In vitro maturation of Arbacia punctulata oocytes and initiation of heavy body formation

Summary An ultrastructural study of in vitro maturation of A. punctulata oocytes was undertaken to determine when heavy body formation was initiated. No heavy bodies were seen in germinal vesicle oocytes or in oocytes undergoing germinal vesicle breakdown or polar body formation. Heavy bodies were only observed in ova examined one to two hours after pronuclear formation. Several small heavy bodies were seen in sections of eggs fixed as early as three hours after the pronucleus had formed. The number of these structures in the egg cytoplasm increased with time. Therefore it is concluded that heavy body formation in sea urchin ova is a phenomenon following nuclear maturation.Contribution No. 222 from the Institute for Molecular and Cellular Evolution. Supported in part by grants from the National Institutes of Health (5-T01-HD00026-09 to the Fertilization and Gamete Physiology Training Program at the Marine Biological Laboratory and predoctoral fellowship 1-F01-GM-36,719-01A1 to C. M. Conway) and the National Science Foundation (GB3899 to C. B. Metz).The authors are grateful to Drs. A. F. Conway, Giovanni Giudice, and Gertrude W. Hinsch for consultation and criticism of this work.     

Applications and limitations of measurement of 15-keto,13,14-dihydro prostaglandin E2 in human blood by radioimmunoassay

It has been anticipated that the inherent limitations of radioimmunoassays for prostaglandin E (PGE) would be obviated by assays for its major circulating metabolite, 15-keto, 13,14-dihydro PGE2) which has a longer half-life in blood. We examined the effects of PGE2 infusion and alterations in lipolysis in vivo, and of clotting, prolonged storage and hemolysis in vitro, on KH2-PGE2 immunoreactivity in unextracted human plasma and serum samples. Indeed KH2-PGE2 levels rose several hundred fold during infusions of PGE2 at doses which cause little or no increment in peripheral PGE levels. During stimulation of lipolysis by infusions of epinephrine, apparent KH2-PGE2 levels rose five-fold. However, the dilution curve of plasma obtained during stimulation of lipolysis was not parallel to the standard curve; furthermore, apparent KH2-PGE2 levels were correlated strongly with free fatty acid (FFA) levels, suggesting that FFA's cross-reacted in the RIA weakly but significantly due to their very high molar concentration in blood. Clotting and prolonged storage of samples, but not hemolysis, also caused marked apparent increments in KH2-PGE2 levels. Competition curves using dilutions of such samples were again not parallel to the standard curves in plasma or buffer, but resembled dilution curves of samples containing high levels of FFA. These results suggest that handling of human blood samples for KH2-PGE2 measurement must be carefully standardized to avoid significant artifacts which presumably are due in part to fatty acids released from triglyceride stores in vivo or from disrupted membrane phospholipids in vitro. Unextracted plasma appears to be unsatisfactory for use in this RIA.     

Identification of a chloroplast membrane polypeptide associated with the oxidizing side of photosystem II by the use of select low-fluorescent mutants of Scenedesmus

Comparison of photochemical activities and variable fluorescence yield characteristics of whole cells and isolated chloroplast particles of low-fluorescent, photosystem II mutants of $\text{Scenedesmus}$$\text{obliquus}$ to those of the wild-type showed that several strains were affected primarily on the oxidizing side of photosystem II. In strains LF-1, LF-3, and LF-5 analysis of the manganese content of isolated chloroplast membranes showed a predominant shift in the $\text{Mn}\text{400}$ Chl from the wild-type value (4.3) to values near 1.5; this difference was also associated with a near total loss of cytochrome b-559 (high potential). Examination of chloroplast membrane polypeptides by gel electrophoresis revealed a decrease only in the mobility of one band in all three mutants; the apparent molecular weight was shifted from 34 kilodalton in the wild-type to 36 kilodalton in the mutants. Evidence is presented suggesting that the 34 kilodalton polypeptide of the wild-type is probably associated with the manganese requiring portion of the water-splitting apparatus of photosystem II.     

Heart innervation after ligation of the left anterior descending coronary artery (LAD)

Distribution and amount of neuropeptide Y- and synaptophysin-immunoreactive nervous structures within the heart were investigated in dogs 4 days after ligation of the left anterior descending coronary artery (LAD). In the right atrium and posterior left ventricular regions, which were taken as (non-infarcted) control areas, neuropeptide Y-immunoreactive paravascular nerves and a perivascular nerve plexus running within the adventitia of the coronary arteries and their branches down to the arterioles were observed. Morphometric measurements of the area density revealed 0.099 +/- 0.014% for synaptophysin- and 0.037 +/- 0.0072% for neuropeptide Y-immunoreactivity within the posterior wall of the left ventricular myocardium. Four days after ligation of the LAD only single synaptophysin- and neuropeptide Y-immunoreactive nerve fibers were very rarely detected in the infarcted region of the anterior wall of the left ventricle. Above the ligature larger than normal neuropeptide Y-immunoreactive axons within nerves along the LAD indicated a blockage of the axoplasmic transport of this peptide. When investigating this model of experimental myocardial infarction, mechanical traumatization of peri- and paravascular nerves of the LAD by the ligature has to be considered as a major pathogenetic factor, in addition to ischemia leading to denervation of infarcted as well as non-ischemic myocardium.