Activation of p53/ATM-dependent DNA damage signaling pathway by shiga toxin in mammalian cells.

In this report, we studied the role of DNA damage signaling pathway in shiga toxin (STX)-induced mammalian cell death. Shiga toxin 1 exhibited cytotoxic activity in different mammalian cells such as HeLa cells, mouse embryo fibroblasts, and Caco-2 cells (a human intestinal primary fibroblast cell line). STX-1 was found to induce the release of cytochrome c from the mitochondria, nuclear condensation, and fragmentation of chromosomal DNA.

Prevalence of virulence genes and cytolethal distending toxin production in Campylobacter jejuni isolates from diarrheal patients in Bangladesh.

From 300 stool samples, 58 Campylobacter strains were isolated by standard microbiological and biochemical methods. Of these, 40 strains were identified as Campylobacter jejuni and 5 as Campylobacter coli. The presence of flaA (100%), cadF (100%), racR (100%), dnaJ (100%), pldA (100%), ciaB (95%), virB11 (0%), ceuE (82.

Covalently attached FGF-2 to three-dimensional polyamide nanofibrillar surfaces demonstrates enhanced biological stability and activity.

Activation of fibroblast growth factor receptors (FGFRs) requires the formation of a ternary complex between fibroblast growth factors (FGFs), FGFRs, and heparan sulfate proteoglycans, which are all located on the cell surface and the basement membrane (BM)/extracellular matrix (ECM). Heparan sulfate proteoglycans appear to stabilize FGFs by inhibiting the rapid degradation of FGFs normally observed in solution. Because of the pivotal role of FGFs in proliferative and developmental pathways, a number of recent studies have attempted to engineer microenvironments to stabilize growth factors for use in applications in tissue culture and regenerative medicine.

Role of DNA topoisomerase IIbeta in neurite outgrowth.

Failure to establish neuromuscular junctions is a major phenotype of top2beta knockout mice. However, the precise mechanism for this defect is not known. In the current study, we have investigated the role of TopIIbeta in cultured neurons.

NBK/BIK antagonizes MCL-1 and BCL-XL and activates BAK-mediated apoptosis in response to protein synthesis inhibition.

Ribonucleases, antibiotics, bacterial toxins, and viruses inhibit protein synthesis, which results in apoptosis in mammalian cells. How the BCL-2 family of proteins regulates apoptosis in response to the shutoff of protein synthesis is not known. Here we demonstrate that an Escherichia coli toxin, MazF, inhibited protein synthesis by cleavage of cellular mRNA and induced apoptosis in mammalian cells.

Morphology, cytoskeletal organization, and myosin dynamics of mouse embryonic fibroblasts cultured on nanofibrillar surfaces.

Growth of cells in tissue culture is generally performed on two-dimensional (2D) surfaces composed of polystyrene or glass. Recent work, however, has shown that such 2D cultures are incomplete and do not adequately represent the physical characteristics of native extracellular matrix (ECM)/basement membrane (BM), namely dimensionality, compliance, fibrillarity, and porosity. In the current study, a three-dimensional (3D) nanofibrillar surface composed of electrospun polyamide nanofibers was utilized to mimic the topology and physical structure of ECM/BM.

Living in three dimensions: 3D nanostructured environments for cell culture and regenerative medicine.

Research focused on deciphering the biochemical mechanisms that regulate cell proliferation and function has largely depended on the use of tissue culture methods in which cells are grown on two-dimensional (2D) plastic or glass surfaces. However, the flat surface of the tissue culture plate represents a poor topological approximation of the more complex three-dimensional (3D) architecture of the extracellular matrix (ECM) and the basement membrane (BM), a structurally compact form of the ECM. Recent work has provided strong evidence that the highly porous nanotopography that results from the 3D associations of ECM and BM nanofibrils is essential for the reproduction of physiological patterns of cell adherence, cytoskeletal organization, migration, signal transduction, morphogenesis, and differentiation in cell culture.

Elevated expression of ISG15 in tumor cells interferes with the ubiquitin/26S proteasome pathway.

IFN-stimulatory gene factor 15 (ISG15) is a ubiquitin-like protein, which is conjugated to many cellular proteins. However, its role in protein degradation is unclear. Here, we show that ISG15 is highly elevated and extensively conjugated to cellular proteins in many tumors and tumor cell lines.

Role of Cdc42 in neurite outgrowth of PC12 cells and cerebellar granule neurons.

Inactivation of Rho GTPases inhibited the neurite outgrowth of PC12 cells. The role of Cdc42 in neurite outgrowth was then studied by selective inhibition of Cdc42 signals. Overexpression of ACK42, Cdc42 binding domain of ACK-1, inhibited NGF-induced neurite outgrowth in PC12 cells.

Neurite guidance by the FnC repeat of human tenascin-C: neurite attraction vs. neurite retention.

The alternatively spliced fibronectin type-III repeat C of human tenascin-C (fnC) provides directional cues to elongating neurites in vitro. When given a choice at an interface with poly L-lysine (PLL), rat cerebellar granule neurites preferentially crossed onto fnC (defined herein as neurite attraction) whereas neurites originating on fnC preferentially remained on fnC (defined as neurite retention). Guidance motifs were further refined using synthetic peptides spanning the sequence of fnC.

Three-dimensional nanofibrillar surfaces promote self-renewal in mouse embryonic stem cells.

The regulation of mouse embryonic stem cell (mESC) fate is controlled by the interplay of signaling networks that either promote self-renewal or induce differentiation. Leukemia inhibitory factor (LIF) is a cytokine that is required for stem cell renewal in mouse but not in human embryonic stem cells. However, feeder layers of embryonic fibroblasts are capable of inducing stem cell renewal in both cell types, suggesting that the self-renewal signaling pathways may also be promoted by other triggers, such as alternative cytokines and/or chemical or physical properties of the extracellular matrix (ECM) secreted by feeder fibroblasts.

Requirement of activated Cdc42-associated kinase for survival of v-Ras-transformed mammalian cells.

Activated Cdc42-associated kinase (ACK) has been shown to be an important effector molecule for the small GTPase Cdc42. We have shown previously an essential role for Cdc42 in the transduction of Ras signals for the transformation of mammalian cells. In this report, we show that the ACK-1 isoform of ACK plays a critical role in transducing Ras-Cdc42 signals in the NIH 3T3 cells.

A synthetic nanofibrillar matrix promotes in vivo-like organization and morphogenesis for cells in culture.

The purpose of this study was to design a synthetic nanofibrillar matrix that more accurately models the porosity and fibrillar geometry of cell attachment surfaces in tissues. The synthetic nanofibrillar matrices are composed of nanofibers prepared by electrospinning a polymer solution of polyamide onto glass coverslips. Scanning electron and atomic force microscopy showed that the nanofibers were organized into fibrillar networks reminiscent of the architecture of basement membrane, a structurally compact form of the extracellular matrix (ECM).

Three dimensional nanofibrillar surfaces induce activation of Rac.

Studies to define the mechanisms by which the extracellular matrix (ECM) activates Rho GTPases within the cell have generally focused on the chemistry of the macromolecules comprising the ECM. Considerably less information is available to assess the role of the physical structure of the ECM, particularly its three dimensional (3D) geometry. In this report, we examined the effect of 3D surfaces on the activation states of Rho GTPases within NIH 3T3 fibroblasts and normal rat kidney cells.

Nuclear translocation of cytochrome c during apoptosis.

Release of cytochrome c from mitochondria is a major event during apoptosis. Released cytochrome c has been shown to activate caspase-dependent apoptotic signals. In this report, we provide evidence for a novel role of cytochrome c in caspase-independent nuclear apoptosis.

Cdc42-dependent nuclear translocation of non-receptor tyrosine kinase, ACK.

Ras signals for the transformation of mammalian cells are apparently transduced through Rho GTPases. The Rho GTPase family member Cdc42 generates independent signals that regulate the rearrangement of the actin cytoskeleton and the transcription of genes. However, the molecular mechanism of signal transduction from Cdc42 to the nucleus remains to be understood.

Neurite outgrowth by the alternatively spliced region of human tenascin-C is mediated by neuronal alpha7beta1 integrin.

The region of tenascin-C containing only alternately spliced fibronectin type-III repeat D (fnD) increases neurite outgrowth by itself and also as part of tenascin-C. We previously localized the active site within fnD to an eight amino acid sequence unique to tenascin-C, VFDNFVLK, and showed that the amino acids FD and FV are required for activity. The purpose of this study was to identify the neuronal receptor that interacts with VFDNFVLK and to investigate the hypothesis that FD and FV are important for receptor binding.

DNA damage-mediated apoptosis induced by selenium compounds.

Selenium (Se) compounds, which are the most extensively studied cancer chemopreventive agents, induce apoptotic death of tumor cells. In the current study, we show that selenite-induced apoptosis involves DNA damage. We showed that selenite-induced apoptosis as evidenced by cleavage of poly(ADP-ribose) polymerase was reduced in NIH 3T3 cells treated with ATM small interfering RNA, suggesting the involvement of the DNA damage regulator ATM.

Inactivation of Cdc13p triggers MEC1-dependent apoptotic signals in yeast.

Inactivation of the budding yeast telomere binding protein Cdc13 results in abnormal telomeres (exposed long G-strands) and activation of the DNA damage checkpoint. In the current study, we show that inactivation of Cdc13p induces apoptotic signals in yeast, as evidenced by caspase activation, increased reactive oxygen species production, and flipping of phosphatidylserine in the cytoplasmic membrane. These apoptotic signals were suppressed in a mitochondrial (rho(o)) mutant.

Single-stranded DNA induces ataxia telangiectasia mutant (ATM)/p53-dependent DNA damage and apoptotic signals.

Single-stranded DNA has been speculated to be the initial signal in the DNA damage signaling pathway. We showed that introduction of single-stranded DNA with diverse sequences into mammalian cells induced DNA damage as well as apoptosis signals. Like DNA damaging agents, single-stranded DNA up-regulated p53 and activated the nuclear kinase ataxia telangiectasia mutant (ATM) as evidenced by phosphorylation of histone 2AX, an endogenous ATM substrate.