Sequencing, cloning, and expression of human red cell-type acid phosphatase, a cytoplasmic phosphotyrosyl protein phosphatase.

Low molecular weight phosphotyrosyl protein phosphatases of human placenta and human red cell were purified and sequenced by a combination of Edman degradation and tandem mass spectrometry. Screening of a human placental lambda gt11 cDNA library yielded overlapping cDNA clones coding for two distinct human cytoplasmic low molecular weight phosphotyrosyl protein phosphatases (HCPTPs). The two longest clones, designated HCPTP1-1 and HCPTP2-1, were found to have identical nucleotide sequences, with the exception of a 108-base pair segment in the middle of the open reading frame. Polymerase chain reaction studies with human genomic DNA suggest that the difference between HCPTP1-1 and HCPTP2-1 does not result from alternative RNA splicing. Studies with a human chromosome 2-specific library confirmed that these sequences are located on chromosome 2, which is known to be the location of red cell acid phosphatase locus ACP1. The coding sequences of HCPTP1-1 and HCPTP2-1 were placed downstream from a bacteriophage T7 promoter and the proteins were expressed in Escherichia coli. The resulting recombinant enzymes (designated HCPTP-A and HCPTP-B, respectively) showed molecular weights of 18,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and both of them exhibited immunoreactivity with antisera raised against authentic human placental and bovine heart enzymes. The expressed proteins were highly active towards the phosphatase substrates p-nitrophenyl phosphate, beta-naphthyl phosphate, and O-phospho-L-tyrosine, but not alpha-naphthyl phosphate, threonine phosphate, or O-phospho-L-serine. HCPTP-A and -B possessed effectively identical amino acid compositions, immunoreactivities, inhibition by formaldehyde, and kinetic properties when compared with two human red cell acid phosphatase isoenzymes. It is concluded that HCPTP-A and -B are the fast and slow forms of red cell acid phosphatase, respectively, and that this enzyme is not unique to the red cell but is instead expressed in all human tissues.     

6‐O‐(3″, 4″‐di‐O‐trans‐cinnamoyl)‐α‐l‐rhamnopyranosylcatalpol and verbascoside: Cytotoxicity,cell cycle kinetics,apoptosis, and ROS production evaluation in tumor cells

The aim of this study was to evaluate the impact that 6‐O‐(3″, 4″‐di‐O‐trans‐cinnamoyl)‐α‐ l ‐rhamnopyranosylcatalpol (Dicinn) and verbascoside (Verb), two compounds simultaneously reported in Verbascum ovalifolium, have on tumor cell viability, apoptosis, cell cycle kinetics, and intracellular reactive oxygen species (ROS) level. At 100 µg/mL and 48 hours incubation time, Dicinn and Verb produced good cytotoxic effects in A549, HT‐29, and MCF‐7 cells. Dicinn induced cell‐cycle arrest at the G₀/G₁ phase and apoptosis, whereas Verb increased the population of subG₁ cells and cell apoptosis rates. Furthermore, the two compounds exhibited time‐dependent ROS generating effects in tumor cells (1‐24 hours). Importantly, no cytotoxic effects were induced in nontumor MCF‐10A cells by the two compounds up to 100 µg/mL. Overall, the effects exhibited by Verb in tumor cells were more potent, which can be correlated with its structural features, such as the presence of phenolic hydroxyl groups.     

A cross-platform public domain PC image-analysis program for the comet assay

The single-cell gel electrophoresis, also known as the comet assay, has gained wide-spread popularity as a simple and reliable method to measure genotoxic and cytotoxic effects of physical and chemical agents as well as kinetics of DNA repair. Cells are generally stained with fluorescent dyes. The analysis of comets--damaged cells which form a typical comet-shaped pattern--is greatly facilitated by the use of a computer image-analysis program. Although several image-analysis programs are available commercially, they are expensive and their source codes are not provided. For Macintosh computers a cost-free public domain macro is available on the Internet. No ready for use, cost-free program exists for the PC platform. We have, therefore, developed such a public domain program under the GNU license for PC computers. The program is called CASP and can be run on a variety of hardware and software platforms. Its practical merit was tested on human lymphocytes exposed to gamma-rays and found to yield reproducible results. The binaries for Windows 95 and Linux, together with the source code can be obtained from: http://www.casp.of.pl.     

Inhibition of bovine α-chymotrypsin by cyclic trypsin inhibitor SFTI-1 isolated from sunflower seeds and its two acyclic analogues

Summary Trypsin inhibitor SFTI-1 isolated from sunflower seeds (comprising 14 amino acid residues and two cycles: head-to-tail cyclisation and disulfide bridge) is the smallest naturally occurring plant serine proteinase inhibitor. In our recent paper we have shown that the elimination head-to-tail cyclisation did not change trypsin inhibitory activity as judged by measured by association equilibrium constants K _a . The removal of disulfide bridge produced 2.4-fold lower activity. In the present paper we described chymotrypsin inhibitory activity. SFTI-1 inhibits significantly lower bovine α-chymortypsin (K _a =(5.20±1.56)×10⁶ M⁻¹). The activity of the analogue with disulfide bridge only was practically the same, whereas the K _a value determined for homodetic peptide was almost 3-fold lower. Considering the results obtained and the recent literature data we postulate the lower inhibitory activity against both enzymes of the analogue with head-to-tail cyclisation only reflect its lower proteolytic stability.     

Regulation of mouse oocyte microtubule and organelle dynamics by PADI6 and the cytoplasmic lattices

Organelle positioning and movement in oocytes is largely mediated by microtubules (MTs) and their associated motor proteins. While yet to be studied in germ cells, cargo trafficking in somatic cells is also facilitated by specific recognition of acetylated MTs by motor proteins. We have previously shown that oocyte-restricted PADI6 is essential for formation of a novel oocyte-restricted fibrous structure, the cytoplasmic lattices (CPLs). Here, we show that α-tubulin appears to be associated with the PADI6/CPL complex. Next, we demonstrate that organelle positioning and redistribution is defective in PADI6-null oocytes and that alteration of MT polymerization or MT motor activity does not induce organelle redistribution in these oocytes. Finally, we report that levels of acetylated microtubules are dramatically suppressed in the cytoplasm of PADI6-null oocytes, suggesting that the observed organelle redistribution failure is due to defects in stable cytoplasmic MTs. These results demonstrate that the PADI6/CPL superstructure plays a key role in regulating MT-mediated organelle positioning and movement.     

Can a stench be beautiful? – Osmophores in stem-succulent stapeliads (Apocynaceae-Asclepiadoideae-Ceropegieae-Stapeliinae)

Carrion flower stapeliads are examples of olfactory mimicry, forming sapromyiophilous flowers, which mimic food sources or oviposition sites to attract fly pollinators. The aim of this work was to investigate the ultrastructure of osmophores involved in the release of the carrion odor of Orbea variegata and Boucerosia indica flowers. In spite of their similar architecture (epidermal epithelium+subepidermal secretory layers), the osmophores of stapeliads feature some differences in morphology and ultrastructure. The epidermal epithelial cells of O. variegata and B. indica differ in shape, but both are extremely rich in endoplasmic reticulum and flocculent material in the vacuole. Unlike the Orbea, Boucerosia has starchless leucoplasts in the epidermal epithelium. Orbea features a cuticle with microchannels, while Boucerosia has a different mechanism for the pathway of scent substances to the cell exterior. They are released by rupturing of the outer layer of cuticle at the apex of the papillae. The epidermal cells of the adaxial corolla differ even between parts of the corolla, the corolla lobes and the annulus in the flower. This diversity may be connected with an odor gradient. The morphological and anatomical features of stapeliad (subtribe Stapeliinae) osmophores are generally similar to osmophores of members of subtribe Ceropegiinae (Ceropegia), thus, we suggest that this model of osmophores evolved before early diversification of Ceropegieae. The ultrastructural features of stapeliad osmophores are generally similar to those of Araceae, Orchidaceae and Passifloraceae.     

Antisense hairpin loop oligonucleotides as inhibitors of expression of multidrug resistance-associated protein 1: their stability in fetal calf serum and human plasma

Multidrug resistance-associated protein (MRP1) is a transmembrane pump protein responsible for the efflux of chemotherapeutic drugs, an important cause of anticancer treatment failure. Trying to circumvent MRP-mediated resistance we designed and synthesized hairpin loops forming antisense oligodeoxyribonucleotides (ODNs), both phosphodiesters (PO-ODNs) and their phosphorothioate analogues (PS-ODNs), to reduce the protein expression by targeting its mRNA in a sequence specific manner. Melting temperature measurements as well as polyacrylamide gel electrophoresis supported the preferential formation of a secondary structure, which was expected to protect ODNs against 3'-exonuclease degradation. ODNs and PS-ODNs designed in this work were successfully tested as antisense inhibitors of the expression of MRP1 in the leukaemia HL60/ADR cell line. Foreseeing the necessity to perform clinical studies with such ODNs we investigated their stability against the 3'-exonuclease activity of fetal calf serum and human plasma. Under the conditions, corresponding to physiological ones, we observed high stability of hairpin loop forming ODNs, especially those containing longer (e.g. 7 base pair) stems. Comparative studies on the stability of chemically unmodified hairpin loop forming ODNs and their PS-counterparts indicated that endonuclease activity did not play any important role in the process of their nucleolytic degradation. Our studies provide strong evidence for high stability of chemically unmodified hairpin loop ODNs, making them an attractive alternative to phosphorothioate analogues commonly used in antisense strategy.     

Sensitivity of antioxidant-deficient yeast Saccharomyces cerevisiae to peroxynitrite and nitric oxide

Sensitivity of Saccharomyces cerevisiae strains deficient in superoxide dismutases and catalases and of decreased level of glutathione to peroxynitrite and a nitric oxide donor, S-nitrosoglutathione was compared. Moderate but significant differences observed point to increased sensitivity to both agents of yeast deficient in antioxidant defense, the superoxide dismutase-deficient strain showing the highest sensitivity, The sequence of sensitivity of various strains to peroxynitrite and nitric oxide was the same. The results are compatible with the view that cytotoxic effects of peroxynitrite involve formation of secondary reactive oxygen species.     

Changes in plasma membrane fluidity of immortal rodent cells induced by anticancer drugs doxorubicin, aclarubicin and mitoxantrone

The aim of this study was to examine the effect of three structurally different anticancer drugs-the pro-oxidative anthracyclines doxorubicin (DOX) and aclarubicin (ACL), and antioxidative anthraquinone mitoxantrone (MTX) on the fluidity of plasma membrane of immortalized rodent fibroblasts using fluorescence spectroscopy and electron spin resonance (ESR) techniques. Two kinds of fluorescent probes (TMA-DPH and 12-AS) and spin labels (5-DS and methyl-12-DS) were used to monitor fluidity in the hydrophobic core and in the polar headgroup region of the lipid bilayer. Immortalized hamster B14 and NIH 3T3 mouse fibroblasts were exposed to DOX, ACL and MTX. We demonstrate that these drugs influence predominantly the hydrophobic core of the lipid bilayer, inducing significant decrease in its fluidity at low concentrations (2-5 microM). A decreased membrane fluidity at the surface of the lipid bilayer was observed only at a higher concentration (20 microM) of the drugs, which indicates that DOX, ACL and MTX intercalate mainly into the hydrophobic core of the membrane, thereby perturbing its structure.