Interrelationship between estrogen and thyroxine on the release of luteinizing hormone and gonadotropin-releasing hormone in vitro

The present experiments were designed to study the interaction between estradiol benzoate (EB) and thyroxine (T4) given in vivo on the responsiveness of pituitary luteinizing hormone (LH) to gonadotropin-releasing hormone (GnRH) and the release of GnRH in vitro. Ovariectomized-thyroidectomized (Ovx-Tx) rats were injected s.c. with saline or T4 (2 micrograms/100 g b.wt), and oil or EB (0.1 microgram) once daily for 40 days following a 2 x 2 factorial design. All animals were then decapitated and blood samples were collected. Anterior pituitaries (APs) were incubated in vitro with and without 0.1 ng GnRH at 37 degrees C for 4 h. Mediobasal hypothalami (MBHs) were excised and then incubated with and without APs from Ovx donor rats. Concentrations of LH and GnRH in the medium and that of LH in the serum were measured by radioimmunoassay. The LH level in media containing MBHs and donor APs was used as the index of bioactive GnRH release. In Ovx-Tx rats, T4 injections reduced the serum LH concentration, the pituitary LH response to GnRH, and the bioactive as well as the immunoreactive GnRH release. The serum LH levels and the spontaneous as well as the GnRH-stimulated release of LH in vitro were suppressed in Ovx-Tx rats following administration of EB. By contrast, the serum LH concentration, as well as pituitary LH response to GnRH and GnRH release in vitro, were higher in the group treated with both T4 and EB than in that treated with saline and EB. These results suggest that the differential changes in the LH secretion after thyroidectomy of Ovx versus non-Ovx rats are due to an antagonistic effect between T4 and estrogen on the response of pituitary LH to GnRH, and the release of GnRH.     

Genetic mechanisms of tumor-specific loss of 11p DNA sequences in Wilms tumor.

Wilms tumor, a common childhood renal tumor, occurs in both a heritable and a nonheritable form. The heritable form may occasionally be attributed to a chromosome deletion at 11p13, and tumors from patients with normal constitutional chromosomes often show deletion or rearrangement of 11p13. It has been suggested that a germinal or somatic mutation may occur on one chromosome 11 and predispose to Wilms tumor and that a subsequent somatic genetic event on the normal homologue at 11p13 may permit tumor development. To study the frequency and mechanism of such tumor-specific genetic events, we have examined the karyotype and chromosome 11 genotype of normal and tumor tissues from 13 childhood renal tumor patients with different histologic tumor types and associated clinical conditions. Tumors of eight of the 12 Wilms tumor patients, including all viable tumors examined directly, show molecular evidence of loss of 11p DNA sequences by somatic recombination (four cases), chromosome loss (two cases), and recombination (two cases) or chromosome loss and duplication. One malignant rhabdoid tumor in a patient heterozygous for multiple 11p markers did not show any tumor-specific 11p alteration. These findings confirm the critical role of 11p sequences in Wilms tumor development and reveal that mitotic recombination may be the most frequent mechanism by which tumors develop.     

The isolation,characterization and application in the Triticeae of a set of wheat RFLP probes identifying each homoeologous chromosome arm

Summary To investigate the use of RFLP analysis in the Triticeae, a set of low copy number probes has been isolated from a wheat cDNA library. The probes identify each of the 14 homoeologous chromosome arms of wheat as determined by analysis of DNA fragments hybridizing to the probes in aneuploid lines of Chinese Spring. These probes can be used in RFLP analyses both for the assignment of homoeology of alien chromosomes or arms added to wheat, and for the determination of chromosome dosage in wheat aneuploids. Different chromosomes from various Triticeae species can therefore be followed in a wheat genetic background using a single technique. The potential uses of the set in facilitating the transfer of alien segments into wheat are outlined.     

RFLP-based genetic maps of wheat homoeologous group 7 chromosomes

Summary Restriction fragment length polymorphism (RFLP) mapping was attempted using 18 cDNA clones, 14 anonymous and 4 of known function, which had been shown to have homologous DNA sequences on the group 7 chromosomes of wheat. The loci identified by these probes have been mapped on one or more chromosomes in this homoeologous group using linkage data derived from various F₂, random inbred, doubled haploid and single chromosome recombinant populations. The maps also include three isozyme loci, five disease resistance loci, two anthocyanin pigment loci and a vernalisation response locus. The mapping data have been used to determine the extent of map co-linearity over the A, B and D genomes, the degree of RFLP variability in the three genomes and the relative efficiency of various restriction enzymes in detecting RFLPs in wheat. The strategy for future mapping in wheat, particularly the use of alien genomes or segments, such as that from Aegilops ventricosa used here, is discussed.     

Monospecific antibodies against a synthetic peptide predicted from the alpha-3 nicotinic receptor cDNA inhibit binding of [3H]nicotine to rat brain nicotinic cholinergic receptor

Polyclonal antibodies were raised against a synthetic decapeptide (designated S3) predicted from a segment of the alpha-3 subunit cDNA (amino acid residues 130-139) encoding the rat brain nicotinic cholinergic receptor. This segment was selected because it may be proximate to the nicotine/acetylcholine-binding site of the receptor (1). By radioligand binding assays and sucrose density gradient centrifugation, these monospecific antibodies were shown to inhibit the binding of [3H]nicotine to both the large molecular weight rat brain receptor (240 kDa) and to an SDS-disaggregated nicotine-binding subunit species (80 kDa), in a dose-dependent manner. The neutralizing effect of the anti-S3 antibodies supports the view that this region of the protein is closely related to the agonist binding site.     

Acquired immunity in rats against Angiostrongylus cantonensis infection

Acquired immunity against Angiostrongylus cantonensis was induced by immunizing rats with somatic antigens from fifth-stage larvae and adult worms and live third-stage larvae. Rats immunized twice had significantly fewer worms than rats immunized three times. Fewer worms were recovered from rats immunized with 200 live third-stage larvae than from any other groups. Rats immunized with somatic antigens had higher enzyme-linked immunosorbent assay (ELISA) antibody levels than rats immunized with live larvae. Rats immunized with live third-stage larvae of Angiostrongylus cantonensis were more strongly protected against challenge infections (62-92%) than rats immunized with antigens extracted from fifth-stage larvae (0-30%) and adult worms (11-24%).     

A partial map of the barley genome incorporating restriction fragment length polymorphism, polymerase chain reaction, isozyme, and morphological marker loci

Nine low copy number genomic DNA clones, a ribosomal sequence, and seven cDNA clones were found to identify polymorphisms in cultivated barley (Hordeum vulgare L.). An F2 population consisting of 100 plants was produced from a cross between a high-yielding two-rowed feed barley cultivar and a genetic marker stock homozygous for nine recessive and one dominant morphological marker genes. Through the use of these 10 well-distributed marker genes, five previously mapped isozyme loci, and two storage-protein loci, the approximate recombinational location for each of 17 restriction fragment length polymorphism loci was estimated. One clone, pMSU21, identified variation that appeared to be the result of a small insertion-deletion event that differentiated two-rowed and six-rowed genotypes. This difference was characterized, and one allele was sequenced. Oligonucleotide primers that flanked the insertion-deletion event were synthesized, and DNA samples from the F2 population were subjected to polymerase chain reaction sequence amplification. The variation identified by this technique was determined to be allelic to the variation identified using pMSU21 in Southern blot analysis. Maps of previously undescribed informative clones are included.     

Glutathione production in copper-deficient isolated rat hepatocytes.

Dietary copper deficiency has been shown to reduce copper-dependent superoxide dismutase (SOD) activity and to increase lipid peroxidation in rats. Circulating reduced glutathione (GSH) concentrations are elevated in copper-deficient (CuD) rats, which suggests an increased GSH synthesis or decreased degradation, perhaps as an adaptation to the oxidative stress of copper deficiency. GSH synthesis was examined in isolated hepatocytes from CuD rats. Isolated hepatocytes were prepared by collagenase perfusion and incubated in Krebs-Henseleit bicarbonate buffer, pH 7.4, 10 mM glucose, 2.5 mM Ca2+ in the presence and absence of 1.0 mM buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. Cell viability was assessed by trypan blue exclusion. GSH and oxidized glutathione (GSSG) were measured by the glutathione reductase recycling assay. Copper deficiency depressed hepatocyte Cu by greater than 90% and increased intracellular GSH by 41-117% over the 3-h incubation, with a two- to threefold increase in the rate of intracellular GSH synthesis. Intracellular GSSG values were minimally influenced by CuD, with a constant mol% GSSG. Extracellular total glutathione (GSH + 2GSSG) synthesis was increased by approximately 33%. Both intracellular GSH and extracellular total glutathione synthesis were inhibited by BSO. The pattern of food consumption in CuD rats, meal fed versus ad libitum fed, had no effect on glutathione synthesis. The results indicate an increased hepatic GSH synthesis as a response to dietary copper deficiency and suggest an interrelationship between the essential nutrients involved in oxyradical metabolism.     

The antigen of hepatitis delta virus: examination of in vitro RNA-binding specificity.

The only known protein of hepatitis delta virus (HDV), the delta antigen, is found both within virus particles and within the nucleus of the infected cell, where it has one or more roles essential for RNA genome replication. Others have demonstrated that the antigen has the ability, in vitro, to specifically bind HDV RNA species. We report a further examination of this phenomenon, using partially purified recombinant protein, expressed as a fusion with the staphylococcal protein A. From Northwestern (RNA-immunoblot) analyses with both complete and various subdomains of HDV genomic and antigenomic RNAs, we found that a necessary feature for specific binding was that the RNA be able to fold to some extent into the so-called rodlike structure; this structure is a predicted intramolecular partial base-pairing of the circular RNA, with about 70% of all bases involved, so as to produce an unbranched rodlike structure. Six different subregions of the HDV rodlike structure, three on the genomic RNA and three on its complement, the antigenomic RNA, were tested and found to be sufficient for antigen binding. However, features in addition to the rodlike structure may also be necessary for specific binding, because we found that a similar structure present in the RNA of the potato spindle tuber viroid did not allow binding.