Seasonal histochemical changes in the cambium ofTectona grandis L. f. andgmelina arborea roxb

Starch, lipids and proteins are localized histochemically in fusiform and ray initials ofTectona grandis andGmelina arborea during the periodicity of cambium in the year 1978. The histochemical variations are correlated with the seasonal activity and dormancy of the cambium and phenology. Starch grains appear in the cambial cells with the development of young leaves after defoliation. Lipid and protein bodies are significantly high in dormant cambial cells and scarce in active ones.     

Bleomycin treatment of chick fibroblasts causes an increase of polysomal type I procollagen mRNAs. Reversal of the bleomycin effect by dexamethasone

Bleomycin treatment of primary chick skin fibroblasts and chick lung fibroblasts resulted in a selective dose-dependent increase of cell layer procollagen synthesis. Solid support hybridization of total cellular RNA to 32P-labeled pro-alpha 1(I) and pro-alpha 2(I) cDNAs did not indicate an increase of total cellular procollagen type I mRNAs in bleomycin-treated cells. However, bleomycin treatment of chick skin fibroblasts causes a redistribution of procollagen type I mRNAs within the nuclear, cytoplasmic, and polysomal subcellular fractions. Both the nuclear and cytoplasmic procollagen type I mRNAs are significantly decreased in concentration after bleomycin administration. In contrast, the polysomal procollagen type I mRNAs are significantly increased in both chick skin and lung fibroblasts treated with bleomycin. Administration of dexamethasone to bleomycin-treated fibroblasts resulted in a reversal of the bleomycin-induced increase in cell layer procollagen synthesis. The increased amounts of polysomal procollagen type I mRNAs in bleomycin-treated cells were also reduced by subsequent administration of dexamethasone. These data indicate that bleomycin treatment of chick skin and chick lung fibroblasts results in a specific increase in procollagen synthesis in the cell layer which is mediated by elevated levels of polysomal type I procollagen mRNAs via a repartitioning of these mRNAs within the fibroblast. Furthermore, dexamethasone reverses the bleomycin-induced elevations of both cell layer procollagen synthesis and polysomal type I procollagen mRNAs.     

Morphology of isolated triads

The triad is the junctional association of transverse tubule with sarcoplasmic reticulum terminal cisternae. A procedure for the isolation of highly enriched triads from skeletal muscle has been described in the previous paper. In the present study, the structural features of isolated triads have been examined by thin-section, negative-staining, and freeze-fracture electron microscopy. In isolated triads, key features of the structure observed in situ have been retained, including the osmiophilic "feet," junctional structures between the transverse tubule and terminal cisternae. New insight into triad structure is obtained by negative staining, which also enables visualization of feet at the junctional face of the terminal cisternae, whereas smaller surface particles, characteristic of calcium pump protein, are not visualized there. Therefore, the junctional face is different from the remainder of the sarcoplasmic reticulum membrane. Junctional feet as viewed by thin section or negative staining have similar periodicity and extend approximately 100 A from the surface of the membrane. Freeze-fracture of isolated triads reveals blocklike structures associated with the membrane of the terminal cisternae at the junctional face, interjunctional connections between the terminal cisternae and t-tubule, and intragap particles. The intragap particles can be observed to be closely associated with the t-tubule. The structure of isolated triads is susceptible to osmotic and salt perturbation, and examples are given regarding differential effects on transverse tubules and terminal cisternae. Conditions that adversely affect morphology must be considered in experimentation with triads as well as in their preparation and handling.     

Purification of morphologically intact triad structures from skeletal muscle

A procedure has been devised for isolation of triads (t-tubule/sarcoplasmic reticulum (SR) junctional complexes) from rabbit skeletal muscle. The procedure consists of preparation of a heavy microsomal fraction followed by two sequential 90-min sucrose gradient centrifugations to enrich the triads. A pyrophosphate/phosphate/magnesium buffer system was introduced to decrease aggregation in order to achieve effective separation. The preparation time is 12 h. Some differences between purified triads isolated by two variants of this method are noted. The purity of the triad fractions has been estimated by particle counting to be in the vicinity of 50%. There is good retention of morphology and Ca++-loading activity and enrichment in Na+,K+-ATPase and adenylate cyclase. The triads are practically devoid of contractile elements, mitochondria, and free plasmalemma, and low in content of light SR. The method for obtaining enriched triads is reproducible, and sufficient yields are obtained for structural, biochemical, and functional characterization.     

Cellular glutathione depletion by diethyl maleate or buthionine sulfoximine: no effect of glutathione depletion on the oxygen enhancement ratio

The hypoxic and euoxic radiation response for Chinese hamster lung and A549 human lung carcinoma cells was obtained under conditions where their nonprotein thiols, consisting primarily of glutathione (GSH), were depleted by different mechanisms. The GSH conjugating reagent diethylmaleate (DEM) was compared to DL-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutathionine biosynthesis. Each reagent depleted cellular GSH to less than 5% of control values. A 2-hr exposure to 0.5 mM DEM or a 4- or 24-hr exposure to BSO at 10 or 1 mM, respectively, depleted cellular GSH to less than 5% of control values. Both agents sensitized cells irradiated under air or hypoxic conditions. When GSH levels are lowered to less than 5% by both agents, hypoxic DEM-treated cells exhibited slightly greater X-ray sensitization than hypoxic BSO-treated cells. The D0's for hypoxic survival curves were as follows: control, 4.87 Gy; DEM, 3.22 Gy; and BSO, 4.30 Gy for the V79 cells and 5.00 Gy versus 4.02 Gy for BSO-treated A549 cells. The D0's for aerobic V79 cells were 1.70 Gy versus 1.13 Gy, DEM, and 1.43 Gy for BSO-treated cells. The D0's for the aerobic A549 were 1.70 and 1.20 for BSO-treated cells. The aerobic and anoxic sensitization of the cells results in the OER's of 2.8 and 3.0 for the DEM- and BSO-treated cells compared to 2.9 for the V79 control A549. BSO-treated cells showed an OER of 3.3 versus 3 for the control. Our results suggest that GSH depletion by either BSO or DEM sensitizes aerobic cells to radiation but does not appreciably alter the OER.     

Maximum shortening velocity of smooth muscle: zero load-clamp vs. afterloaded method

Previous reports from this laboratory of force-velocity relationships of canine tracheal smooth muscle (TSM) have presented maximum shortening velocities (Vmax) mathematically derived from the linearized transformation of the Hill equation (A. V. Hill, Proc. Roy. Soc. London, Ser. B., 126:136-195, 1938). Recent technical advances enable us to measure Vmax directly using an electromagnetic lever system that can instantaneously clamp to a zero load, thus we compared values of Vmax derived mathematically and those directly measured on the same TSM strips. Derived Vmax values from afterloaded isotonic shortening curves for loads greater than preload were 0.328 +/- 0.021 optimal length (lO)/s and were not significantly different from zero load-clamp measurements of 0.301 +/- 0.022 lO/s from the same (n = 15) muscles. These data indicate that Vmax values mathematically derived for TSM from conventional isotonic afterloaded force-velocity curves are valid estimates of zero load velocity, because they were not significantly different from values obtained by direct measurement using the zero load-clamp technique.     

The effect of various inhibitors of DNA synthesis on the repair of DNA photoproducts

The effect on DNA repair of several inhibitors of DNA synthesis has been investigated in CHO cells. Three assays were employed following ultraviolet irradiation of G₁ cells: unscheduled DNA synthesis, removal of antibody binding sites and alkaline elution. Cytosine arabinoside and aphidicolin were found to reduce unscheduled DNA synthesis in a dose-dependent manner without affecting the removal of antibody-binding sites. Strand rejoining was also inhibited. These results are consistent with the hypothesis that inhibition is due to premature chain termination during repair synthesis some time after excision of the lesion. Conversely, inhibition of unscheduled DNA synthesis by novobiocin is paralleled by inhibition of excision of the lesion. However, no inhibition of incision was apparent. Since nalidixic acid, an inhibitor of topoisomerase II, did not inhibit excision, it is unlikely that the primary site of action of novobiocin is this topoisomerase. The possibility that a second topoisomerase and/or a polymerase are affected is discussed in the light of previously published data.     

A computer program for the statistical analysis of disease prevalence data from survival/sacrifice experiments

This paper presents a computer program for analyzing disease prevalence data from animal survival experiments in which there may also be some serial sacrifice. The method has been described in Biometrics 35 (1979) 221-234. The user is interrogated about the details of particular models he wishes to fit. Then a generalized EM algorithm is used to compute maximum likelihood estimates of various quantities of interest concerning the effects of treatment, time and presence of other diseases on the prevalences and lethalities of specific diseases of interest.     

Loss and resynthesis of a developmentally regulated membrane protein (gp80) during dedifferentiation and redifferentiation in Dictyostelium

When developing cultures of Dictyostelium discoideum are disaggregated and resuspended in nutrient medium, they lose the capacity to rapidly reaggregate after 90 min, in a rapid and synchronous step referred to as the "erasure event." They then proceed to lose remaining developmentally acquired functions in a program of dedifferentiation culuminating with the loss of EDTA-resistant cohesion roughly 5 hr later. Immediately following the erasure event, cells can be stimulated to reenter the developmental program even though they still possess a number of developmentally acquired functions. These cells therefore appear to undergo dedifferentiation and redifferentiation simultaneously (D. R. Soll and L. H. Mitchell, 1982, Dev. Biol. 91, 183-190). In this report, we have employed an antiserum made against a developmentally acquired membrane glycoprotein, gp80, to examine whether gp80 is lost during dedifferentiation and whether it is either reutilized or resynthesized during redifferentiation. Results are presented which demonstrate that (1) when 9-hr developing cells are disaggregated and resuspended in nutrient medium, gp80 continues to accumulate for several hours after the erasure event, then is lost at roughly the same time as EDTA-resistant cohesion; (2) when cells are stimulated to reenter the developmental program immediately after the erasure event, both gp80 and EDTA-resistant cohesion are still lost according to the program of dedifferentiation, but are then reacquired soon afterwards according to the program of redifferentiation; (3) during redifferentiation, cells do not reutilize gp80 which had been synthesized during initial development; rather they synthesize gp80 de novo; and (4) developing cells of a dedifferentiation-defective variant, HI4, when disaggregated and resuspended in nutrient medium, retain gp80, EDTA-resistant cohesion, and the capacity to rapidly reinitiate aggregation for at least 12 hr. This last result indicates that the loss of gp80 is regulated by the dedifferentiation process and is not an independent response to disaggregation or the reintroduction of nutrients. Together, these results reinforce the conclusion that dedifferentiation and redifferentiation can function independently and simultaneously in the same cells.