Luteal progesterone and prostaglandin F2α production invitro during fluprostenol-induced luteolysis in the mare

Prostaglandin F₂α (PGF₂α) release $\text{in}$$\text{vitro}$ by luteal tissue from mares was quantified to determine if exogenous prostaglandin analog increased endogenous luteal PGF₂α production during induced luteolysis. On day 8 after ovulation, luteal tissue was collected by flank laparotomy and endometrium was collected by uterine biopsy. Mares were assigned to one of four treatments: (1) no intramuscular injection at 0-hr (n = 5), (2) 250 μg Fluprostenol (ICI 81008 PGF₂α analog) at 4-hr (n = 4), (3) 250 μg Fluprostenol at 12-hr (n = 5), or (4) 250 μg Fluprostenol at 28-hr (n = 5) prior to tissue collection at laparotomy. Blood was collected from a jugular vein at laparotomy. Luteal and endometrial tissues (100-mg minces) were incubated in duplicate in 5 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) in an ice bath in an air atmosphere or at 37°C in an atmosphere of 95% O₂:5% CO₂. The incubation treatments consisted of: no treatment, indomethacin 1.3 × 10^?4M, 1 μg/ml of arachidonic acid, 10 μg/ml of Fluprostenol, and 100 μM dbc-AMP (Fluprostenol was not added to endometrial tissue incubations). The injection of Fluprostenol induced luteolysis in these mares as indicated by decreased plasma progesterone and luteal tissue progesterone production (P<0.01). Luteal PGF₂α production was only detectable in tissue from mares that had been injected with Fluprostenol; production reached a maximum by 12 hr post-injection and had returned to pre-treatment levels by 28 hr (P<0.01). Endometrial tissue produced PGF₂α, but this activity was not significantly affected by injection of mares with Fluprostenol. Increased production of PGF₂α by luteal tissue of mares during PGF₂α analog induced luteolysis was similar to that observed in the pig and ewe.     

Enzyme studies on TPPase-reactive cytoplasmic structures observed in early meiotic prophase I of the hamster oocyte

Summary Ovaries of immature and adult hamsters were incubated in medium containing thiamine pyrophosphate (TPP) to determine the age at which TPPase-reactive cytoplasmic structures first appear in the germ cells, and at what age the structures cease to be present. The structures were found only in oocytes from animals 8–15 days of age. They occur in predictyate germ cells in polyovular follicles and in very early dictyate oocytes in unilaminar follicles. The TPPase-reactive structures were never observed in atretic oocytes, in unilaminar follicles of adult animals, nor in multilaminar follicles of animals at any age.Ovaries of 8–12-day-old animals and adults were then incubated in media in which one of the following substrates was substituted for TPP: uridine diphosphate (UDP), inosine diphosphate (IDP), and adenosine monophosphate (AMP). Half of the samples in each experiment were incubated in medium containing the inhibitor L-p-bromotet-ramisole. -Glycerophosphate was used in control incubations, or the substrate was omitted entirely.The cytoplasmic structures were found to be reactive after incubation in UDP-containing media, but not after incubation in media containing AMP. With IDP as substrate, reactions were atypical and confined to peripheral regions of the cytoplasm.Other sites of enzyme activity after incubation with the various substrates (cell membranes, zona pellucida, endoplasmic reticulum and Golgi apparatus) are also described and discussed.     

Patterns of cell division and interkinetic nuclear migration in the chick embryo hindbrain

Early in its development, the chick embryo hindbrain manifests an axial series of bulges, termed rhombomeres. Rhombomeres are units of cell lineage restriction, and both they and their intervening boundaries form a series that reiterates various features of neuronal differentiation, cytoarchitecture, and molecular character. The segmented nature of hindbrain morphology and cellular development may be related to early patterns of cell division. These were explored by labeling with BrdU to reveal S-phase nuclei, and staining with basic fuchsin to visualise mitotic cells. Whereas within rhombomeres, S-phase nuclei were located predominantly toward the pial surface of the neuroepithelium, at rhombomere boundaries S-phase nuclei were significantly closer to the ventricular surface. The density of mitotic figures was greater toward the centres of rhombomeres than in boundary regions. Mitotic cells did not show any consistent bias in the orientation of division, either in the centres of rhombomeres, or near boundaries. Our results are consistent with the idea that rhombomeres are centres of cell proliferation, while boundaries contain populations of relatively static cells with reduced rates of cell division.     

Postmortem Changes in Rat Brain Extracellular Opioid Peptides Revealed by Microdialysis

Microdialysis combined with a solid-phase radioimmunoassay was used to monitor changes in extracellular opioid peptide levels in the rat globus pallidus/ventral pallidum as a result of terminal brain ischemia. Ischemia was induced by anesthetic overdose or by severance of blood vessels supplying the brain. In control animals the recovered immunoreactivity increased an average of 13-fold in the 30-min sample following anesthetic overdose. Perfusion of a calcium-free, 10 mM EGTA-containing medium through the dialysis probe significantly attenuated the amplitude of this response, with the average increase being only threefold. Shorter sampling intervals (5 min) indicated that release of opioid peptide material into the extracellular environment occurs within the first 5 min of ischemia resulting from severance of the blood supply to the brain. HPLC analysis identified the majority of the postmortem-induced immunoreactive material as Met- and Leu-enkephalin.     

Epitope mapping of monoclonal antibodies toEscherichia coli ribosomal protein S3

The antigenic structure ofEscherichia coli ribosomal protein S3 has been investigated by use of monoclonal antibodies. Six S3-specific monoclonal antibodies secreted by mouse hybridomas have been identified by immunoblotting of two-dimensional ribosomal protein separation gels. By using a competitive enzyme-linked immunosorbent assay, we have divided these monoclonal antibodies into three mutual inhibition groups, members of which are directed to three distinct regions of the S3 molecule. The independence of these monoclonal antibody-defined regions was confirmed by the failure of pairs of monoclonal antibodies from two inhibition groups to block the binding of biotinylated monoclonal antibodies of the third group. To determine the regions recognized by these monoclonal antibodies, chemically cleaved S3 peptides were fractionated by gel filtration and reverse-phase high-performance liquid chromatography. The fractionated peptides were coated on plates and examined for specific interaction with monoclonal antibody by enzyme immunoassay. In this manner, two epitopes have been mapped at the ends of the S3 molecule: one, in the last 22 residues, is recognized by three monoclonal antibodies; and the second, in the first 21 residues, is defined by two monoclonal antibodies. The third S3 epitope, recognized by a single monoclonal antibody, has been localized in a central segment of about 90 residues by gel electrophoresis and immunoblotting. These epitope-mapped monoclonal antibodies are valuable probes for studying S3 structurein situ.     

A Comparison of 2-Hydroxyestradiol and U-0521 (3'4'-Dihydroxy-2-methylpropiophenone, Upjohn) as In Situ and In Vitro Inhibitors of Tyro sine Hydroxylase

Abstract: Feedback inhibition of tyrosine hydroxylase by catechols was evaluated using in situ and in vitro enzyme assays. The three catechol compounds used were norepinephrine, 2-hydroxyestradiol, and 3'4'-dihydroxy-2-methylpropiophenone (U-0521, Upjohn); representing endogenous catechol-amines, catechol estrogens, and a synthetic catechol, respectively. The in situ experiments were performed with dissociated retinal cells from rats and with stationary phase adrenergic-like neuroblastoma cells (N1E-115). The catechol estrogen, 2-hydroxyestradiol, resembled the endogenous catecholamines in its potency to inhibit in vitro and in situ tyrosine hydroxylations with IC₅₀ values of 10 μM in vitro and 100 μM in situ. The drug U-0521, which has been used as an inhibitor of catechol- O -methyltransferase (COMT), was also found to be an inhibitor of tyrosine hydroxylase. Further, it was shown to be more potent than the natural catechols, both in vitro and in situ , with IC₅₀ values of 30–600 nM.     

Azido analogs of acridine: Photoaffinity probes for frameshift mutagenesis in Salmonella typhimurium

In order to identify a photoaffinity probe for 9-aminoacridine frameshift mutagenesis, 20 azido analogs of acridine were synthesized and tested in Ames' Salmonella tester strains, TA1535, TA1537, TA1538 and their corresponding excision-repair-proficient strains TA1975, TA1977, and TA1978, to determine their mutagenicity and toxicity relative to 9-aminoacridine. The substituent-mutagenicity patterns observed for these compounds agree very well with those obtained previously for non-azidoacridines. The results presented here show that the 2-azido-analog of 9-aminoacridine demonstrates biological activity similar to 9-aminoacridine prior to photolytic activation. With light activation, however, the 9-amino-2-azido derivative becomes more effective at producing frameshift mutations characteristics of 9-aminoacridine. Furthemore, this photolytic enhancement of mutagenesis appears to be due to the repairable lesion suggesting that covalent attachment of the drug occurs.     

Cytochemistry of the Golgi apparatus in developing ovarian germ cells of the Syrian hamster

Summary A cytochemical study of the Golgi apparatus in the developing oocyte of the golden hamster was carried out using the TPPase, AcPase and zinc iodide-osmium tetroxide (ZnOs) techniques. Tissue from both immature and sexually mature animals was investigated.Peak TPPase activity was found in pre-growth oocytes in ovaries from sexually mature adults. Some activity was also present in SER in the peripheral cytoplasm of growing oocytes. AcPase activity was found only after the onset of oocyte growth. It was present in Golgi cisternae and associated vesicles and in some profiles of peripheral SER. No structures corresponding to GERL were identified. Strong staining with ZnOs was seen, at all stages studied, in certain Golgi vesicles and short tubules but not in the cisternae unless the oocyte was atretic. Weaker ZnOs staining was characteristic of ER throughout the oocyte.With all techniques there was a falling off of reactivity as oocyte size increased. Within a single oocyte some Golgi bodies were negative while others were positive, with both TPPase and AcPase techniques. This suggests that two or more functional types of this organelle are present within the developing oocytes.We would like to thank Dr. K.N. Christie for his interest and helpful suggestions regarding the enzyme techniques     

Crystallization of the B800-820 light-harvesting complex from Rhodopseudomonas acidophila strain 7750.

The B800-820 light-harvesting complex, an integral membrane protein, from Rhodopseudomonas acidophila strain 7750 has been crystallized. The tabular plates have a hexagonal unit cell of a = b = 121.8 A and c = 283.1 A and belong to the space group R32. X-ray diffraction data have been collected to 6 A resolution, using an area detector on a rotating anode source. The B800-820 light-harvesting complex is comprised of four low molecular weight apoproteins (B800-820 alpha 1, B800-820 alpha 2, B800-820 beta 1 and B800-820 beta 2). Polyacrylamide gel electrophoresis shows that the complex exists as an oligomeric assembly, with an apparent molecular weight of 92,000.