Isolation and characterization of cytoskeletons from cotton fiber cytoplasts

Summary Over the last 25 yr, success in characterizing the individual protein components of animal cytoskeletons was possible, in part, due to technical advances in the isolation and purification of anucleate cytoskeletons from animal cells. As a step towards characterizing protein components of the plant cytoskeleton, we have isolated cytoskeletons from cytoplasts (anucleate protoplasts) prepared from cotton fiber cells grown in ovule culture. Cytoplasts isolated into a hypertonic, Ca²⁺-free medium at pH 6.8 retained internal structures after extraction with the detergent, Triton X-100. These structures were shown to include microtubule and microfilament arrays by immunofluorescence and electron microscopy. Actin and tubulin were the only abundant proteins in these preparations, suggesting that microfilaments and microtubules were the major cytoskeleta elements in the isolated cytoskeletons. The absence of additional, relatively abundant proteins suggests that (a) other cytoskeletal arrays potentially present in fiber cells (e.g., intermediate filaments) were either lost during detergent extraction or were minor components of the fiber cell cytoskeleton; and (b) high ratios of individual cytoskeletal-associated proteins relative to actin and tubulin were not required to maintain microtubules and microfilaments in organized structures.     

Cryopreservation and long-term storage of pear germplasm

Summary Germplasm collections of vegetatively propagated crops are usually maintained as plants in fields or potted in greenhouses or screened enclosures. Safety duplication of these collections, as duplicate plants or separate collections, is costly and requires large amounts of space. Cryopreservation techniques which were recently developed for long-term storage of pear germalasm may offer an efficient alternative to conventional germplasm collection maintenance. Pear (Pyrus L.) germplasm may now be stored as seeds (species), dormant buds or pollen from field-grown trees, or shoot tips fromin vitro-grown plants (cultivars). Pear germplasm may now be cryopreserved and stored for long periods (> 100 yr) utilizing slow-freezing or vitrification ofin vitro-grown shoot-tips. Dormant bud freezing, pollen, and seed cryopreservation of other lines are being developed to complete the base collection forPyrus. This cryopreserved collection provides base (long-term) storage for the field-grown pear germplasm collection at the National Clonal Germplasm Repository, Corvallis, Oregon. Based on a presentation at the 1997 Congress on In Vitro Biology held in Washington, D.C., June 14–18, 1997.     

Lumenal and Transmembrane Domains Play a Role in Sorting Type I Membrane Proteins on Endocytic Pathways

Previous studies have shown that when the cytosolic domains of the type I membrane proteins TGN38 and lysosomal glycoprotein 120 (lgp120) are added to a variety of reporter molecules, the resultant chimeric molecules are localized to the trans-Golgi network (TGN) and to lysosomes, respectively. In the present study we expressed chimeric constructs of rat TGN38 and rat lgp120 in HeLa cells. We found that targeting information in the cytosolic domain of TGN38 could be overridden by the presence of the lumenal and transmembrane domains of lgp120. In contrast, the presence of the transmembrane and cytosolic domains of TGN38 was sufficient to deliver the lumenal domain of lgp120 to the trans-Golgi network. On the basis of steady-state localization of the various chimeras and antibody uptake experiments, we propose that there is a hierarchy of targeting information in each molecule contributing to sorting within the endocytic pathway. The lumenal and cytosolic domains of lgp120 contribute to sorting and delivery to lysosomes, whereas the transmembrane and cytosolic domains of TGN38 contribute to sorting and delivery to the trans-Golgi network.     

The Activity of Differentiation Factors Induces Apoptosis in Polyomavirus Large T-Expressing Myoblasts

It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G0₀ arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis.     

An Arsenic Exposure Model: Probabilistic Validation Using Empirical Data

This paper describes development of a multi-pathway arsenic exposure model. The model uses information on arsenic concentrations in food, water, soil, and dust, combined with estimates of intake and medium-specific absorption. Urinary arsenic is predicted assuming that 60% of absorbed arsenic is excreted in urine under steady state conditions. Fecal arsenic is predicted assuming all unabsorbed arsenic is excreted in feces. We applied this model at a former copper smelter site. Site specific distributions were available for the following parameters: soil and dust arsenic concentration (geometric mean approximately 100 to 200?ppm and 50 to 100?ppm, respectively); the combined childhood soil and dust ingestion rate (geometric mean of 20?mg/d); soil and dust arsenic relative bioavailability (geometric mean 0.20 and 0.28, respectively); exposure duration; water arsenic concentration; air arsenic concentration; and total arsenic in food. Monte Carlo simulation was used to predict daily arsenic uptake and excretion in urine and feces for children. Predicted urine arsenic levels were less than measured levels (73% to 88% of measured values, depending on region of site). On the other hand, predicted fecal arsenic levels exceeded measured levels by a factor of 1.7 to 4.6. We were able to improve the correspondence between predicted and measured arsenic excretion rates by decreasing the assumed value of the combined soil and dust ingestion rate, and increasing the assumed bioavailability of arsenic in soil and dust.     

Detection of mitogen induced stimulation of leukocytes from the rainbow trout (Oncorhynchus mykiss) by flow cytometric analysis of intracellular calcium

Flow cytometric analysis of the forward/side light scatter (FSC/SSC) of density gradient-separated head kidney cells of the rainbow trout revealed three distinctly separated populations, which we defined as population 1, 2 and 3. In spleen cells, populations 1 and 2 were also found, whereas population 3 was not detected. Further characterization regarding the surface Ig (sIg) revealed that population 2 of the head kidney and spleen contained 37.4 and 34.4% sIg⁺-cells, respectively. Incubation of the head kidney and spleen cells with different concentrations of concanavalin A (ConA), phytohemagglutinin (PHA) and [PWM] induced a pronounced intracellular calcium increase only in cells of population 2. This reaction was concentration dependent and caused by a release of intracellular Ca²⁺-stores. FMLP, a chemotactic peptide, had no effect on intracellular calcium response in all three populations. Similarly, the stimulation with PMA had no effect. This indicates that population 2 of the head kidney as well as the spleen is characterized by a high forward and low side light scatter and contains both subpopulation of lymphocytes, B- and T-cells. We demonstrated that the analysis of intracellular calcium increase due to mitogens is a suitable approach to identify lymphocytes in fish and enables further functional studies in these cells.     

Stress symposium in Johannesburg: plants versus humans

On 18 February 1998, a ‘Stress symposium’ was held at the Rand Afrikaans University (RAU) in Johannesburg, South Africa. The meeting brought together people from both the plant and the human oxidative stress field, which was exemplified by a talk entitled ‘Heat shock proteins in host-pathogen interactions: plants versus humans’. There were moments when it appeared as if the main difference between plants and humans was, as sung by Julos Beaucarne, that ‘the human plant is the only one to be able to water itself…’     

Aerobic exercise maintains regional bone mineral density during weight loss in postmenopausal women

This studyexamines the effects of weight loss by caloric restriction (WL) andaerobic exercise plus weight loss (AEx+WL) on total and regional bonemineral density (BMD) in older women. Healthy,postmenopausal women [age 63 ± 1 (SE) yr] not onhormone-replacement therapy underwent 6 mo of WL(n = 15) consisting of dietarycounseling one time per week with a caloric deficit (250-350kcal/day) or AEx+WL (n = 15)consisting of treadmill exercise three times per week in addition tothe weight loss. Maximal aerobic capacity increased only in the AEx+WLgroup (P < 0.001). Body weight,percent fat, and fat mass decreased similarly in both groups(P < 0.005), with no changesin fat-free mass. Total body BMD (by dual-energy X-rayabsorptiometry) decreased in both groups(P < 0.05). Femoral neck, Ward'striangle, and greater trochanter BMD decreased in the WL group(P  0.05) but were not significantlydifferent after AEx+WL.L₂-L₄BMD did not significantly change in either group. Thus WL andAEx+WL both result in losses of totalbody BMD; however, AEx+WL appears to prevent the loss in regional BMDseen with WL alone in healthy, older women. This suggests that theaddition of exercise to weight-loss programs may reduce the risk forbone loss.