Radioiodination and characterization of the plasma membrane of sea urchin sperm

Two methods were used to radioiodinate sea urchin sperm: lactoperoxidase-glucose oxidase and Iodo-Gen. Following iodination the sperm are viable, they undergo the acrosome reaction, and they fertilize eggs. Of the radioactivity associated with the labeled sperm, 28–50% is presumed to be free ¹²⁵I^?, 37–47% is incorporated in lipid, and 8–15% is in trypsin-digestible material believed to be protein. Digestion of the labeled, living sperm with trypsin removes 95.6–99.5% of the macromolecular label (the cells are alive after digestion) suggesting that almost all the protein label is on the external surface of the cell. Thin-layer chromatography of the lipid fraction shows that the major membrane phospholipids and cholesterol are labeled. SDS-PAGE analysis shows the protein-incorporated ¹²⁵I is distributed among four glycoproteins of >250K, 84K, 64K, and 52K dalton apparent molecular weight. Twenty-eight percent of the total protein (trypsin-digestible) label is in the 84K component and 46% in the 64K band. Although both molecules contain much of the label, they are relatively minor components of the TX-100 extract of sperm. The methods outlined will be useful in determining the role of sperm surface components in fertilization.     

Intrinsic vs. extrinsic influences on life history expression: metabolism and parentally induced temperature influences on embryo development rate

Intrinsic processes are assumed to underlie life history expression and trade‐offs, but extrinsic inputs are theorised to shift trait expression and mask trade‐offs within species. Here, we explore application of this theory across species. We do this based on parentally induced embryo temperature as an extrinsic input, and mass‐specific embryo metabolism as an intrinsic process, underlying embryonic development rate. We found that embryonic metabolism followed intrinsic allometry rules among 49 songbird species from temperate and tropical sites. Extrinsic inputs via parentally induced temperatures explained the majority of variation in development rates and masked a relationship with metabolism; metabolism explained a minor proportion of the variation in development rates among species, and only after accounting for temperature effects. We discuss evidence that temperature further obscures the expected interspecific trade‐off between development rate and offspring quality. These results demonstrate the importance of considering extrinsic inputs to trait expression and trade‐offs across species.     

Frost resistance of winter rape leaves as related to the changes in water potential and growth capability

Differential thermal analysis indicated that the frost resistance of winter rape leaves ( Brassica napus L. var. oleifera L. cv. Gòrczanski), collected from plants grown in the cold (5/2°C), relies mainly on their ability to supercool to −9 to −11°C, i.e. consists in freezing avoidance. Initiation of ice formation in the cold-acclimated leaves resulted in the death of more than 50% of the cells as determined with a conductivity method. The development of freezing tolerance appeared to be an attribute of the second stage of plant hardening and was induced by the exposure of plants to a slightly subzero temperature (−5°C) for 18 h. Such a treatment brought about a sudden and persistent water potential decrease in the leaves, despite the fact that they had reabsorbed water from the medium prior to water potential measurements. Water potential changes were associated with a higher growth capability of the leaves as checked by determinations of disk area increments. It is suggested that the increased frost tolerance of the cold-grown winter rape leaves, subjected to subfreezing temperature, is related to the decreased water potential of the tissue caused by changes in turgor and/or in osmotic pressures of the cells.     

Electrostatic calculations and model-building suggest that DNA bound to CAP is sharply bent

Two observations suggest that DNA, upon binding to E. coli catabolite gene activator protein (CAP), is sharply bent by a total angle of at least 100-150 degrees: (1) The electrostatic potential field of CAP shows regions of positive potential that form a ramp on 3 sides of the protein. (2) The DNA binding site size as determined by DNA ethylation interference with binding, (Majors: "Control of the E. coli Lac Operon at the Molecular Level." Ph.D. Thesis, Harvard University, Cambridge, 1977) and by relative affinities of DNA fragments of various lengths (Liu-Johnson et al.: Cell 47:995-1005, 1986) requires severe bending of the DNA to maintain its favorable electrostatic contact with the protein.     

Role of xanthine oxidase inhibitor as free radical scavenger: a novel mechanism of action of allopurinol and oxypurinol in myocardial salvage

Xanthine oxidase (XO) has been hypothesized to be a potential source of oxygen-derived free radicals during reperfusion of ischemic myocardium based on the fact that allopurinol, a XO-inhibitor, can reduce reperfusion injury. In this communication we report that both allopurinol and oxypurinol, the principle metabolite of allopurinol, prevent the reperfusion injury in isolated pig heart. However, we found that neither pig heart nor pig blood contain any XO activity. Our study showed a direct free radical scavenging action of these XO-inhibitors during ischemia and reperfusion, as judged by the reduction of free radical signals when compared using an Electron Paramagnetic Resonance Spectrometer. Using a Luminometer, we also confirmed that both allopurinol and oxypurinol can scavenge ClO2, HOCl, and significantly inhibit free radical signals generated by activated neutrophils. These XO-inhibitors, however, failed to scavenge O2. and OH. radicals. Our results suggest that these XO-inhibitors salvaged the ischemic-reperfused myocardium by scavenging free radicals, and not by inhibiting XO in the pig heart.     

Oxygen-derived free radicals and hemolysis during open heart surgery

Reperfusion injury occurs during open-heart surgery after prolonged cardioplegic arrest. Cardiopulmonary bypass also is known to cause hemolysis. Since reperfusion of ischemic myocardium is associated with the generation of oxygen free radicals, and since free radicals can attack a protein molecule, it seems reasonable to assume that hemolysis might be the consequence of free radical attack on hemoglobin protein. The results of this study demonstrated that reperfusion following ischemic arrest caused an increase in free hemoglobin and free heme concentrations, simultaneously releasing free iron and generating hydroxyl radicals. In vitro studies using pure hemoglobin indicated that superoxide anion generated by the action of xanthine oxidase on xanthine could release iron from the heme ring and cause deoxygenation of oxyhemoglobin into ferrihemoglobin. This study further demonstrated that before the release of iron from the heme nucleus, oxyhemoglobin underwent deoxygenation to ferrihemoglobin. The released iron can catalyze the Fenton reaction, leading to the formation of cytotoxic hydroxyl radical (OH·). In fact, the formation of OH. in conjunction with hemolysis occurs during cardiac surgery, and when viewed in the light of the in vitro results, it seems likely that oxygen-derived free radicals may cause hemolysis during cardiopulmonary bypass and simultaneously release iron from the heme ring, which can catalyze the formation of OH·.     

Effects of cold on CO2 exchange in winter rape leaves

A viable wheat hybrid intermediate of the same height as the parents was obtained by crossing the female parent of tall variety NP4 with the male parent of the dwarf variety HD2160. Seeds of the hybrid and its parents were germinated and their growth pattern as well as the activities of peroxidase, indolyl-3-acetic acid oxidase and amylase in extracts made from them were studied at the early seedling stages i.e. up to 96 h.
A positive correlation existed between the length of the axis at the early seedling stage and at mature plant height as far as the parental varieties are concerned but no such correlation was observed with the hybrid. Growth of the hybrid seedlings was less than of its parents. Light appeared to stimulate the longitudinal growth of the axis to different extents in the parents and hybrid. Higher activities of peroxidase, indolyl-3-acetic acid oxidase and amylase were observed in the hybrid as compared to both of its parents. Lethal wheat hybrid also exhibits increased activities of amylase, indolyl-3-acetic acid oxidase and peroxidase. Therefore, it appears that seedling growth and enzyme activities at the seedling stage are not always correlated with hybrid vigour.     

Effects of Phosfon-D and of low temperature on the morphology of cell protoplasts

Summary The adventitious roots of onion (Allium cepa L.) or onion bulb scales were treated either with growth retardant Phosfon-D (2,4-dichlorobenzyltributylphosfonium chloride), or with low temperature (5° C). Low temperature, likewise Phosfon-D treatment, resulted in the appearance of membranous structures within the cytoplasm of the root cells fixed in chromaceto-formalin (CrAF) and in the occurrence of minute vacuoles in the living onion bulb scale cells. The cytochemical analysis revealed the membrane-like elements to contain basic and acid proteins as well as nucleic acids.It seems that the observed effect of Phosfon-D and that of low temperature were due to the local accumulation of protein resulting in changes of the physical properties of the cytoplasm. The fact that membranous structures do contain nucleic acid may suggest their important, metabolic role especially in the process of cold acclimation of plant tissue.This work was partly supported by Grant No. FG-PO-225 from U.S. Department of Agriculture, Agricultural Research Program, Public Law 480.     

Histochemische Untersuchungen über den Involutionsmechanismus der Milchdrüse

Zusammenfassung Es wurden mit histochemischen Methoden Milchdrüsen von Meerschweinchen in physiologischer und durch Kastration während der Laktationsperiode hervorgerufener Involution untersucht. Nachgewiesen wurden alkalische und saure Phosphatase (APh und SPh), unspezifische Esterase, Sukzinodehydrase, SH-Gruppen, Nukleinsäuren und Lipide. — Die Aktivität der SPh nimmt während der Involution zu. Die Bedeutung dieses Enzyms für den Rückbildungsprozeß des Drüsengewebes wird diskutiert. — Das Vorkommen der APh-Aktivität in Drüsenendstücken im Involutionsstadium kann auf die Beteiligung dieses Enzyms an der Rückresorption der mit der Milch ausgeschiedenen Substanzen hinweisen. — Zwischen der Aktivität der unspezifischen Esterase und dem Gehalt sowie der Lokalisation von Lipiden besteht eine Abhängigkeit. — Es konnten keine Unterschiede in der Sukzinodehydraseaktivität und dem DNS-Gehalt aufgezeigt werden. — Die Verminderung von SH-Gruppen und RNS hängt mit dem Aufhören der Sekretionsproduktion durch die Zellen der Drüsenendstücke der Milchdrüsen zusammen.

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Histochemical studies on the involution mechanism of the mammary gland

Summary Histochemical methods were used to study mammary glands of guinea pigs in the course of physiologic involution and that induced by castration during lactation. Alkaline and acid phosphatases, unspecific esterase, succinic dehydrogenase, SH groups, nucleic acids and lipids were determined. Acid phosphatase activity was found to be increased in mammary glands, subject to involution. The participation of the enzyme in the involutionary process of the gland tissue is discussed. The distribution of alkaline phosphatase in the secretory sections of the gland during involution would suggest the participation of the enzyme in the reabsorption of the substance secreted with milk. A correlation existing between the activity of unspecific esterase, the level and distribution of lipids in the mammary gland could be established. No differences were detected in the activity of succinic dehydrogenase and DNA level. A decrease in SH groups and RNA content is related to cessation of milk secretion.