The effect of intrinsic strain on the thermal expansion behavior of Janus MoSSe nanotubes: a molecular dynamic simulation.

In this study, we conducted molecular dynamic simulations to investigate the thermal expansion behavior of Janus MoSSe nanotubes. We focused on understanding how the intrinsic strain in these nanotubes affects their thermal expansion coefficient (TEC). Interestingly, we found that Janus MoSSe nanotubes with sulfur (S) on the outer surface (MoSeS) exhibit a different intrinsic strain compared to those with selenium (Se) on the outer surface (MoSSe). In light of this observation, we explored the influence of this intrinsic strain on the TEC of the nanotubes. Our results revealed distinct trends for the TEC along the radial direction (TEC-r) and the axial direction (TEC-lx) of the MoSSe and MoSeS nanotubes. The TEC-rof MoSeS nanotubes was found to be significantly greater than that of MoSSe nanotubes. Moreover, the TEC-lxof MoSeS nanotubes was smaller than that of MoSSe nanotubes. Further analysis showed that the TEC-rof MoSeS nanotubes decreased by up to 37% as the radius increased, while that of MoSSe nanotubes exhibited a slight increase with increasing radius. On the other hand, the TEC-lxof MoSeS nanotubes increased by as much as 45% with increasing radius, whereas that of MoSSe nanotubes decreased gradually. These opposite tendencies of the TECs with respect to the radius were attributed to the presence of intrinsic strain within the nanotubes. The intrinsic strain was found to play a crucial role in inducing thermally induced bending and elliptization of the nanotubes' cross-section. These effects are considered key mechanisms through which intrinsic strain influences the TEC. Overall, our study provides valuable insights into the thermal stability of Janus nanotubes. By understanding the relationship between intrinsic strain and the thermal expansion behavior of nanotubes, we contribute to the broader understanding of these materials and their potential applications.

Anti-ceramide antibody prevents the radiation gastrointestinal syndrome in mice.

Radiation gastrointestinal (GI) syndrome is a major lethal toxicity that may occur after a radiation/nuclear incident. Currently, there are no prophylactic countermeasures against radiation GI syndrome lethality for first responders, military personnel, or remediation workers entering a contaminated area. The pathophysiology of this syndrome requires depletion of stem cell clonogens (SCCs) within the crypts of Lieberkühn, which are a subset of cells necessary for postinjury regeneration of gut epithelium. Recent evidence indicates that SCC depletion is not exclusively a result of DNA damage but is critically coupled to ceramide-induced endothelial cell apoptosis within the mucosal microvascular network. Here we show that ceramide generated on the surface of endothelium coalesces to form ceramide-rich platforms that transmit an apoptotic signal. Moreover, we report the generation of 2A2, an anti-ceramide monoclonal antibody that binds to ceramide to prevent platform formation on the surface of irradiated endothelial cells of the murine GI tract. Consequently, we found that 2A2 protected against endothelial apoptosis in the small intestinal lamina propria and facilitated recovery of crypt SCCs, preventing the death of mice from radiation GI syndrome after high radiation doses. As such, we suggest that 2A2 represents a prototype of a new class of anti-ceramide therapeutics and an effective countermeasure against radiation GI syndrome mortality.

Mitochondrial ceramide-rich macrodomains functionalize Bax upon irradiation.

BACKGROUND: Evidence indicates that Bax functions as a "lipidic" pore to regulate mitochondrial outer membrane permeabilization (MOMP), the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM), which we visualize and isolate, into which Bax integrates. METHODOLOGY/PRINCIPAL FINDINGS: MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1-2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight "pore-forming" oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax. CONCLUSIONS/SIGNIFICANCE: Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

Kinase suppressor of Ras transphosphorylates c-Raf-1.

Whether kinase suppressor of Ras1 (KSR1) is an active kinase that phosphorylates c-Raf-1 or a scaffold that coordinates signaling along the Ras/ERK1 signaling module is actively debated. In this study, we generated a monoclonal antibody against a c-Raf-1 peptide containing phosphorylated Thr(269), the putative target for KSR1 kinase activity. We show that this antibody detects Thr(269)-phosphorylated c-Raf-1 in A431 cells upon epidermal growth factor (EGF) stimulation, preceding MEK1 activation. Furthermore, this antibody detects in vitro phosphorylation of FLAG-c-Raf-1 and kinase-dead FLAG-c-Raf-1(K375M) by immunopurified KSR1, but fails to detect phosphorylation of FLAG-c-Raf-1(K375M/T269V), engineered with a Thr(269) to valine substitution. To provide unequivocal evidence that KSR1 is a legitimate kinase, we purified KSR1 to homogeneity, confirmed by mass spectrometry, renatured it in-gel, and demonstrated that it phosphorylates BSA-conjugated c-Raf-1 peptide at Thr(269). These studies add to emerging data validating KSR1 as a kinase that phosphorylates c-Raf-1.

A ceramide-binding C1 domain mediates kinase suppressor of ras membrane translocation.

Genetic and biochemical data support Kinase Suppressor of Ras 1 (KSR1) as a positive regulator of the Ras-Raf-MAPK pathway, functioning as a kinase and/or scaffold to regulate c-Raf-1 activation. Membrane translocation mediated by the KSR1 CA3 domain, which is homologous to the atypical PKC C1 lipid-binding domain, is a critical step of KSR1-mediated c-Raf-1 activation. In this study, we used an ELISA to characterize the KSR1 CA3 domain as a lipid-binding moiety. Purified GST-KSR1-CA3 protein effectively binds ceramide but not other lipids including 1,2-diacylglyceol, dihydroceramide, ganglioside GM1, sphingomyelin and phosphatidylcholine. Upon epidermal growth factor stimulation of COS-7 cells, KSR1 translocates into and is activated within glycosphingolipid-enriched plasma membrane platforms. Pharmacologic inhibition of ceramide generation attenuates KSR1 translocation and KSR1 kinase activation in COS-7 cells. Disruption of two cysteines, which are indispensable for maintaining ternary structure of all C1 domains and their lipid binding capability, mitigates ceramide-binding capacity of purified GST-KSR1-CA3 protein, and inhibits full length KSR1 membrane translocation and kinase activation. These studies provide evidence for a mechanism by which the second messenger ceramide can target proteins to subcellular compartments in the process of transmembrane signal transduction.

Ceramide biogenesis is required for radiation-induced apoptosis in the germ line of C. elegans.

Ceramide engagement in apoptotic pathways has been a topic of controversy. To address this controversy, we tested loss-of-function (lf) mutants of conserved genes of sphingolipid metabolism in Caenorhabditis elegans. Although somatic (developmental) apoptosis was unaffected, ionizing radiation-induced apoptosis of germ cells was obliterated upon inactivation of ceramide synthase and restored upon microinjection of long-chain natural ceramide. Radiation-induced increase in the concentration of ceramide localized to mitochondria and was required for BH3-domain protein EGL-1-mediated displacement of CED-4 (an APAF-1-like protein) from the CED-9 (a Bcl-2 family member)/CED-4 complex, an obligate step in activation of the CED-3 caspase. These studies define CEP-1 (the worm homolog of the tumor suppressor p53)-mediated accumulation of EGL-1 and ceramide synthase-mediated generation of ceramide through parallel pathways that integrate at mitochondrial membranes to regulate stress-induced apoptosis.

Downregulation of KSR1 in pancreatic cancer xenografts by antisense oligonucleotide correlates with tumor drug uptake.

While antisense oligonucleotide (AS-ODN) technology holds promise for the treatment of cancer, to date there have been no clinical successes. Unfortunately, current assays are not sufficiently sensitive to measure tissue ODN levels. Hence it has not been possible to ascertain whether treatment failures result from failure of drug delivery. To investigate the relationship between drug uptake and therapeutic effect, we developed an ultrasensitive noncompetitive hybridization-ligation enzyme-linked immunosorbent assay (NCHL-ELISA) to quantify Kinase Suppressor of Ras1 (KSR1) AS-ODN drug uptake in plasma and tumor tissues. In mice harboring PANC-1 pancreatic cancer xenografts and continuously infused with AS-ODN, our ELISA detects plasma and tumor KSR1 AS-ODN levels over an extended range, from 0.05 nM to 20 nM. Using this sensitive assay, we demonstrate that KSR1 repression in pancreatic cancer xenografts correlates highly with AS-ODN uptake into tumor tissues. In contrast, plasma drug levels do not correlate with tumor drug content or target downregulation. These studies indicate the efficacy of our ELISA, and suggest that tumor biopsy material will need to be procured to estimate the potential of this antisense technology.

Caenorhabditis elegans ABL-1 antagonizes p53-mediated germline apoptosis after ionizing irradiation.

c-Abl, a conserved nonreceptor tyrosine kinase, integrates genotoxic stress responses, acting as a transducer of both pro- and antiapoptotic effector pathways. Nuclear c-Abl seems to interact with the p53 homolog p73 to elicit apoptosis. Although several observations suggest that cytoplasmic localization of c-Abl is required for antiapoptotic function, the signals that mediate its antiapoptotic effect are largely unknown. Here we show that worms carrying an abl-1 deletion allele, abl-1(ok171), are specifically hypersensitive to radiation-induced apoptosis in the Caenorhabditis elegans germ line. Our findings delineate an apoptotic pathway antagonized by ABL-1, which requires sequentially the cell cycle checkpoint genes clk-2, hus-1 and mrt-2; the C. elegans p53 homolog, cep-1; and the genes encoding the components of the conserved apoptotic machinery, ced-3, ced-9 and egl-1. ABL-1 does not antagonize germline apoptosis induced by the DNA-alkylating agent ethylnitrosourea. Furthermore, worms treated with the c-Abl inhibitor STI-571 (Gleevec; used in human cancer therapy), two newly synthesized STI-571 variants or PD166326 had a phenotype similar to that generated by abl-1(ok171). These studies indicate that ABL-1 distinguishes proapoptotic signals triggered by two different DNA-damaging agents and suggest that C. elegans might provide tissue models for development of anticancer drugs.

Trihydrophobin 1 is a new negative regulator of A-Raf kinase.

Our previous work indicated that instead of binding to B-Raf or C-Raf, trihydrophobin 1 (TH1) specifically binds to A-Raf kinase both in vitro and in vivo. In this work, we investigated its function further. Using confocal microscopy, we found that TH1 colocalizes with A-Raf, which confirms our former results. The region of TH1 responsible for the interaction with A-Raf is mapped to amino acids 1-372. Coimmunoprecipitation experiments demonstrate that TH1 is associated with A-Raf in both quiescent and serum-stimulated cells. Wild type A-Raf binds increasingly to TH1 when it is activated by serum and/or upstream oncogenic Ras/Src compared with that of "kinase-dead" A-Raf. The latter can still bind to TH1 under the same experimental condition. The binding pattern of A-Raf implies that this interaction is mediated in part by the A-Raf kinase activity. As indicated by Raf protein kinase assays, TH1 inhibits A-Raf kinase, whereas neither B-Raf nor C-Raf kinase activity is influenced. Furthermore, we observed that TH1 inhibited cell cycle progression in TH1 stably transfected 7721 cells compared with mock cells, and flow cell cytometry analysis suggested that the TH1 stably transfected 7721 cells were G(0)/G(1) phase-arrested. Taken together, our data provide a clue to understanding the cellular function of TH1 on Raf isoform-specific regulation.

The beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3)-linked bond have similar stimulatory effects on the mouse spleen as Lentinan.

The stimulatory effects of the synthetic beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3)-linked bond on the mouse spleen were studied for elucidation of the mechanism of their antitumor activity, and their stimulatory effects were compared with Lentinan. The mouse spleen's weight was increased after the intraperitoneal (i.p.) injection of the oligosaccharides compared with the saline group. In addition, routinely hematoxylin and eosin (HE)-stained spleen sections showed that the injection also changed the spleen's histopathology. RNA samples were isolated from splenocytes of oligosaccharides, Lentinan or saline-injected mice. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot showed that the administration of the oligosaccharides or Lentinan enhanced mouse spleen mRNA production of TNF-alpha but not IL-2. The injection also enhanced Concanavalin A (Con A)-induced mouse splenocytes proliferation, but the in vitro administration of the oligosaccharides did not have the proliferation-enhancing effect. Taken together, these results suggest that the synthetic beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3)-linked bond have similar stimulatory effects as Lentinan. Additionally, they may exert their antitumor effects through the induction of splenocytes mediated immune responses.

Constitutively active PKB/Akt inhibited apoptosis and down-regulated beta1,4-galactosyltransferase 1 in hepatocarcinoma cells.

Beta1,4-galactosyltransferase1 (beta1,4GT1) is localized both in the Golgi complex and on the cell surface. In our previous study, we first reported that beta1,4GT1 was associated with cycloheximide-induced apoptosis in human hepatocarcinoma cells. In this study, we transfected constitutively active protein kinase B (Gag-PKB), a central mediator of anti-apoptotic signals transduced by the PI3-kinase, into SMMC-7721 human hepatocarcinoma cells, and examined its effect on apoptosis and beta1,4GT1 activity. Flow cytometry analysis showed that apoptosis was inhibited in Gag-PKB transfected SMMC-7721 cells. At the same time, beta1,4GT1 mRNA level and enzyme activities were downregulated in these cells, consistent with which, the content of beta1,4 Gal branch in the glycoconjugates was decreased in stably transfected cells. Cotransfection of beta1,4GT1 promoter/luciferase reporter and Gag-PKB decreased the luciferase reporter activity in a dose-dependent manner, indicating that the differences in mRNA levels might be regulated through promoter function. All these findings suggested that changes of beta1,4GT1 activity might be involved in apoptotic pathway in hepatocarcinoma cells.

The C-terminal kinase domain of the p34cdc2-related PITSLRE protein kinase (p110C) associates with p21-activated kinase 1 and inhibits its activity during anoikis.

The PITSLRE protein kinases are parts of the large family of p34cdc2-related kinases. During apoptosis induced by some stimuli, specific PITSLRE isoforms are cleaved by caspase to produce a protein that contains the C-terminal kinase domain of the PITSLRE proteins (p110C). The p110C induces apoptosis when it is ectopically expressed in Chinese hamster ovary cells. In our study, similar induction of this p110C was observed during anoikis in NIH3T3 cells. To investigate the molecular mechanism of apoptosis mediated by p110C, we used the yeast two-hybrid system to screen a human fetal liver cDNA library and identified p21-activated kinase 1 (PAK1) as an interacting partner of p110C. The association of p110C with PAK1 was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscope analysis. The interaction of p110C with PAK1 occurred within the residues 210-332 of PAK1. Neither association between p58PITSLRE or p110PITSLRE and PAK1 nor association between p110C and PAK2 or PAK3 was observed. Anoikis was increased and PAK1 activity was inhibited when NIH3T3 cells were transfected with p110C. Furthermore, the binding of p110C with PAK1 and inhibition of PAK1 activity were also observed during anoikis. Taken together, these data suggested that PAK1 might participate in the apoptotic pathway mediated by p110C.