sequence similarity with other Ca2+ binding pollen allergens, namely Bet v 4 from birch and Bra r 1 from oilseed rape. Approximately 10% of allergic sera tested showed IgE reactivity to this allergen. IgE cross-reactivity was observed between this allergen and Bra r 1 of oilseed rape. IgE reactivity of this allergen requires protein-bound Ca2+. Using IgE affinity-purified from the recombinant allergen to probe Western blots of pollen extracts Cyn d 7 has been identified as a 12 kDA protein.

Allergens↗

Technical note: stocking equivalents and stocking rate-gain relationships for steers and cow-calf pairs grazing oversown bermuda grass.

Grazing experiments may use steers or cow-calf pairs for measuring animal performance on pasture treatments, but the validity of extrapolation between these classes of cattle has not been verified. A grazing study was conducted in the spring and summer of both 1988 and 1989 to determine stocking equivalents and stocking rate-weight gain relationships for steers and cow-calf pairs grazing Coastal bermuda grass (Cynodon dactylon [L.] pers.) oversown with rye (Secale cereale L.) and ryegrass (Lolium multiflorum Lam.). Average daily gain and stocking rate (SR; 3.2, 4.2, 6.2, and 7.4 animals per hectare for steers and 1.7, 2.5, 3.7, and 4.9 pairs per hectare for cow-calf pairs) were both adjusted so that comparisons could be made on an equal BW basis. Disk meter height readings were used as measurements of forage accessibility. Disk meter height responses to SR did not differ (P greater than .10) between steer and cow-calf paddocks. There was a linear (P less than .001) decrease in ADG as SR increased, but this decline was steeper (P less than .001) for steers than for cows or suckling calves. Steers tended to be more productive than calves at low SR but less productive at high SR. Disk meter heights for the range of SR used in the study did not differ (P greater than .10) for steers and cow-calf pairs at equivalent BW per hectare. Our study suggests that live BW is a reasonable basis for determining forage requirements of steers and cow-calf pairs under grazing conditions, but extrapolation of production between classes of livestock will not be reliable.

Animal Feed↗

Effects of supplemental Bermuda grass hay or corn on intake, digestion and performance of cattle consuming endophyte-infected fescue.

Effects of supplemental Bermuda grass hay (BG) or ground corn on intake, digestion and performance of cattle consuming endophyte-infected fescue (I) were studied. In Exp. 1, a Latin square study, five growing Holstein steers (158.1 kg) consumed I ad libitum and were offered 0, .3, .6, .9 or 1.2% body weight (BW) of BG daily. Total dry matter (DM) intake rose linearly (P less than .05) with increasing BG, although intake was numerically similar with .6, .9 and 1.2% BW of BG. Digestibility was constant with diet (P greater than .10). Six growing Holstein steers used in Exp. 2, a Latin square with a 2 x 3 factorial arrangement of treatments, ingested I or noninfected (NI) fescue hay ad libitum with 0, .5 or 1.0% BW of ground corn. Total DM intake increased linearly as the level of corn rose (P less than .05). Total intake with I increased more with the first than with the second addition of corn, and the opposite occurred with NI (interaction between fescue infection and the quadratic effect of corn level, P less than .10). Organic matter digested (g/d) was greater for NI than for I and rose linearly with increasing corn ingestion (P less than .05). Ninety-six crossbred beef heifers and steers (184.2 kg avg initial live weight) were used in a 77-d fall grazing experiment (Exp. 3) with a 2 x 3 factorial treatment arrangement. Cattle grazed I or NI paddocks and were given no supplement or .34% BW of BG or .65% BW of ground corn on a daily basis (DM).(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Feed↗

Identification of oviposition attractants for Culex quinquefasciatus from fermented Bermuda grass infusions.

Compounds which attract and stimulate oviposition by gravid Culex quinquefasciatus were isolated and identified from a fermented Bermuda grass infusion. The neutral portion of the ether extract of the aqueous infusion contained the stimulatory materials. Fractionation by liquid chromatography yielded an active fraction containing phenol, 4-methylphenol, 4-ethylphenol, indole and 3-methylindole. A blend of the 5 compounds strongly stimulated oviposition, as did blends of any 4 of them. Bioassays with individual compounds showed that only 3-methylindole consistently induced oviposition, in concentrations spanning 5 orders of magnitude.

Animals↗

Isolation and partial characterization of Bermuda grass-pollen allergen: BG-60b.

Our earlier studies have shown that the pollen of Bermuda grass (Cynodon dactylon)-pollen contain at least 12 IgE-binding proteins which can be analyzed by the immunoblot technique. One of the highly active components was found to be a basic protein with a molecular weight of 60,000 daltons, designated as BG-60. This component was showed to consist of a group of proteins. One of them, BG-60a (pI 9.7), has been isolated and characterized. In this study, we have further isolated and characterized the second component of the antigen, designated as BG-60b. Its purity was demonstrated by gel electrophoresis experiment and antigen-antibody precipitation studies. The antigen is of glycoprotein nature with a pI of 10.0. It exhibits IgE-binding activity and shows cross-reactivity to antigen BG-60a in double diffusion. Its chemical and physical properties are similar to antigen BG-60a.

Allergens↗

Rush immunotherapy with a standardized Bermuda grass pollen extract.

We carried out a double-blind clinical trial in 30 patients who were sensitized to Bermuda grass pollen. Before and after immunotherapy we performed in vivo tests (skin tests and standardized tests of specific and nonspecific bronchial hyperreactivity), in vitro tests (histamine release), and specific IgE and IgG antibodies to BGP. We found a significant decrease (P less than .001) in specific bronchial hyperreactivity and skin sensitivity to BGP in the group of patients treated with immunotherapy, a decrease in the delayed responses (P less than .05) in histamine release (P less than .01), and an increase (P less than .001) in specific IgE and IgG antibodies to BGP. The placebo group showed no changes in these parameters.

Adolescent↗