Tracing of cell lineage in embryonic development of Phallusia mammillata (ascidia) by vital staining of mitochondria

Vital staining of mitochondria with a fluorescent dye 3,3′-diethyloxacarbocyanine was used to follow cell lineage in embryos of Phallusia mammillata. The results agree in general with the plan established by Conklin in 1905. Strong fluorescence migrated after fertilization similarly to the pigment of the “yellow crescent” in Styela. Later, fluorescence segregated into muscle cell primordia, but not into mesenchyme cells. An animal hemisphere cell, b 8.17 also exhibited strong fluorescence and joined a group of muscle primordia, very likely becoming a muscle cell itself. In the tadpole, all the tail muscle cells were fluorescent. Fluorescence was also noticed in nerve cell primordia of the vegetal hemisphere, particularly in the cell A 8.16 whose descendants appeared to become part of the sensory vesicle which was strongly fluorescent in the tadpole. The usefulness of this type of vital staining in following cell lineage of colorless embryos is stressed.     

A rapid increase in lipoprotein (a) levels after ethanol withdrawal in alcoholic men.

Plasma concentrations of lipoprotein (a) (Lp(a)) were studied in 11 male alcoholics at the end of a drinking period and monitored during subsequent abstinence. Lp(a) levels showed a daily increase for four consecutive days after the beginning of abstinence, the values for the third and the fourth day being significantly higher than those of the first day (p less than 0.05 and p less than 0.01, respectively). The changes in Lp(a) showed no association with the changes in low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol levels. In one alcoholic subject with a heterozygous form of familial hypercholesterolemia who was monitored for 11 days, the Lp(a) levels rose up to the fourth day and remained at a high level thereafter. These results suggest that ethanol ingestion may be associated with a lowering of Lp(a) levels, which may contribute to the delayed progression of atherosclerosis observed in alcohol drinkers. Ethanol intake may be added to the short list of factors that affect the quite stable, genetically determined Lp(a) concentrations in the plasma.     

The aspartic proteinase of barley is a vacuolar enzyme that processes probarley lectin in vitro.

Previous work suggested that the aspartic proteinase from Hordeum vulgare (HvAP) would be a vacuolar protein in plant cells. Based on N-terminal sequencing we show that the in vitro-translated protein was translocated into the lumen of microsomal membranes, causing a concomitant removal of 25 amino acid residues from the protein. Vacuoles were purified from barley leaf protoplasts and were shown to contain all of the aspartic proteinase activity found in the protoplasts. This vacuolar localization of HvAP was confirmed with immunocytochemical electron microscopy using antibodies to HvAP in both barley leaf and root cells. In an attempt to discern a function for this protease, we investigated the ability of HvAP to process the C-terminal proregion of barley lectin (BL) in vitro. Prolectin (proBL), expressed in bacteria, was processed rapidly when HvAP was added. Using several means, we were able to determine that 13 amino acid residues at the C terminus of proBL were cleaved off, whereas the N terminus stayed intact during this incubation. Immunohistochemical electron microscopy showed that HvAP and BL are co-localized in the root cells of developing embryos and germinating seedlings. Thus, we propose that the vacuolar HvAP participates in processing the C terminus of BL.     

Bacterioplankton interactions with Daphnia and algae in experimental enclosures

Development of bacterioplankton was studied by manipulation of planktivorous fish and/or nutrients in experimental enclosures in a fish pond. Grazing pressure exerted by large zooplankton (Daphnia galeata and Daphnia pulicaria) strongly influenced the counts and size distribution of bacterial populations. Morphometric analyses by scanning electron microscope revealed a shift in size distribution from larger mainly rod-type bacteria under low grazing pressure towards smaller mainly coccus-type under strong grazing pressure. The metabolic activity of bacteria measured as glucose uptake was higher under strong grazing pressure. After removal of large daphnids, the increase in bacterial density was probably the result of two additive factors: low grazing pressure and high level of dissolved organic matter (DOM) due to photosynthetic activity of more abundant algae. Composition of bacterial populations shifted toward larger, rod-type bacteria, and their metabolic efficiency measured by uptake, was lowered. The basic dimensionality of the system and interactions between variables was describe by R-mode factor analysis. The manipulated enclosures were relate with factor score.     

Exercise enhancement by RGS14 disruption is mediated by brown adipose tissue

Enhanced exercise capacity is not only a feature of healthful aging, but also a therapy for aging patients and patients with cardiovascular disease. Disruption of the Regulator of G Protein Signaling 14 (RGS14) in mice extends healthful lifespan, mediated by increased brown adipose tissue (BAT). Accordingly, we determined whether RGS14 knockout (KO) mice exhibit enhanced exercise capacity and the role of BAT in mediating exercise capacity. Exercise was performed on a treadmill and exercise capacity was assessed by maximal running distance and work to exhaustion. Exercise capacity was measured in RGS14 KO mice and their wild types (WT), and also in WT mice with BAT transplantation from RGS14 KO mice or from other WT mice. RGS14 KO mice demonstrated 160 ± 9% increased maximal running distance and 154 ± 6% increased work to exhaustion, compared to WT mice. RGS14 KO BAT transplantation to WT mice, resulted in a reversal of phenotype, with the WT mice receiving the BAT transplant from RGS14 KO mice demonstrating 151 ± 5% increased maximal running distance and 158 ± 7% increased work to exhaustion, at three days after BAT transplantation, compared to RGS14 KO donors. BAT transplantation from WT to WT mice also resulted in increased exercise performance, but not at 3 days, but only at 8 weeks after transplantation. The BAT induced enhanced exercise capacity was mediated by (1) mitochondrial biogenesis and SIRT3; (2) antioxidant defense and the MEK/ERK pathway, and increased hindlimb perfusion. Thus, BAT mediates enhanced exercise capacity, a mechanism more powerful with RGS14 disruption.     

Variation in life history responses of Daphnia to toxic Microcystis aeruginosa

Three clones of Daphnia pulex and two clones of Daphnia longispinawere exposed to toxic Microcystis aeruginosa for 21 days ina lifetable experiment. The growth and reproduction of individualdaphnids were followed daily to study the long-term effectsof toxic Microcystis. Exposure to Microcystis increased mortality,decreased growth, delayed maturation and decreased offspringproduction, indicating nutritional deficiency and toxic effects.We found variation in life history responses between speciesand among clones. Our results suggest that toxic cyanobacteriamay act as a modifying agent in zooplankton communities at boththe species and clonal level.     

HMG-GoA reductase and terpenoid phytoalexins: Molecular specialization within a complex pathway

Terpenoid phytoalexins and other defense compounds play an important role in disease resistance in a variety of plant families but have been most widely studied in solanaceous species. The rate-limiting step in terpenoid phytoalexin production is mediated by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), which catalyzes mevalonic acid synthesis. HMGRs are involved in the biosynthesis of a broad array of terpenoid compounds, and distinct isoforms of HMGR may be critical in directing the flux of pathway intermediates into specific end products. Plant HMGRs are encoded by a small gene family, and genomic or cDNA sequences encoding HMGR have been isolated from several plant species. In tomato, four genes encode HMGR; these genes are differentially activated during development and stress responses. One gene, hmg 2 , is activated in response to wounding and a variety of pathogenic agents suggesting a role in sesquiterpene phytoalexin biosynthesis. In contrast, expression patterns of tomato hmg l suggest a role in sterol biosynthesis and cell growth. Other plant species show an analogous separation of specific HMGR isoforms involved in growth and/or housekeeping function and inducible isoforms associated with biosynthesis of phytoalexins or other specialized "natural products". We are applying a variety of cell and molecular techniques to address whether subcellular localization and/or differential expression of these isoforms are key factors in determining end product accumulation during development and defense.     

A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder

A preparative procedure for obtaining highly purified plasmid DNA from bacterial cells is described. The method is adapted from our earlier procedure, which gave partially purified plasmid in a form suitable for rapid screening of a large number of samples. In the present method, all detectable RNA, chromosomal DNA, and protein are removed without the use of enzymes, phenol extraction, dialysis, or equilibrium centrifugation. Binding of plasmid DNA to glass powder in the presence of 6 m sodium perchlorate is used for the final purification step.