Membrane and cytoplasmic resistivity properties of normal and sickle red blood cells

The cytoplasmic resistivities and membrane breakdown potentials of normal (AA), sickle-cell-trait (AS), and sickle (SS) red blood cells have been measured by the biophysical methodology of resistive pulse spectroscopy over a range of osmolalities. At isotonicity, the average membrane breakdown potentials are virtually identical for the three types of cells occurring at about 1150 V/cm. Average isotonic cytoplasmic resistivities are somewhat higher for the SS cells (166.7±7.49 ohm-cm) compared to the AA (147.6±1.98 ohm-cm) or AS cells (148.7±1.79 ohm-cm). As medium osmolality is varied, the differences in resistive properties become enlarged, especially at very low and very high osmolalities. At high osmolalities, both types of sickle cells show a large increase in internal resistivity compared to the normals; at low osmolality, the SS samples exhibit a distinctly different membrane breakdown characteristic, decreasing in this parameter, whereas the other two groups increase. Of the 15 SS samples tested, three displayed much higher cytoplasmic resistivities at isotonicity: 218.2±5.25 ohm-cm, compared to an average of 153.5±3.46 ohm-cm for the other 12. The relationship between these high resistivities and the subfraction of irreversibly sickled cells in the sample is discussed.     

Cytoplasmic suppression of malignancy

Summary Using both normal and transformed rat liver epithelial cells to prepare cytoplasmic hybrids (cybrids) we have found evidence to support the theory that the cytoplasm from a normal cell can suppress tumorigenicity. A unique aspect of this study is that all of the cells utilized, both normal and malignantly transformed, were derived from an original cloned cell. We found that fusing cytoplasts from normal cells to malignantly transformed whole cells resulted in cybrid clones which, when injected into newborn rat pups, isogenic with those from which the cell culture was initiated, yielted tumors in 51% of the animals injected compared to 92% of the animals injected with the tumorigenic parent. Those animals that did develop tumors from the cybrid cells survived longer than those injected with cells from the tumorigenic parent. Thus, the cybrid, formed of cytoplasm from both parents, was less tumorigenic than the malignantly transformed parent cell. When reconstituted cells were prepared by fusing cytoplasts from normal cells with karyoplasts from malignantly transformed cells, a situation in which essentially all of the cytoplasm of the reconstituted cell is derived from normal cells, the tumorigenic phenotype was extinguished. This work was supported in part by United States Public Health Service grant CA12056, and grant CA09100 from the National Cancer Institute, Bethesda, MD. This work is partial fulfillment for the degree of Doctor of Philosophy for B.A.I.     

Effect of calcium ion on cytoplasmic streaming during turgor regulation in a brackish water charophyte Lamprothamnium

Abstract The brackish water charophyte Lamprothamnium succinctum regulates its turgor pressure against changes in the external osmotic pressure. Upon hypotonic treatment, the rate of cytoplasmic streaming in the internodal cells fell to almost zero, and then recovered to the original value within 20 min. The decrease could be inhibited by lowering the external Ca²⁺ concentration in the hypotonic medium. Also, cytoplasmic streaming in tonoplast-free cells of L. succintum was sensitive to Ca²⁺ like freshwater charophyte. Thus, the concentration of free Ca²⁺ in the cytoplasm seems to increase transiently upon hypotonic treatment.     

Virus particle packaging in baculovirus and cytoplasmic polyhedrosis virus inclusion bodies

The structure of the inclusion bodies (IBs) of three multiply enveloped nuclear polyhedrosis viruses (MNPVs), one singly enveloped NPV (SNPV), two granulosis viruses (GVs) and one cytoplasmic polyhedrosis virus (CPV) were compared. A method was devised to calculate the numbers of virus particles and nucleocapsids in IBs using data from light microscopy and thin sections. The three MNPVs, from Agrotis segetum (English and Polish virus isolates) and Mamestra brassicae had similar concentrations of virus particles ranging from 17.3 to 19.6 per μm³ of IB. Plusia gamma SNPV had a higher density of 59.6 virus particles per μm³ of IB, which partly compensated for its having smaller IBs (mean volume 0.65 μm³) than the MNPVs (2.60–9.71 μm³). The English A. segetum MNPV isolate had the most nucleocapsids in each virus particle (mean, 4.04) and the largest IBs (mean volume, 9.71 μm³), giving 674 nucleocapsids per IB on average. The GVs, from A. segetum and Pieris brassicae, mainly contained one nucleocapsid per IB. P. gamma CPV IBs had a much higher density of virus particles than the baculoviruses (260 per μm³ compared with 17–60 per μm³). These data are discussed in relation to the biological properties of these viruses, and possible adaptational advantages of alternative IB designs are considered.     

Morphological anomalies induced by antimicrotubule agents inBryopsis plumosa

Summary Antimicrotubule agents, colchicine, vinblastine, and griseofulvin, induced conspicuous morphological anomalies inBryopsis plumosa. First, following cessation of protoplasmic streaming within 15 minutes, elongation stopped in a few hours. Second, innumerable protrusions or new growth points generated over the cell flank in a few days. Similar phenomena were observed in the cells which were subjected to high pressure or low temperature both of which are known to disrupt microtubule.These phenomena were investigated with light and electron microscopy. It is suggested that inhibition of microtubule dependent protoplasmic streaming which may function as an intracellular transport system causes such morphological anomalies.     

水稻不育花药中H_2O_2的积累与膜脂过氧化的加剧

水稻7017、二九矮细胞质雄性不育系及其保持系花药的POD,CAT和SOD活性研究的结果表明,单核早期时不育及可育花药的酶活性差异不明显,单核晚期、二核及三核期的不育花药显著低于可育花药。在不育花药中缺少两条Cu-Zn SOD同工酶带,而且O_2~ 产生效率为可育的4.1～5.5倍,并有H_2O_2和MDA的积累。不育花药中H_2O_2的积累和膜脂过氧化的加剧可能与花粉败育有关。     

Effect of cytoplasmic acidification on the membrane potential of T-lymphocytes: Role of trace metals

Summary The effect of lowering intracellular pH on the membrane potential (E _m ) of rat thymic lymphocytes was studied using the potential-sensitive dyebis-oxonol. Cells were acid loaded by addition of the electroneutral K⁺/H⁺ exchanging ionophore nigericin. Acidification to pH 6.3 in Na⁺-free solution resulted in a biphasic change inE _m : an early transient hyperpolarization followed by a sustained depolarization. These changes were associated with a rise in cytosolic free Ca²⁺ ([Ca²⁺] _i ). The hyperpolarization was eliminated when the change in [Ca²⁺] _i was prevented using BAPTA, an intracellular Ca²⁺ chelator. Moreover, a similar hyperpolarization was elicited by elevation of [Ca²⁺] _i at physiological pH _i using ionomycin, suggesting involvement of Ca²⁺-activated K⁺ channels. In contrast, the depolarization phase could not be mimicked by raising [Ca²⁺] _i with ionomycin. However, intracellular BAPTA effectively inhibited the acidificationinduced depolarization. Inhibition was also obtained by extracellular addition of EGTA or dithiothreitol, even when the external free Ca²⁺ concentration remained unaltered. These observations suggested a possible role of contaminating trace metals. Cytosolic acidification is envisaged to induce intracellular accumulation of one or more trace metals, which induces the observed changes inE _m . Accordingly, similar changes inE _m can be induced without acidification by the addition of small amounts of Cu²⁺ to the medium. The ionic basis of theE _m changes induced by acidification and the significance of these observations are discussed.     

Measurement of motive force for cytoplasmic streaming in characean internodes

Summary With an attempt to measure the motive force responsible for cytoplasmic streaming in characean internodal cells, the difference between densities of cytoplasm and vacuolar sap was heightened by about 10 times (density of vacuolar sap was made larger than that of cytoplasm) by replacing the natural vacuolar sap ofChara corallina with an artificial one of higher density. Endoplasmic flow contiguous to the peripheral actin cables (peripheral flow of endoplasm) in the centrifugal direction was not influenced at all by the application of centrifugal acceleration up to 1400 g. We thus concluded that the motive force for the peripheral flow should be much larger than 12dyn/cm², a figure more than 10 times larger than that for bulk endop lasmic flow so far reported.Dedicated to Emeritus Professor Noburo Kamiya on the occasion of his 80th birthday     

Expression of sunflower low-molecular-weight heat-shock proteins during embryogenesis and persistence after germination: localization and possible functional implications

We isolated and sequenced Ha hsp 17.9, a DNA complementary (cDNA) of dry-seed stored mRNA that encodes a low-molecular-weight heat-shock protein (LMW HSP). Sequence analysis identified Ha hsp17.9, and the previously reported Ha hsp17.6, as cDNAs encoding proteins (HSP17.6 and HSP17.9) which belong to different families of cytoplasmic LMW HSPs. Using specific antibodies we observed differential expression of both proteins during zygotic embryogenesis under controlled environment, and a remarkable persistence of these LMW HSPs during germination. Immuno-blot analysis of HSP17.9 proteins in two-dimensional gels revealed that the polypeptides expressed in embryos were indistinguishable from LMW HSPs expressed in vegetative tissues in response to water deficit; but they appeared different from homologeous proteins expressed in response to thermal-stress. Tissue-print immunolocalization experiments showed that HSP17.9 and HSP17.6 were homogeneously distributed in every tissue of desiccation-tolerant dry seeds and young seedlings under non-stress conditions. These results demonstrate developmental regulation of specific, cytoplasmic, plant LMW HSPs, suggesting also their involvement in water-stress tolerance.