A new method of in situ hybridization

A new method for gene mapping at the chromosome level using in situ hybridization and scanning electron microscopy is described and has been applied to mapping the rRNA genes of Drosophila melanogaster. Biotin is covalently attached to Drosophila rRNA via a cytochrome c bridge at a ratio of one cytochrome-biotin per 130 nucleotides by a chemical procedure. Polymethacrylate spheres with a diameter of ca. 60 nm are prepared by emulsion polymerization and are covalently attached to the protein avidin at a ratio of 5–20 avidins per sphere. The biotin-labeled rRNA is hybridized to denatured DNA in a chromosome squash. Upon incubation with a sphere solution, some of the biotin sites become labeled with spheres because of the strong non-covalent interaction between biotin and avidin. The chromosome squash is examined in the scanning electron microscope (SEM). Polymer spheres, which are visible in the SEM, are observed to label the nucleolus, where the rRNA genes are located.Contribution number 5121 from the Department of Chemistry.     

Xanthine dehydrogenase accumulation in developing Drosophila eyes

Xanthine dehydrogenase activity is assayed by following the oxidation of pterin to isoxanthopterin by spectrofluorometry at the reaction's wavelength peaks: excitation, 344 nm; emission, 412 nm. The method is sensitive to less than 0·1 μU of activity (0·1 pmol/min) and allows the assay of Drosophila imaginal disk homogenates.While the larval eye disk contains less than 0·1 per cent of the individual's XDH, the developing eye becomes a major store, with 30 per cent of the individual's activity by the time of eye pigmentation. The data suggest a basis for the well-known non-autonomous action of the gene rosy, the structural gene for XDH: the enzyme is synthesized in an organ of primary gene expression, and transported through the haemolymph to the eye of the pupa and pharate adult.     

Severe parasitism in an opossum.

Chronic debilitation and anemia were observed in a free-living opossum (Didelphis marsupialis) heavily parasitized by Physaloptera turgida, Brachylaima virginianum, and Cruzia americana. Chronic interstitial pneumonia associated with Capillaria aerophila and a metastrongyloid nematode also was present.     

The location of proteins labeled by the 125I-lactoperoxidase system in the NIL 8 hamster fibroblast.

NIL 8 hamster fibroblast cells were labeled by lactoperoxidase-catalyzed iodination. Their membranes were fractionated by sedimentation-rate and isopycnic zonal centrifugation. All the iodinated proteins except the very prominently labeled high molecular weight protein (greater than 200,000 daltons) were located in a fraction identified enzymically and compositionally as plasma membrane. The high molecular weight protein that was previously shown to be sensitive to virus transformation (Hynes, 1973) is concentrated in a very high density particle (rho equals 1.253-1.259) which contains mainly carbohydrate and protein and very low levels of lipid. 5'-nucleotidase was the only enzyme reproducibly demonstrated in this fraction, and electron micrographs revealed a predominantly amorphous morphology together with a few membraneous structures. The iodine label in this fraction was very sensitive to trypsinization prior to homogenization. All the available evidence indicates that this fraction is derived from the surface coat. Mitochondria, nuclei, and soluble protein were labeled to an insignificant extent. The presence of the iodinated surface proteins associated with the endoplasmic reticulum fraction is discussed in the light of these results.     

Proteolytic activity within lysosomes and turnover of pinocytic vesicles a kinetic analysis

In previous studies, in vitro digestion of [^{1 2 5}I] ribonuclease by lysosomes of mouse kidney was limited because breakdown, which was rapid at first slowed markedly so that most of the labeled protein escaped degradation. We now describe incubation conditions which allow digestion to proceed until approximately 70% of the exogenous protein label is released in acid-soluble from, after 30–45 min at 37°C. Such activity is seen with either the addition of EDTA or incubation of concentrated cell particle suspensions. EDTA is effective in low concentrations and shows the same stimulation of digestion over a range of approximately 10⁻⁶−10⁻³ M. Other chelating agents have similar effects; dipyridyl and hydroxquinoline are as effective as EDTA, o-phenanthroline and diethyldithiocarbamate are slightly less effective. When the incubation medium had been treated with a chelating resin, Chelex 100, dilute suspensions of lysosomes were as active as those in EDTA. These results lead to the conclusion that metal ions, present as contaminants in very small concentraions, inhibit the activity of mouse kidney lysosomes.The effect of the metal ions is to diminish lysosomal stability, leading to release of intact labeled ribonuclease in non-sedimentable form. Interaction between lysosomes and metal, leading to inhibition of digestion upon heating occurs at low temperature, but breakdown requires incubation at 37°C and may be autolytic. In contrast to chelators, mercaptoethanol is without marked effect on stability; the stimulation in digestion rate caused by this agent is due either to a direct effect on the lysosomal enzymes or to a non-destructive influence on the lysosomal structure.     

Photosynthetic responses of Eucalyptus nitens (Deane and Maiden) Maiden to green pruning

 Three-year-old Eucalyptus nitens (Deane and Maiden) Maiden trees and 1-year-old ramets of a single clone of E. nitens were pruned to remove 0, 50% or 70% of the green crown length. This was equivalent to removal of 0, 55% or 88% of foliage area of trees, and 0, 77% or 94% of foliage area of ramets. CO₂ assimilation (A) and stomatal conductance (g_s) were measured at constant illumination in five height zones and three foliage-age classes of trees over a 16-month period following pruning. Foliar nitrogen (N) and phosphorus (P) concentrations were determined for each measurement time during the first 12 months of the experiment. In ramets A and g_s were measured in four height zones and two foliage-age classes over a six-week period, and N and P concentrations were measured only once, at the end of the experiment. Rates of A increased by up to 175% following pruning. This response occurred throughout the canopy irrespective of position in the crown or foliage age. The magnitude of the response was generally greater in ramets than in trees, and increased with increasing severity of pruning. The initiation of the response was later, and the duration of the response was longer, in trees than ramets. In the lower crown of trees there was evidence of delayed senescence following pruning. Photosynthetic enhancement was not related to changes in foliar N concentrations. The ratio of A/N increased in many zones following pruning, especially after more severe defoliation. There was no evidence that changes in P concentrations were responsible for the result. The increases in A may have been related to changes in g_s, as maximum values of g_s were greater, and the ratio of A/g_s was generally lower, in pruned than unpruned plants. Received: 31 December 1996 / Accepted: 19 August 1997     

Methods for simultaneous measurement of apoptosis and cell surface phenotype of epithelial cells in effusions by flow cytometry

We describe here a protocol for the detection of epithelial cells in effusions combined with quantification of apoptosis by flow cytometry (FCM). The procedure described consists of the following stages: culturing and induction of apoptosis by staurosporine in control ovarian carcinoma cell lines (SKOV-3 and OVCAR-8); preparation of effusion specimens and cell lines for staining; staining of cancer cells in effusions and cell lines for cell surface markers (Ber-EP4, EpCAM and CD45) and intracellular/nuclear markers of apoptosis (cleaved caspase-3 and caspase-8, and incorporated deoxyuridine triphosphates); and FCM analysis of stained cell lines and effusions. This protocol identifies a specific cell population in cytologically heterogeneous clinical specimens and applies two methods to measure different aspects of apoptosis in the cell population of interest. The cleaved caspase and deoxyuridine triphosphate incorporation FCM assays are run in parallel and require (including sample preparation, staining, instrument adjustment and data acquisition) 8 h. The culturing of cell lines requires 2-3 days and induction of apoptosis requires 16 h.     

Molecular mechanisms for environmentally induced and evolutionarily rapid redistribution (plasticity) of meiotic recombination

It has long been known (circa 1917) that environmental conditions, as well as speciation, can affect dramatically the frequency distribution of Spo11/Rec12-dependent meiotic recombination. Here, by analyzing DNA sequence-dependent meiotic recombination hotspots in the fission yeast Schizosaccharomyces pombe, we reveal a molecular basis for these phenomena. The impacts of changing environmental conditions (temperature, nutrients, and osmolarity) on local rates of recombination are mediated directly by DNA site-dependent hotspots (M26, CCAAT, and Oligo-C). This control is exerted through environmental condition-responsive signal transduction networks (involving Atf1, Pcr1, Php2, Php3, Php5, and Rst2). Strikingly, individual hotspots modulate rates of recombination over a very broad dynamic range in response to changing conditions. They can range from being quiescent to being highly proficient at promoting activity of the basal recombination machinery (Spo11/Rec12 complex). Moreover, each different class of hotspot functions as an independently controlled rheostat; a condition that increases the activity of one class can decrease the activity of another class. Together, the independent modulation of recombination rates by each different class of DNA site-dependent hotspots (of which there are many) provides a molecular mechanism for highly dynamic, large-scale changes in the global frequency distribution of meiotic recombination. Because hotspot-activating DNA sites discovered in fission yeast are conserved functionally in other species, this process can also explain the previously enigmatic, Prdm9-independent, evolutionarily rapid changes in hotspot usage between closely related species, subspecies, and isolated populations of the same species.     

Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100

Apolipoprotein (apo) B is an obligatory component of very low density lipoprotein (VLDL), and its cotranslational and posttranslational modifications are important in VLDL synthesis, secretion, and hepatic lipid homeostasis. ApoB100 contains 25 cysteine residues and eight disulfide bonds. Although these disulfide bonds were suggested to be important in maintaining apoB100 function, neither the specific oxidoreductase involved nor the direct role of these disulfide bonds in apoB100-lipidation is known. Here we used RNA knockdown to evaluate both MTP-dependent and -independent roles of PDI1 in apoB100 synthesis and lipidation in McA-RH7777 cells. Pdi1 knockdown did not elicit any discernible detrimental effect under normal, unstressed conditions. However, it decreased apoB100 synthesis with attenuated MTP activity, delayed apoB100 oxidative folding, and reduced apoB100 lipidation, leading to defective VLDL secretion. The oxidative folding–impaired apoB100 was secreted mainly associated with LDL instead of VLDL particles from PDI1-deficient cells, a phenotype that was fully rescued by overexpression of wild-type but not a catalytically inactive PDI1 that fully restored MTP activity. Further, we demonstrate that PDI1 directly interacts with apoB100 via its redox-active CXXC motifs and assists in the oxidative folding of apoB100. Taken together, these findings reveal an unsuspected, yet key role for PDI1 in oxidative folding of apoB100 and VLDL assembly.     

Pulmonary hemodynamics at birth: effect of acute cyclooxygenase inhibition in lambs

The effect of acute cyclooxygenase (CYO) inhibition on the cardiopulmonary adjustments at birth was examined in chronically instrumented, unanesthetized, term lambs before, during, and after cesarean section (spontaneous respiration). One of three infusions was started 20 min before birth: saline control (C, n = 6), indomethacin (I, n = 6), or meclofenamate (M, n = 3). The stable metabolite of prostacyclin, plasma 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha, aorta), was measured by radioimmunoassay as an index of CYO activity. Indomethacin blocked the rise of 6-keto-PGF1 alpha observed in control lambs after birth and indomethacin-treated lambs exhibited an attenuation of the postnatal decrease in mean pulmonary arterial pressure. Pulmonary arterial pressure (Ppa) was 53 +/- 2 and 47 +/- 2 Torr (mean +/- SE) at 15 min and 40 +/- 3 and 34 +/- 2 Torr at 120 min in I and C groups, respectively. There were no serial or group differences in cardiac output and cardiac right to left shunt (indicator dilution) from 15 to 120 min after birth. Arterial PO2 (PaO2) was not different between groups: 37 +/- 4 Torr at 15 min and 47 +/- 5 min at 120 min after birth (control lambs). The results for I and M were similar for all measurements.(ABSTRACT TRUNCATED AT 250 WORDS)