A Characeae Cells Plasma Membrane as a Model for Selection of Bioactive Compounds and Drugs: Interaction of HAMLET-Like Complexes with Ion Channels of Chara corallina Cells Plasmalemma.

Interaction of a HAMLET-like La-OA cytotoxic complex (human α-lactalbumin-oleic acid) and its constituents with the excitable plasmalemma of giant Chara corallina cells was investigated. The voltage-clamp technique was used to study Ca2+ and Cl- transient currents in the plasmalemma of intact cells. The action of the complex and OA on the target cell membrane has a dose-dependent character. It was found that the La-OA complex has an inhibiting effect on Ca2+ current across the plasmalemma, while α-lactalbumin alone does not affect the electrophysiological characteristics of the cellular membrane. However, oleic acid blocks Ca2+ current across the plasmalemma. This is accompanied by the induction of a non-selective conductivity in the cellular membrane, a decrease in the resting potential and plasma membrane resistance of algal cells. We propose that the cytotoxicity of La-OA and other HAMLET-like complexes is determined by oleic acid acting as a blocker of potential-dependent Ca2+ channels in the plasma membrane of target cells. The presented results show that the study model of green algae C. corallina cells plasmalemma is a convenient tool for the investigation of ion channels in many animal cells.

Expression of exogenous genes under the control of endogenous HSP70 and CAB promoters in the Closterium peracerosum-strigosum-littorale complex.

A unicellular charophyte alga, Closterium peracerosum-strigosum-littorale complex (C. psl. complex), has been studied in order to obtain basic information regarding sexual reproduction in plants. Systems for gene introduction and transient expression were developed for endogenous genes using phleomycin resistance (ble) and Chlamydomonas green fluorescent protein (cgfp) genes as selection markers. These genes have codon usage similar to that of genes in the C. psl. complex. To drive these genes strongly into C. psl. complex cells, two native promoters of the C. psl. complex genome-CpHSP70 and CpCAB1-were linked to a ble::cgfp fusion gene and introduced into the cells by particle bombardment. Following 2 d of incubation, we found 500 cells expressing GFP under the control of the CpHSP70 promoter, which were identified following heat shock treatment at 42 degrees C, and 100 cells expressing GFP under the control of the CpCAB1 promoter, which were observed in lit conditions. In contrast, the GFP signal was only detected in two cells when ble::cgfp under control of the cauliflower mosaic virus 35S promoter was introduced. The ble::cgfp fusion protein was detected in the nucleus, whereas the single cgfp protein was detected in the cytoplasm. Our results indicate that the newly isolated native promoters of CpHSP70 and CpCAB1 are useful tools for inducing exogenous gene expression under heat shock and lit conditions, respectively. In addition, this strategy can be used for transient assays, such as the intracellular localization of unknown gene products in the C. psl. complex.

[The roles of microtubule in internodal cell elongation (Nitellopsis obtusa)].

The relationship between cell elongation and microtubules (MTs) was investigated in characean internodal cells (Nitellops obtusa). First, we examined the immunofluorescent localization of MTs in different living stages under confocal laser scanning microscope. In young, rapidly elongating cells, MTs were predominantly transverse to the long axis of the cell. As the relative growth rate fell, transverse MTs gradually decreased, and in non-growing cells, longitudinally oriented cortical MTs became most pronounced. Moreover, cells in different living stages responded to the treatment of oryzalin (microtubule-disrupting agent) differently, young active internodal cells seemed to be more sensitive. After 40 min incubation of 10 micromol/L oryzalin, nearly all cortical MTs in the elongating cells depolymerized. However, in the old, non-growing cells, some MT fragments still remained after 3 h treatment of oryzalin. Second, we measured the cell growth rates with and without the treatment of oryzalin. In young growing cells treated with 10 micromol/L oryzalin, the elongation rates were inhibited obviously. When the oryzalin was removed, the elongation rates could be recovered to some extent. Interestingly, a time-gap existed between microtubule disassembly (40 min) and cessation of cell elongation (100 min). Our data confirmed the evidence that MTs are involved in cell elongation.

Cytoskeletal 10 nm filaments in cells of the algal phyla Chlorophyta, Charophyta, and Chrysophyta and their developmentally regulated and species-specific association with prosomes.

10 nm diameter filaments were observed in whole-mount preparations of algae of diverse phyla: Acetabularia acetabulum and A. major (Chlorophyta), Chara australis and Nitella flexilis (Charophyta), and Poterioochromonas malhamensis (Chrysophyta). A polyclonal antibody raised against a basic, 50 kDa DNA-binding protein of A. acetabulum stains the filaments of A. acetabulumand and A. major as well as of C. australis and N. flexilis. While in the perinuclear region of A. acetabulumand and A. major and throughout the cytoplasm of P. malhamensis the 10 nm filaments have a smooth appearance, in the stalk of A. acetabulumand and A. major they are densely covered by globular structures; in C. australis and N. flexilis they are less frequently associated with such material. The morphology of a part of the globular particles is quite reminiscent of prosomes. A monoclonal antibody elicited against prosomes isolated from A. acetabulum indeed decorates the globular particles on the A. acetabulum and A. major filaments. The possible role of these filament-particle associations is discussed.