Kinetics of insulin action on protein synthesis in isolated adipocytes. Ability of glucose to selectively desensitize the glucose transport system without altering insulin stimulation of protein synthesis

When adipocytes were exposed to [3H]leucine for times ranging from 5 to 180 s, leucine was found to enter cells rapidly and equilibrate with the cell interior within 5 s. After an additional 15-30 s [3H]leucine was incorporated into nascent protein, and the rate of incorporation was linear for up to 6 h in both control and insulin-treated cells. Since treatment of adipocytes with 10 ng/ml insulin enhanced the rate of leucine incorporation 2-3-fold with minimal or no effect on the rate of protein degradation or leucine uptake, we conclude that the predominant effect of insulin is on enhancement of protein synthesis. To assess the time required for insulin to stimulate protein synthesis, we preincubated cells with 10 ng/ml of insulin for various times from 2 to 30 min and then measured [3H]leucine incorporation into protein during a 4-min assay. These results revealed that the insulin stimulation of protein synthesis is rapid (t 1/2 of 4.4 min), but 9-fold slower than insulin activation of glucose transport (t 1/2 less than 0.5 min under identical conditions). In contrast to the rapidity of insulin activation, we found that deactivation proceeded at much slower rates (t 1/2 of 32 and 21 min for protein synthesis and glucose transport, respectively). Desensitization of the glucose transport system has previously been shown to occur after adipocytes are exposed to high glucose and insulin. To examine the specificity of desensitization, we treated cells for 6 h with 20 mM glucose and 25 ng/ml insulin and then examined insulin sensitivity and maximal insulin responsiveness of both the glucose transport and protein synthesis systems. After treatment, the glucose transport was markedly insulin-resistant (60% loss in maximal insulin responsiveness and a marked loss in insulin sensitivity), whereas the protein synthesis system exhibited neither diminished insulin responsiveness nor loss of insulin sensitivity. In fact, insulin sensitivity actually increased, as indicated by the finding that less insulin was required to stimulate protein synthesis (insulin ED50 values of 0.25 and 18 ng/ml at 0 and 6 h of treatment). From these studies we conclude that desensitization of the glucose transport system by glucose and insulin treatment appears to be specific for this particular effector system and does not reflect a state of generalized cellular insulin resistance.     

The compact domain conformation of human Glu-plasminogen in solution.

A complete understanding of the accelerating mechanisms of plasminogen activation and fibrinolysis necessarily requires structural information on the conformational forms of plasminogen. Given the absence of high-resolution structural data on plasminogen the use of lower resolution approaches has been adopted. Two such approaches have previously indicated a compact conformation of Glu-plasminogen (Tranqui, L., Prandini, M., and Chapel, A. (1979) Biol. Cellulaire, 34, 39-42; Bányai, L. and Patthy, L. (1985) Biochim. Biophys. Acta, 832, 224-227) whereas a third has suggested a fairly extended conformation (Mangel, W., Lin, B. and Ramakrishnan, V. (1990) Science, 248, 69-73). Native Glu-plasminogen has been investigated using small-angle X-ray scattering (SAXS) experiments. It is concluded that this molecule in solution is compact (radius of gyration, RG 3.05 +/- 0.02 nm and maximum intramolecular distance, Im 9.1 +/- 0.3 nm) and that the data are consistent with the right-handed spiral structure observed using electron microscopy by Tranqui et al. (1979). A spiral structure of native plasminogen would have important implications for the conformational response of plasminogen to fibrin and concomitant stimulation of plasminogen activation.     

Purification and properties of pyruvate kinase from Dictyostelium discoideum

Pyruvate kinase (EC 2.7.1.40) from aggregating Dictyostelium discoideum cells has been purified to homogeneity. It has a monomeric molecular weight of 66kD and is tetrameric in low ionic strength buffers. The enzyme is not regulated by fructose 1,6-bisphosphate or by alanine and appears to resemble the M1 isoenzyme from rat liver most closely, although its activity is not inhibited by ATP.     

Interrelationships among fluorometric analyses of spermatozoal function, classical semen quality parameters and the fertility of frozen-thawed bovine spermatozoa

Cryopreserved spermatozoa from 8 bulls were used to examine the interrelationships among flow cytometric spermatozoal quality assessments and classical semen quality parameters and nonreturn rate estimates of fertility. The integrity of the sperm cell membrane and the functional capacity of the mitochondria were quantified by flow cytometry after concurrent staining with carboxydimethylfluorescein diacetate (CDMFDA), propidium iodide (PI), and rhodamine 123 (R123). For each sample a total of 10,000 stained spermatozoa were simultaneously quantified for the intensity of their green and red fluorescence. Three straws from each bull were each examined initially and following incubation at 37 degrees C for 3 hours to assess the rate of senescence. The proportion of spermatozoa retaining membrane integrity and having functional mitochondria, as determined by CDMFDA and R123 staining, were compared with classical semen quality assessments (sperm motility, acrosomal status, cellular and head morphology, presence of vacuoles/craters and cytoplasmic droplets) and with fertility (nonreturn to estrus rates). For individual ejaculates nonreturn rates, the range was from 61.8 to 78.8%, whereas the cumulative rates of several ejaculates for each bull ranged from 71.3 to 83.5%. The proportion of spermatozoa with functional membranes and mitochondria were positively correlated with the percentage of spermatozoa with normal morphology (r=0.82; P=0.01) and motility after 4 hours of incubation (r=0.78; P=0.02), but not with the estimates of fertility. The actual number of spermatozoa per straw staining with CDMFDA and R123 after 4 hours of incubation at 37 degrees C was correlated with the percentage of spermatozoa with normal morphology (r=0.73; P=0.04). Multiple regression equations indicated that combinations of semen quality measurements could be useful in estimating fertilizing potential.     

Plant clonality: Biology and diversity

The current approaches to the study of clonal plants are reviewed. Most studies concentrate at the level of the ramet and clonal fragment exploring the “microscopic” view of clonal plants, dealing with the translocation of resources, clonal integration, plasticity of growth etc. The information gained, by this approach can be used in the understanding of higher levels of organization within the clonal system either with the help of spatially explicit modelling techniques, or by using means and distributions of size within a population instead of studying individual ramets separately. Plant scientists use the term clone with two meanings, viz. (a) a set of physiologically connected, but potentially independent ramets, and (b) a set of genetically identical, but potentially physically separated individuals. The overlap of these terms differs between individual plant species, depending on the extent of physical separation of the ramets and the degree of physiological integration between the ramets; the lower the frequency of ramet separation, the closer are the physiological and genetic concepts of the clone. Three critical areas seem to be neglected in clonal plant research: (a) the interrelationship between hierarchical levels in clonal plants, (b) the particular spatial structure of their environment, and (c) the importance of clonal plants in different ecological communities.     

A comparison of phytoplankton assemblages in the Chesapeake and Delaware estuaries (USA), with emphasis on diatoms

The Delaware Bay is characterized as having greater nutrient and turbidity levels than the Chesapeake Bay. In reference to these differences, a one year study was conducted to identify any similarities and differences in the phytoplankton populations in these estuaries. The results indicated patterns of similarity in the diatom composition, with the total phytoplankton assemblage forming two site groups along a salinity gradient in each bay. These site groups were associated with stations located in the tidal fresh-oligohaline and meso-polyhaline regions of both estuaries. The seasonal concentrations of diatoms and total phytoplankton in both of these regions were higher in the Chesapeake Bay.Subtle differences between the two estuaries include a more diversified and abundant assemblage of neritic phytoplankters (including dinoflagellates) are present in the lower Chesapeake Bay. In contrast, a diatom dominated community is more characteristic of Delaware Bay. It is suggested the entry of neritic species into lower regions of the estuaries was enhanced by the reduced amount of rainfall and flow rates that occurred during the study period. The greater success of neritic species in the Chesapeake Bay is attributed to the lower turbidity of that estuary compared to Delaware Bay.     

Nuclear envelope assembly after mitosis

In higher eukaryotes, the entire nucleus disassembles during prometaphase of the cell cycle and later reassembles around daughter chromosomes. Remarkably, the complex events that occur to create a functional nucleus in vivo can be duplicated in vitro by using cell-free extracts. Current experiments are aimed at understanding the molecular mechanisms of assembly and disassembly of the nuclear pore complexes and nuclear membranes, and the functional roles of four identified inner membrane proteins, two of which bind to both chromatin and the nuclear lamina.     

Microspectrophotometric studies of Romanowsky stained blood cells. IV. Maturation of myeloid and erythroid cell lines in bone marrow

A quantitative characterization has been made of azure B/eosin stained cells from bone marrow. Two cell lines were followed: the myeloid line (white cell blast, promyelocyte, neutrophilic myelocyte, neutrophilic metamyelocyte, neutrophilic band, neutrophilic segmented) and the erythroid line (rubriblast, prorubricyte, rubricyte, metarubricyte, diffusely basophilic erythrocyte, erythrocyte). A consensus scheme was used to obtain the "true" classification of the cells. Cell types were characterized by three methods: absorbance spectra, dye binding, and chromaticities. Within both cell lines nuclear maturation is accompanied by an overall increase in peak absorbance with little shift in the position of the maximum. Generally, binding of azure B and eosin increases; azure B dimer/monomer ratios show a slight downward trend during maturation. Changes in chromaticities are to bluish purples of increasing saturation. Cytoplasmic changes accompanying maturation are much more striking than nuclear changes, and again the two cell lines show similarities. Generally, there is decreased binding of azure B during maturation. In the erythroid line, the Soret band of hemoglobin becomes increasingly prominent. Chromaticities change from bluish purples to purplish pinks, particularly in the erythroid line.