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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Characterization of insect calmodulin during oogenesis and embryogenesis of Blattella germanica

Zhang, Yujun 01 January 1992 (has links)
A Ca$\sp{2+}$-binding protein has been purified and characterized from Blattella germanica eggs. This protein has biochemical features in common with calmodulin. These common features include a relatively low molecular weight of $\sim$19 KDa, thermal stability, an acidic pI of 4.0, a low specific absorbance (E$\sb{\rm 277nm}\sp{1\%}$ = 2.8), an altered electrophoretic mobility in SDS-polyacrylamide gels in the presence of 1 mM Ca$\sp{2+},$ and calcium-dependent binding to the calmodulin antagonist W-7. These features, considered together with activation of calmodulin-dependent phosphodiesterase in a Ca$\sp{2+}$-dependent manner and cross-reactivity with anti-bovine brain calmodulin antibody, are sufficient to define this protein as bone fide calmodulin. A rabbit antibody specific for the B. germanica calmodulin cross-reacts with bovine brain calmodulin. The calmodulin levels during oogenesis and embryogenesis were estimated by densitometric analysis of immunoblots using anti-Blattella germanica egg calmodulin antibody as a probe. A high concentration of calmodulin is present in vitellogenic follicles and early embryonic eggs (about 15 ng calmodulin/$\mu$g protein). During oogenesis calmodulin accumulates in the oocyte throughout the yolk deposition phase. During embryogenesis calmodulin is present at uniformly high levels until vitellin utilization starts, then it is undetectable until the pharate larval stage at the end of embryogenesis. $\sp{14}$C-labeled egg calmodulin in a gel-overlay technique binds to vitellin, the major yolk protein of B. germanica eggs. The calmodulin-binding site of vitellin is located on the 95 KDa subunit before degradation, and on the 53 KDa fragment after 95 KDa subunit breakdown. There is sufficient calmodulin to bind stoichiometrically (1:1) with the vitellin trimer. Circumstantial evidence suggests that this CaM is derived from outside the oocyte. In vitro experiments with ($\sp{35}$S) -methionine showed that the highly abundant calmodulin accumulating in vitellogenic follicles was not synthesized by the oocyte. On the other hand, isolated follicle cells rapidly synthesize large amounts of calmodulin. No calmodulin is detectable in serum. These facts suggest that calmodulin produced by follicle cells is most likely the source of calmodulin in the vitellogenic oocyte. Indirect immunofluorescent staining with anti-egg calmodulin demonstrated that in early- and mid-vitellogenic follicles calmodulin is localized in the cytoplasm of follicle cells and the cytoplasmic compartment surrounding yolk granules but not inside yolk granules. Immunofluorescence was most intense in the cortex of the oocyte and outside the membranes of yolk granules. Transport of calmodulin into the cytoplasmic compartment of the oocyte is not via binding to vitellogenenin.
2

Modulation of microtubule dynamics in living cells

Iyengar, Rama Dhamodharan 01 January 1995 (has links)
The effect of brain microtubule associated proteins and low concentrations of the drug vinblastine have been used to study the modulation of MT dynamic instability in living BSC-1 cells using low-light-level fluorescence microscopy and quantitative MT tracking methods. Heat stable brain MAPs (hs MAPs) and microtubule associated protein 2 (MAP-2) were microinjected into cultured BSC-1 cells which had been previously injected with rhodamine labeled tubulin. Both MAP preparations suppressed MT dynamics in vivo, by reducing the average rate and extent of both growth and shortening events. Both hs MAPs and MAP-2 did not significantly alter frequency of rescue per unit time but significantly increased the frequency of rescue per unit distance. Hs MAPs decreased the catastrophe frequency per unit time by two fold, while MAP-2 reduced this parameter to a lesser extent. When calculated as events per unit distance, both MAP preparations increased the frequency of rescue, without altering the frequency of catastrophe. Both MAP-2 and hs MAPs decreased the percentage of time spent shortening, increased the percentage of time spent paused, and had no effect on percentage of time spent growing. Hs MAPs increased the average pause duration, decreased the average number of events/min/MT and increased the probability that a paused MT switch to growing rather than shortening. The results demonstrate that addition of MAPs to living cells reduces the dynamic behavior of individual MTs primarily by suppressing the magnitude of dynamic events and increasing the time spent in pause, where no change in the MT length can be detected. The results further suggest that the expression of molecules like MAPs directly contributes to cell type specific MT dynamic behavior. Vinblastine treatment suppresses MT dynamics in vivo, by reducing the average distance and rate of growth and shortening events. Vinblastine treatment increased the average pause duration and the percent time the MTs spent in the paused state; the percent time in shortening was decreased. Vinblastine markedly reduced the frequency of catastrophe measured as events/unit time or unit distance. The frequency of rescue was increased when measured as events/unit distance. The probability of transition from pause to growth phase increased with increasing concentration of vinblastine. These results suggest that low concentration of vinblastine decreases the dynamicity of microtubules in vivo, consistent with the results reported in vitro and indicates that vinblastine might actively cap the ends of the MTs thereby reducing the exchange of tubulin dimers at the ends, consistent with earlier studies (Wilson et al., 1982; Jordan and Wilson, 1990).

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