Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Role of the Lymphoreticular System in Prion Neuroinvasion from the Oral and Nasal Mucosa

Prion neuroinvasion from peripheral tissues involves agent replication in the lymphoreticular system (LRS) prior to entry into the nervous system. This study investigated the role of the LRS in prion neuroinvasion from the oral and nasal mucosa in wild-type and immunodeficient mice and in hamsters infected with the HY and DY strains of the transmissible mink encephalopathy (TME) agent. Following inoculation at neural sites, all hosts were susceptible to prion disease and had evidence of prion infection in the brain, but infection of the LRS was found only in scrapie-infected wild-type mice and HY TME-infected hamsters. In the LRS replication-deficient models, prion neuroinvasion was not observed following intraperitoneal or oral inoculation. However, immunodeficient mice, which have impaired follicular dendritic cells, were susceptible to scrapie following intratongue and intranasal inoculation despite the absence of PrP^Sc in the tongue or the nasal cavity. For DY TME, hamsters were susceptible following intratongue but not intranasal inoculation and PrP^Sc was limited to nerve fibers of the tongue. These findings indicate that neuroinvasion from the tongue and nasal cavity can be independent of LRS infection but neuroinvasion was partially dependent on the strain of the prion agent and/or the host species. The paucity of PrP^Sc deposition in the oral and nasal mucosa from LRS replication-deficient hosts following neuroinvasion from these tissues suggests an infection of nerve fibers that is below the threshold of PrP^Sc detection and/or the transport of the prion agent along cranial nerves without agent replication.In natural and experimental prion infections originating in the periphery, prion agent replication in the lymphoreticular system (LRS) precedes agent entry and spread in the peripheral nervous system. In the LRS, follicular dendritic cells (FDCs) are the major target of prion infection, and blocking or reversing FDC maturation can prevent scrapie agent replication in the LRS (25, 26, 28, 30, 32). Other migrating cell populations may also influence the progression of experimental prion disease (27, 36). From the LRS, centripetal spread of the prion agent to the spinal cord or brain occurs by spread along nerve fibers of the peripheral nervous system. In the central nervous system, prion agent replication can induce neurodegeneration and disease after an incubation period that can last from weeks to years. For example, in lambs from flocks with endemic scrapie, agent replication is initially detected in the gut-associated lymphoid tissues prior to proximal and distal spread in the LRS, infection of peripheral nerves that innervate the LRS, and subsequent spread to the spinal cord (19, 42). In addition, scrapie agent infection of the vagal nerve, which innervates many peripheral organs including the digestive tract, results in axonal transport directly to the dorsal motor nucleus of the vagus in the brain stem (29, 41). The role of scrapie infection in the LRS in the latter pathway of neuroinvasion is unknown. A similar pathway of prion neuroinvasion occurs in mule deer experimentally infected with the chronic wasting disease agent with the exception that early infection is also established in the lymph nodes of the upper gastrointestinal tract (37, 38). Recent studies indicate that a similar pathway of neuroinvasion occurs in natural and experimental bovine spongiform encephalopathy (BSE) following oral exposure except that agent replication in the LRS is greatly reduced and appears restricted to portions of the gut-associated lymphoid tissues (13, 20, 39).There are natural prion diseases in sheep and cattle that do not exhibit the typical distribution of the prion agent in the brain and LRS that are presumably acquired via oral prion exposure (5, 33). The absence of the abnormal isoform of the prion protein, PrP^Sc, in the LRS and dorsal motor nucleus of the vagus in atypical scrapie and the H type or L type of BSE raises the question as to whether these cases are due to infection by an alternate route(s) other than ingestion or whether these cases have an etiology that is distinct from that of acquired prion diseases. Direct prion infection of nerve fibers or terminals in highly innervated tissues, such as the mucosa in the head, has been suggested to represent potential sites of prion agent entry that would not require prior agent replication in the LRS (4, 12, 31). The presence of scrapie or BSE infection in the retina, sensory fibers of the tongue, and nasal mucosa of sheep, goat, and/or cattle suggests that the eye, tongue, or nasal cavity could be alternate sites of prion agent entry into hosts (8, 11, 15, 16, 40). Experimental prion inoculation at these mucosal sites can cause prion disease and in some cases rapid neuroinvasion (4, 9, 17, 18). Another explanation for this distribution of infection is that centrifugal spread of the prion agent away from the brain and along cranial nerves could serve as a pathway for prion infection and accumulation in these mucosal tissues (4, 10, 43).In this work, we investigated the role of the LRS in prion neuroinvasion from the oral and nasal cavities. In order to investigate neuroinvasion following neural and extraneural routes of inoculation in which prion replication is blocked in the LRS, we used two rodent models for prion infection. In muMT mice, which lack mature B cells, and in lymphotoxin-α (LTα) null mice, FDCs do not undergo maturation, and as a result, these mice do not develop clinical disease following intraperitoneal inoculation of the scrapie agent but are susceptible following direct inoculation into the brain (23, 30). In a second model, the HY and DY strains of the transmissible mink encephalopathy (TME) agent were used to investigate neuroinvasion in Syrian hamsters. The HY and DY TME agents can replicate in the nervous system, but the DY TME agent does not replicate in the LRS, and therefore, the DY TME agent is not pathogenic following intraperitoneal (i.p.) inoculation (2, 3). Following intratongue (i.t.) or intranasal (i.n.) inoculation, prion neuroinvasion was independent of scrapie agent replication in the LRS of immunodeficient mice, but evidence for scrapie infection of peripheral nerve fibers or olfactory neurons at these mucosa was lacking. In hamsters, i.t. inoculation of the HY or DY TME agent resulted in PrP^Sc deposition in nerve fibers and prion disease, but only the HY TME agent caused disease following i.n. inoculation. These findings suggest that neuroinvasion from the oral and nasal mucosa in LRS replication-deficient rodents can be independent of LRS infection, but the paucity of PrP^Sc at these mucosal sites of exposure in immunodeficient mice and DY TME-infected hamsters suggests that neuroinvasion is due to either a low-level prion infection of the nervous system at the site of inoculation or transport of the prion agent in axons in the absence of agent replication at the site of prion entry. These findings indicate that these mucosal tissues may not exhibit early evidence of infection and therefore will prove difficult to identify as a portal for agent entry.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

ST segment body surface isointegral maps in patients with arterial hypertension

In this retrospective study we analysed changes of the ST segment in patients with arterial hypertension using multi-lead body surface mapping of the electric heart field as the ST segment often shows non-specific changes and is influenced by many different conditions. We constructed isointegral maps (IIM) of chosen intervals (the first 35 ms, the first 80 ms, and the whole ST segment) in 42 patients with arterial hypertension (with and without left ventricular hypertrophy) and in the control group involving 23 healthy persons. We analysed the position and values of map extrema. Spatial distribution of voltage integrals was similar in the control group and in the "pure" hypertensives. Patients with the left ventricular hypertrophy exhibited shifts of the integral minima. Despite our expectations, the highest extrema values were found in the control group and not in the left ventricular hypertrophy group. The extrema values were similar in all hypertensives, with or without left ventricular hypertrophy. Differences could be explained neither by the influence of the age, nor by the body habitus.     

Growth,anatomy and enzyme activity changes in maize roots induced by treatment of seeds with low-temperature plasma

The seeds of Zea mays L. cv. KWS were exposed to low-temperature plasma (LTP) by using Diffuse Coplanar Surface Barrier Discharge (DCSBD) for 60 and 120 seconds respectively. Growth parameters, anatomy of roots and activity of some enzymes (CAT, G-POX, SOD and DHO) isolated from roots grown from the seeds treated by LTP were evaluated. Our results indicate that LTP treatment of maize seeds affects post-germination growth of seedlings and this effect depends on the duration of LTP treatment. LTP treatment in duration of 60 seconds significantly increased the length, fresh and dry weight of the roots. However, the increase in time of LTP treatment to 120 seconds had inhibitive effect on the studied growth parameters. The activities of all studied antioxidant enzymes significantly increased with the age of maize seedlings in control conditions. On the other hand the application of LTP resulted in small, mostly non significant changes in the activity of antioxidant enzymes. Significant decrease in CAT activity was observed both in 3 and 6-day old maize roots and G-POX activity in 3-day old maize roots grown from seeds exposed to LTP for 60 seconds. A small, significant increase was detected only in SOD activity in 3-day old maize roots grown from seeds treated with LTP for 120 seconds and in 6-day old maize roots treated with LTP for 60 seconds. Significantly higher DHO activity was determined in embryos isolated from seeds treated with LTP for 60 seconds. On the contrary, in roots the DHO activity decreased with the time of LTP treatment. LTP treatment of seeds did not affect the anatomy of maize roots and caused only minor changes in the isoenzyme composition of G-POX and SOD.     

Cadmium uptake,localization and stress-induced morphogenic response in the fern Pteris vittata

Cadmium uptake, tissue localization and structural changes induced at cellular level are essential to understand Cd tolerance in plants. In this study we have exposed plants of Pteris vittata to different concentrations of CdCl₂ (0, 30, 60, 100 μM) to evaluate the tolerance of the fern to cadmium. Cadmium content determination and its histochemical localization showed that P. vittata not only takes up, but also transports and accumulates cadmium in the aboveground tissues, delocalizing it mainly in the less bioactive tissues of the frond, the trichomes and the scales. Cadmium tolerance in P. vittata was strictly related to morphogenic response induced by the metal itself in the root system. Adaptive response regarded changes of the root apex size, the developmental pattern of root hairs, the differentiation of xylem elements and endodermal suberin lamellae. All the considered parameters suggest that, in our experimental conditions, 60 μM of Cd may represent the highest concentration that P. vittata can tolerate; indeed this Cd level even improves the absorbance features of the root and allows good transport and accumulation of the metal in the fronds. The results of this study can provide useful information for phytoremediation strategies of soils contaminated by Cd, exploiting the established ability of P. vittata to transport, delocalize in the aboveground biomass and accumulate polluting metals.     

High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups

We recently proposed that patterns of evolution of non-LTR retrotransposable elements can be used to study patterns of spontaneous mutation. Transposition of non-LTR retrotransposable elements commonly results in creation of 5' truncated, "dead-on-arrival" copies. These inactive copies are effectively pseudogenes and, according to the neutral theory, their molecular evolution ought to reflect rates and patterns of spontaneous mutation. Maximum parsimony can be used to separate the evolution of active lineages of a non-LTR element from the fate of the "dead-on-arrival" insertions and to directly assess the relative frequencies of different types of spontaneous mutations. We applied this approach using a non-LTR element, Helena, in the Drosophila virilis group and have demonstrated a surprisingly high incidence of large deletions and the virtual absence of insertions. Based on these results, we suggested that Drosophila in general may exhibit a high rate of spontaneous large deletions and have hypothesized that such a high rate of DNA loss may help to explain the puzzling dearth of bona fide pseudogenes in Drosophila. We also speculated that variation in the rate of spontaneous deletion may contribute to the divergence of genome size in different taxa by affecting the amount of superfluous "junk" DNA such as, for example, pseudogenes or long introns. In this paper, we extend our analysis to the D. melanogaster subgroup, which last shared a common ancestor with the D. virilis group approximately 40 MYA. In a different region of the same transposable element, Helena, we demonstrate that inactive copies accumulate deletions in species of the D. melanogaster subgroup at a rate very similar to that of the D. virilis group. These results strongly suggest that the high rate of DNA loss is a general feature of Drosophila and not a peculiar property of a particular stretch of DNA in a particular species group.      

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.