High-energy femtosecond amplifier-similariton Er-doped fiber oscillator.

We demonstrated high-energy femtosecond amplifier-similariton oscillators with predominant Er-doped fibers of normal dispersion. Stably mode-locked pulses of ~3 ps, 33 nJ were produced at 720 mW pump power, while a double-pass grating pair of 36% efficiency compressed the pulses to 156 fs and 47 kW peak power (a new record). Broad mode-locked spectra supporting transform-limited pulsewidths down to 60 fs were obtained by adjusting the intracavity waveplates and filter. Continuous wave (CW) mode-locked pulses up to 53 nJ were generated by increasing the pump power to 1.5 W and by introducing significant spectral phase modulation via an intracavity pulse shaper. However, weak subpulses or pedestal could arise along with increased shot-to-shot fluctuation under this extreme operation mode.

Prenatal Exposure of Rats to Staphylococcal Enterotoxin B Alters the Expression of Th1 and Th2 Cytokines via Decreased Methylation in the Spleen of Adult but not Neonatal Offspring.

BACKGROUND: The methylation of IFN-γ and IL-4 genes is regarded as an epigenetic regulation that maintains the Th1 or Th2 phenotype. OBJECTIVE: To explore the influence of prenatal administration of the staphylococcal enterotoxin B (SEB) in pregnant rats, on the IFN-γ or IL-4 expression in the offspring spleen. METHODS: The SEB or PBS was administered intravenously to pregnant rats on the embryo-day 16. After normal delivery, the spleens from the fifth-day neonates and adult offspring were isolated under anesthesia. Quantitative PCR, western blot, ELISA and MeDIP-qPCR were applied to determine the levels of the splenic IFN-γ or IL-4 mRNAs, their protein levels, and methylation status, respectively. RESULTS: Prenatal administration of the SEB in pregnant rats decreased the levels of the splenic IFN-γ and IL-4 proteins in neonates, but increased their mRNA levels. However, prenatal administration of the SEB significantly augmented both mRNA and protein levels of the IFN-γ and IL-4 in the adult spleen. In addition, the prenatal SEB administration decreased the methylation of the splenic IFN-γ and IL-4 in adult but not neonatal offspring. CONCLUSION: The prenatal administration of SEB in pregnant rats can cause a mixed Th1 and Th2 cytokines response in the offspring spleen, and alter the cytokine expression of the Th1 and Th2 via decreasing the methylation in adult but, not neonatal offspring spleen.

Exposure of pregnant rats to staphylococcal enterotoxin B increases offspring splenic Treg number and function via decreasing FoxP3 methylation.

Staphylococcus aureus is an infectious pathogen that is relatively common, but that can cause severe disease in pregnant women and their fetus. We previously demonstrated that exposing pregnant rats to staphylococcal enterotoxin B (SEB) altered splenic CD4/CD8 T cell frequencies in their offspring. Whether prenatal SEB exposure impacts Tregs in these offspring, however, remains to be determined. As such, in this study, we intravenously injected pregnant rats with 15 µg of SEB on gestational day 16. Splenic tissue was then harvested from 1-, 3-, and 5-day-old neonatal rats and analyzed via flow cytometry to assess Treg numbers. In addition, FoxP3 expression levels were assessed via qPCR and western blotting, while FoxP3 methylation status was evaluated via methyl-DNA immunoprecipitation qPCR. Immunosuppression assays were additionally used to gauge Treg suppressive functionality. We found that exposing pregnant rats to SEB resulted in a significant increase in Treg numbers, FoxP3 expression, and Treg suppressive capacity in the spleens of both neonatal and adult offspring. In addition, total T cell, CD4+T cell, and non-Treg CD4+ T cell numbers were elevated in the spleens of offspring following prenatal SEB exposure. We additionally determined that SEB exposure resulted in a significant reduction in FoxP3 DNA methylation. Together, our results indicate that prenatal SEB exposure can markedly enhance offspring splenic Treg numbers and functionality at least in part by decreasing FoxP3 methylation.

The unfolded protein response of B-lymphocytes: PERK-independent development of antibody-secreting cells.

When B-lymphocytes differentiate into plasma cells, immunoglobulin (Ig) heavy and light chain synthesis escalates and the entire secretory apparatus expands to support high-rate antibody secretion. These same events occur when murine B-cells are stimulated with lipopolysaccharide (LPS), providing an in vitro model in which to investigate the differentiation process. The unfolded protein response (UPR), a multi-pathway signaling response emanating from the endoplasmic reticulum (ER) membrane, allows cells to adapt to increasing demands on the protein folding capacity of the ER. As such, the UPR plays a pivotal role in the differentiation of antibody-secreting cells. Three specific stress sensors, IRE1, PERK/PEK and ATF6, are central to the recognition of ER stress and induction of the UPR. IRE1 triggers splicing of Xbp-1 mRNA, yielding a transcriptional activator of the UPR termed XBP-1(S), and activation of the IRE1/XBP-1 pathway has been reported to be required for expansion of the ER and antibody secretion. Here, we provide evidence that PERK is not activated in LPS-stimulated splenic B-cells, whereas XBP-1(S) and the UPR transcriptional activator ATF6 are both induced. We further demonstrate that Perk-/- B-cells develop and are fully competent for induction of Ig synthesis and antibody secretion when stimulated with LPS. These data provide clear evidence for differential activation and utilization of distinct UPR components as activated B-lymphocytes increase Ig synthesis and differentiate into specialized secretory cells.

Activating transcription factor 3 is integral to the eukaryotic initiation factor 2 kinase stress response.

In response to environmental stress, cells induce a program of gene expression designed to remedy cellular damage or, alternatively, induce apoptosis. In this report, we explore the role of a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) in coordinating stress gene responses. We find that expression of activating transcription factor 3 (ATF3), a member of the ATF/CREB subfamily of basic-region leucine zipper (bZIP) proteins, is induced in response to endoplasmic reticulum (ER) stress or amino acid starvation by a mechanism requiring eIF2 kinases PEK (Perk or EIF2AK3) and GCN2 (EIF2AK4), respectively. Increased expression of ATF3 protein occurs early in response to stress by a mechanism requiring the related bZIP transcriptional regulator ATF4. ATF3 contributes to induction of the CHOP transcriptional factor in response to amino acid starvation, and loss of ATF3 function significantly lowers stress-induced expression of GADD34, an eIF2 protein phosphatase regulatory subunit implicated in feedback control of the eIF2 kinase stress response. Overexpression of ATF3 in mouse embryo fibroblasts partially bypasses the requirement for PEK for induction of GADD34 in response to ER stress, further supporting the idea that ATF3 functions directly or indirectly as a transcriptional activator of genes targeted by the eIF2 kinase stress pathway. These results indicate that ATF3 has an integral role in the coordinate gene expression induced by eIF2 kinases. Given that ATF3 is induced by a very large number of environmental insults, this study supports involvement of eIF2 kinases in the coordination of gene expression in response to a more diverse set of stress conditions than previously proposed.

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 is required for activation of NF-kappaB in response to diverse cellular stresses.

Nuclear factor kappaB (NF-kappaB) serves to coordinate the transcription of genes in response to diverse environmental stresses. In this report we show that phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2) is fundamental to the process by which many stress signals activate NF-kappaB. Phosphorylation of this translation factor is carried out by a family of protein kinases that each respond to distinct stress conditions. During impaired protein folding and assembly in the endoplasmic reticulum (ER), phosphorylation of eIF2alpha by PEK (Perk or EIF2AK3) is essential for induction of NF-kappaB transcriptional activity. The mechanism by which NF-kappaB is activated during ER stress entails the release, but not the degradation, of the inhibitory protein IkappaB. During amino acid deprivation, phosphorylation of eIF2alpha by GCN2 (EIF2AK4) signals the activation of NF-kappaB. Furthermore, inhibition of general translation or transcription by cycloheximide and actinomycin D, respectively, elicits the eIF2alpha phosphorylation required for induction of NF-kappaB. Together, these studies suggest that eIF2alpha kinases monitor and are activated by a range of stress conditions that affect transcription and protein synthesis and assembly, and the resulting eIFalpha phosphorylation is central to activation of the NF-kappaB. The absence of NF-kappaB-mediated transcription and its antiapoptotic function provides an explanation for why eIF2alpha kinase deficiency in diseases such as Wolcott-Rallison syndrome leads to cellular apoptosis and disease.

GCN2 phosphorylation of eIF2alpha activates NF-kappaB in response to UV irradiation.

In response to UV irradiation, mammalian cells elicit a gene expression programme designed to repair damage and control cell proliferation and apoptosis. Important members of this stress response include the NF-kappaB (nuclear factor-kappaB) family. However, the mechanisms by which UV irradiation activates NF-kappaB are not well understood. In eukaryotes, a variety of environmental stresses are recognized and remediated by a family of protein kinases that phosphorylate the alpha subunit of eIF2 (eukaryotic initiation factor-2). In the present study we show that NF-kappaB in MEF (murine embryo fibroblast) cells is activated by UV-C and UV-B irradiation through a mechanism requiring eIF2alpha phosphorylation. The primary eIF2alpha kinase in response to UV is GCN2 (general control non-derepressible-2), with PEK/PERK (pancreatic eIF2alpha kinase/RNA-dependent-protein-kinase-like endoplasmic-reticulum kinase) carrying out a secondary function. Our studies indicate that lowered protein synthesis accompanying eIF2alpha phosphorylation, combined with eIF2alpha kinase-independent turnover of IkappaBalpha (inhibitor of kappaBalpha), reduces the levels of IkappaBalpha in response to UV irradiation. Release of NF-kappaB from the inhibitory IkappaBalpha would facilitate NF-kappaB entry into the nucleus and targeted transcriptional control. We also find that loss of GCN2 in MEF cells significantly enhances apoptosis in response to UV exposure similar to that measured in cells deleted for the RelA/p65 subunit of NF-kappaB. These results demonstrate that GCN2 is central to recognition of UV stress, and that eIF2alpha phosphorylation provides resistance to apoptosis in response to this environmental insult.

B-Myb represses vascular smooth muscle cell collagen gene expression and inhibits neointima formation after arterial injury.

OBJECTIVE: The function of B-Myb, a negative regulator of vascular smooth muscle cell (SMC) matrix gene transcription, was analyzed in the vasculature. METHODS AND RESULTS: Mice were generated in which the human B-myb gene was driven by the basal cytomegalovirus promoter, and 3 founders were identified. Mice appeared to develop normally, and human B-myb was expressed in the aortas. Total B-Myb levels were elevated in aortas of adult transgenic versus wild-type (WT) animals and varied inversely with alpha1(I) collagen mRNA expression. However, neonatal WT and transgenic aortas displayed comparable levels of alpha1(I) collagen mRNA, likely resulting from elevated levels of cyclin A, which ablated repression by B-Myb. Aortic SMCs from adult transgenic animals displayed decreased alpha1(I) collagen mRNA levels. To examine the role of B-Myb after vascular injury, animals were subjected to femoral artery denudation, which induces SMC-rich lesion formation. A dramatic reduction in neointima formation and lumenal narrowing was observed in arteries of B-myb transgenic versus WT mice 4 weeks after injury. CONCLUSIONS: Data indicate that B-Myb, which inhibits matrix gene expression in the adult vessel wall, reduces neointima formation after vascular injury. To analyze B-Myb function in the vasculature, mice overexpressing B-myb were generated. Neonates displayed normal alpha1(I) collagen mRNA levels, whereas adults expressed decreased collagen mRNA in aortas and isolated vascular SMCs. On femoral artery denudation, neointima formation was dramatically reduced in B-myb transgenic mice.

Famitinib in metastatic renal cell carcinoma: a single center study.

BACKGROUND: Famitinib is a novel and potent multitargeting receptor tyrosine kinase inhibitor. The phase I clinical study showed that famitinib was well tolerated and had a broad anti-tumor spectrum. The purpose of this study was to examine the efficacy and safety of famitinib for the treatment of metastatic renal cell carcinoma (mRCC). METHODS: The data of famitinib in treating patients with mRCC from the single-center phases I and II clinical trials were analyzed. Famitinib was administered orally at the dose of 13-30 mg once daily until tumor progression, occurrence of intolerable adverse reactions or withdrawal of the informed consent. RESULTS: A total of 24 patients with mRCC were treated including 17 patients at a dose of 25 mg once daily, 4 patients at a dose of 27 mg and 1 patient each at a dose of 13 mg, 20 mg and 30 mg, respectively. Twelve (50.0%) patients achieved partial response (PR) and 9 patients achieved stable disease (SD). Progressive disease was found in 3 (12.5%) patients. The disease control rate was 87.5%. The median follow-up time was 17.6 months; the median progression free survival (PFS) was 10.7 (95% CI 7.0-14.4) months; and the estimated median overall survival (OS) time was 33.0 (95% CI 8.7-57.3) months. The adverse drug reactions mainly included hypertension (54.1%), hand-foot skin reactions (45.8%), diarrhea (33.3%), mucositis (29.2%), neutropenia (45.8%), thrombocytopenia (29.2%), hyperlipidemia (41.7%) and proteinuria (41.7%). The incidence rate of grades 3 and 4 adverse events was low, mainly including hypertension 12.5%, hand-foot skin reactions 4.2%, neutropenia 4.2%, thrombocytopenia 4.2%, hyperlipidemia 4.2% and proteinuria 12.5%. CONCLUSIONS: Famitinib has significant anti-tumor activity in mRCC. The common adverse reactions are generally manageable.

Translational control of C-terminal Src kinase (Csk) expression by PRL3 phosphatase.

Phosphatase of regenerating liver 3 (PRL3) is up-regulated in cancer metastases. However, little is known of PRL3-mediated cellular signaling pathways. We previously reported that elevated PRL3 expression increases Src kinase activity, which likely contributes to the increased tumorigenesis and metastasis potential of PRL3. PRL3-induced Src activation is proposed to be indirect through down-regulation of Csk, a negative regulator of Src. Given the importance of PRL3 in tumor metastasis and the role of Csk in controlling Src activity, we addressed the mechanism by which PRL3 mediates Csk down-regulation. PRL3 is shown to exert a negative effect on Csk protein synthesis, rather than regulation of Csk mRNA levels or protein turnover. Interestingly, the preferential decrease in Csk protein synthesis is a consequence of increased eIF2 phosphorylation resulting from PRL3 expression. Reduced Csk synthesis also occurs in response to cellular stress that induces eIF2 phosphorylation, indicating that this regulatory mechanism may occur in response to a wider spectrum of cellular conditions known to direct translational control. Thus, we have uncovered a previously uncharacterized role for PRL3 in the gene-specific translational control of Csk expression.

The eukaryotic initiation factor-2 kinase pathway facilitates differential GADD45a expression in response to environmental stress.

Phosphorylation of eukaryotic initiation factor-2 (eIF2) regulates general and gene-specific translation in response to diverse environmental stresses. Central to gene expression induced by eIF2 phosphorylation is the preferential translation of ATF4, a basic zipper transcription activator. Phosphorylation of eIF2 and its attendant induction of ATF4 can lead to different patterns of gene expression depending on the environmental stress. This is of fundamental importance because eIF2 kinases can induce the expression of genes involved in survival as well as in apoptosis. In this report, we explore the molecular basis for why there can be differential expression of GADD45a, a stress-responsive protein that regulates genome stability, apoptosis, and immune responses. We find that whereas ATF4 is required for GADD45a transcription during many different environmental stresses, GADD45a protein accumulates only during a limited number of stress arrangements. The basis for this difference between measurable GADD45a mRNA and protein lies in the observation that GADD45a protein is labile. Those stress agents that enhance ATF4-directed GADD45a transcription and impede the turnover of GADD45a protein by blocking ubiquitin/proteasome-mediated degradation elevate GADD45a protein levels. By comparison, those stress arrangements that trigger ATF4 levels and GADD45a transcription, but do not perturb the proteasome pathway, only elevate GADD45a mRNA levels. This study highlights the molecular mechanisms by which environmental stresses can differentially control central regulatory proteins targeted by the eIF2 kinase pathway.

Phosphorylation of the alpha-subunit of the eukaryotic initiation factor-2 (eIF2alpha) reduces protein synthesis and enhances apoptosis in response to proteasome inhibition.

Protein ubiquitination and subsequent degradation by the proteasome are important mechanisms regulating cell cycle, growth and differentiation, and apoptosis. Recent studies in cancer therapy suggest that drugs that disrupt the ubiquitin/proteasome pathway induce apoptosis and sensitize malignant cells and tumors to conventional chemotherapy. In this study we addressed the role of phosphorylation of the alpha-subunit eukaryotic initiation factor-2 (eIF2), and its attendant regulation of gene expression, in the cellular stress response to proteasome inhibition. Phosphorylation of eIF2alpha in mouse embryo fibroblast (MEF) cells subjected to proteasome inhibition leads to a significant reduction in protein synthesis, concomitant with induced expression of the bZIP transcription regulator, ATF4, and its target gene CHOP/GADD153. The primary eIF2alpha kinase activated by exposure of these fibroblast cells to proteasome inhibition is GCN2 (EIF2AK4), which has a central role in the recognition of cytoplasmic stress signals. Endoplasmic reticulum (ER) stress is not effectively induced in MEF cells subjected to proteasome inhibition, with minimal activation of the ER stress sensory proteins, eIF2alpha kinase PEK (PERK/EIF2AK3), IRE1 protein kinase and the transcription regulator ATF6 following up to 6 h of proteasome inhibitor treatment. Loss of eIF2alpha phosphorylation thwarts caspase activation and delays apoptosis. Central to this pro-apoptotic function of eIF2alpha kinases during proteasome inhibition is the transcriptional regulator CHOP, as deletion of CHOP in MEF cells impedes apoptosis. We conclude that eIF2alpha kinases are integral to cellular stress pathways induced by proteasome inhibitors, and may be central to the efficacy of anticancer drugs that target the ubiquitin/proteasome pathway.

Aggregation and porin-like channel activity of a beta sheet peptide.

Beta sheet peptides (e.g., amyloid beta) are known to form ion channels in lipid bilayers possibly through aggregation, though the channel structure is not clear. We have recently reported that a short beta sheet peptide, (xSxG)(6), forms porin-like voltage-gated channels in lipid bilayers [Thundimadathil et al. (2005) Biochem. Biophys. Res. Commun. 330, 585-590]. To account for the porin-like activity, oligomerization of the peptide into a beta barrel-like structure was proposed. In this work, peptide aggregation in aqueous and membrane environments and a detailed study of channel properties were performed to gain insight into the mechanism of channel formation. The complex nature of the channel was revealed by kinetic analysis and the occurrence of interconverting multiple conductance states. Ion channels were inhibited by Congo red, suggesting that the peptide aggregates are the active channel species. Peptide aggregation and fibril formation in water were confirmed by electron microscopy (EM) and Congo red binding studies. Furthermore, oligomeric structures in association with lipid bilayers were detected. Circular dichroism of peptide-incorporated liposomes and peptide-lipid binding studies using EM suggest a lipid-induced beta sheet aggregation. Gel electrophoresis of peptide-incorporated liposomes showed dimeric and multimeric structures. Taken together, this work indicates insertion of (xSxG)(6) as oligomers into the lipid bilayer, followed by rearrangement into a beta barrel-like pore structure. A large peptide pore comprising several individual beta sheets or smaller beta sheet aggregates is expected to have a complex behavior in membranes. A dyad repeat sequence and the presence of glycine, serine, and hydrophobic residues in a repeated pattern in this peptide may be providing a favorable condition for the formation of a beta barrel-like structure in lipid bilayers.

IMPACT, a protein preferentially expressed in the mouse brain, binds GCN1 and inhibits GCN2 activation.

Translational control directed by the eukaryotic translation initiation factor 2 alpha-subunit (eIF2alpha) kinase GCN2 is important for coordinating gene expression programs in response to nutritional deprivation. The GCN2 stress response, conserved from yeast to mammals, is critical for resistance to nutritional deficiencies and for the control of feeding behaviors in rodents. The mouse protein IMPACT has sequence similarities to the yeast YIH1 protein, an inhibitor of GCN2. YIH1 competes with GCN2 for binding to a positive regulator, GCN1. Here, we present evidence that IMPACT is the functional counterpart of YIH1. Overexpression of IMPACT in yeast lowered both basal and amino acid starvation-induced levels of phosphorylated eIF2alpha, as described for YIH1 (31). Overexpression of IMPACT in mouse embryonic fibroblasts inhibited phosphorylation of eIF2alpha by GCN2 under leucine starvation conditions, abolishing expression of its downstream target genes, ATF4 (CREB-2) and CHOP (GADD153). IMPACT bound to the minimal yeast GCN1 segment required for interaction with yeast GCN2 and YIH1 and to native mouse GCN1. At the protein level, IMPACT was detected mainly in the brain. IMPACT was found to be abundant in the majority of hypothalamic neurons. Scattered neurons expressing this protein at higher levels were detected in other regions such as the hippocampus and piriform cortex. The abundance of IMPACT correlated inversely with phosphorylated eIF2alpha levels in different brain areas. These results suggest that IMPACT ensures constant high levels of translation and low levels of ATF4 and CHOP in specific neuronal cells under amino acid starvation conditions.