Small interfering RNA screens reveal enhanced cisplatin cytotoxicity in tumor cells having both BRCA network and TP53 disruptions

RNA interference technology allows the systematic genetic analysis of the molecular alterations in cancer cells and how these alterations affect response to therapies. Here we used small interfering RNA (siRNA) screens to identify genes that enhance the cytotoxicity (enhancers) of established anticancer chemotherapeutics. Hits identified in drug enhancer screens of cisplatin, gemcitabine, and paclitaxel were largely unique to the drug being tested and could be linked to the drug's mechanism of action. Hits identified by screening of a genome-scale siRNA library for cisplatin enhancers in TP53-deficient HeLa cells were significantly enriched for genes with annotated functions in DNA damage repair as well as poorly characterized genes likely having novel functions in this process. We followed up on a subset of the hits from the cisplatin enhancer screen and validated a number of enhancers whose products interact with BRCA1 and/or BRCA2. TP53(+/-) matched-pair cell lines were used to determine if knockdown of BRCA1, BRCA2, or validated hits that associate with BRCA1 and BRCA2 selectively enhances cisplatin cytotoxicity in TP53-deficient cells. Silencing of BRCA1, BRCA2, or BRCA1/2-associated genes enhanced cisplatin cytotoxicity approximately 4- to 7-fold more in TP53-deficient cells than in matched TP53 wild-type cells. Thus, tumor cells having disruptions in BRCA1/2 network genes and TP53 together are more sensitive to cisplatin than cells with either disruption alone.     

Cyclic strain induces expression of specific smooth muscle cell markers in human endothelial cells

The objective of this study was to determine whether cyclic strain could promote human umbilical vein endothelial cells (HUVECs) to express markers in common with the mature smooth muscle cell (SMC) phenotype, suggesting endothelial cell to SMC transdifferentiation. HUVECs were cultured on stretched membranes at 10% stretch and 60 cycles/min for 24-96 hr, and demonstrated elongation with enhanced and organized F-actin distribution. By using real-time polymerase chain reaction analysis, the mRNA levels of five specific SMC markers, SM22-alpha, alpha-smooth muscle actin (alpha-SMA), caldesmon-1, smooth muscle myosin heavy chain (SMMHC), and calponin-1 were significantly increased in cyclic strain-treated HUVECs as compared with those in static control cells. Protein levels of SM22-alpha and alpha-SMA were also substantially increased by Western blot and immunofluorescence staining. In addition, two specific endothelial markers, von Willebrand factor (vWF) and vascular endothelial growth factor receptor-2 (VEGFR-2), showed a reduction in mRNA expression. In addition, cyclic strain-induced increase of SM22-alpha and alpha-SMA expression were reversible when cells were cultured back to the static condition. These results demonstrate a possible endothelial cell to SMC transdifferentiation in response to cyclic strain. Hemodynamic forces in modulating endothelial phenotype may play an important role in the vascular system.     

Variations in soil N cycling and trace gas emissions in wet tropical forests

We used a previously described precipitation gradient in a tropical montane ecosystem of Hawai’i to evaluate how changes in mean annual precipitation (MAP) affect the processes resulting in the loss of N via trace gases. We evaluated three Hawaiian forests ranging from 2200 to 4050 mm year⁻¹ MAP with constant temperature, parent material, ecosystem age, and vegetation. In situ fluxes of N₂O and NO, soil inorganic nitrogen pools (NH₄⁺ and NO₃⁻), net nitrification, and net mineralization were quantified four times over 2 years. In addition, we performed ¹⁵N-labeling experiments to partition sources of N₂O between nitrification and denitrification, along with assays of nitrification potential and denitrification enzyme activity (DEA). Mean NO and N₂O emissions were highest at the mesic end of the gradient (8.7±4.6 and 1.1±0.3 ng N cm⁻² h⁻¹, respectively) and total oxidized N emitted decreased with increased MAP. At the wettest site, mean trace gas fluxes were at or below detection limit (≤0.2 ng N cm⁻² h⁻¹). Isotopic labeling showed that with increasing MAP, the source of N₂O changed from predominately nitrification to predominately denitrification. There was an increase in extractible NH₄⁺ and decline in NO₃⁻, while mean net mineralization and nitrification did not change from the mesic to intermediate sites but decreased dramatically at the wettest site. Nitrification potential and DEA were highest at the mesic site and lowest at the wet site. MAP exerts strong control N cycling processes and the magnitude and source of N trace gas flux from soil through soil redox conditions and the supply of electron donors and acceptors.     

Cytotoxic Alangium alkaloids from Alangium longiflorum

Seven alkaloids (1-7) were isolated from the stem bark of Alangium longiflorum. Compound 1, (-)-10-O-demethylisocephaeline, was isolated for the first time as a naturally occurring product from a plant source. All structures were elucidated by detailed spectroscopic analysis. Biological evaluation showed that 2, 10-O-demethylcephaeline, exhibited potent cytotoxic activity against human lung carcinoma (A549) and breast adenocarcinoma (MCF-7) with ED(50) values of 0.013 and 0.062 microM, respectively. The stereoisomer 1 was less potent than 2, and related compounds with different hydroxy/methoxy substitution patterns were also less potent or inactive. Thus, compound 2 merits attention as a cytotoxic lead for further study.     

Effect of plasma membrane ion permeability modulators on respiration and heat output of wheat roots

A study was made of changes in the rates of respiration, heat production, and membrane characteristics in cells of excised roots of wheat seedlings under the modulation of plasma membrane ion permeability by two membrane active compounds: valinomycin (20 microM (V50)) and chlorpromazine (50 microM (CP50) and 100 microM (CP100)). Both compounds increased the loss of potassium ions, which correlated with the lowering of membrane potential, rate of respiration, and heat production after a 2 h exposure. The differences in alteration of these parameters were due to specific action of either compound on the membrane and to the extent of ion homeostasis disturbance. V20 had a weak effect on the studied parameters. V50 caused an increase of the rate of respiration and heat production, which enhanced following a prolonged action (5 h) and were associated with ion homeostatis restoration. The extent of alteration of membrane characteristics (an increase of potassium loss by roots, and lowering of cell membrane potential) as well as energy expense under the action of CP50 during the first period were more pronounced than in the presence of V50. During a prolonged action of CP50, the increase of respiration intensity and heat production correlated with partial recovery of ion homeostatis in cells. Essential lowering of membrane potential and substantial loss of potassium by cells, starting from the early stages of their response reaction, were followed by inhibition of respiration rate and heat production. Alterations of the structure and functional characteristics of excised root cells indicate the intensification of the membrane-tropic effect of a prolonged action of CP100, and the lack of cell energy resources.     

Mitotic index of meristematic cells and root growth of Pisum sativum is affected by inositol cycle modulators

Relationships between cell division and inositol cycle modulation caused by different effectors in roots of Pisum sativum were studied. Stimulation of the inositol cycle by myoinositol increased the mitotic index of meristematic cells and root length, while the inhibition of the cycle with Li+ and a heavy metal Gd3+ considerably decreased mitotic activity and growth. Exposure of roots to 10 mM CaCl2 and 15 mM myoinositol resulted in the accumulation of chromosome aberrations. Changes in the activity of inositol cycle are assumed to be involved in the root growth control.     

Mandibular size and shape variation in the hominins at Dmanisi, Republic of Georgia

The hominin fossils of Dmanisi, Republic of Georgia, present an ideal means of assessing levels of skeletal size and shape variation in a fossil hypodigm belonging to the genus Homo because they have been recovered from a spatially and temporally restricted context. We compare variation in mandible size and shape at Dmanisi to that of extant hominoids and extinct hominins. We use height and breadth measurements of the mandibular corpus at the first molar and the symphysis to assess size, and analyze shape based on size-adjusted (using a geometric mean) versions of these four variables. We compare size and shape variation at Dmanisi relative to all possible pairs of individuals within each comparative taxon using an exact resampling procedure of the ratio of D2600 to D211 and the average Euclidean distance (AED) between D2600 and D211, respectively. Comparisons to extant hominoids were conducted at both the specific and subspecific taxonomic levels and to extinct hominins by adopting both a more, and less speciose, hominin taxonomy. Results indicate that the pattern of variation for the Dmanisi hominins does not resemble that of any living species: they exhibit significantly more size variation when compared to modern humans, and they have significantly more corpus shape variation and size variation in corpus heights and overall mandible size than any extant ape species. When compared to fossil hominins they are also more dimorphic in size (although this result is influenced by the taxonomic hypothesis applied to the hominin fossil record). These results highlight the need to re-examine expectations of levels of sexual dimorphism in members of the genus Homo and to account for marked size and shape variation between D2600 and D211 under the prevailing view of a single hominin species at Dmanisi.     

Genetic distinctions among clinical and environmental strains of Vibrio vulnificus

Vibrio vulnificus causes rare but frequently fatal septicemia associated with raw oyster consumption by persons with underlying hepatic or immune system dysfunction. The virulence potential of environmental reservoirs appears widely distributed, because most strains are virulent in animal models; however, several investigations recently demonstrated genetic divergence among strains from clinical versus environmental origin at independent genetic loci. The present study used PCR to screen DNA polymorphisms in strains from environmental (n = 35) or clinical (n = 33) sources, and genomic relationships were determined by repetitive extragenic palindromic DNA PCR (rep-PCR) typing. Significant (P < 0.01) association was observed for typical "clinical" or "environmental" polymorphism profiles based on strain origin. Most oyster isolates (88%), including all of those with the "environmental" profile, also formed a single rep-PCR genogroup. Clinical isolates within this group did not have the typical "clinical" profile. On the other hand, clinical isolates with the typical polymorphism profile were distributed among multiple rep-PCR genogroups, demonstrating greater genetic diversity than was evident by profiling genetic polymorphisms. Wound isolates were genetically distinct from typical blood isolates by all assays. Strains from an outbreak of wound infections in Israel (biotype 3) were closely related to several U.S. strains by rep-PCR, indicating potential reservoirs of emerging disease. Strains genetically related to blood isolates appeared to be relatively rare in oysters, as only one had the "clinical" polymorphism profile or clustered by rep-PCR. However, this study was not an extensive survey, and more sampling using rep-PCR for sensitive genetic discrimination is needed to determine the virulence potential of environmental reservoirs.     

Direct evidence that receptor site-4 of sodium channel gating modifiers is not dipped in the phospholipid bilayer of neuronal membranes

In a recent note to Nature, R. MacKinnon has raised the possibility that potassium channel gating modifiers are able to partition in the phospholipid bilayer of neuronal membranes and that by increasing their partial concentration adjacent to their receptor, they affect channel function with apparent high affinity (Lee and MacKinnon (2004) Nature 430, 232-235). This suggestion was adopted by Smith et al. (Smith, J. J., Alphy, S., Seibert, A. L., and Blumenthal, K. M. (2005) J. Biol. Chem. 280, 11127-11133), who analyzed the partitioning of sodium channel modifiers in liposomes. They found that certain modifiers were able to partition in these artificial membranes, and on this basis, they have extrapolated that scorpion beta-toxins interact with their channel receptor in a similar mechanism as that proposed by MacKinnon. Since this hypothesis has actually raised a new conception, we examined it in binding assays using a number of pharmacologically distinct scorpion beta-toxins and insect and mammalian neuronal membrane preparations, as well as by analyzing the rate by which the toxin effect on gating of Drosophila DmNa(v)1 and rat brain rNa(v)1.2a develops. We show that in general, scorpion beta-toxins do not partition in neuronal membranes and that in the case in which a depressant beta-toxin partitions in insect neuronal membranes, this partitioning is unrelated to its interaction with the receptor site and the effect on the gating properties of the sodium channel. These results negate the hypothesis that the high affinity of beta-toxins for sodium channels is gained by their ability to partition in the phospholipid bilayer and clearly indicate that the receptor site for scorpion beta-toxins is accessible to the extracellular solvent.