Energy requirement of bovine spermatozoa for in vitro capacitation

The oxidative energy requirements of bovine spermatozoa capacitated with dilauroil-phosphatidylcholine liposomes (PC 12) and the effect of these liposomes on acrosome reaction necessary for in vitro fertilization were studied. Mitochondrial respiration was measured using 3 different substrates (pyruvate-lactate-glucose) and endogenous substrates. The samples were either treated with PC 12 or were left untreated and used as the control. A 2.8-fold increase in the consumption of oxygen was observed in the PC 12 treated spermatozoa in the presence of the 3 combined substrates (pyruvate-lactate-glucose). Respiration changes were not observed when the spermatozoa were capacitated with only 2 of the 3 substrates or with glucose alone. When endogenous substrates were used, the consumption of oxygen increased 1.7 times, and mitochondrial uncoupling was observed in the treated samples. The hypermotility characteristic of the capacitation process was not observed when glucose or endogenous substrates were used. When the percentage of intact acrosomes was determined using differential-interferential contrast (DIC) microscopy, it was found that in the presence of oxidative substrates there was a 26% decrease compared with that of the control sample. The proportion of reacted acrosomes was in the range of 41.3 to 49.6%, as measured by the chlortetracycline epifluorescence method in the presence of calcium ionophore A23187. Only 4% of the spermatozoa showed acrosome reaction with endogenous substrates. A higher percentage of fertilized oocytes were observed when the spermatozoa were capacitated in the presence of the 3 substrates (pyruvate-lactate-glucose), confirming that the success of in vitro fertilization depends on the energy conditions associated with the capacitation process. The results of these experiments indicate that the presence of oxidative energy is necessary to produce capacitation and the hyperactivation characteristic in frozen-thawed bovine spermatozoa treated with liposomes.     

Inducible nitric oxide synthase and guinea-pig ileitis induced by adjuvant

We sought to establish a model of inflammatory bowel disease by augmenting the activity of the local immune system with Freund's complete adjuvant, and to determine if inducible nitric oxide synthase (iNOS) expression and peroxynitrite formation accompanied the inflammatory condition. In anaesthetized guinea-pigs, a loop of distal ileum received intraluminal 50% ethanol followed by Freund's complete adjuvant. Control animals were sham operated. When the animals were killed 7 or 14 days later, loop lavage fluid was examined for nitrite and PGE(2) levels; mucosal levels of granulocyte and macrophages were estimated by myeloperoxidase (MPO) and N-acetyl-D-glucosaminidase (NAG) activity, respectively. Cellular localization if iNOS and peroxynitrite formation were determined by immunohistochemistry with polyclonal antibodies directed against peptide epitopes of mouse iNOS and nitrotyrosine, respectfully. Adjuvant administration resulted in a persistent ileitis, featuring gut thickening, crypt hyperplasia, villus tip swelling and disruption, and cellular infiltration. Lavage levels of PGE(2) and nitrite were markedly elevated by adjuvant treatment. Immunoreactive iNOS and nitrotyrosine bordered on detectability in normal animals but were markedly evident with adjuvant treatment at day 7 and particularly day 14. Immunohistochemistry suggested that enteric neurons and epithelia were major sites of iNOS activity and peroxynitrite formation. We conclude that local administration of adjuvant establishes a chronic ileitis. Inducible nitric oxide synthase may contribute to the inflammatory process.     

Low Temperature Effects on Soybean (Glycine max [L.] Merr. cv. Wells) Mitochondrial Respiration and Several Dehydrogenases during Imbibition and Germination

The influence of low temperature on soybean (Glycine max [L.] Merr. cv. Wells) energy transduction via mitochondrial respiration and dehydrogenases was investigated in this study during imbibition and germination. Mitochondria were isolated from embryonic axes of seeds treated at 10 and 23 C (control) by submergence in H₂O for 6 hours and maintenance for an additional 42 hours in a moist environment. Arrhenius plots of initial respiration rates revealed that those from cold-treated axes had respiratory control (RC) ratios of near 1.0 above an inflection in the plot at 8 C. Arrhenius plots of control axes mitochondrial respiration showed RC ratios of 2.8 above and 5.0 below an inflection temperature of 12.5 C. Energies of activation for mitochondrial respiration between 20 and 30 C for the cold and control treatments were 7.8 and 15.6 kcal/mole, respectively. These data indicate possible differences in mitochondrial membranes, degree of mitochondrial integrity, and mitochondrial enzyme complement between the two treatments.     

Host-Pathogen Interactions: XIV. Isolation and Partial Characterization of an Elicitor from Yeast Extract

An elicitor of glyceollin accumulation in soybeans (Glycine max L.) has been isolated from a commercially available extract of brewers' yeast. Yeast is not a known pathogen of plants. The elicitor was isolated by precipitation in 80% (v/v) ethanol followed by column chromatography on DEAE-cellulose, sulfopropyl-Sephadex, and concanavalin A-Sepharose. Compositional and structural analysis showed the elicitor to be a glucan containing terminal, 3-, 6-, and 3,6-linked glucosyl residues. The yeast elicitor stimulates the accumulation of glyceollin in the cotyledons and hypocotyls of soybeans when as little as 15 nanograms or 100 nanograms of the elicitor is applied to the respective tissues. The yeast elicitor is very similar in both structure and absolute elicitor activity to an elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae, a pathogen of soybeans. These and other results of this laboratory suggest that plants are able to respond to the presence of a wide range of fungi by recognizing, as foreign to the plant, structural polysaccharides of the mycelial walls of the fungi.     

Isolation and preliminary characterization of a casein kinase from cauliflower nuclei

A casein-type protein kinase has been isolated from cauliflower (Brassica cauliflora Gars.) nuclei and purified to a specific activity of 23,000 units/milligram of protein (1 unit is defined as the transfer of 1 picomole of ³²Pi from γ-[³²P]ATP to substrate per minute at 28 C). The enzyme has a molecular weight of approximately 39,000 as judged by sucrose density gradient sedimentation. The casein kinase requires ATP as the phosphate donor and will phosphorylate casein and phosvitin, but not histones. The enzyme activity is not affected by cAMP or cGMP. The casein kinase appears to be analogous to casein kinases described in other plant and animal systems.     

Localization of Nitrate Absorption and Translocation within Morphological Regions of the Corn Root

The absorption of NO₃⁻ was characterized in six regions of a 7-d-old corn root (Zea mays L. cv W64A × W182E) growing in a complete nutrient solution. Based on changing rates of ¹⁵N accumulation during 15-min time courses, translocation of the concurrently absorbed N through each region of the intact root was calculated and distinguished from direct absorption from the medium. Of the ¹⁵N accumulated in the 5-mm root tip after 15 min, less than 15 and 35% had been absorbed directly from the external solution at 0.1 and 10 mm NO₃⁻ concentration of the external solution, respectively. The characterization of the apical portion of the primary root as a sink for concurrently absorbed N was conconfirmed in a pulse-chase experiment that showed an 81% increase of ¹⁵N in the 5-mm root tip during a 12-min chase (subsequent to a 6-min labeling period). The lateral roots alone accounted for 60% of root influx and 70% of 15-min whole root ¹⁵N accumulation at either 0.1 or 10 mm. NO₃⁻ concentration of the external solution. Because relatively steady rates of ¹⁵N accumulation in the shoot were reached after 6 min, the rapidly exchanging pools in lateral roots must have been involved in supplying ¹⁵N to the shoot. The laterals and the basal primary root also showed large decreases (24 and 17%) in ¹⁵N during the chase experiment, confirming their role in rapid translocation.     

Ion distribution in roots of barley seedlings measured by electron probe x-ray microanalysis

The distribution of ions, particularly K and Na, was studied in roots of barley seedlings grown on various ionic solutions. Analyses were made by means of electron probe x-ray microanalysis using frozen, fractured bulk specimens. By this technique, it was demonstrated that there can be variability in the ratio K/Na measured in the vacuoles of cortical cells, with this ratio often being lower in epidermal cells of the root than in the inner cortex. A sharp difference in the K/Na ratio was also found between cells of the endodermis and those of the adjacent cortex, and generally higher ratios of K/Na occurred in the stele than in the cortex. Estimation of the concentrations in the cytoplasm was at the limit of resolution of this technique, but it can be shown that the K/Na ratio in the cytoplasm was higher than that in the vacuole. In low salt roots, the K concentration in the cytoplasm was higher than that in the vacuoles. The results with the x-ray microprobe confirm other measurements based on flux analysis or analysis of small samples of the root.     

Use of Tetraethylthiuram Disulfide to Discriminate between Alternative Respiration and Lipoxygenase

Mitochondria from axes of Glycine max (L.) Merr. cv. Chippewa 64 seedlings purified on discontinuous Percoll gradients exhibited classical cyanide-resistant respiration. These mitochondria also possessed lipoxygenase activity, as determined by O(2) uptake in the presence of 0.8 millimolar linoleic acid. This activity is inhibited by most known inhibitors of alternative respiration (i.e. hydroxamates and propyl gallate). Tetraethylthiuram disulfide (disulfiram) at 50 micromolar inhibited cyanide-resistant succinate oxidation by 90 per cent, whereas concentrations as high as 100 micromolar had no effect on lipoxygenase activity. Use of tetraethylthiuram disulfide allows discrimination between alternative respiration and lipoxygenase activity in mitochondria.     

FIRST HEART SOUND ALTERNANS IN MASSIVE PERICARDIAL EFFUSION

First heart sound alternans in the absence of pulsus alternans, or variation in heart rate (i.e., variation in PR interval), occurring in a patient with massive neoplastic pericardial effusion is described. The mechanism appears to be similar to that in electrical alternans and is shown by echocardiography to be related to the cardiac position within the pericardium, with the first heart sound intensity varying according to the distance of the heart from the chest wall. First heart sound alternans, electrical alternans, and alternating cardiac motion within the pericardium disappeared following pericardiocentesis.