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Induction of systemic and mucosal immune responses in cotton rats immunized with human adenovirus type 5 recombinants expressing the full and truncated forms of bovine herpesvirus type 1 glycoprotein gD.

We generated both replication-incompetent (HAd5-gD-E1 and HAd5-tgD-E1) and replication-competent (HAd5-gD-E3 and HAd5-tgD-E3) human adenovirus type 5 (HAd5) recombinants expressing the full (gD) or truncated form (tgD) of the glycoprotein gD gene of bovine herpevirus type 1 (BHV-1). Recombinant gD and tgD expressed by HAd5-gD-E1 and HAd5-gD-E3 and by HAd5-tgD-E1 and HAd5-tgD-E3, respectively, were recognized by gD-specific monoclonal antibodies (MAbs) directed against linear and conformational epitopes, suggesting that antigenicity of recombinant gD and tgD was similar to that of the native gD expressed in BHV-1 infected cells. In HAd5-gD-E1- or HAd5-gD-E3-inoculated cotton rats there was a strong gD- and HAd5-specific IgG and IgA antibody response. The immune response was significantly lower in animals similarly immunized with HAd5-tgD-E1 or HAd5-tgD-E3, indicating that live adenovirus vaccine vectors may be better suited to the full-length form of glycoprotein gD than its truncated form. After a BHV-1 challenge, no infectious BHV-1 virions were isolated from the trachea of cotton rats previously immunized with HAd5-gD-E1 or HAd5-gD-E3. These results suggest that adenovirus E1 insertion (replication-incompetent) and E3 insertion (replication-competent) vectors have excellent potential for use in developing live recombinant virus vaccines and provide evidence that the cotton rat model can be used in BHV-1 vaccination-challenge trials.

Adenoviruses, Human↗

Insertion of HIV-1 genes into Ad4DeltaE3 vector abrogates increased pathogenesis in cotton rats due to E3 deletion.

Adenovirus type 5 (Ad5) E3 region proteins abrogate Ad pathogenicity in the lungs of cotton rats. Our use of Ad4-HIV E3-deleted (DeltaE3) recombinants as vaccines necessitates further examination of these viruses for enhanced pathogenesis. Equivalent infectious doses of Ad4 wild-type (Ad4WT), Ad4DeltaE3, and two recombinants: Ad4DeltaE3HIVenv and Ad4DeltaE3HIVgag, were inoculated intranasally into cotton rats. Ad4 viruses did not replicate in the lungs, but caused mild pathologic effects, including peribronchiolitis, bronchitis, alveolitis, and interstitial pneumonia. As found previously for Ad5, deletion of Ad4 E3 genes resulted in increased lung pathology. Surprisingly, insertion of HIV genes into this region significantly restored protection attributed to E3 gene products, diminishing overall pathologic effects to Ad4WT levels (P < 0.0001). Similarly, following administration of equivalent particle numbers of the four viruses, only Ad4DeltaE3 caused increased overall pathology, while the two HIV recombinant viruses showed effects comparable to Ad4WT (P < 0.01). Our observation that Ad4DeltaE3HIV recombinants are as safe in cotton rats as Ad4WT encourages their continued development as AIDS vaccines.

AIDS Vaccines↗

Structural characterization of genes corresponding to cotton fiber mRNA, E6: reduced E6 protein in transgenic plants by antisense gene.

Two genes, each corresponding to fiber mRNA E6, were isolated from cotton cultivars Coker 312 (Gossypium hirsutum L.) and Sea Island (G. barbadense L.). E6 is one of the predominant fiber-specific mRNAs present during early fiber development. The distinguishing feature of nucleotide-derived E6 protein is the presence of a motif where a dimer, Ser-Gly, is repeated several times. Two of the Sea Island genes contained a pentameric motif, Ser-Gly, while one of the Coker genes had one and the other had four motifs each. cDNA clones containing one or five Ser-Gly motifs were also identified. Thus, it appears that the strict conservation of this motif may not be critical to E6 protein function. Sequence characterizations of the genes and cDNAs showed that multiple members of the E6 family are transcribed in fiber and may result in proteins 238 to 246 amino acids long. The 3' ends of the genes and cDNAs showed considerable heterology among them. Transgenic plants containing antisense genes were generated to decipher E6 function. Transgenic cotton with reduced E6 protein levels in the range of 60 to 98% were identified. However, no discernible phenotypic changes in fiber development or properties were apparent. This result leads to the conclusion that E6 is not critical to the normal development or structural integrity of cotton fibers.

Amino Acid Sequence↗

Characterization and expression of metallothionein-like genes in cotton.

We have characterized cotton (Gossypium hirsutum L.) genes encoding type 1 metallothionein-like proteins that are highly expressed in roots. Little or no expression of these genes was detected in other organs and tissues. The deduced amino acid sequences have a high degree of similarity with type 1 metallothionein-like proteins from other plants, including a central hydrophobic domain flanked by conserved cysteine-rich motifs. The type 1 metallothionein-like genes of cotton are encoded by a small gene family. One gene (MT1-A) was analyzed in detail and found to have three exons which are 52, 83 and 397 bp long, and two introns 130 and 1042 bp in length. Three of the type 1 metallothionein-like genes are organized in a tandom array, and the 5'-flanking regions of these genes share a high degree of sequence similarity. Two of the clustered genes (MT1-A and MT1-B) are expressed at about equal levels in roots and use the same transcription start site. A 640 bp promoter fragment from the MT1-A gene was sufficient to direct expression of beta-glucuronidase (GUS) in transformed cotton roots. The expression was highest near the root tip.

Amino Acid Sequence↗

The cotton-top tamarin revisited: Mhc class I polymorphism of wild tamarins, and polymorphism and allelic diversity of the class II DQA1, DQB1, and DRB loci.

Cotton-top tamarins (Saguinus oedipus) in captivity are unusual in that they exhibit low levels of polymorphism and allelic diversity at the major histocompatibility complex (Mhc) class I loci. Since the polymorphism has previously only been examined in captive tamarins, we analyzed the Mhc class I alleles of a population of wild tamarins. These wild tamarins, like their captive counterparts, exhibited limited class I polymorphism. We also assessed the levels of polymorphism and allelic diversity at the Mhc class II DQA1, DQB1, DQB2, and the DRB loci in captive populations of cotton-top tamarins. In contrast to the extensive polymorphism in Old World monkeys, only two alleles were detected at each of DQA1 and DQB1. Also, the DQB2 locus was monomorphic and conserved between New and Old World monkeys. Sequences derived from four putative DRB loci were obtained, and extensive polymorphism was found at all four loci. Phylogenetic analysis did not indicate that any of the tamarin DRB loci, with the possible exception of Saoe-DRB3, were orthologous to the human DRB loci. At three of the DRB loci (Saoe-DRB11, Saoe-DRB*W12, Saoe-DRB*W22), the number of nonsynonymous changes was higher than the number of synonymous changes in the putative antigen recognition sites, indicative of positive selection. We found no support for a restriction on the polymorphism at the cotton-top tamarin class II loci. However, the allelic diversity at some of the Saoe-DRB loci is more limited than for the HLA-DRB1, consistent with a restriction imposed by the bone marrow-chimerical lifestyle.

Alleles↗

Intragenome distribution of 5-methylcytosine in DNA of healthy and wilt-infected cotton plants (Gossypium hirsutum L.).

Fractionation of DNA of healthy and wilt-infected cotton plants has been carried out according to the reassociation kinetics and the content of GC and 5-methylcytosine in the resulting fractions has been studied. The genome of cotton plant was found to be methylated quite unevenly. The GC rich (GC equals 64.7 mole%) fraction of highly reiterated sequences (Cot equals 0-3.7 times 10- minus 2) has a high content of 5-methylcytosine (5.8 mole%), whereas the methylation degree of the fraction of unique sequences (Cot larger than or equal to 487) is very low (the 5-methylcytosine content is about 0.5 mole%). In plants being infected with wilt, the 5-methylcytosine content in DNA or cotton leaves decreases two-fold; no changes in the structure and molecular population of DNA has been found. The sharp change in the 5-methylcytosine content in DNA of infected plants takes place at the expense of the decrease in the 5-methylcytosine content in fractions of highly reiterated sequences. The methylation degree of unique sequences (structural genes) remains unchanged.

Base Sequence↗

Residues of toxaphene, DEF, and paraquat in plant parts and gin waste from a treated cotton field.

The origin and fate of the gin waste residues of three chemicals--toxaphene, DEF, and paraquat--used frequently in cotton production in California were studied. Residues were determined for foliage and bolls in the live plants, for lint and seed in harvested seed cotton, and for gin waste and its lint and non-lint components, following commercial treatments to the cotton crop. Gin waste residues were followed during five months of open storage to ascertain dissipation rates in a typical waste sample. Relatively high residues of each of the three chemicals persisted at harvest, during storage prior to ginning, and in stored gin waste. The major proportion of gin waste residue was associated with the non-lint components--leaf parts, burrs, stems and other foreign matter. Average stored gin residues in the lint and non-lint components were 13 and 60, 11 and 58, and 5 and 10 ppm for toxaphene, DEF, and paraquat, respectively, during the open storage period.

Chromatography, Gas↗

[Parameters of collagen biosynthesis in cotton pellet granuloma of rats on iron deficiency (author's transl)].

Rats of Wistar strain, of same age, were kept on iron free diet up to nearly absolute iron deficiency in iron depots. Afterwards granuloma was produced by implantation of cotton pellets subcutaneously. For characterization of the collagen biosynthesis in the granuloma the neutral salt soluble collagen and the activity of the prolyl hydroxylase were measured in the granuloma 4, 8 and 12 days after cotton pellet implantation. At the same time "collagen-like" protein was determined in the serum of the animals. The enzyme activity was statistically significant lower in the granuloma of animals on iron deficiency than in the granuloma of the comparable groups on normal diet. Statistically significant higher concentration of neutral salt soluble collagen was found 8 and 12 days after cotton pellet implantation in the group on iron free diet. There was no significant difference as to the serum levels of "collagen-like" protein in serum.

Anemia, Hypochromic↗

The evaluation of cotton twine as a new anti-inflammatory assay.

A new anti-inflammatory assay is described, utilizing a subcutaneously implanted elongated section of cotton twine to induce granuloma in rats. The cotton twine irritant is easily introduced under the abdominal skin and after seven days a well formed granuloma is established. The procedure works effectively for both steroidal and non-steroidal anti-inflammatory drug evaluation. Consistency of the cotton twine eliminates individual weighing of each section and uniformity in compactness affords reproducible results.

Animals↗

Genes encoding small GTP-binding proteins analogous to mammalian rac are preferentially expressed in developing cotton fibers.

In animals, the small GTP-binding proteins, Rac and Rho, of the ras superfamily participate in the signal transduction pathway that regulates the organization of the actin cytoskeleton. We report here on the characterization of two distinct cDNA clones isolated from a cotton fiber cDNA library that code for homologs of animal Rac proteins. Using gene-specific probes, we have determined that amphidiploid cotton contains two genes that code for each of the two Rac proteins, designated Rac13 and Rac9, respectively. The gene for Rac13 shows highly enhanced expression in developing cotton fibers, with maximal expression occurring at the time of transition between primary and secondary wall synthesis. This is also the time at which reorganization of the cytoskeleton occurs, and thus the pattern of expression of Rac13 is consistent with its possible role, analogous to animal Rac, in the signal transduction pathway that controls cytoskeletal organization.

Amino Acid Sequence↗

Ultrastructural effects of cellulose biosynthesis inhibitor herbicides on developing cotton fibers.

Cotton fibers are often utilized as a model system to investigate cellulose biosynthesis and cell wall elongation. In this study, we grew cotton fibers in vitro, with ovules dissected at day zero post anthesis as the explant source, in the presence of three herbicides that inhibit cellulose biosynthesis. Cultures were sampled for electron microscopy and immunocytochemistry 1-2 days after beginning the treatments. After dichlobenil treatment, the fibers were much shorter than the controls and assumed a variety of abnormal shapes, from shortened versions of the control fiber to nearly spherical. The inner layers of the fiber wall often contained juxtaposed electron-translucent and -transparent areas; this layer reacted strongly with antibodies to callose. Cellulase-gold labeling in these newly developed fibers grown in the presence of dichlobenil was present at only about 3% of the control labeling. After treatment with either isoxaben or flupoxam, the fibers assumed spherical shapes and frequently (more than 60% of fibers) exhibited a new cell plate within the fiber, indicating that cell division had occurred, a process that rarely occurred in the controls. Unlike the dichlobenil-treated fibers, fibers grown in the presence of isoxaben or flupoxam contained an extensive accumulation of chiefly deesterified pectins, replacing the entire wall with an elaborated version of the pectin sheath found in control cotton fibers. These data indicate that all three herbicides are effective disrupters of cellulose biosynthesis and cause radical changes in cell wall structure and composition. Moreover, these data indicate that the composition of the walls may influence indirectly cell cycle kinetics, keeping these fiber cells in a more meristematic mode.

Benzamides↗

An optimized gossypol high-performance liquid chromatography assay and its application in evaluation of different gland genotypes of cotton.

A comparative study on gossypol content of various genetic types of pigment glands of cotton varieties was conducted through an optimized high-performance liquid chromatography (HPLC) on a C18 column (4.6 mm x 250 mm, 5 microm particle) with methanol-0.5% acetic acid aqueous solution, 90 : 10 (v/v), as mobile phase, at a flow rate of 0.8 ml/min and UV detection at 254 nm. The method was shown to be highly reproducible, with precision [as relative standard deviation (RSD)] and accuracy [as relative mean error (RME)] < 10%, both intra-day and inter-day. Absolute recoveries were > 94%. The results revealed major differences among the different gland varieties or species of cotton, including the special and ordinary glandless and glanded Gossypium hirsutum, G. barbadense, and displayed the precious resources of different glands of extraordinary cotton.

Calibration↗

Changes in the levels of bioactive components of cotton bracts accompanying senescence.

A series of bracts from Deltapine 41 variety cotton were harvested weekly from October 16 through November 25, 1982, and examined for changes in biologically active components accompanying senescence. Aqueous extracts of cotton bracts (CBE) were tested for their ability to cause 5-hydroxytryptamine (5-HT) release from human platelets and contraction of isolated canine tracheal smooth muscle (TSM). In addition, the amounts of soluble tannins (the platelet-activating factor found in CBE) and endotoxin were determined for each CBE. The amount of CBE decreased significantly from October 16 through November 25, whereas endotoxin levels rose dramatically from October 23 to November 6 and declined thereafter. The amount of soluble tannin and platelet 5-HT release both decreased from highs on October 23 to unmeasurable levels on November 25. Smooth muscle contraction was attributed to two primary factors contained in CBE. The major constrictor was a 5-HT receptor agonist and its levels remained stable from October 16 through October 30, but then decreased to unmeasurable levels by November 13. In contrast, CBE also contained lesser amounts of an acetylcholine receptor agonist whose levels paralleled the changes in endotoxin levels. We conclude that several important biochemical changes occur in cotton bracts during senescence and that these changes profoundly influence their biological activity in various assays.

Animals↗

Effect of spent cotton stalks on color removal and chemical oxygen demand lowering in olive oil mill wastewater by white rot fungi.

Wastewater from olive oil mill was decolorized (and its chemical oxygen demand reduced in static cultivation) using the fungi Coriolus versicolor, Funalia trogii, Phanerochaete chrysosporium and Pleurotus sajor-caju. The effect of cotton stalk on decolorizing and COD removing capability was demonstrated. P. chrysosporium (in 20% medium with cotton stalk) reduced the COD by 48% and color by 58%, F. trogii (in 30% medium with cotton stalk)) by 51 and 55%, respectively.

Biodegradation, Environmental↗

Cotton smoke inhalation primes alveolar macrophages for tumor necrosis factor-alpha production and suppresses macrophage antimicrobial activities.

The present study determined the effects of cotton smoke inhalation on the functioning of alveolar macrophages (mphi). Smoke inhalation led to dose-dependent impairment of respiratory gas exchange by 48 h postexposure and pulmonary edema by 96 h. Maximal effects were observed in animals ventilated with 54 breaths of cotton smoke (3-min exposure, 18 breaths/min). Macrophages were obtained at 48 h postexposure by bronchoalveolar lavage of rabbits subjected to 54 breaths of smoke or room air (control). Phagocytosis of opsonized bacteria and adherence to solid substratum were reduced in smoke-exposed mphi. Smoke inhalation primed mphi for release of tumor necrosis factor-alpha (TNF-alpha) induced by lipopolysaccharide (LPS). Smoke-exposed mphi were also primed for TNF-alpha release induced by phorbol myristate acetate, which suggests that the priming event occurred downstream of protein kinase C activation in the signal transduction pathway. Further, smoke exposure attenuated the inhibitory effects of phosphodiesterase inhibitors on LPS-induced TNF-alpha release. Thus, the priming event may be mediated through cAMP and/or protein kinase A. The data indicate that cotton smoke inhalation suppresses the antimicrobial activities of alveolar mphi and can lead to excessive mphi production of TNF-alpha. These mphi effects would be expected to contribute to the pathophysiological abnormalities associated with smoke inhalation injury.

Acute Disease↗

A cost of alarm pheromone production in cotton aphids, Aphis gossypii.

The sesquiterpene, (E)-beta-farnesene, is used by many aphid species as an alarm pheromone to warn related individuals of predation. Disturbed cotton aphids, Aphis gossypii Glover, released (E)-beta-farnesene into the air as detected by solid phase microextraction and gas chromatography mass spectrometry (GC-MS). Solvent extracts of cotton aphids of various life stages and weights also were analyzed by GC-MS for sums of ions 69 and 93, which discriminated (E)-beta-farnesene from coeluting compounds. Aphids of all life stages and sizes reared on cotton plants in both an environmental chamber and glasshouse contained (E)-beta-farnesene in amounts ranging from 0.1 to 1.5 ng per individual. The quantities of (E)-beta-farnesene in aphids increased in relation to increasing body weight, and variation in individual weights explained about 82% of the variation in alarm pheromone. However, the concentrations (ng/mg fresh weight) declined exponentially with increasing body weight. These findings indicate that aphid nymphs try to compensate for their smaller size by producing relatively more pheromone per weight than adults but still cannot approach an evolutionary optimal load, as assumed in adults with the greatest total amounts. This suggests that young aphids need to balance costs of growth and maturation with costs of producing the alarm pheromone.

Animals↗

Genetic mapping of a cross between Gossypium hirsutum (cotton) and the Hawaiian endemic, Gossypium tomentosum.

The existence of five tetraploid species that derive from a common polyploidization event about 1 million years ago makes Gossypium (cotton) an attractive genus in which to study polyploid evolution and offers opportunities for crop improvement through introgression. To date, only crosses (HB) between the cultivated tetraploid cottons Gossypium hirsutum and G. barbadense have been genetically mapped. Genetic analysis of a cross (HT) between G. hirsutum and the Hawaiian endemic G. tomentosum is reported here. Overall, chromosomal lengths are closely correlated between the HB and HT maps, although there is generally more recombination in HT, consistent with a closer relationship between the two species. Interspecific differences in local recombination rates are observed, perhaps involving a number of possible factors. Our data corroborate cytogenetic evidence that chromosome arm translocations have not played a role in the divergence of polyploid cottons. However, one terminal inversion on chromosome (chr.) 3 does appear to differentiate G. tomentosum from G. barbadense; a few other apparent differences in marker order fall near gaps in the HT map and/or lack the suppression of recombination expected of inversions, and thus remain uncertain. Genetic analysis of a discrete trait that is characteristic of G. tomentosum, nectarilessness, mapped not to the classically reported location on chr. 12 but to the homoeologous location on chr. 26. We propose some hypotheses for further study to explore this incongruity. Preliminary quantitative trait locus (QTL) analysis of this small population, albeit with a high probability of false negatives, suggests a different genetic control of leaf morphology in HT than in HB, which also warrants further investigation.

Chromosome Mapping↗

Physical mapping of the Rf1 fertility-restoring gene to a 100 kb region in cotton.

Cytoplasmic male sterility (CMS) plays an important role in crop heterosis exploitation. Determining one or more nuclear genes that can restore male fertility to CMS is essential for developing hybrid cultivars. Genetic and physical mapping is the standard technique required for isolating these restoration genes. By screening 2,250 simple sequence repeat (SSR) primer pairs in cotton (Gossypium hirsutum L.), we identified five new SSR markers that are closely linked to the Rf1 gene, a fertility restorer gene of cotton for CMS-D2. Based on our previous fine mapping of the Rf1 gene and assemblage of three published STS markers, we constructed a high-resolution genetic map of Rf1 containing 13 markers in a genetic distance of 0.9 cM. The 13 molecular markers were used to screen a bacterial artificial chromosome (BAC) library from a restorer line 0-613-2R containing Rf1 gene, which yielded 50 single positive clones. There was an average of 3.8 clones ranging from 1 to 12 BAC clones per PCR marker. These 50 clones produced an average insert size of 120 kb (ranging between 80 and 225 kb). Thirty-five primer pairs were designed based on 38 sequences of BAC ends, and two new STS markers tightly linked to Rf1 gene have been tagged and integrated into this map. The physical map for the Rf1 gene was constructed by fingerprinting the positive clones digested with the HindIII enzyme. We were able to delimit the possible location of the Rf1 gene to a minimum of two BAC clones spanning an interval of approximately 100 kb between two clones designated 081-05K and 052-01N. Further work using these two BAC clones will lead to isolation of the Rf1 gene in cotton.

Chromosome Walking↗