Graphene Oxide as a Multifunctional Platform for Intracellular Delivery, Imaging, and Cancer Sensing.

Graphene oxide (GO), the most common derivative of graphene, is an exceptional nanomaterial that possesses multiple physical properties critical for biomedical applications. GO exhibits pH-dependent fluorescence emission in the visible/near-infrared, providing a possibility of molecular imaging and pH-sensing. It is also water soluble and has a substantial platform for functionalization, allowing for the delivery of multiple therapeutics. GO physical properties are modified to enhance cellular internalization, producing fluorescent nanoflakes with low (<15%) cytotoxicity at the imaging concentrations of 15 µg/mL. As a result, at lower flake sizes GO rapidly internalizes into HeLa cells with the following 70% fluorescence based clearance at 24 h, assessed by its characteristic emission in red/near-IR. pH-dependence of GO emission is utilized to provide the sensing of acidic extracellular environments of cancer cells. The results demonstrate diminishing green/red (550/630 nm) fluorescence intensity ratios for HeLa and MCF-7 cancer cells in comparison to HEK-293 healthy cells suggesting a potential use of GO as a non-invasive optical sensor for cancer microenvironments. The results of this work demonstrate the potential of GO as a novel multifunctional platform for therapeutic delivery, biological imaging and cancer sensing.

Alleviation of the effects of endotoxin exposure on behavior and hippocampal IL-1beta by a selective non-peptide antagonist of corticotropin-releasing factor receptors.

Previous research has shown that lipopolysaccharide (LPS) or interleukin-1beta (IL-1beta) administration produces learning/memory deficits in a variety of paradigms. In our laboratory, we have consistently observed LPS-induced behavioral alterations in a two-way active avoidance conditioning paradigm. Following LPS administration, one factor that affects cytokine production is corticotropin-releasing factor (CRF). CRF has well known anti-inflammatory effects, via stimulation of ACTH and corticosterone release. However, CRF acting directly on immune cells or within the CNS may potentiate proinflammatory effects. The current experiments explored the potential of antalarmin, a CRF-R1 non-peptide antagonist, to diminish or negate deficits observed with LPS administration. On the first day of testing, four-month-old male C57BL/6J mice received an intraperitoneal (i.p.) injection of antalarmin, followed 90min later by a second i.p. injection of LPS 4h prior to two-way active avoidance conditioning testing. As hypothesized, LPS administration altered performance. However, pretreatment with antalarmin attenuated the adverse effects of LPS administration. Moreover, evidence indicates that antalarmin attenuated hippocampal, but not peripheral, cytokine release. The behavioral results cannot be explained by alterations in the HPA axis, as antalarmin did not affect the LPS-induced rise in corticosterone. The current research contributes preliminary evidence that CRF may be an important factor in the development of LPS-induced behavioral effects, and that blocking the activity of CRF may be sufficient to alleviate some of the effects of endotoxin exposure, possibly due to diminished LPS-induced IL-1beta release in the dorsal hippocampus.

TAK1 is a ubiquitin-dependent kinase of MKK and IKK.

TRAF6 is a signal transducer that activates IkappaB kinase (IKK) and Jun amino-terminal kinase (JNK) in response to pro-inflammatory mediators such as interleukin-1 (IL-1) and lipopolysaccharides (LPS). IKK activation by TRAF6 requires two intermediary factors, TRAF6-regulated IKK activator 1 (TRIKA1) and TRIKA2 (ref. 5). TRIKA1 is a dimeric ubiquitin-conjugating enzyme complex composed of Ubc13 and Uev1A (or the functionally equivalent Mms2). This Ubc complex, together with TRAF6, catalyses the formation of a Lys 63 (K63)-linked polyubiquitin chain that mediates IKK activation through a unique proteasome-independent mechanism. Here we report the purification and identification of TRIKA2, which is composed of TAK1, TAB1 and TAB2, a protein kinase complex previously implicated in IKK activation through an unknown mechanism. We find that the TAK1 kinase complex phosphorylates and activates IKK in a manner that depends on TRAF6 and Ubc13-Uev1A. Moreover, the activity of TAK1 to phosphorylate MKK6, which activates the JNK-p38 kinase pathway, is directly regulated by K63-linked polyubiquitination. We also provide evidence that TRAF6 is conjugated by the K63 polyubiquitin chains. These results indicate that ubiquitination has an important regulatory role in stress response pathways, including those of IKK and JNK.

Compartmentalized protein kinase C activation in ovarian carcinoma cells.

Protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinases, plays a cardinal role in malignancy (1-5). PKC isozymes can be categorized into three groups: Group A or conventional (c) PKC: α, ßI, ßII, and γ; Group B or novel (n) PKC: δ, ε, η, θ, and µ, and Group C or atypical (a) PKC: ζ and λ (ι) (1,3,4). Whereas cPKCs require Ca(2+)and diacylglycerol (DAG) phorbol esters for their activities, nPKCs and aPKCs are Ca(2+)-independent. aPKCs are also insensitive to diacylglycerol and phorbol esters (1,3,4).

Overexpression of protein kinase C-eta attenuates caspase activation and tumor necrosis factor-alpha-induced cell death.

The protein kinase C (PKC) signal transduction pathway regulates cell death by tumor necrosis factor-alpha (TNF). We previously showed that the induction of novel PKC eta isozyme by PKC activators correlated with their ability to protect MCF-7 breast cancer cells against TNF cytotoxicity. In the present study, we have transfected PKC eta in MCF-7 cells to directly examine its involvement in cell death by TNF. Overexpression of PKC eta delayed TNF-induced cell death in MCF-7 cells. TNF caused a rapid activation of caspase-8 and -7 in cells transfected with a vector. The activation of these caspases was potentiated by the PKC inhibitor bisindolylmaleimide (BIM) which downregulates PKC eta and sensitizes cells to TNF. Overexpression of PKC eta delayed the activation of caspase-8 and -7 by both TNF and the combination of BIM and TNF. These results suggest that PKC eta protects MCF-7 cells against TNF-induced cell death by preventing the activation of caspases.

Regulation of caspase activation and cis-diamminedichloroplatinum(II)-induced cell death by protein kinase C.

Activation of caspases is critical for the induction of apoptosis. We have shown previously that cell death mediated by the anticancer agent cis-diamminedichloroplatinum(II) (cDDP) is influenced by the protein kinase C (PKC) signal transduction pathway. In the present study, we have examined whether regulation of cDDP sensitivity by PKC involves caspase activation. cDDP caused a time- and concentration-dependent increase in the generation of the catalytic fragment (CF) of novel (n) PKCdelta, nPKCepsilon, and atypical (a) PKCzeta but had little effect on conventional (c) PKCalpha. Cleavage of PKC isozymes was associated with the activation of caspase-3 and -7 but not of caspase-2. PKC activators enhanced cDDP-induced cleavage of these isozymes and activation of caspase-3. Rottlerin, an inhibitor of nPKCdelta, blocked caspase-3 activation and proteolytic cleavage of nPKCdelta by cDDP. Bryostatin 1, which elicits a biphasic concentration-response in potentiating cell death by cDDP, exhibited a similar biphasic effect on cDDP-induced activation of caspase-3 and caspase-7 and the cleavage of poly(ADP-ribose) polymerase; while 1 nM bryostatin 1 induced maximum activation of these caspases, 1 microM bryostatin 1 had little effect. z-DEVD-fmk, an inhibitor of caspase-3-like proteases, prevented cDDP-induced cell death. Bryostatin 1 also induced a similar biphasic down-regulation of nPKCdelta but not of cPKCalpha or nPKCepsilon. These results suggest that nPKCdelta not only acts downstream of caspases but also regulates the activation of caspases and that the biphasic concentration response of bryostatin 1 on cDDP-induced cell death could be explained by its distinct effect on nPKCdelta down-regulation and caspase activation.

Herpes simplex virus vector-mediated dystrophin gene transfer and expression in MDX mouse skeletal muscle.

BACKGROUND: Duchenne muscular dystrophy (DMD) results from mutations that prevent the expression of functional dystrophin in muscle fibers. Herpes simplex virus type-1 (HSV-1) represents a potentially useful vector for treatment of DMD because it has the capacity to accommodate the 14-kb full-length dystrophin cDNA and can efficiently transduce muscle cells. We have tested the ability of first- and second-generation replication-defective HSV vectors to deliver full-length dystrophin to dystrophin-deficient mdx muscle cells in vitro and in vivo. METHODS: First-generation replication-defective HSV vectors harboring full-length or truncated (Becker) dystrophin expression cassettes and lacking a single viral immediate-early (IE) gene were constructed and tested by immunofluorescence and immunoblotting for their ability to direct dystrophin expression in infected mdx cells in culture. To reduce vector cytotoxicity and safety concerns, a second-generation dystrophin vector missing additional IE genes was constructed and tested in vitro and in vivo. RESULTS: Dystrophin expression was observed in infected mdx myotubes in vitro in all cases. Confocal microscopy showed exclusive localization of full-length dystrophin to the cell membrane whereas the Becker variant was also found abundantly throughout the cytoplasm. Dystrophin expression in mdx mice was restored in muscle cells near the site of vector injection. CONCLUSION: Highly defective HSV-1 vectors which lack the ability to spread systemically and are greatly reduced in toxicity for infected cells, thus removing an impediment to prolonged transgene expression, can direct the delivery and proper expression of full-length dystrophin whose considerable size is compatible with few other modes of delivery. These vectors may offer a legitimate opportunity toward the development of effective gene therapy treatments for DMD.

Increase in eukaryotic initiation factor 2B activity following fertilization reflects changes in redox potential.

One of the factors involved in the postfertilization activation of protein synthesis in the sea urchin, Strongylocentrotus purpuratus, is the activation of eIF-2B, the initiation factor responsible for guanine nucleotide exchange on eIF-2. Cell-free translation systems from unfertilized eggs are stimulated by added eIF-2B, although this dependency is rapidly lost in translation systems prepared at various times following fertilization. Cell-free translation systems prepared from unfertilized eggs show significantly lower eIF-2B activities than those prepared from 2-h embryos. However, the provision of an NADPH regeneration system significantly stimulates eIF-2B activity in egg extracts and, in addition, stimulates both binding of initiator tRNA to the small ribosomal subunit and protein synthetic activity. These data suggest that the activation of eIF-2B following fertilization reflects the fertilization-induced increase in NADPH levels.

Evaluation of protein phosphorylation state by a combination of vertical slab gel isoelectric focusing and immunoblotting.

Conditions have been established for one-dimensional isoelectric focusing using vertical slab gel electrophoresis, followed by immunoblotting, for the measurement of the phosphorylation state of proteins. The method provides a less time-consuming alternative to two-dimensional gel electrophoresis combined with radiolabeling or immunoblotting. The main advantage of the method is that many samples can be analyzed simultaneously. The technique is applied here to the study of a mammalian initiation factor for protein synthesis, eukaryotic initiation factor 2 (eIF-2). The method allows good separation and quantitation of the different phosphorylated forms of the alpha subunit of eIF-2, when used to analyze either purified eIF-2 or eIF-2 contained in complex mixtures. The method is shown to be well adapted to the measurement of rapid phosphorylation/dephosphorylation kinetics in cell extracts, as well as the measurement of the phosphorylation state of eIF-2 in cultured cells. In addition, the method is shown to confirm the existence of a second phosphorylation site on eIF-2. Although eIF-2 has been used for this demonstration of the efficacy of the method, the technique is applicable to a study of the regulation of covalent modification of any polypeptide for which antibodies are available.

Vaccinia specific kinase inhibitory factor prevents translational inhibition by double-stranded RNA in rabbit reticulocyte lysate.

Mouse L-cells infected with vaccinia virus produce a specific kinase inhibitory factor (SKIF) which inhibits the activation of the interferon-induced, double-stranded (ds)RNA-dependent, eukaryotic initiation factor (eIF)-2 alpha-specific protein kinase in L-cell extracts (Whitaker-Dowling, P., and Younger, J. S., (1984) Virology 137, 171). The effects of a partially purified preparation of SKIF have been examined in cell-free extracts of rabbit reticulocytes. Both the phosphorylation state of eIF-2 and protein synthetic activity have been determined. SKIF inhibits the phosphorylation of the alpha subunit of eIF-2 by dsRNA-dependent eIF-2 alpha-kinase in reticulocyte lysate, but does not affect phosphorylation of eIF-2 by the heme-sensitive kinase. In addition to its effects on eIF-2 alpha-PKds activity, SKIF prevents dsRNA-induced inhibition of protein synthesis in reticulocyte lysate. In contrast, SKIF does not prevent the translational inhibition caused by hemin depletion. These data provide a direct correlation between the effects of SKIF on eIF-2 alpha phosphorylation and on protein synthetic activity and demonstrate the specificity of SKIF. The results also show that SKIF does not abolish dsRNA sensitivity, but increases the concentration of dsRNA required to activate the kinase and phosphorylate eIF-2.