ICE1 demethylation drives the range expansion of a plant invader through cold tolerance divergence.

Cold tolerance adaption is a crucial determinant for the establishment and expansion of invasive alien plants into new cold environments; however, its evolutionary mechanism is poorly understood. Crofton weed (Ageratina adenophora), a highly invasive alien plant, is continuously spreading across subtropical areas in China, north-eastward from the first colonized south-western tropical regions, through cold tolerance evolution. Close relations between the cold tolerance levels of 34 populations, represented by 147 accessions, and the latitude, extreme lowest temperature, coldest month average temperature, and invasion period have provided direct insight into its cold tolerance divergence. A comparative study of the CBF pathway, associated with the cold tolerance enhancement of cold-susceptible CBF1-transgenic plant, among four geographically distinct crofton weed populations revealed that the CBF pathway plays a key role in the observed cold tolerance divergence. Four epialleles of the cold response regulator ICE1 ranged from 66 to 50 methylated cytosines, representing a 4.4% to 3.3% methylation rate and significantly corresponding to the lowest to highest cold tolerance levels among these different populations. The significant negative relation between the transcription levels of the primary CBF pathway members, except for CBF2, and the methylation levels among the four populations firstly demonstrates that the demethylation-upregulated transcription level of CBF pathway is responsible for this evolution. These facts, combined with the cold tolerance variation and methylation found among three native and two other introduced populations, indicate that the ICE1-demethylated upregulation of cold tolerance may be the underlying evolutionary mechanism allowing crofton weed to expand northward in China.

Elevated PLGF contributes to small-cell lung cancer brain metastasis.

Brain metastasis (BM) is a major cause of mortality in small-cell lung cancer (SCLC) patients; however, the molecular pathway of SCLC BM remains largely unknown because of a lack of investigation. Here we screen the levels of some candidate-soluble factors in the serum of SCLC patients and find that SCLC patients with high levels of placental growth factor (PLGF) are prone to BM. Using in vitro blood-brain barrier model, we show that PLGF derived from SCLC cells triggers vascular endothelial growth factor receptor-1-Rho-extracellular regulated protein kinase 1/2 signaling axis activation, results in disassembly of tight junction in brain endothelial cells and promotes SCLC cell transendothelial migration. Furthermore, the downregulation of PLGF suppresses SCLC cell metastasis to the brain in an experimental BM model. These data suggest that PLGF is a potential signature of SCLC BM and a prospective therapeutic target for SCLC BM.

Characteristics of AMPA receptor-mediated responses of cultured cortical and spinal cord neurones and their correlation to the expression of glutamate receptor subunits, GluR1-4.

Electrophysiological recordings have been used to characterize responses mediated by AMPA receptors expressed by cultured rat cortical and spinal cord neurones. The EC(50) values for AMPA were 17 and 11 microM, respectively. Responses of cortical neurones to AMPA were inhibited competitively by NBQX (pK(i)=6.6). Lower concentrations of NBQX (< or =1 microM) also potentiated the plateau responses of spinal cord neurones to AMPA, which could be attributed to a depression of desensitization to AMPA. GYKI 52466 inhibited responses of spinal cord neurones to AMPA to about twice the extent of responses of cortical neurones. Blockade of AMPA receptor desensitization by cyclothiazide (CTZ) potentiated responses of spinal cord neurones (6.8 fold) significantly more than responses of cortical neurones (4.8 fold). Responses of cortical neurones to KA were potentiated 3.5 fold by CTZ, while responses of spinal cord neurones were unaffected. Ultra-fast applications of AMPA to outside-out patches showed responses of spinal cord neurones desensitized by 97.5% and exhibit marked inward rectification, whereas cortical neurones desensitized by 91% and exhibited slight outward rectification. The time constants of deactivation and desensitization were about twice as fast in spinal cord than cortical neurones. In cortical neurones, single-cell RT - PCR showed GluR2 and GluR1 accounted for 91% of all subunits and were expressed together in 67% of neurones, predominantly as the flip variants (78%). GluR2 was detected alone in 24% of neurones. GluR3 and GluR4 were present in only 14 and 29% of neurones, respectively. For spinal cord neurones, GluR4(o) was detected in 81% of neurones, whereas predominantly flop versions of GluR1, 2 and 3 were detected in 38, 13 and 13% of neurones, respectively. These expression patterns are related to the respective pharmacological and mechanistic properties.

Larger intercellular variation in (Q/R) editing of GluR6 than GluR5 revealed by single cell RT-PCR.

RNA editing of the pre-mRNA encoding the kainate receptor subtypes determines the Ca2+ permeability and the rectifying properties of the receptors in which these are assembled. GluR6 pre-mRNA contains three characterized editing sites: Q/R, IN and the Y/C, whereas GluR5 pre-mRNA contains only the (Q/R) site. Single cell RT-PCR was used on cultured cortical neurons to determine the relative expression and editing levels of the kainate receptor subunits encoding mRNA. The analysis showed a large intercellular variation in editing efficiency. The overall lower level of GluR5 editing, in the culture, compared to GluR6 editing is a result of an approximately 60% lower editing efficiency of GluR5 pre-mRNA, within single cells, compared with GluR6.

Bifunctional 2-naphthyl propargylic sulfones exhibiting high DNA intercalating and alkylating activity.

A number of novel 2-naphthyl propargylic sulfones were synthesized as nucleic base alkylating agents. Extremely high DNA cleavage activity was observed for the sulfones with a free omega-hydroxyl group in the carbon chain in contrast to the ester conjugates possessing an additional intercalating unit.

Evidences for adduct formation between intracellular non-protein thiols and nitroazoles possessing an alpha,beta-unsaturated carbonyl side chain and the effects on radiosensitization of hypoxic cells.

Reactivity of a number of nitroazole derivatives bearing an alpha,beta-unsaturated carbonyl group on the side chain toward non-protein thiols (NPSH) was examined both in the phosphate buffer solution and in the biological system. These alpha,beta-unsaturated compounds reacted with NPSH, such as glutathione (GSH) and L-cysteine (Cys), in the buffer solution to afford the 1,4-addition products. The reaction gave a second-order rate constant. The adducts of methyl 4-(2'-nitroimidazol-1'-yl)crotonate (1) with GSH and Cys were isolated and characterized as two diastereomers (7a,b and 8a,b) in ca. 1:1 ratio, respectively. Similarly, exposure of EMT6/KU cells to 1 at 1.0 mM for 1 h resulted in depletion of the intracellular NPSH by more than 80%. Over 50% of the depleted NPSH was attributed to the formation of the conjugated diastereomeric adducts. On the other hand, incubation of EMT6/KU cells with 1 at 1.0 mM under hypoxic conditions before X-ray irradiation caused concurrently a sharp reduction of the shoulder of the dose-survival curves (reduced the extrapolation number (n) from 8.0 to ca. 1.0) and an increase in the slope (decreased the mean lethal dose (Do) to ca. 50% of the control level). The observed effects of 1 on the dose-survival curves were due to the NPSH depletion through the Michael addition occurred in the cellular system. A fairly linear relationship was obtained between the n value and the reduced intracellular NPSH level. It indicated that the shoulder effect of the dose-survival curves of hypoxic cells should be the result of the NPSH depletion by the alpha,beta-unsaturated carbonyl group attached to the nitroazoles.

Studies of the antagonist actions of (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl] propionic acid (ATPO) on non-NMDA receptors in cultured rat neurones.

Whole-cell patch-clamp recordings from single cultured cortical neurones have been used to study the action of (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl+ ++]propionic acid (ATPO), which has previously been proposed to be a potent selective antagonist of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors. ATPO competitively reduced peak responses evoked by semi-rapid applications of AMPA (Ki = 16 microM) but had variable effects on plateau responses, which were on average unchanged. Following blockade of AMPA receptor desensitization by cyclothiazide (CTZ, 100 microM), the plateau responses were reduced by ATPO to a similar extent as the peak responses, indicating that ATPO reduces desensitization of AMPA receptors. Semi-rapid application of kainic acid (KA) and the KA receptor-selective agonist, (2S,4R)-4-methylglutamic acid (MeGlu) evoked non-desensitizing responses which were competitively antagonized by ATPO (Ki values: 27 and 23 microM, respectively). Responses to MeGlu were unaffected by CTZ (100 microM), but potentiated 3 fold following blockade of KA receptor desensitization by concanavalin A (Con A, 300 microg ml(-1)). Responses of spinal cord neurones to MeGlu were blocked by ATPO to a similar extent before and after blockade of KA receptor desensitization by Con A. Although selectively potentiated by Con A, plateau responses to MeGlu were reduced by 69.6% by the AMPA selective antagonist, GYKI 53655 (10 microM). The remaining component was further reduced by ATPO with a Ki of 36 microM, which was not significantly different from that in the absence of GYKI 53655, but was greater than that on responses to AMPA. It is concluded that ATPO is a moderate-potency competitive inhibitor of naturally expressed non-NMDA receptors.

Remarkable tethering effect on DNA cleavage of propargylic sulfone conjugates with intercalating moieties.

A number of novel propargylic sulfone conjugates 3 and 4 with intercalating moieties were synthesized and evaluated for DNA cleavage activity through nucleic base alkylation. A remarkable enhancement in DNA cleaving potency was observed with those conjugates 3 possessing a suitable spacer, a right attachment point at the aromatic ring, and a good intercalator.

Designed enediynes: a new class of DNA-cleaving molecules with potent and selective anticancer activity.

The rational design and biological actions of a new class of DNA-cleaving molecules with potent and selective anticancer activity are reported. These relatively simple enediyne-type compounds were designed from basic chemical principles to mimic the actions of the rather complex naturally occurring enediyne anticancer antibiotics, particularly dynemicin A. Equipped with locking and triggering devices, these compounds damage DNA in vitro and in vivo on activation by chemical or biological means. Their damaging effects are manifested in potent anticancer activity with remarkable selectivities. Their mechanism of action involves intracellular unlocking and triggering of a Bergman reaction, leading to highly reactive benzenoid diradicals that cause severe DNA damage. The results of these studies demonstrate the potential of these de novo designed molecules as biotechnology tools and anticancer agents.