Calculated calibrations for ion chambers fabricated from plastics simulating air and muscle; determination of W and tauRa

An experiment is described utilizing two 16-liter ionization chambers, fabricated from electrically conducting plastics which closely simulate air and human muscle, designed to minimize most of the errors inherent in the use of cavity chambers. A careful calibration was done, using a 226Ra source in 0.5 mm platinum, in an almost scatter-free environment which permitted the derivation of accurate corrections for scatter and air attenuation. Calibrations calculated from the physical measurements of the ion chambers are compared with the experimental calibrations. Values of Wbeta for air, and muscle gas and of tauRa are derived.     

Increased free-cystine content of fibroblasts cultured from patients with cystinosis

The presence of a significantly increased content of free-cystine in skin fibroblasts from both homozygotes and heterozygotes for cystinosis emphasizes the central role of cystine in this disease, even though the primary defect responsible for cystine accumulation is yet to be determined. The studies described in this communication provide evidence that cystine is compartmentalized in a subcellular location in cystinotic cells. In fact, the very growth of cystinotic fibroblasts in the presence more than 100 times the usual content of free-cystine is evidence that the accumulated cystine is not freely dispersed throughout the cell, since would otherwise inhibit many enzymes requiring free sulfhydryl groups for activity (Patrick, 1965). We have no evidence as to whether the cystine is located in a known subcellular organelle or in a previously unrecognized location. Skin fibroblasts may provide a convenient tool to pursue these questions.     

Localization of the ca-mediated apparent ion selectivity in the cross sectional volume of soybean roots

A major portion of the calcium in a soybean root appears to be in less than 10% of the root volume. Specifically, Ca is considered to be almost entirely localized in the epidermal cell layer. This relationship was established from consideration of rates and extent of ion absorption and ion interactions during the absorption process.

Presence of calcium at the root-solution interface was associated with a change in the apparent selectivity of K over Mg by soybean roots. Accumulation of calcium by soybean roots was negligible.

     

Monoclonal antibody technology for mycotoxins

Specific monoclonal antibodies (MABs) against aflatoxins, ochratoxin A, zearalenone, diacetoxyscirpenol and T-2 toxin have been prepared in various laboratories by the application of hybridoma technology to mycotoxins. These antibodies can be selected for sensitivity, reduced cross-reactivity, reliability and ease of production. When a suitable antibody is chosen it can then be used in a rapid immunological method such as an enzyme-linked or radio-immunoassay or immunoaffinity chromatography system. These assays have a lower limit of mycotoxin detection in the ng/ml range and have been applied to the determination of mycotoxins in samples such as maize, peanuts, peanut butter, milk and porcine kidneys. Using these immunoassay techniques, sample preparation has generally been simplified to a matter of solvent extraction of mycotoxins from the sample followed by dilution; under these conditions, levels of 1-5ug of mycotoxins/kg of sample can be found. The application and advantages of MABs to mycotoxins and the use of these antibodies in various assay techniques is discussed.     

Starch Grain Distribution in Taproots of Defoliated Medicago sativa L

Defoliation of alfalfa (Medicago sativa L.) results in a cyclic pattern of starch degradation followed by reaccumulation in taproots. Characterization of changes in anatomical distribution of starch grains in taproots will aid our understanding of biochemical and physiological mechanisms involved in starch metabolism in taproots of this species. Our objectives were to determine the influence of defoliation on starch grain distribution and size variation in taproots of two alfalfa lines selected for contrasting concentrations of taproot starch. In addition, we used electron microscopy to examine the cellular environment of starch grains, and computer-based image optical analysis to determine how cross-sectional area of tissues influenced starch accumulation. Taproots of field-grown plants were sampled at defoliation and weekly thereafter over a 28-day period. Taproot segments were fixed in glutaraldehyde and prepared for either light or electron microscopy. Transverse sections were examined for number and size of starch grains and tissue areas were measured. Starch grains were located throughout bark tissues, but were confined primarily to ray parenchyma cells in wood tissues. During the first week of foliar regrowth after defoliation, starch grains in ray cells near the cambium disappeared first, while degradation of those near the center of the taproot was delayed. During the third and fourth weeks of regrowth, there was a uniform increase in number of starch grains per cell profile across the rays, but by 28 days after defoliation there were more starch grains in ray cells near the cambium than in cells near the center of the taproot (low starch line only). Bark tissues from both lines showed synchronous degradation and synthesis of starch grains that was not influenced greatly by cell location. Diameter of starch grains varied with cell location in medullary rays during rapid starch degradation, but was not influenced by cell position in bark tissues. Therefore, during foliar regrowth there is a spatial separation in starch degradation and synthesis in alfalfa taproots. Amyloplasts from alfalfa taproots contained numerous starch grains, prolamellar-, and electron-dense bodies. The high starch line had 23% more cross-sectional area as ray cells in wood tissues when compared to the low starch line, which may explain part of the difference in starch accumulation between these alfalfa lines.     

Characteristics of a membrane-associated lipoxygenase in tomato fruit

Microsomal membranes isolated from the pericarp of maturegreen tomato (Lycopersicon esculentum) fruit rapidly metabolize exogenous radiolabeled linoleic acid into fatty acid oxidation products at 22°C. The reaction is strongly inhibited by n-propyl gallate, an inhibitor of lipoxygenase. The membranes also rapidly metabolize 16:0/18:2^* phosphatidylcholine into radiolabeled oxidation products that comigrate on TLC plates with those formed from free linoleic acid. At 30°C, the formation of fatty acid oxidation products from 16:0/18:2^* phosphatidylcholine is slower, and there is an initial accumulation of radiolabeled linoleic acid that is not evident at 22°C, which can be attributed to the action of lipolytic acyl hydrolase. Radiolabeled phosphatidic acid and diacylglycerol are also formed during metabolism of 16:0/18:2^* phosphatidylcholine by the microsomal membranes, and there is no breakdown of either linoleic acid or phosphatidylcholine by heat-denatured membranes. When Triton X-100 treated membranes were used, the same patterns of metabolite formation from radiolabeled linoleic acid and 16:0/18:2^* phosphatidylcholine were observed. Thus, the enzymes mediating the breakdown of these radiolabeled compounds appear to be tightly associated with the membranes. Collectively, the data indicate that there is a lipoxygenase associated with microsomal membranes from tomato fruit that utilizes free fatty acid substrate released from phospholipids. The microsomal lipoxygenase is strongly active over a pH range of 4.5 to 8.0, comprises approximately 38% of the total (microsomal plus soluble) lipoxygenase activity in the tissue, has an apparent K_m of 0.52 millimolar and an apparent V_max of 0.186 millimoles per minute per milligram of protein. The membranous enzyme also cross-reacts with polyclonal antibodies raised against soybean lipoxygenase-1 and has an apparent molecular mass of 100 kilodaltons.     

Site of action for endotoxin-induced cortisol release in the suppression of preovulatory luteinizing hormone surges

The study was conducted to identify the mechanisms of endotoxin/cortisol action in the suppression of preovulatory LH surges in heifers infused with Escherichia coli (E. coli ) endotoxin. The hypotheses tested were that 1) endotoxin stimulates the release of progesterone, possibly from the adrenal leading to the LH blockade; 2) cortisol released in response to endotoxin infusion blocks the synthesis of estradiol at the ovarian level, culminating in a failure of the LH surge. Eight Holstein heifers were given two injections of prostaglandin F(2alpha) (PG), 11 d apart, to synchronize estrus. Starting from 25 h after the second injection of PG (PG-2), the uterus of each heifer was infused either with 5 ml of pyrogen-free water (control, n = 3) or with E. coli endotoxin (5 mug/kg of body weight) in 5 ml of pyrogen-free water (treated, n = 5), once every 6 h for 10 treatments. Blood samples were obtained every 15 min for 1 h before infusion and again 2 h after each infusion, then hourly until 1 h before the next infusion. After the tenth infusion, blood was collected daily until estrus. Serum progesterone concentrations remained at baseline values (< 1 ng/ml) in control and treated heifers. The total amount of progesterone measured starting 24 to 84 h after PG-2 injection was not different between control and treated heifers (P 0.05). In the control heifers, serum estradiol concentrations remained basal (< 10 pg/ml) until 4 h before the LH surge. Serum estradiol concentrations increased to 20 +/- 5.6 pg/ml, 4 h before the LH surge in control heifers (LH surge occurred 60 to 66 h after the PG-2 injection). There were no changes in serum estradiol concentrations in treated heifers during the sampling period, and the concentrations remained < 10 pg/ml. The total amount of estradiol measured in control heifers was higher (P < 0.05) than in treated heifers. The results if this study suggest that increases in cortisol concentrations after the infusion of endotoxin might block the synthesis of estradiol at the ovarian level, resulting in the failure of a preovulatory LH surge to occur.     

Comparison of six-day bovine embryo development in uterine tube (oviduct) epithelial cell co-culture versus in vivo development in the cow

A study was designed to evaluate and compare the appearance of embryos recovered from donor cows on Day 6 to embryos from in vivo fertilized cow zygotes developed to Day 6 on uterine tube (oviduct) epithelial cell co-culture using serum-free CZB medium. Embryo stage of development and quality score were assessed. Hoechst 33342 DNA stain was then used to determine the total number of blastomeres, the number of poor nuclei and the number of nuclei in mitosis. Mean cell counts did not differ for the 70 embryos evaluated in each group (65 cells in vivo, 61 cells in vitro). The percentage of transferable emryos (excellent, good or fair quality), in each group also did not differ (57% in vivo, 56% in vitro). There were no significant differences in any of the measured parameters. Our findings suggest that co-culture of in vivo produced cow zygotes can result in embryos comparable in developmental stage and quality to embryos developed in vivo in the cow for 6 d.     

Maturation, fertilization and development of bovine oocytes in vitro using TCM199 and a simple defined medium with co-culture

Bovine oocytes obtained from ovarian follicles (2 to 5 mm in diameter) from slaughtered cattle were cultured in TCM199 with 10% heat-inactivated estrous cow serum (ECS) for 24 to 25 h at 39 degrees C under 5% CO(2) in air. The 10% ECS was selected on the basis of preliminary studies in which in vitro fertilization rates of oocytes with 10, 15 and 20% ECS in the medium were 46, 30 and 31%, respectively (P<0.05). Of 120 oocytes cultured for 24 to 25 h, 63% were classified as being in Metaphase II. The rate of oocytes matured in vitro was 55% (69 125 ), the proportion of penetrated oocytes which contained male and female pronuclei was 94% (65 69 ), and the incidence of polyspermy was very low (0 to 9%). Of 122 oocytes fertilized in vitro and cultured in TCM199 medium with 10% fetal bovine serum for 7 d, 53% were cleaved, but only 2% developed beyond the 16-cell block. However, in simple semi-defined Chatot-Ziomek-Bavister medium co-cultured with bovine oviduct epithelial cells (BOEC), 75% of 138 oocytes cleaved, and 38% of those which cleaved developed into morulae or blastocysts. The results of this study indicate that co-culture with BOEC exerted a pronounced beneficial effect on development of in vitro fertilized bovine oocytes through the 16-cell block. The medium required in the co-culture system was simple and semi-defined.     

Influence of mineral supplementation on bovine serum, liver and endometrium at day 1 and day 12 of the estrous cycle

The effects of diet on mineral concentrations in serum, liver and endometrium were determined at points in the reproductive cycle in heifers. Dietary treatments extended 134 d and included feeding a basal hay (negative control), basal hay with concentrate (feed control), feed control with phosphorus and feed control with both phosphorus and trace minerals. Samples of serum and liver were taken at the beginning and end of the trial. Within an estrous cycle during the trial (endometrial biopsy) the cows were sampled either at Day 1 or Day 12 for determining progesterone levels and mineral elements in the blood, liver and endometrial tissue. Trace element concentrations in serum and liver did not differ among collection periods pretrial, endometrial biopsy and post breeding nor among treatment groups. However, endometrial tissue concentrations of copper, manganese and zinc were higher at Day 1 than at Day 12 (P < 0.05) in reverse of serum progesterone, which was higher at Day 12 (P < 0.05). Supplemental trace minerals appeared to increase concentrations of copper (P < 0.20), manganese (P < 0.10) and zinc (P < 0.20) at Day 1 but decrease concentrations of these same elements at Day 12 (P < 0.05, P < 0.10 and P < 0.05, respectively). The large differences in trace element concentrations observed in endometrial tissue at the estral phases and under different diets suggest the possible importance of trace elements and trace element nutrition in fertilization and (or) embryo survival.