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Cycloheximide treatment of cotton ovules alters the abundance of specific classes of mRNAs and generates novel ESTs for microarray expression profiling.

Fibres of cotton (Gossypium hirsutum L.) are single elongated epidermal cells that start to develop on the outer surface of cotton ovules on the day of anthesis. Little is known about the control of fibre initiation and development. As a first step towards discovering important genes involved in fibre initiation and development using a genomics approach, we report technical advances aimed at reducing redundancy and increasing coverage for anonymous cDNA microarrays in this study. Cotton ovule cDNA libraries (both normalised and un-normalised) from around the time of fibre initial formation have been prepared and partially characterised by sequencing. Re-association-based normalisation partially reduced library redundancy and increased representation of novel sequences. However, another library generated from in vitro cultured cotton ovules treated with the protein synthesis inhibitor, cycloheximide, showed a significantly altered gene representation including a greater proportion of protein phosphorylation genes, transport genes and transcription factors and a much reduced proportion of protein synthesis genes than were identified in the conventional types of libraries. Over 10,000 expressed sequence tag (EST) clones randomly selected from the three libraries were printed on microarray slides and used to assess gene expression in tissue cultured ovules with and without cycloheximide treatment. The microarray results showed that cycloheximide had a dramatic effect in modifying the pattern of the gene expression in cultured ovules, affecting the same types of genes identified in the preliminary analysis on relative EST abundance in the different ovule cDNA libraries. Cycloheximide clearly provided a simple and useful method for enriching novel gene sequences for genomic studies.

Base Sequence↗

Transgenic tobacco expressing geminiviral RNAs are resistant to the serious viral pathogen causing cotton leaf curl disease.

Cotton, the major cash crop in Pakistan, suffers 30% losses to cotton leaf curl disease, caused by the geminivirus, cotton leaf curl virus DNA A, plus a satellite component, DNA beta responsible for symptom development with plants failing to produce cotton bolls. We constructed transgenic tobacco expressing sense and antisense RNAs representing: [i] the 5' half of the viral DNA replication gene, AC1, [ii] the 3' half of AC1, [iii] two overlapping genes, AC2, a transcription activator, and AC3, a replication enhancer. In contrast to controls, 25% of 72 transgenic tobacco lines tested showed heritable resistance [T(1) - T(3) generations]: symptom-free and no replication of DNA A or DNA beta even after 120 days of continuous exposure to viruliferous whiteflies. As geminiviral and transgene RNAs are not detected in resistant lines following infection, and selected uninfected resistant tobacco sense lines reveal double-stranded and small interfering RNAs, the most likely mechanism is via post-transcriptional gene silencing.

DNA Replication↗

False-positive results caused by cotton swabs in commercial Aspergillus antigen latex agglutination test.

While testing for Aspergillus spp. using a commercially available latex agglutination test on a brain biopsy wrapped in a cotton swab, false-positive results were obtained. Subsequent application of the test on lavage samples obtained using cotton and synthetic swabs resulted positive with the cotton swabs only. This suggests that epitopes cross-reactive with Aspergillus galactomannan may be present in cotton.

Antigens, Fungal↗

Population dynamics of cotton rats (Sigmodon hispidus) inhabiting abandoned petroleum landfarms in Oklahoma, USA.

Previous work by our group has shown bioaccumulation of contaminants and alterations in the immune system of hispid cotton rats (Sigmodon hispidus) inhabiting petrochemical waste sites (landfarms). We studied populations of cotton rats inhabiting petrochemical landfarms or uncontaminated reference sites. Cotton rat populations inhabiting abandoned landfarms experienced reduced summer population densities and lower mean monthly survival, with maximum densities 65% that of populations inhabiting non-contaminated grassland (reference) sites. Survival was lower in populations from landfarms (0.62+/-0.04) compared to reference sites (0.75+/-0.04), with differences most notable during summer months. Cotton rat populations sampled from landfarms had even sex ratios and a lower proportion of juveniles compared to populations from reference sites. No differences were observed in the weight of epididymides, testes, uteri, or ovaries, and no differences in fecundity were detected among sites.

Animals↗

Persistence and dissipation kinetics of spiromesifen in chili and cotton.

In a field study carried out at three different locations, the dissipation of spiromesifen on cotton and chili was studied and its DT50, and DT99 were estimated at each location. Spiromesifen was sprayed on chili at 96 and 192 g a.i. ha(-1) and cotton at 120 and 240 g a.i. ha(-1). Samples of chili fruits were drawn at 0, 1, 3, 5, 7, 10, 15, 21, 30 days after treatment and that of cotton seed and lint at first picking and harvest. Soil samples were drawn 30 days after treatment from 0 to 15 and 15 to 30 cm layer. Quantification of residues was done on GC-MS in Selected Ion Monitoring (SIM) mode in mass range 271-274 m/z. The LOQ of this method was found 0.033 microg g(-1), LOD being 0.01 microg g(-1). The DT50 of spiromesifen when applied at recommended doses in chili fruits was found to be 2.18-2.40 days. Ninety-nine percent degradation was found to occur within 14.5-16.3 days after application. Residues of spiromesifen were not detected in cotton seed and lint samples at the first picking. In soil, no residues of spiromesifen were detectable 15 days after treatment.

Capsicum↗

Stable transformation of the cotton plastid genome and maternal inheritance of transgenes.

Chloroplast genetic engineering overcomes concerns of gene containment, low levels of transgene expression, gene silencing, positional and pleiotropic effects or presence of vector sequences in transformed genomes. Several therapeutic proteins and agronomic traits have been highly expressed via the tobacco chloroplast genome but extending this concept to important crops has been a major challenge; lack of 100 homologous species-specific chloroplast transformation vectors containing suitable selectable markers, ability to regulate transgene expression in developing plastids and inadequate tissue culture systems via somatic embryogenesis are major challenges. We employed a 'Double Gene/Single Selection (DGSS)' plastid transformation vector that harbors two selectable marker genes (aph A-6 and npt II) to detoxify the same antibiotic by two enzymes, irrespective of the type of tissues or plastids; by combining this with an efficient regeneration system via somatic embryogenesis, cotton plastid transformation was achieved for the first time. The DGSS transformation vector is at least 8-fold (1 event/2.4 bombarded plates) more efficient than 'Single Gene/Single Selection (SGSS)' vector (aph A-6; 1 event per 20 bombarded plates). Chloroplast transgenic lines were fertile, flowered and set seeds similar to untransformed plants. Transgenes stably integrated into the cotton chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. Cotton is one of the most important genetically modified crops (120 billion US dollars US annual economy). Successful transformation of the chloroplast genome should address concerns about transgene escape, insects developing resistance, inadequate insect control and promote public acceptance of genetically modified cotton.

Blotting, Southern↗

Recovery of soluble proteins from glanded cotton tissues with amines.

A simple soluble protein extraction method was developed for glanded cotton (Gossypium hirsutum L.) tissues. Gossypol, a major component of glands, is known to crosslink and precipitate proteins in cotton tissue homogenates. Established phenolic removal reagents were evaluated as gossypol binding agents and found to be less than effective in enhancing cotton leaf-soluble protein recovery. Several other amines, including a number of affinity support bound amines, were tested and found relatively ineffectual when compared with urea as cotton protein protectants. Urea and (NH4)2SO4, the next most active agent found in the study, were compared on both whole-leaf homogenates and artificial mixtures containing known quantities of poly-L-lysine and a clathrate of gossypol and acetic acid. Urea treatment resulted in both an increased number of stained bands and more concentrated staining of bands than any other treatment on polyacrylamide gels. Selected enzymes demonstrated increased activities in urea homogenates compared with other treatments.

Amines↗

Ulcerative colitis-linked antineutrophil cytoplasmic antibody in the cotton-top tamarin model of colitis.

The presence of antineutrophil cytoplasmic antibodies (ANCA) in serum has recently been shown to be a good disease marker for ulcerative colitis. An animal model of colitis expressing serum ANCA would allow prospective studies of the relationship between ANCA expression and development of colitis. The cotton-top tamarin model of spontaneous colitis was investigated for presence of ANCA to evaluate its potential as a model for the study of the immune response in human ulcerative colitis. Results show that cotton-top tamarin neutrophils have antigens recognized by immunoglobulin G (IgG) from the ANCA-positive serum of patients with ulcerative colitis. However, cotton-top tamarins do not generate serum IgG reactive to cotton-top tamarin or human neutrophils even when equal amounts of IgG are used for antigen detection. As new animal models of spontaneous and induced chronic colitis are developed, the presence of ANCA in serum, using the species' neutrophils, will be useful in relating the immune response of the model to that in human ulcerative colitis.

Animals↗

Effect of superior cervical ganglionectomy on the ultrastructure of the pinealocyte in the cotton rat (Sigmodon hispidus).

The ultrastructure of pinealocytes was examined morphometrically in superior cervical ganglionectomized and sham-operated cotton rats. Following denervation, the pinealocytes decreased in size. Reduced areas of the nucleoli, Golgi apparatus, granular endoplasmic reticulum, mitochondria, and vacuoles containing a flocculent material as well as a decreased number of dense-core vesicles were noted. In contrast, the numbers of "synaptic" ribbons and ribbon fields increased. The nature of ultrastructural changes observed in the cotton rat pinealocyte after sympathectomy may indicate a diminution of the presumptive secretory processes of this cell. The presence of a small number of myelinated and unmyelinated axons in the pineal gland of the cotton rat following superior cervical ganglionectomy suggests that the sympathetic nerve fibers from the superior cervical ganglia do not comprise the only source of innervation to the pineal. Additionally, the presence of striated muscle fibers has been observed in the present study in the pineal gland of the cotton rat which heretofore has not been reported in this species.

Animals↗

Effects of wood and cotton smoke on the surface properties of pulmonary surfactant.

The effects of wood and cotton smoke and several known smoke components on the dynamic surface activity of pulmonary surfactant were characterized with a modified Wilhelmy balance. Surfactant was harvested by saline lavage from dog lungs, placed in the balance and a control surface tension/area isotherm (y-A) and surface tension at minimum area (control y/min = 6.6 +/- 1.6 dynes/cm) measured. Hysteresis area (HA), recruitment index (RI), and stability index (SI) were calculated. Following control measurements, smoke (wood or cotton) was gently blown over the surfactant in the balance. Similarly, each of the individual smoke components or Liquid smoke (prepared by bubbling wood smoke through saline) were injected onto the balance. Wood smoke significantly (P < 0.05) altered all surface properties measured, increasing ymin (22.0 +/- 1.6 dynes/cm) and decreasing HA, RI, and SI as compared to control; cotton smoke exposure had almost no effect on surfactant function. A supplementary dose of surfactant was added to the balance, following wood smoke exposure, which decreased ymin (9.4 +/- 2.6 dynes/cm, P = NS vs control) but not the other parameters to control. Acrolein, formaldehyde, and HCl had little effect on any of the surface properties measured whereas isobutyraldehyde and liquid smoke altered the y-A curve but did not increase ymin. These data demonstrate that wood but not cotton smoke inhibit surfactant function, however, surfactant function can be restored, following deactivation by smoke, suggesting that surfactant replacement therapy for victims of severe smoke inhalation may be of benefit.

Animals↗

Pulmonary reactions to inhaled cotton dust: an animal model for byssinosis.

Groups of guinea pigs were exposed to respirable particles of cotton dust obtained from opening and carding rooms of a textile mill. The bulk cotton dust was resuspended with a modified sonic sifter. Exposures were conducted 6 hr/day at concentrations varying between 16 and 25 mg/m3. The particle size for these exposures varied around a 3 micron aerodynamic equivalent diameter. In animals exposed for 5 days, a pulmonary effect began to develop 3 hr after the start of exposure on the first day and was maximal 18 hr postexposure. The pulmonary effect faded during the ensuing weekdays as daily exposures continued. In one group exposed for 6 weeks, 5 days/week, the pulmonary effect was apparent on each Monday and faded during the week. The pulmonary response can best be characterized as a reflex restriction of breathing from stimulation of vagal nerve endings, resulting in rapid shallow breathing. This effect was exaggerated upon challenge of the animals with air containing 10% CO2. In humans this pattern of rapid shallow breathing is associated with symptoms of shortness of breath, dyspnea, breathlessness, and fatigue upon exertion. Such symptoms are characteristic of those reported by workers exposed to cotton dust. This animal model is proposed for further evaluation of the possible relationship between the acute responses to cotton dust and the development of a chronic response.

Animals↗

Evaluation of the toxicity and antiviral activity of carbocyclic 3-deazaadenosine against respiratory syncytial and parainfluenza type 3 viruses in tissue culture and in cotton rats.

The toxicity and antiviral efficacy of carbocyclic 3-deazaadenosine (Cc3Ado) against respiratory syncytial (RSV) and parainfluenza type 3 (PIV3) virus infections were tested in tissue culture and in cotton rats. The mean median efficacious dose (ED50) of Cc3Ado in HEp2 cells against RSV and PIV3 was 9 and 14 micrograms/ml, respectively. These values were 85- and 55-fold less than the median inhibitory (toxic) dose (ID50) of Cc3Ado in this cell line (750 micrograms/ml), and similar to values obtained for ribavirin. Cc3Ado exhibited no significant antiviral activity against influenza A, influenza B, adeno type 5 or adeno type 7 viruses (all ED50 were greater than 1000 micrograms/ml). In cotton rats, animals given greater than or equal to 1 mg/kg/day Cc3Ado intraperitoneally on days 1, 2 and 3 after experimental challenge with virus, consistently had significant reductions in pulmonary RSV and PIV3 titers compared to pulmonary virus titers in comparably treated control animals. The minimum efficacious dose of ribavirin given under the same conditions was 30 mg/kg/day. Cc3Ado was also efficacious in cotton rats when given orally by gavage, or when different administration schedules were used. The median efficacious dose of Cc3Ado when given orally was 10 mg/kg/day. No significant toxic effects were noted in cotton rats, even in animals given 20 mg/kg daily for eight consecutive days.

Animals↗

Comparison of cytobrush with cotton swab for endocervical cytologic sampling.

This study was designed to compare the Ayres spatula/cytobrush combination with the Ayres spatula/cotton swab combination as to the quality of the Pap smear in adolescent females. We evaluated 111 Pap smears, of which 60 (54%) were from cotton applicators/spatula combination and 51 (46%) were from cytobrush/spatula combination. Seventy-nine percent of the specimens with the cytobrush showed good cellular yield, compared to only 50% of the specimens obtained with a cotton applicator (p less than 0.05). The adequate specimen rate for endocervical cell content was 68% for the cytobrush versus 56% for the cotton applicator (p = not significant). The adequacy rate was less for trainees than for attending physicians, but the difference was less marked for those using the cytobrush. The cytobrush does have some impact on improving adequacy rates for Pap smears. However, this study was unable to determine whether this is a cost-effective addition to the Ayres spatula or cotton swab. The problem of inadequate Pap smears deserves more scrutiny among various health care systems.

Female↗

Physiological and growth responses of differentially irrigated cotton to ozone.

This study was conducted to determine the physiological and growth responses of cotton (Gossypium hirsutum L.) to the interaction of ozonee (O3) and drought stress. Cotton (cv SJ-2) was grown in open-top chambers in the field at three levels of soil water and exposed to charcoal-filtered air (CF), nonfiltered air (NF), and NF x 1.25, and NF x 1.5 ambient O3 concentrations in Riverside, CA, from June to October 1986. Ozone reduced carbon fixation an average of 74.6% in optimally watered (OW) plots, 63.4% in suboptimal (SO) plots, but only 19.3% in severely water-stressed (SS) plots. Leaf and stem biomass in OW and SO plots showed similar linear reductions in mass response to increased O3 concentrations, but SS plots showed no response to O3 except at the highest O3 treatment (seasonal 12-h O3 mean of 0.111 ppm 218 microm(-3)). These results showed that moderately water-stressed cotton had similar physiological and growth responses to O3 as well-watered plants, but severely water-stressed cotton showed little response to O3 at ambient O3 concentrations.

Journal Article↗

Spontaneous lytic activity against heterologous erythrocytes in cotton rat (Sigmodon hispidus) serum.

Although innate immunity has been well studied in laboratory animal models, no such documentation exists for wild species possessing a diversity of physiological adaptations to their environment. We examined the blood sera of 188 hispid cotton rats (Sigmodon hispidus) for naturally occurring hemolytic activity against heterologous erythrocytes. Ninety-two percent of the blood sera samples from cotton rats lysed sheep erythrocytes. All sera tested against chicken erythrocytes showed hemolytic activity, while only 44% of the same sera could lyse bovine erythrocytes. No hemolytic activity was present in cotton rat sera against erythrocytes from other rodent species (Eastern woodrat, Neotoma floridana, and pine vole, Microtus pinetorum). Hemolytic activity was heat labile and appeared to be mediated through the classical complement pathway. The protective nature of this hemolytic factor is unclear but it is probably directed at a more relevant molecule. These data, along with other reports of naturally occurring target specific serum factors in the cotton rat, may reflect the importance of innate protective mechanisms to small mammal populations.

Aging↗

Antiviral efficacy of VP14637 against respiratory syncytial virus in vitro and in cotton rats following delivery by small droplet aerosol.

VP14637, the lead compound in a series of substituted bis-tetrazole-benzhydrylphenols developed by ViroPharma Incorporated, was evaluated for antiviral efficacy against respiratory syncytial virus (RSV) in vitro in cell culture and in vivo in cotton rats. A selective index of >3000 (> or =2000 times greater than that observed for ribavirin) was determined in the in vitro studies for this compound against both RSV A and B subtypes. In cotton rats, animals given as little as 126 microg drug/kg by small droplet aerosol in divided doses starting 1 day after experimental virus infection with either a RSV A or B subtype consistently had significantly lower mean pulmonary RSV titers and reduced histopathological findings than mock-treated animals or cotton rats given placebo (vehicle-treated animals). No cotton rat treated with aerosols of VP14637 during these studies manifested any evident untoward responses. Thus, VP14637 exhibited good selective antiviral efficacy both in vitro and in vivo.

Aerosols↗

GhHb1: a nonsymbiotic hemoglobin gene of cotton responsive to infection by Verticillium dahliae.

Verticillium wilt of cotton is a widespread and destructive disease that is caused by the fungus pathogen Verticillium dahliae. Although no cotton cultivar is immune to the disease, some genotypes exhibit superior wilt tolerance. To gain an insight into the molecular mechanisms responsible for wilt tolerance, we employed the method of suppression subtractive hybridization (SSH) to isolate genes whose expression is up-regulated after inoculation of the pathogen in a wilt-tolerant cotton cultivar (Gossypium hirsutum cv. BD18). Among the identified candidate ESTs, a cDNA representing a nonsymbiotic hemoglobin gene (designated GhHb1) was further characterized in this study. Northern blot hybridization demonstrated that GhHb1 shares similar characteristics to some other nonsymbiotic hemoglobin genes including the hypoxic stress-induced expression. Sub-cellular localization analysis indicated that GhHb1 proteins were predominantly present in the nucleus with a minor amount appearing in the cytoplasm. Two novel features of GhHb1 were also identified, indicating that GhHb1 expression is activated in the cotton roots after inoculation with V. dahliae and that exogenous hydrogen peroxide induces GhHb1 expression. These results suggest that the GhHb1 may play a role in the defense response of G. hirsutum against V. dahliae invasion.

Amino Acid Sequence↗

Ultrastructural changes and location of beta-1, 3-glucanase in resistant and susceptible cotton callus cells in response to treatment with toxin of Verticillium dahliae and salicylic acid.

Calli from two cotton cultivars susceptible and resistant to Verticillium wilt, were treated with a crude toxin of Verticillium dahliae (VD-toxin) plus salicylic acid (SA). Cells treated with VD-toxin showed distinct ultrastructural changes. Cells from the susceptible cultivar displayed damage to plasma membrane and cytoplasm. The deleterious effect on cells of the resistant cultivar, with an accumulation of electron-dense precipitate in the vacuoles, was less noticeable. Exogenous SA protected callus cells from VD-toxin. We also report the localization of beta-1,3-glucanase in callus cells with immunofluorescence Labeling. Stronger fluorescence was observed in the extracellular space in resistant than in susceptible cotton; strongest in resistant cotton after 5 days of treatment with VD-toxin plus SA. The findings reported here indicate an important role of exogenous salicylic acid in the induction of resistance to VD-toxin in cotton. Coupled with an increase in beta-1,3-glucanase, cellular integrity is maintained and damage to cell wall and plasma membrane is avoided.

Cellulases↗