bluuebunny/crossref_metadata_2025 huggingface dataset:
A first attempt to use a Fusarium subglutinans culture filtrate for the selection of pineapple cultivars resistant to fusariose disease
Authors: Borrás O. , Santos R. , Matos A. P. , Cabral R. S. , Arzola M.
Published: March 2003

Pineapple, Ananas comosus L. Merr., cultivars differing in resistance to fusariose were examined for the phytotoxic effect of culture nitrate (CF), and fusaric acid (FA) isolated from Fusarium subglutinans (Wollew & Reinking) Nelson, Toussoun & Marasas. The possibility of using these substances on F1 hybrids as selection agents for resistance was also evaluated. The phytotoxic effect of these substances was assessed in pineapple plantlets from tissue culture by placing them on wounded leaf segments. The resistance level of the F1 hybrids and cultivars under natural conditions was measured in greenhouse field tests. The susceptible cultivars proved to be the most sensitive in each test, whereas resistant cultivars showed resistance to CF. FA affected all the cultivars independently of the degree of resistance of the cultivars The presence of other metabolites in CF was responsible for the screening effect between the varieties. Using CF as selection criteria allowed the selection of resistant plants with behaviour quite similar to plants where the fungus itself was used as the selection agent.
Pleiotropic effects of the ea7 photoperiod response gene on the morphology and agronomic traits in barley
Authors: Buck‐Sorlin G. H. , Börner A.
Published: March 2003

The photoperiod‐insensitive barley mutant ‘Atsel’, carrying the recessive gene ea7, was studied together with the donor variety ‘Atlas’ (wild‐type, Ea7) under different daylengths with the aim of analysing pleiotropic effects. Grown under long and short photoperiods ‘Atsel’ flowered about 10 days and 34 days, respectively, earlier than ‘Atlas’. The significantly shorter life‐cycle of the photoperiod‐insensitive mutant resulted in several changes of plant morphology. Tillering, plant height, number of leaves and number of internodes were reduced. A lower number of florets per main spike was observed for ‘Atsel’, but only in the long photoperiod experiment. Finally, photoperiod insensitivity combined with a lower grain yield per plant was most pronounced under long‐day treatment. The data are comparable with results obtained from single chromosome recombinant lines of wheat that have differences in their photoperiod response caused by the genes Ppd1 or Ppd2.
The use of AFLP markers for the identification of carrot breeding lines and F1 hybrids
Authors: GRZEBELUS D. , SENALIK D. , JAGOSZ B. , SIMON P. W. , MICHALIK B.
Published: March 2003

Four inbred lines of carrot (cytoplasmic male‐steriles and corresponding maintainers) and eight of their F1 hybrids were studied with the amplified fragment length polymorphism (AFLP) technique to examine their genetic relationship and produce markers useful for testing hybrid seed purity. Eighty‐six polymorphic amplicons were identified in bulked DNA samples using eight primer pair combinations. Genetic distance was estimated on the basis of the presence or absence of polymorphic bands. The dendrogram plotted on the basis of the AFLP data closely represented the pedigree relationships of the lines and their hybrids. From one to six amplicons specific for a breeding line were identified. Most of them were also present in the DNA bulks of respective F1 hybrids. However, screening performed on individual plants of two parental lines and the corresponding hybrid indicated insufficient uniformity of parental lines, limiting the applicability of AFLP markers for testing hybrid seed purity.
Inheritance of high palmitic acid content in the sunflower mutant CAS‐12 and its relationship with high oleic content
Authors: Pérez‐Vich B. , Garcés R. , Fernández‐Martínez J. M.
Published: March 2003

CAS‐12 is a sunflower mutant with increased levels of palmitic (C16: 0 = 30%) and oleic (C18: 1 = 55%) acids in its seed oil, hence it has a reduced linoleic acid content (C18: 2 < 5%). This study was conducted to determine the inheritance of high C16: 0 content and its relationship with high C18: 1 content in CAS‐12. Reciprocal crosses involving CAS‐12, CAS‐5 (high C16: 0 content), HAOL‐9 (high C18: 1 content) and HA‐89 (standard fatty acid profile) were made. The F1, F2 and BC1F1 generations were obtained. The genetic control of the high C16: 0 trait in CAS‐12 was partially recessive and gametophytic. In all cases, this character segregated in the ratio 19: 38: 7 (low: intermediate: high C16: 0 content) in the F2 generation. These results, together with the lack of segregation for C16: 0 content in crosses between CAS‐12 and CAS‐5, indicated that the genetic control of the high C16: 0 trait in CAS‐12 was similar to that in CAS‐5 in being controlled by partially recessive alleles (p1, p2, and p3) at three loci. Crosses between HA‐89 and CAS‐12, and HAOL‐9 and CAS‐5 (segregating for C16: 0 and C18: 1) demonstrated that the high C16: 0 and the high C18: 1 traits were independently inherited. However, C18: 1 segregation in these crosses exhibited reversal of dominance. Apparently, the low C18: 1 parental lines carried modifier genes causing the deviation.
Evaluation of aluminium tolerance in a spring rye collection by hydroponic screening
Authors: Hede A. R. , Skovmand B. , Ribaut J.‐M. , González‐de‐León D. , Stølen O.
Published: March 2003

Aluminium (A1) tolerance of 63 rye accessions from a world spring rye collection was evaluated using the haematoxylin method and the root growth method. The haematoxylin method is based on the ability of A1‐tolerant seedlings to continue root growth following a short pulse treatment with a high Al concentration, while the root growth method uses the root growth and root tolerance index to judge A1 tolerance. Significantly higher levels of A1 tolerance were found in rye than in the A1‐tolerant bread wheat cultivar ‘Maringa’. Under the assumption that the ability of roots to grow under A1 stress is a combination of root vigour (long roots) and A1 tolerance, a hypothesis allowing for the differentiation of five genotype classes was suggested. This study demonstrated that the haematoxylin method and the root growth parameter identify genotypes with long root growth under A1 stress, but failed to detect A1 tolerance in genotypes with poor root vigour. These genotypes can only be identified using the root tolerance index parameter. However, the haematoxylin method is highly suitable for screening large segregating populations derived from improved germplasm that has been preselected for agronomically preferable traits, including plant vigour.
Characterization and mapping of gene Rph19 conferring resistance to Puccinia hordei in the cultivar ‘Reka 1’ and several Australian barleys
Authors: Park R. F. , Karakousis A.
Published: March 2003

Previous studies established that the Australian barley cultivar ‘Prior’ possessed resistance to Puccinia hordei (RphP), displaying the same specificity as an uncharacterized resistance in the differential cultivar ‘Reka 1’ (also possessing Rph2). Multipathotype tests confirmed the presence RphP in nine additional barley cultivars and indicated that RphP differed in specificity to the genes Rph1 to Rph15 and Rph18, plus the gene RphX present in the barley cultivar ‘Shyri’. RphP was inherited as a single dominant gene. Mapping studies using a doubled haploid population derived from ‘Chebec’/‘Harrington’ located RphP to the long arm of chromosome 7H, and demonstrated linkage with an restriction fragment length polymorphism marker (pTAG732), a resistance gene analogue marker (RLch4(Nc)), and two microsatellite markers (HVM11 and HVM49) at genetic distances of about 4‐10 cM. RphP showed linkage of 28 ± 4.3 cM with Rph3. RphP was designated Rph19, with the allele designation Rph19.ah. Previous studies have established that virulence for Rph19 occurs in many barley growing regions of the world.
Genotyping apricot cultivars for self‐(in)compatibility by means of RNases associated with S alleles
Authors: Alburquerque N. , Egea J. , Pérez‐Tornero O. , Burgos L.
Published: March 2003

In previous work the existence of proteins with RNase activity associated with S alleles in apricot was demonstrated. These proteins were inherited as described previously for the inheritance of self‐compatibility in this species. In this study, new cultivars have been genotyped for self‐compatibility using this method and it has been demonstrated that in all self‐compatible cultivars examined, the self‐compatibility allele is the same and is associated with an RNase with high activity. Homozygous self‐compatible individuals have been detected among established cultivars as well as among seedlings following breeding activity. This germplasm is of great value within the breeding programme because only self‐compatible seedlings will be produced. The number of S alleles in apricot appears to be low and only eight different alleles have been found in the large number of different cultivars screened. Furthermore, there are alleles present in the Spanish population that are also found in the genetic pool of North American cultivars. The screening of a progeny from the cross between the American cultivar ‘Goldrich’ and the Spanish cultivar ‘Pepito’ demonstrated the existence of the common allele S2 (detected previously by examining RNases), which was confirmed by the segregation of self‐compatibility in the progeny.
An ms2 male‐sterile, female‐fertile soybean sharing phenotypic expression with other ms mutants
Authors: Bione N. C. P. , Pagliarini M. S. , de Almeida L. A. , Seifert A. L.
Published: March 2003

Genetic and cytological studies of microsporogenesis and microgametogenesis were conducted with a natural male‐sterile, female‐fertile soybean mutant (BR97‐17971) found in an F4 breeding line of the Brazilian soybean breeding programme. Allele tests with ms Type Collection from USDA/ARS showed that the gene was allelic to ms2. Conventional analysis of microsporogenesis revealed a differential meiotic behaviour from the normal one reported in ms2 male‐sterile plants. Sterile plants have different levels of meiotic abnormalities related to chromosome segregation. Despite segregational abnormalities in the first and second divisions leading to micronuclei formation, the main meiotic cause of pollen sterility was absent or the defective cytokinesis following telophase II. Absent or defective cytokinesis has never been reported in ms2 mutants, but it was reported in the ms1 and ms4 mutants. After telophase II, one to four nucleate microspores underwent degeneration. Cross‐sections of sterile plant anthers showed that, at telophase II, the tapetal cells with large vacuoles degenerated. Despite callose deposition around meiocytes, sterile plants did not form tetrads. They degenerated and collapsed after callose dissolution, forming an amorphous mass. Anthers from male‐sterile plants were shrivelled.
Identifying AFLP and microsatellite markers for vernalization response gene Vrn‐B1 in hexaploid wheat using reciprocal mapping populations
Authors: Barrett B. , Bayram M. , Kidwell K. , Weber W. E.
Published: March 2003

Marker‐assisted selection may be useful for combining specific vernalization response (Vrn) alleles into a single wheat genotype for yield enhancement; however, DNA markers are only available for two of the three genes identified to date. The objectives of this study were to investigate reciprocal effects on days to heading using F2 populations generated by cross‐hybridizing near‐isogenic lines (NILs) carrying spring (Vrn‐B1; TDB) and winter (vrn‐B1; TDC) alleles, and to identify markers linked to Vrn‐B1 through genetic linkage analysis. Heading data were recorded for 91 and 89 progeny from reciprocal mapping populations TDB/TDC and TDC/TDB, respectively, and significant (P < 0.0001) reciprocal and dominance effects were detected. Among 207 amplified fragment length polymorphisms primer pairs and seven wheat microsatellite markers screened, two and one, respectively, were linked distally to Vrn‐B1 on wheat chromosome 5BL. Microsatellite Xgwm408 was most closely linked to Vrn‐B1 at 3.9 and 1.1 cM in the TDB/TDC and TDC/TDB map, respectively. Reciprocal differences in recombination distances emphasize the importance of female parent choice when generating mapping populations. Molecular markers are now available for three Vrn loci in wheat.
Uterine Responses to Exogenous Oxytocin Before and After Pre‐partum Luteolysis in the Cow
Authors: Taverne MAM , De Schwartz NCM , Kankofer M , Bevers MM , Van Oord HA , Schams D , Gutjahr St , Van Der Weijden GC
Published: March 2003

The aim of this study was to test the functional status of uterine oxytocin receptors in cows in vivo around parturition. The animals received consecutive, intra‐arterial injections of 800, 1600 and 3200 mU of oxytocin at three different stages: during late gestation (days 260–274), at 12 h and at 24 h after intramuscular injection of a prostaglandin F2α analogue at day 275 to induce parturition. Cows (n=6) had been provided with myometrial electrodes and a catheter had been installed in the aorta and in a branch of the uterine vein (UV). Regular blood samples were obtained from the UV from 5 min before until 45 min after each oxytocin injection to measure plasma levels of prostaglandin F2α (PGF2α) and oxytocin. Uterine electromyographic (EMG) activity was registered continuously during each experiment. The increase of oxytocin levels in UV plasma after intra‐arterial injections was dose dependent (p < 0.02). Pre‐ and post treatment oxytocin levels at 24 h after induction of parturition were significantly increased (p=0.0313). Both during late pregnancy and at 12 h after induction of parturition, oxytocin caused a significant increase in EMG activity (p=0.022). After the 3200 mU dose the increase was significantly higher than with the other 2 doses (p=0.004). After each dose, EMG activity returned to baseline levels within some 15 min. At 24 h after induction of parturition, the pre‐treatment level of EMG activity had increased. Doses of 800 mU and 1600 mU of oxytocin produced a significant (p=0.022) increment of EMG activity, which was of the same magnitude as during the preceding stages; after 3200 mU of oxytocin the response was significantly higher than before (p=0.008). No significant increases of PGF2α levels in UV plasma could be measured after oxytocin injections at any of the three stages. It is concluded that the myometrium of the pregnant cow responds in vivo to physiological doses of oxytocin. At 24 h after induction of parturition, when luteolysis has occurred and a parturient pattern of parturient myometrial activity has already started to develop, the response is enhanced. Physiological doses of oxytocin did not evoke a spurt release of PGF2α in uterine venous blood during the peripartal period.
Follicle Wave Growth in Cattle
Authors: Mihm M , Crowe MA , Knight PG , Austin EJ
Published: March 2003

Contents

Ovarian follicle growth in cattle culminates in the selection of a single dominant follicle which attains the ability for final maturation and ovulation once or twice during the luteal phase and at the end of the oestrous cycle, as well as during other reproductive states. This review will describe in detail the first follicle wave of the cycle leading to selection of the first wave dominant follicle, indicating the specific gonadotrophin dependencies of cohort and dominant follicles, and relating follicle fate to steroidogenesis. As a differential gonadotrophin response of growing antral follicles during the follies‐stimulating hormone (FSH) decline may determine which follicle becomes selected, first wave follicles are also characterized in relation to intrafollicular growth factors, which may modify the gonadotrophin response, such as inhibins and members of the insulin‐like growth factor (IGF) family. Subsequently, the follicular control of the transient FSH rise and decline so crucial to dominant follicle selection will be discussed. It is concluded that successful hormonal manipulation of follicle wave growth and dominant follicle selection will depend on our detailed understanding of the gonadotrophin requirements of differentiating wave follicles.
Effects of Serum‐Free Culture Media on in vitro Development of Domestic Cat Embryos Following In Vitro Maturation and Fertilization
Authors: Murakami M , Otoi T , Karja NWK , Ooka A , Suzuki T
Published: March 2003

Contents

This study was conducted to determine the adequate medium for a serum‐free culture system of domestic cat embryos produced by in vitro maturation (IVM) and fertilization (IVF). Cumulusoocyte complexes recovered from cat ovaries were matured in vitro for 24 h, and then inseminated in vitro for 12 h. After insemination, the oocytes were cultured in five media [Ham's F10, Waymouth 752/1 (Waymouth), TCM199, modified Earle's balanced salt solution (MK‐1) and CR1aa], each of which contained 0.4% bovine serum albumin. There were no significant differences among the rates of fertilization of oocytes cultured in five media following IVF. The rate of oocytes/embryos developed to at least the morula stage was significantly lower (p < 0.05) in Waymouth than in MK‐1, TCM199 and CR1aa. Moreover, none of the embryos cultured in Ham's F10 and Waymouth developed to the blastocyst stage. There were no differences among the rates of development to the blastocyst stage of oocytes/embryos cultured in MK‐1, TCM199 and CR1aa. These results indicate that the type of serum‐free medium has a major impact on in vitro development of domestic cat embryos derived from IVM/IVF, and MK‐1, TCM199 and CR1aa media are suitable for in vitro culture of cat embryos in a serum‐free culture system.
Studies on the Intracellular Ca2+ Concentration of Frozen–Thawed Dog Spermatozoa: Influence of Equex from Different Sources, Two Thawing Diluents and Post‐thaw Incubation in Capacitating Conditions
Authors: Peña AI , López‐Lugilde L , Barrio M , Becerra JJ , Quintela LA , Herradón PG
Published: March 2003

Contents

The addition of 0.5% (v/v) of Equex STM Paste (Nova Chemical Sales, Scituate Inc., MA, USA), whose active ingredient is sodium dodecyl sulphate (SDS), to a Tris–egg yolk extender was demonstrated to improve the longevity of frozen–thawed dog spermatozoa during in vitro incubation at 38°C. The aim of the first experiment was to compare the effects of two SDS‐containing compounds, Equex STM Paste and Equex Pasta (Minitüb, Tiefenbach, Germany), when added to a Tris–egg yolk based extender, on the post‐thaw longevity of dog spermatozoa, as well as on the intracellular Ca2+ concentration of spermatozoa, during post‐thaw incubation at 38°C. The post‐thaw sperm survival and longevity, as well as the quality of the sperm movement, were significantly better when using Equex STM Paste. Such prolonged sperm longevity, however, was associated to a higher intracellular Ca2+ concentration in a large subpopulation of the live spermatozoa. A second experiment was aimed to evaluate the effects of sperm dilution immediately post‐thaw with a Tris buffer containing glucose or fructose. The two Tris buffers were no different for any of the sperm parameters studied. The aim of a third experiment was to evaluate the sperm longevity, motility patterns and intracellular Ca2+ concentration of cryopreserved dog spermatozoa during post‐thaw incubation in capacitating conditions [canine capacitating medium (CCM) with or without 5 μg/ml of heparin]. Heparin had no significant effects on any of the sperm parameters evaluated. During the first 8 h of incubation, the majority of the live spermatozoa had a high intracellular Ca2+ content. However, after 8–10 h of incubation, it had significantly declined. The highest proportion of fast motile sperm, and the highest curvilinear velocity, average path velocity and amplitude of lateral head displacement for the total motile sperm were observed during the 2–4‐h incubation period. It was concluded that: (a) the addition of 0.5% (v/v) of Equex STM Paste to a Tris–egg yolk based extender significantly improved the post‐thaw longevity of dog spermatozoa, but the same concentration of Equex Pasta had no significant beneficial effects; (b) sperm dilution after thawing with a Tris buffer containing glucose or fructose made no difference in post‐thaw sperm longevity; (c) the addition of 5 μg/ml of heparin to CCM had no significant capacitating effects on frozen–thawed dog spermatozoa.
Analysis of In vitro Fertilizing Capacity to Evaluate the Freezing Procedures of Boar Semen and to Predict the Subsequent Fertility
Authors: Sellés E , Gadea J , Romar R , Matás C , Ruiz S
Published: March 2003

Contents

A porcine in vitro fertilization (IVF) system and seminal quality parameters of frozen–thawed boar semen were used to assess the effectiveness of two different thawing rates of frozen boar semen, and to address the question of whether differences between fertility of ejaculates could be predicted in a limited field trial.

In the first experiment, two thawing procedures were analysed (37°C, 30 s; 50°C, 12 s) and no differences in sperm quality were found. However, when the procedure was 50°C, 12 s the IVF results showed a higher number of sperm per penetrated oocyte and a near 10 points higher rate of pronuclear formation.

In the second experiment, the fertility results obtained in the limited field trial show to be efficient enough for application in a commercial use, especially for three of the employed boars (fertility ≥80%). In this limited study, the conventional seminal parameters are not accurate enough to discriminate good and bad boars in relation to fertility. On the contrary, parameters of in vitro penetrability are more precise to predict subsequent fertilities.

As conclusion, the IVF fertilization system seems to be a good tool to evaluate the quality of frozen–thawed boar semen previous to its commercial way, to verify the bank semen storage quality and a good way to assay new sperm freezing procedures, as it is the more precise evaluating method in estimating the potential fertilizing ability.
Phytoplankton variability in Lake Bourget: Phytoplankton dynamics and meteorology
Authors: Vinçon‐Leite Brigitte , Tassin Bruno , Druart Jean‐Claude
Published: March 2003

This paper presents the phytoplankton succession during two successive years in Lake Bourget, a deep subalpine lake recovering from eutrophication. A drastic reduction of the watershed phosphorus loading in 1980 stopped lake eutrophication. At present, the lake has nearly reached constant winter (January–February) nutrient concentrations. However, during the 2 years of the study, the phytoplankton data set shows a great interannual variability, which is analysed on the basis of the meteorological forcing variables.
Oral tolerance as therapy for experimental autoimmune encephalomyelitis and multiple sclerosis: Demonstration of T cell anergy
Authors: Jewell Scott D , Gienapp Ingrid E , Cox Karen L , Whitacre Caroline C
Published: March 2003

Experimental autoimmune encephalomyelitis (EAE) is an important model for developing therapies for multiple sclerosis (MS). The oral administration of the central nervous system antigen, myelin basic protein (MBP), to Lewis rats and susceptible mouse strains prior to MBP immunization prevents the induction of EAE. Clinical trials administering myelin orally to MS patients have met with only partial success, and thus require that oral tolerance be further studied to improve this treatment strategy. Clonal anergy, clonal deletion, immune deviation from Th1 to Th2 T cell subsets, and active suppression by TGF‐β‐secreting T cells have all been implicated as possible mechanisms in oral tolerance. Which mechanism predominates depends on antigen dosage, frequency of feeding, and timing of antigen administration. In this study, we have characterized T cells derived from MBP‐fed rats and determined the level of their unresponsiveness. Myelin basic protein‐specific T cells are indeed present although in reduced numbers in lymphoid tissue of orally tolerized animals. Following several cell divisions in the presence of IL‐2, these MBP‐specific T cells undergo a dramatic reversal of unresponsiveness, proliferate in response to MBP and are capable of transferring EAE. These results support clonal anergy as an important mechanism for oral tolerance. Recent developments in clinical trials of oral tolerance are described.
Characterization of the mitogenic response of murine CD5+ and conventional B lymphocytes to lipopolysaccharide
Authors: Philips JR , Massey WLK , Hunter N
Published: March 2003

The nature of the response of conventional and CD5+ B cells to stimulation in vitro with optimal mitogenic concentrations of LPS was examined to elucidate the contributions of these B cell subsets in polyclonal B lymphocyte responses. Stimulation of murine splenic lymphocytes with LPS resulted in an increase in total biomass, peaking at 72 h of culture. The viability of the cultures remained high (>90%) until 48 h of culture. A combination of trypan blue and 7‐aminoactinomycin D (7AAD) exclusion in conjunction with PE‐anti‐CD5 and FITC‐anti‐B220 enabled more detailed analysis of the cultures. The total number of conventional B cells, viable and non‐viable, increased until 48 h of culture and then decreased when stimulated with LPS, while CD5+ B cells increased over the culture period. The numbers of conventional B cells in the control cultures decreased, but the CD5+ B cell numbers remained stable. An examination of the modes of death of the B cell subsets using 7AAD showed that unstimulated conventional B cells were apoptotic rather than degenerate but, following stimulation with LPS, apoptotic and degenerate cells were found. Apoptotic and degenerate CD5+ B cells were found in both stimulated and unstimulated cultures, but the percentage of these apoptotic and degenerate cells was increased significantly only at 72 h and 96 h of culture in stimulated cultures compared with 24 h onwards in the control cultures. Morphological analysis and gel electrophoretic studies of extracted DNA reflected these findings. It was also found that the increase in the number and percentage of non‐viable cells in the cultures was not equal to the decrease in the number and percentage of viable cells. Activation of B cells was examined using expression of B7‐1 (CD80) as a marker. When stimulated with LPS a greater proportion of conventional B cells expressed B7‐1 after 24 h of culture than in the control cultures; however, only at 72 h and 96 h of culture was the proportion of CD5+ B cells expressing B7‐1 significantly higher than in the control cultures. These results show that conventional B cells are stimulated to proliferate and to become activated by LPS and that death is apoptotic rather than degenerate or necrotic. CD5+ B cells were also shown to be stimulated by LPS; they became activated and death was delayed. The data suggest that in addition to the proliferative role, LPS acts to delay death and to activate conventional and CD5+ B cells.
Signal transmission in ligand‐gated receptors
Authors: Gage Peter W
Published: March 2003

At synapses, a transmitter released from a pre‐synaptic terminal binds to specific, ligand‐gated receptors in the post‐synaptic membrane to open up ion channels through the receptor molecules. The flow of ions through these channels generates electrical signals. Electrophysiological techniques have been used over the past 50 years to understand transmission of these signals at synapses. The most recent of these, the patch‐clamp technique, allows very small picoamp currents through single‐channel molecules to be recorded but gives little information about receptor structure or how drugs influence their function. Now, the subunits of most ligand‐gated ion channels have been cloned and sequenced. Cryo‐electronmicroscopy has revealed the structure of the ion channel activated by nicotinic agonists. It is pentameric and only a small part of it is in the membrane. In spite of this simple structure, the conductance of chloride channels activated by gamma‐aminobutyric acid (GABAA channels) is very variable and can be increased markedly by drugs such as diazepam. Site‐directed mutagenesis and labelling of cysteine residues in the open and the closed states are being used to define the residues that line the ion channel. Similar methods are being used to find the way in which drugs such as general anaesthetics modulate the function of GABAA receptors.
Cellular and molecular regulation of eosinophil trafficking to the lung
Authors: Hogan Simon P , Mould Arne W , Young Janine M , Rothenberg Marc E , Ramsay Alistair J , Matthaei Klaus , Young Ian G , Foster Paul S
Published: March 2003

Airway inflammation is central to the pathogenesis of allergic asthma, and molecules that mediate this process obviously represent targets for therapy. In the present article, we discuss our experiments, which point to CD4+ T cells and IL‐5‐driven eosinophilia as potential targets for the relief of bronchial hyperractivity in late‐phase asthma.
Antibodies against peripheral myelin glycolipids in people with HIV infection
Authors: Petratos Steven , Turnbull Victor J , Papadopoulos Roula , Ayers Margaret , Gonzales Michael F
Published: March 2003

Plasma samples from 35 individuals with HIV infection but without clinical peripheral neuropathy were screened by ELISA for IgM and IgG antibodies against peripheral myelin. Eighteen of the 35 samples (51%) showed IgM reactivity and 11 (31%) showed IgG reactivity. By comparison, none of 48 samples from healthy blood donors showed IgM or IgG reactivity. Epitopes reacting with these antibodies were identified by TLC immunostaining as sulphatide (GalS) and the gangliosides GM1, GD1a and GD1b. Plasma samples from four people with HIV infection and neuropathy (HIV+PN), six HIV‐seronegative individuals with IgM paraproteinaemic demyelinating neuropathy (IgMPDN) and 12 HIV‐seronegative individuals with a variety of other neurological disorders (HIV–OND) were also investigated. Two of the four HIV+PN samples showed IgM reactivity with GalS; and two showed IgG reactivity against GalS. Of the six IgMPDN samples, three showed IgM reactivity with GalS. These data indicate that antibodies against peripheral myelin glycolipids, in particular GalS, occur more frequently in HIV infection than in HIV‐seronegative individuals with and without neurological disease, and may contribute to subclinical neuropathy in HIV infection.
Immortalized cell lines derived from mice lacking both type I and type II IFN receptors unify some functions of immature and mature dendritic cells
Authors: Nunez R , Grob P , Baumann S , Zuniga A , Ackermann M , Suter M
Published: March 2003

Cells with dendritic morphology obtained from several organs of mice lacking both type I and II IFN receptors were immortalized by a retrovirus and analysed for their phenotype and for their function to induce cognate immune responses in vitro and in vivo. Two cell lines called AG101 (skin) and AG116 (brain) were cloned and analysed in more detail. They constitutively expressed the cell surface markers CD45, CD11b, MHC class II, F4/80, N418, B7–2 and ICAM1 but were CD8‐ and B220‐negative. Cells from both lines were capable of taking up ovalbumin (OVA). The processed protein was presented to the OVA‐specific T cell hybridoma BO97.105 which responded specifically with the production of IL‐2. AG101 and AG116 cells were able to induce a mixed lymphocyte reaction as shown by a 50‐fold increase of IL‐2 production over background. Naive T cells were stimulated by antigen‐primed AG101 and AG116, resulting in a T cell proliferation which was 20–30 times over background, and in IL‐2 production it was 10 times the background. The capacity of AG101 or AG116 cells to prime naive T cells was directly compared with freshly isolated and cultured cutaneous dendritic cells (DC) from 129 Sv/Ev mice (wtDC). After cognate T cell interaction, IL‐6 (20–100‐fold) and IL‐12 p40 (100–1000‐fold) were similarly up‐ regulated in either AG101, AG116 or mature wtDC. To analyse the capacity of the immortalized DC to induce antibodies in vivo, cell line AG116 was permanently infected with Borna disease virus (BDV) which is unable to replicate in adult mice. One hundred and twenty‐nine Sv/Ev mice injected with different cell numbers of AG116 carrying BDV (but not control cells) produced antibodies against the viral BDVp40 and BDVp24 protein. Therefore, the cell lines AG101 and AG116 appear to unify some functions of immature and mature DC. They are able to pick up antigen and process it. In the absence of externally added cytokines, the antigen presented on AG101 or AG116 cells drives T cells with an efficiency similar to mature DC. The cloned cell lines may prove to be useful to study both immune response and replication of infectious agents in the absence of functional interferon receptors.
Natural cytotoxic and antibody‐dependent cellular cytotoxic activity of cells in the decidua basales and metrial glands of pseudopregnant rats with deciduomata
Authors: Peel S , Stewart I J
Published: March 2003

Cytotoxic cells are present in the uterine wall of pregnant rats. To determine if the cytotoxic activity arises in response to semen or the products of conception, the profile of cytotoxic activity in deciduomata of pseudopregnant rats was examined. To examine NK activity, Yac‐1 cells were used as targets in chromium release cytotoxicity assays and an antibody to Yac‐1 cells was included in some assays to determine antibody‐dependent cellular cytotoxic (ADCC) activity. Cells from the metrial glands and deciduae of deciduomata of rats at days 10 and 13 of pseudopregnancy did not show NK activity but ADCC activity was present. To examine natural cytotoxic (NC) activity, Wehi 164 cells were used as targets in chromium release cytotoxicity assays. Cells isolated from the metrial glands and deciduae of rats at day 10 of pseudopregnancy were able to kill Wehi 164 cells after 21 h assays, thus demonstrating NC activity. The profile of cytotoxic activity in the uterine wall of pseudopregnant rats with deciduomata is similar to that found in pregnancy and is thus independent of semen or the products of conception.
Elevated plasminogen receptor expression occurs as a degradative phase event in cellular apoptosis
Authors: O’Mullane Matthew J , Baker Mark S
Published: March 2003

Plasminogen activation (PA) is involved in a variety of extracellular proteolytic events, such as fibrinolysis, cell migration (e.g. angiogenesis, tumour cell invasion, inflammation, wound healing, bacterial invasion), ovulation, tissue remodelling and the activation of other protease classes and growth factors. These diverse roles are due to the specific localization of components of the PA system to extracellular matrices, basement membranes, fibrin and cell surfaces. We have previously reported that PA is dramatically elevated during cycloheximide (CHX)‐induced apoptosis in U937 cells due to a concomitant increase in both plasminogen receptors (PLG‐R; i.e. specific PLG binding) and cell–surface urokinase plasminogen activator. We now extend this study by showing that the increase in PLG‐R (resulting in an increase in specific PLG binding) is a late apoptotic event coincident with propidium iodide uptake and internucleosomal DNA fragmentation but occurring after elevations in phosphatidylserine exposure. Plasminogen was also observed to dramatically increase the rate of CHX‐induced apoptosis. We conclude that PA may play a role in the degradative (i.e. late‐stage) events of cellular apoptosis.
Characterization of a bioassay for detection of recombinant and native ovine interleukin‐5
Authors: Bosward Katrina , Emery David L , McWaters Peter W , Husband Alan J , Bendixsen Tuan
Published: March 2003

In order to analyse Th2‐type immune responses in sheep by the assay of interleukin (IL)‐5 in biological fluids, the ovine IL‐5 gene was cloned and expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression vector system. The recombinant product was purified as BAC‐OV‐IL‐5 from the supernatant fluid. The ovine IL‐5 was biologically active in a bioassay using IL‐5‐dependent Baf cells, which have been used previously to specifically detect human IL‐5. The specificity of Baf cells for ovine IL‐5 was examined by two methods. First, Baf cells only proliferated in response to BAC‐OV‐IL‐5 and did not respond to addition of recombinant ovine cytokines granulocyte–macrophage colony stimulating factor (GM‐CSF), IL‐1β, IL‐2, IL‐3, IL‐6, IL‐8, stem cell factor (SCF) or IFN‐γ at doses from 0.01 to 1 μg/well. Second, the rat monoclonal antibody to murine IL‐5, TRFK‐5, neutralized murine, but not ovine, IL‐5. However, rabbit antisera to BAC‐OV‐IL‐5 neutralized murine and ovine recombinant IL‐5 and abolished responses of Baf cells to IL‐5 activity in supernatant fluids from mesenteric lymph node cells (MLNC) of parasitized sheep. The bioassay had a sensitivity to detect 8 ng in a 200 μL assay (40 ng/mL). Thus, the specificity of Baf cells to detect human IL‐5 also extends to ovine IL‐5 and therefore provides a method for monitoring the production of Th2 immune reactivity in sheep.
The effect of molecular weight and β‐1,6‐linkages on priming of macrophage function in mice by (1,3)‐β‐d‐glucan
Authors: Cleary Janelle A , Kelly Graham E , Husband Alan J
Published: March 2003

1,3‐β‐d‐glucans (glucans) are structural elements in the cell walls of yeast and fungi with immunomodulatory properties, mediated through their ability to activate macrophages. This study assessed the activation of cells of the peritoneal cavity between 3 and 90 days after i.p. injection of particulate yeast glucan differing in molecular weight (MW) and degree of (1,6)‐linkages. Female QS mice, 7–9 weeks of age, were injected, i.p., with varying doses of low (< 5 × 105), medium (1–2 × 106) or high (> 3 × 106) MW glucans, all with low (< 5%) β‐(1,6)‐linkages, or high MW (> 3 × 106) glucan with high 1,6‐linkages (> 20%). All glucans induced a transient increase in the proportion of neutrophils and eosinophils and a reduction in mast cell numbers in the peritoneal cavity. Peritoneal macrophages showed an altered morphology, increased intracellular acid phosphatase, increased LPS‐stimulated NO production and increased PMA‐stimulated superoxide production. There were no significant changes in serum lysozyme levels. Most macrophage activities returned to control levels by 28 days post injection of 1,3‐β‐d‐glucan. There was a trend for higher MW or (1,6)‐linked, (1,3)‐β‐d‐glucans to be more stimulatory. It was concluded that particulate yeast (1,3)‐β‐d‐glucan is an effective stimulator of immune function, the efficiency of which may be influenced by the MW and degree of (1,6)‐linkages.
High‐titre IgM anti‐sulfatide antibodies in individuals with IgM paraproteinaemia and associated peripheral neuropathy
Authors: Petratos Steven , Turnbull Victor J , Papadopoulos Roula , Ayers Margaret , Gonzales Michael F
Published: March 2003

The common association between monoclonal gammopathy and peripheral neuropathy was studied in seven patients with demyelinating polyneuropathy and IgM paraproteinaemia. Plasma samples from these individuals were thoroughly tested for antiperipheral nerve myelin (PNM) antibodies and then screened for glycoprotein and glycolipid reactivity by western immunoblotting and thin‐layer chromatography (TLC) immunostaining. Three of the seven samples showed strong IgM anti‐PNM and antisulfatide (GalS) antibody reactivity. Two of these three plasma samples showed extraordinarily high antisulfatide IgM antibody titres, ranging from 1 × 104 to 1 × 106 arbitrary units/L. These same samples also showed intense myelin staining of sciatic nerve sections (paraffin and cryostat) and teased nerve fibres. No axonal immunoreactivity was observed. These results suggest that high titre IgM antisulfatide antibodies may play a pathogenetic role in the immune demyelination associated with IgM paraproteinaemia.
Studying heterotrimeric G‐protein‐linked signal transduction using replication‐deficient adenoviruses
Authors: Cummins Michelle M , Poronnik Philip , O’Mullane Lauren M , Cook David I
Published: March 2003

Plasma membrane‐spanning G‐protein‐linked receptors transduce approximately 60% of all extracellular stimuli in higher animals. Many G‐protein‐linked receptor pathways are yet to be elucidated, with the receptor, G‐protein or effector system as yet unidentified. In addition, many fundamental issues pertaining to G‐protein signalling remain unresolved, such as the factors governing the specificity of G‐protein receptor coupling and the control of signal amplitude in response to G‐protein activation. In order to address some of these issues, the use of replication‐deficient adenoviruses as gene transfer vectors for investigations of G‐protein signalling has been developed, facilitating dissection of G‐protein‐linked signal transduction pathways in an extensive range of cultured cells, as well as in vivo. The present review focuses on the versatility and utility of adenoviruses for the investigation of signalling by heterotrimeric G‐proteins and explores some of the recent advances in adenoviral technology as they relate to the study of signal transduction.
Recruitment of mononuclear leucocytes to osteoarthritic human synovial xenografts in the ears of SCID mice
Authors: Cleland LG , Fusco M , Proudman SM , Wing SJ , Spargo LDJ , Mayrhofer G
Published: March 2003

A system has been established to assess the recruitment of 99mTc‐hexamethylpropylene amine oxamine (99mTc‐HMPAO)‐labelled PBMC and [125I]iododeoxyuridine‐labelled Con A stimulated lymphoblasts to allogeneic human synovial xenografts in the ears of SCID mice. Successful engraftment of osteoarthritic synovium was achieved in approximately 90% of cases and a connection between the human microvasculature of the xenograft and the circulation of the mouse was shown. Cells were delivered to the xenograft by a system of regional vascular perfusion, thus avoiding the major murine vascular beds. The accumulation of 99mTc‐HMPAO‐labelled PBMC in mouse ears was monitored in real time. Direct injection of xenograft‐bearing ears with recombinant human TNF‐α, 7 h prior to perfusion, increased the accumulation of both PBMC and lymphoblasts in cytokine‐injected ears compared to contralateral control‐injected ears. Autoradiography revealed the presence of [125I]iododeoxyuridine‐labelled lymphoblasts associated with human microvasculature within the xenograft. However, the increased accumulation of lymphoblasts in cytokine‐injected ears occurred in the tissues surrounding the xenograft, where lymphoblasts were associated more often with murine than human vessels. Although the system described offers advantages over similar models, the propensity for mouse endothelium to interact with human leucocytes is likely to be a generic disadvantage for models of human leucocyte recruitment to xenografts in immunodeficient mice.
Structural model of human IL‐13 defines the spatial interactions with the IL‐13Rα/IL‐4Rα receptor
Authors: Zuegg J , Webb DC , Foster PS , Casarotto MG
Published: March 2003

Interleukin‐13 (IL‐13) plays a key role in immune responses and inflammation. A structural model of human IL‐13 (HuIL‐13) based on the nuclear magnetic resonance and X‐ray structure of IL‐4 is put forward. Unlike previous models, this model is based on new sequence alignments that take into account the formation of the two disulfide linkages that have been determined experimentally. The proposed structure of human IL‐13 is similar to IL‐4, consisting of a four helix bundle with hydrophobic residues lining the core of the molecule and surface polar residues showing a high degree of solvent accessibility. Regions of HuIL‐13 that are critical for the interaction with its receptors are explored and discussed in relation to existing mutagenic studies. From these studies we predict that helices A and C of HuIL‐13 interact with the IL‐4 receptor alpha (IL‐4Rα) region and helix D is responsible for the interaction with the IL‐13 receptor alpha 1 (IL‐13Rα1) receptor.
Immuno‐informatics: Mining genomes for vaccine components
Authors: De Groot Anne S , Sbai Hakima , Aubin Caitlin Saint , McMurry Julie , Martin William
Published: March 2003

The complete genome sequences of more than 60 microbes have been completed in the past decade. Concurrently, a series of new informatic stools, designed to harness this new wealth of information, have been developed. Some of these new tools allow researchers to select regions of microbial genomes that trigger immune responses. These regions, termed epitopes, are ideal components of vaccines. When the new tools are used to search for epitopes, this search is usually coupled with in vitro screening methods; an approach that has been termed computational immunology or immuno‐informatics. Researchers are now implementing these combined methods to scangenomic sequences for vaccine components. They are thereby expanding the number of different proteins that can be screened for vaccine development, while narrowing this search to those regions of the proteins that are extremely likely to induce an immune response. As the tools improve, it may soon be feasible to skip over many of the in vitro screening steps, moving directly from genome sequence to vaccine design. The present article reviews the work of several groups engaged in the development of immuno‐informatic stools and illustrates the application of these tools to the process of vaccine discovery.
Synergy of type I interferon‐A6 and interferon‐B naked DNA immunotherapy for cytomegalovirus infection
Authors: Bartlett Emmalene J , Cull Vanessa S , Brekalo Natalie L , Lenzo Jason C , James Cassandra M
Published: March 2003

Delivery of type I IFN transgenes by naked DNA immunization can protect against cytomegalovirus infection and myocarditis. Here, we investigate IFN transgene expression, antiviral efficacy, and immunomodulation of myocarditis using various treatment regimes in a mouse CMV model. In vivo expression of the IFN transgene was observed in the sera for 35 days post‐DNA inoculation. Prophylactic IFN‐A6 and IFN‐B DNA treatment for 14 days prior to murine cytomegalovirus (MCMV) infection was more efficacious in significantly reducing viral titres, than 2 days prior to or 2 days post‐virus infection. Similarly, IFN‐A6 DNA treatment commencing 14 days prior to virus infection was superior in suppressing both acute and chronic myocarditis. Furthermore, reduction of autoantibody titres was more pronounced when IFN was administered 14 days prior to viral infection. Combinational IFN gene therapy was assessed for synergy between IFN subtypes. Combination treatment with either IFN‐A6/A9 or IFN‐A6/B greatly reduced spleen viral titres while IFN‐A6/B and IFN‐A9/B reduced virus replication in the liver. Only IFN‐A6/A9 and IFN‐A9/B reduced acute viral myocarditis, whereas IFNA6/B treatment was most efficacious for autoimmune chronic myocarditis. Finally, treatment with IFN‐A6 DNA 2 weeks post‐MCMV infection proved effective at inhibiting the development of chronic autoimmune myocarditis. These findings suggest that immunomodulation of both antiviral and autoimmune responses by IFN DNA immunization may be an avenue for improved viral immunotherapy.
Chemokines and dendritic cells: A crucial alliance
Authors: McColl Shaun R
Published: March 2003

Dendritic cells (DC) are bone marrow‐derived professional antigen‐presenting cells that function as sentinels of the immune system. Their importance in immunity resides in their unique ability to prime or tolerize T lymphocytes, thereby initiating or inhibiting immune responses. They reside in all tissues and organs and upon appropriate activation, migrate to secondary lymphoid organs to present antigen to T lymphocytes in the T cell zones. Because of this central role in T cell activation, there is a great deal of interest in using DC therapeutically to deliver positive or negative signals to the immune system. The DC system is critically dependent on the ability of DC at different stages of maturation to respond to a range of soluble and cell‐bound signals, including members of the chemokine gene superfamily. This review will describe the interactions between DC and the chemokine system.