Time of ovulation in the South Australian Merino ewe following synchronization of estrus. 2. Efficacy of GnRH treatment and its relevance to insemination programs utilizing frozen-thawed semen

In a study of the time of ovulation following synchronization of estrus in the ewe, the effect of time of treatment with GnRH (24 vs 36 h after pessary removal) and dosage (6.25 to 100 ug per ewe) were examined. All treatments synchronized the time of ovulation irrespective of when untreated ewes commenced to ovulate. As part of an evaluation of GnRH treatment in artificial insemination programs, an assessment was made of the quality of eggs obtained from control ewes and ewes treated with GnRH at either 24 or 36 h after pessary removal. Treatment at 24 h increased the number of retarded embryos (P < 0.01) and unfertilized ova (P < 0.01) collected per ewe, reduced the number of embryos collected per ewe (P < 0.01), and reduced (P < 0.05) the percentage of pregnant ewes compared with other groups. However, there were no differences between control ewes and ewes treated with GnRH at 36 h. GnRH treatment at 36 h was consequently examined as a means of improving conception rates following the intrauterine insemination of frozen-thawed semen. Insemination of GnRH-treated ewes 8 to 12 h before the median time of ovulation resulted in a nonsignificant increase (range 5.7 to 7.3%) in the percentage of ewes of mature age which became pregnant. Insemination 0 to 4 h before the median time of ovulation resulted in a nonsignificant decrease in the percentage of pregnant ewes. GnRH treatment did not influence the number of fetuses per ewe. Reasons for the failure of this treatment to significantly improve ewe fertility are discussed.     

The OBF1 protein and its DNA-binding site are important for the function of an autonomously replicating sequence in Saccharomyces cerevisiae.

The autonomously replicating sequence ARS121 was cloned as a 480-base-pair (bp) long DNA fragment that confers on plasmids autonomous replication in Saccharomyces cerevisiae. This fragment contains two OBF1-binding sites (sites I and II) of different affinities, as identified by a gel mobility shift assay and footprint analysis. Nucleotide substitutions (16 to 18 bp) within either of the two sites obliterated detectable in vitro OBF1 binding to the mutagenized site. Linker substitution (6 bp) mutations within the high-affinity site I showed effects similar to those of the complete substitution, whereas DNA mutagenized outside the binding site bound OBF1 normally. We also tested the mitotic stability of centromeric plasmids bearing wild-type and mutagenized copies of ARS121. Both deletion of the sites and the extensive base alterations within either of the two OBF1-binding sites reduced the percentage of plasmid-containing cells in the population from about 88% to 50 to 63% under selective growth and from about 46% to 15 to 20% after 10 to 12 generations of nonselective growth. Furthermore, linker (6 bp) substitutions within site I, the high-affinity binding site, showed similar deficiencies in plasmid stability. In contrast, plasmids containing linker substitutions in sequences contiguous to site I displayed wild-type stability. In addition, plasmid copy number analysis indicated that the instability probably resulted not from nondisjunction during mitosis but rather from inefficient plasmid replication. The results strongly support the notion that the OBF1-binding sites and the OBF1 protein are important for normal ARS function as an origin of replication.     

Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.     

Effect of thyroidectomy on cardiovascular responses to hypoxia and tyramine infusion in fetal sheep

Fetal sheep were thyroidectomized at 80 days' gestation and reoperated at 118-122 days for insertion of vascular catheters. The effects of hypoxaemia and intravenous tyramine infusion on plasma catecholamine concentrations, blood pressure and heart rate were then determined in experiments at 125-135 days' gestation. Age matched intact fetuses were also studied. Thyroidectomy was associated with increased concentrations of noradrenaline, adrenaline and dopamine in some thoracic and abdominal organs, increased noradrenaline concentrations in the cerebellum, and decreased adrenaline concentrations in the hypothalamus, cervical spinal cord, and superior cervical and inferior mesenteric ganglia. Arterial pressure was significantly lower in the thyroidectomized fetuses (34.0 +/- 0.15 mmHg) than in intact fetuses (44.7 +/- 0.2 mmHg; p less than 0.001). In contrast, plasma noradrenaline concentrations were significantly higher in the thyroidectomized fetuses (2.04 +/- 0.25 ng/ml) compared to the intact fetuses (0.99 +/- 0.08 ng/ml; P less than 0.001). In the intact fetuses there was a significant increase in plasma noradrenaline concentration and blood pressure during hypoxaemia, and bradycardia at the onset of hypoxaemia. In contrast, in the thyroidectomized fetuses hypoxaemia did not cause significant change in plasma catecholamine concentrations, blood pressure or heart rate. Infusion of tyramine produced a 1.9-fold increase of plasma noradrenaline in thyroidectomized fetuses compared to a 9.2-fold increase in the intact fetuses (P less than 0.05). Tyramine infusion caused a similar proportional increase of blood pressure in both thyroidectomized and intact fetuses. Heart rate decreased during the tyramine-induced hypertension in the intact fetus, but increased in the thyroidectomized fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immobilization of Penicillium chrysogenum: spore growth on Celite

Summary Penicillium chrysogenum spores have been immobilized by adsorption on two grades of wet or dry diatomaceous earth particles, Chromosorb-W and Celite R-633. Almost 90% of the spores were adsorbed within 2 h and those remaining in suspension were removed by washing to minimise the growth of free mycelia. After germination the immobilized biomass was almost independent of the spore loading on the particles and whether or not the spore suspension was added to wet or dry particles. The free biomass obtained was less than 5% of the immobilized biomass.     

Developmental changes in galactosyltransferase activity in the rat small intestine

During postnatal development, UDP-Gal: GlcNAc(beta 1-4)-galactosyltransferase (4 beta-GT) and UDP-Gal:GalNAc(beta 1-3)-galactosyltransferase (3 beta-GT) activities were increased by 17- and 24-fold, respectively, in the rat small intestine. The injection of cortisone into suckling rats resulted in precocious induction of distal 4 beta- and 3 beta-GT activities by 2.7- and 1.8-fold, respectively. Injection of phorbol-12-myristate-13-acetate (PMA) resulted in precocious induction of distal 3 beta-GT by 2.7-fold. These results suggest that intestinal galactosyltransferase activities are under developmental regulation and can be modified by cortisone and PMA.     

Use of culture fluorescence as a sensor for on-line discrimination of host and overproducing recombinant Escherichia coli

The culture fluorescence of two recombinant Escherichia coli strains with high plasmid copy number were studied and compared to both the host and low copy number varieties of the corresponding strains. Culture fluorescence data are related to the concentration of reduced intracellular nicotinamide adenine dinucleotide within a cell, and can therefore be used as a means for detecting changes in metabolic states. Correlation curves relating culture fluorescence to biomass show that the recombinant system maintains a larger pool of intracellular NADH at high plasmid copy numbers than either the host or the recombinant system at low copy numbers. These results demonstrate the ability of a fluorescence probe to detect differences in the metabolic demands made on an over-producing recombinant organism.     

Transposition of the Responder Element (Rsp) of the Segregation Distorter System (Sd) to the X Chromosome in Drosophila Melanogaster

In order to test whether the meiotic drive system Segregation distorter (SD) can operate on the X chromosome to exclude it from functional sperm, we have transposed the Responder locus (Rsp) to this element. This was accomplished by inducing detachments of a compound-X chromosome in females carrying a Y chromosome bearing a Rsps allele. Six Responder-sensitive-bearing X chromosomes, with kappa values ranging from 0.90 to 1.00, were established as permanent lines. Two of these have been characterized more extensively with respect to various parameters affecting meiotic drive. SD males with a Responder-sensitive X chromosome produce almost exclusively male embryos, while those with a Rsp-Y chromosome produce almost exclusively female embryos. This provides a genetic system of great potential utility for the study of early sex-specific differentiation events as it allows the collection of large numbers of embryos of a given sex.     

Cell death by apoptosis during involution of the lactating breast in mice and rats

The role of cell death in involution of lactating breast was investigated in mice and rats by light and electron microscopy. Apoptosis, recognized by sharply demarcated compaction of chromatin against the nuclear envelope and by shrinkage and budding of the whole cell to form membrane-bounded apoptotic bodies, was responsible for major loss of cells in both species. In the mouse, rapid involution during the first 2 days was associated with shedding of large numbers of apoptotic bodies derived from alveolar epithelial cells into alveolar lumens. This was followed by more gradual regression, during which the bodies were mostly phagocytosed by macrophages within the epithelium. In the rat, glandular involution was a more gradual and uniform process, with shedding of apoptotic epithelial cells into alveolar lumens being much less conspicuous. Apoptosis of myoepithelial cells was observed in mice, the resulting apoptotic bodies being phagocytosed by intraepithelial macrophages, but was not detected in rats. Apoptosis of capillary endothelial cells caused rapid regression of the capillary beds in both mice and rats. Intraepithelial macrophages increased in number during involution, developed cytoplasmic lipofuscin pigment, and either remained within the epithelium or migrated to the interstitium and regional nodes. Cell loss by apoptosis has been demonstrated during involution and atrophy of a variety of other glands. It characteristically results in shrinkage of a tissue without disruption of its basic architecture.     

Two bovine genes for mitochondrial ADP/ATP translocase expressed differences in various tissues

Two different bovine cDNAs have been characterized that encode closely related homologues of the mitochondrial membrane carrier protein ADP/ATP translocase. One of them codes for the protein that has been characterized previously from bovine heart mitochondria, and the other codes for a protein that differs from it in 33 amino acids out of 297. Including the base substitutions required to bring about these changes in amino acid sequence, the coding regions of the cDNAs differ at 184 positions. In addition, they are extensively diverged in their 3' noncoding sequences, which differ greatly in both length and sequence, and these segments of the cDNAs have been used as hybridization probes to demonstrate that the expression of the two genes giving rise to the two proteins is very different in various bovine tissues. Expression of one gene predominates in heart muscle and that of the other in intestine. Hybridization experiments with digests of genomic DNA have shown the presence of numerous sequences related to the two cDNAs in both the bovine and human genomes. Some of these probably arise from pseudogenes, but three expressed genes have been detected in the human genome. The study of the regulation of the expression of these genes may help to illuminate the basis of tissue-specific human mitochondrial diseases which arise because of defects in mitochondrial enzymes only in the affected tissue and not in other tissues of the same individual.