Analysis of heat transfers inside counterflow plate heat exchanger augmented by an auxiliary fluid flow.

Enhancement of heat transfers in counterflow plate heat exchanger due to presence of an intermediate auxiliary fluid flow is investigated. The intermediate auxiliary channel is supported by transverse conducting pins. The momentum and energy equations for the primary fluids are solved numerically and validated against a derived approximate analytical solution. A parametric study including the effect of the various plate heat exchanger, and auxiliary channel dimensionless parameters is conducted. Different enhancement performance indicators are computed. The various trends of parameters that can better enhance heat transfer rates above those for the conventional plate heat exchanger are identified. Large enhancement factors are obtained under fully developed flow conditions. The maximum enhancement factors can be increased by above 8.0- and 5.0-fold for the step and exponential distributions of the pins, respectively. Finally, counterflow plate heat exchangers with auxiliary fluid flows are recommended over the typical ones if these flows can be provided with the least cost.

Trophic factor withdrawal: p38 mitogen-activated protein kinase activates NHE1, which induces intracellular alkalinization.

Trophic factor withdrawal induces cell death by mechanisms that are incompletely understood. Previously we reported that withdrawal of interleukin-7 (IL-7) or IL-3 produced a rapid intracellular alkalinization, disrupting mitochondrial metabolism and activating the death protein Bax. We now observe that this novel alkalinization pathway is mediated by the pH regulator NHE1, as shown by the requirement for sodium, blocking by pharmacological inhibitors or use of an NHE1-deficient cell line, and the altered phosphorylation of NHE1. Alkalinization also required the stress-activated p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK activity with pharmacological inhibitors or expression of a dominant negative kinase prevented alkalinization. Activated p38 MAPK directly phosphorylated the C terminus of NHE1 within a 40-amino-acid region. Analysis by mass spectroscopy identified four phosphorylation sites on NHE1, Thr 717, Ser 722, Ser 725, and Ser 728. Thus, loss of trophic cytokine signaling induced the p38 MAPK pathway, which phosphorylated NHE1 at specific sites, inducing intracellular alkalinization.

Interleukin-7: physiological roles and mechanisms of action.

Interleukin-7 (IL-7), a product of stromal cells, provides critical signals to lymphoid cells at early stages in their development. Two types of cellular responses to IL-7 have been identified in lymphoid progenitors: (1) a trophic effect and (2) an effect supporting V(D)J recombination. The IL-7 receptor is comprised of two chains, IL-7R alpha and gamma(c). Following receptor crosslinking, rapid activation of several classes of kinases occurs, including members of the Janus and Src families and PI3-kinase. A number of transcription factors are subsequently activated including STATs, c-myc, NFAT and AP-1. However, it remains to be determined which, if any, previously identified pathway leads to the trophic or V(D)J endpoints. The trophic response to IL-7 involves protecting lymphoid progenitors from a death process that resembles apoptosis. This protection is partly mediated by IL-7 induction of Bcl-2, however other IL-7-induced events are probably also involved in the trophic response. The V(D)J response to IL-7 is partly mediated through increased production of Rag proteins (which cleave the target locus) and partly by increasing the accessibility of a target locus to cleavage through chromatin remodeling.

Aberrant expression of the NF-kappaB and IkappaB proteins in B cells from viable motheaten mice.

In viable motheaten mice, a mutation in the gene encoding the phosphatase, SHP1, causes severe immunodeficiency and autoimmunity. A defective phosphatase may result in modified phosphorylation of proteins involved in gene regulation. Since the NFkappaB/IkappaB proteins are regulated through phosphorylation, we wished to understand if the expression of these proteins was altered by the SHP1 defect. Splenic B cells from viable motheaten mice were isolated and assessed for purity by flow cytometry. Levels of each protein in isolated B cells were examined by Western blot analyses. Measurement of RNA levels for each protein was assessed by semi-quantitative RT-PCR. Western blots revealed that, in me(v) whole cell lysates, there were reduced levels of RelA and RelB proteins and increased levels of p50 and c-Rel. Furthermore, we analyzed the protein levels of IkappaBalpha and found that, in me(v), this inhibitor was significantly reduced, while the level of another member of the IkappaB family, IkappaBbeta, was not. To determine if these findings in me(v) were secondary to the autoimmune process, we evaluated NF-kappaB/IkappaB expression in the BXSB murine model of autoimmunity. Unlike me(v), B cells from BXSB/Yaa mice had NF-kappaB complexes composed of the RelA submit, and IkappaBalpha was readily detected. In addition, RNA for the RelA and IkappaBalpha proteins in me(v) and control littermates was detected by RT-PCR, indicating that the reduced amounts of these proteins was not exclusively due to transcriptional defects. We conclude that the differences in NF-kappaB/IkappaB proteins that we have described in me(v) are likely a consequences of the SHP1 defect and could contribute to the clinical disorder that characterizes me(v) mice.

Analysis of oscillatory flow disturbances and thermal characteristics inside fluidic cells due to fluid leakage and wall slip conditions.

The effects of both fluid leakage and wall slip conditions are studied analytically and numerically on the fluctuation rate in the flow inside non-isothermal disturbed thin films supported by soft seals within a fluidic cell. Flow disturbances due to internal pressure pulsations and external squeezing are considered in this work. The main controlling parameters are found to be the dimensionless leakage parameter, softness of the seal, squeezing number, dimensionless slip parameter, the thermal squeezing parameter and the power law index. Accordingly, their influences on the fluctuation rate and heat transfer characteristics inside disturbed thin films are determined and discussed. It is found that an increase in the dimensionless leakage parameter, softness of the seal-upper plate assembly and the wall slip parameter result in more cooling and an increase in the fluctuation level in the flow. However, an increase in the squeezing number and the fluid power index decrease flow fluctuations. Finally, a suggested design to alleviate a number of problems in fluidic cells is presented.

The CT20 peptide causes detachment and death of metastatic breast cancer cells by promoting mitochondrial aggregation and cytoskeletal disruption.

Metastasis accounts for most deaths from breast cancer, driving the need for new therapeutics that can impede disease progression. Rationally designed peptides that take advantage of cancer-specific differences in cellular physiology are an emerging technology that offer promise as a treatment for metastatic breast cancer. We developed CT20p, a hydrophobic peptide based on the C terminus of Bax that exhibits similarities with antimicrobial peptides, and previously reported that CT20p has unique cytotoxic actions independent of full-length Bax. In this study, we identified the intracellular actions of CT20p which precede cancer cell-specific detachment and death. Previously, we found that CT20p migrated in the heavy membrane fractions of cancer cell lysates. Here, using MDA-MB-231 breast cancer cells, we demonstrated that CT20p localizes to the mitochondria, leading to fusion-like aggregation and mitochondrial membrane hyperpolarization. As a result, the distribution and movement of mitochondria in CT20p-treated MDA-MB-231 cells was markedly impaired, particularly in cell protrusions. In contrast, CT20p did not associate with the mitochondria of normal breast epithelial MCF-10A cells, causing little change in the mitochondrial membrane potential, morphology or localization. In MDA-MB-231 cells, CT20p triggered cell detachment that was preceded by decreased levels of α5ß1 integrins and reduced F-actin polymerization. Using folate-targeted nanoparticles to encapsulate and deliver CT20p to murine tumors, we achieved significant tumor regression within days of peptide treatment. These results suggest that CT20p has application in the treatment of metastatic disease as a cancer-specific therapeutic peptide that perturbs mitochondrial morphology and movement ultimately culminating in disruption of the actin cytoskeleton, cell detachment, and loss of cell viability.

Homeostasis and the importance for a balance between AKT/mTOR activity and intracellular signaling.

The AKT family of serine threonine kinases is of critical importance with regard to growth factor signaling, cell proliferation, survival and oncogenesis. Engagement of signaling receptors induces the lipid kinase, phosphatidylinositol 3-kinase (PI3K), which enables the activation of AKT. Responsive to the PI3K/AKT pathway is the mammalian target of rapamycin (mTOR), a major effector that is specifically implicated in the regulation of cell growth as a result of nutrient availability and cellular bioenergetics. These kinases mediate the activity of a multitude of intracellular signaling molecules and intersect with multiple pathways that regulate cellular processes. Elucidating the role of AKT/mTOR in metabolism and in hallmark signaling pathways that are aberrantly affected in cancer has provided a solid foundation of discoveries. From this, new research directions are emerging with regard to the role of AKT/mTOR in diabetes and T cell-mediated immunity. As a result, a new perspective is developing in how AKT/mTOR functions within intracellular signaling pathways to maintain cellular homeostasis. An appreciation is emerging that altered equilibrium of AKT/mTOR pathways contributes to disease and malignancy. Such new insights may lead to novel intervention strategies that may be useful to reprogram or reset the balance of intracellular signaling.

The use of therapeutic peptides to target and to kill cancer cells.

Peptide therapeutics is a promising field for emerging anti-cancer agents. Benefits include the ease and rapid synthesis of peptides and capacity for modifications. An existing and vast knowledge base of protein structure and function can be exploited for novel peptide design. Current research focuses on developing peptides that can (1) serve as tumor targeting moieties and (2) permeabilize membranes with cytotoxic consequences. A survey of recent findings reveals significant trends. Amphiphilic peptides with clusters of hydrophobic and cationic residues are features of anti-microbial peptides that confer the ability to eradicate microbes and show considerable anti-cancer toxicity. Peptides that assemble and form pores can disrupt cell or organelle membranes and cause apoptotic or necrotic death. Cell permeable and tumor-homing peptides can carry biologically active cargo to tumors or tumor vasculature. The challenge lies in developing the clinical application of therapeutic peptides. Improving delivery to tumors, minimizing non-specific toxic effects and discerning pharmacokinetic properties are high among the needs to produce a powerful therapeutic peptide for cancer treatment.

Cell cycling through Cdc25A: transducer of cytokine proliferative signals.

A balance between survival and proliferative signals maintains a constant number of T lymphocytes that populate the mammalian immune system, a process termed "homeostasis". Central to this process is the availability of a stromal cell product--the cytokine interleukin-7 (IL-7). We recently showed that IL-7, in addition to protecting cells from apoptosis, drives the cell cycling of lymphocytes through regulation of the stability of the phosphatase, Cdc25A, a key activator of cyclin-dependent kinases (cdks). IL-7 achieves this by controlling the activity of p38 MAP kinase (MAPK), which can phosphorylate Cdc25A, triggering its degradation. Sustained expression of Cdc25A had diverse effects: it promoted cell cycling, even in presence of cell cycle inhibitors such p27Kip1, and prevented cell shrinkage in response to cytokine deprivation. Herein we show a role for Cdc25A as a transducer of cytokine-driven proliferation and discuss novel implications for cell growth from the perspective of the requirements for maintenance of lymphocyte homeostasis.

Use of phosphorothioate-modified oligodeoxynucleotides to inhibit NF-kappaB expression and lymphocyte function.

NF-kappaB is a potential target for immunosuppressive therapy. Two methods were evaluated to inhibit NF-kappaB: the antisense (AS) approach in which single-stranded oligodeoxynucleotides (ODNs) bind the mRNA for the RelA subunit of NF-kappaB and the transcription factor decoy (TFD) approach in which double-stranded ODNs bind the NF-kappaB protein. AS and TFD inhibited NF-kappaB binding and decreased total IgG and anti-dsDNA antibody production in splenocytes from the BXSB/Yaa autoimmune mouse strain. TNF-alpha expression was reduced by AS and TFD, as were the levels of IL-2. But AS effects did not last beyond 24 h, whereas TFD inhibited cytokine production after 72 h. AS had no effect upon IL-6, while the TFD reduced the secretion of IL-6. Therefore, the suppression of immune response mediators by AS or TFD, through inhibition of NF-kappaB, is substantial. These inhibitors can serve as novel choices for therapy in the treatment of autoimmune disorders.

Functional consequences of the SHP-1 defect in motheaten viable mice: role of NF-kappa B.

To define the functional consequences of the src-homology domain-1 protein (SHP-1) defect, we examined cytokine production and NF-kappa B activity in motheaten viable (Mev) mice. We found elevated levels of interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor (TNF), and interferon-gamma (IFN-gamma) in Mev mice sera and cultured B and T cells compared to littermate control adult mice. The levels of interleukin-2 (IL-2) detected in Mev sera and activated Mev T cells were decreased, but IL-2 receptor expression was increased. We then evaluated the activity of NF-kappa B and found that this protein is highly expressed in Mev B and T cells. To determine if NF-kappa B had a role in causing the elevated levels of cytokines in Mev mice, we treated activated Mev T cells with an NF-kappa B decoy and found that cell culture treatment with the decoy resulted in significant reduction of the secretion of IL-6, GM-CSF, and TNF, but not IFN-gamma. Therefore, our data show that Mev mice secrete elevated levels of inflammatory cytokines, which can be mediators in the development of the Mev clinical disorder, and that NF-kappa B has an important role in this process, impacting upon the regulation of the immune response.

Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH.

IL-7 functions as a trophic factor during T lymphocyte development by a mechanism that is partly based on the induction of Bcl-2, which protects cells from apoptosis. Here we report a mechanism by which cytokine withdrawal activates the prodeath protein Bax. On loss of IL-7 in a dependent cell line, Bax protein translocated from the cytosol to the mitochondria, where it integrated into the mitochondrial membrane. This translocation was attributable to a conformational change in the Bax protein itself. We show that a rise in intracellular pH preceded mitochondrial translocation and triggered the change in Bax conformation. Intracellular pH in the IL-7-dependent cells rose steadily to peak over pH 7.8 by 6 hr after cytokine withdrawal, paralleling the time point of Bax translocation (a similar alkalinization and Bax translocation was also observed after IL-3 withdrawal from a dependent cell line). The conformation of Bax was directly altered by pH of 7.8 or higher and was demonstrated by increased protease sensitivity, exposure of N terminus epitopes, and exposure of a hydrophobic domain in the C terminus. Eliminating charged amino acids at the C or N termini of Bax induced a conformational change similar to that induced by raising pH, implicating these residues in the pH effect. Therefore, we have shown that by either cytokine withdrawal, experimental manipulation of pH, or site-directed mutagenesis, Bax protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the cascade of events leading to apoptotic death.

Inhibition of the p50 (NKkappaB1) subunit of NF-kappaB by phosphorothioate-modified antisense oligodeoxynucleotides reduces NF-kappaB expression and immunoglobulin synthesis in murine B cells.

NF-kappaB is a regulatory protein of immune response genes and a candidate for targeting in immunosuppressive therapy. NF-kappaB proteins are formed from components of which p50 (NFkappaB1) is a subunit. By targeting p50 gene expression with specific antisense 3' phosphorothioate-oligodeoxynucleotides (3' PS-ODNs), an effect upon NF-kappaB regulation and immunoglobulin synthesis in murine B cells was achieved. A 49% decrease in p50 protein was induced by treatment of WEHI 231 B cells with p50 antisense 3' PS-ODNs and not by control 3' PS-ODNs. p50 antisense specifically reduced the expression of NF-kappaB by 51%, but not the transcription factor, Oct-1. In the BXSB murine model of autoimmunity, p50 antisense inhibited NF-kappaB expression and total IgM and IgG synthesis, but, more importantly, dsDNA antibodies were reduced 90%. These results validate the use of p50 antisense to reduce NF-kappaB expression and, by downregulating the immune response, has application in the treatment of autoimmune disorders.

Bax translocation and mitochondrial fragmentation induced by Helicobacter pylori.

BACKGROUND AND AIMS: Previous in vitro and in vivo studies have revealed an association between Helicobacter pylori infection and apoptosis in gastric epithelial cells. Although involvement of the Bcl-2 family of proteins as well as cytochrome c release has been demonstrated in H pylori induced cell death, the exact role of the mitochondria during this type of programmed cell death has not been fully elucidated. Therefore, we sought to determine whether or not Bax translocation and mitochondrial fragmentation occur on exposure of gastric epithelial cells to H pylori, resulting in cell death. METHODS: Experiments were performed with human gastric adenocarcinoma (AGS) cells, AGS cells transfected with the HPV-E6 gene (which inactivates p53 function), AGS-neo cells (transfected with the backbone construct), mouse embryonic fibroblasts (MEFs), and p19(ARF) null (ARF(-/-)) MEFs. Cells were incubated with a cag positive H pylori strain for up to 24 hours, lysed, and cytoplasmic and mitochondrial membrane fractions were analysed by western blot for Bax translocation. RESULTS: Bax translocation was detected in AGS, AGS-neo, and normal MEF cells after exposure to H pylori for three hours, but not in ARF(-/-) MEFs cells. Translocation of Bax after H pylori incubation was also detected in AGS-E6 cells (inactive p53 gene) but to a lesser degree than in AGS-neo cells. In parallel studies, the mitochondrial morphology of living cells infected with H pylori was assessed by confocal microscopy. Mitochondrial fragmentation was detectable after 10 hours of H pylori incubation with AGS cells and after seven hours with MEF cells. In wild-type MEFs, mitochondrial fragmentation was significantly increased in comparison with ARF null MEFs (43% v 10.4%, respectively). Furthermore, mitochondrial depolarisation and caspase-3 activity were initiated within four hours in cells incubated with H pylori, and these events were inhibited by forced expression of Bcl-2. CONCLUSIONS: These data suggest that during H pylori induced apoptosis, Bax translocates to the mitochondria which subsequently undergo depolarisation and profound fragmentation. Functional ARF and p53 proteins may play an important role in H pylori induced mitochondrial modification.

Interleukin-3 withdrawal induces an early increase in mitochondrial membrane potential unrelated to the Bcl-2 family. Roles of intracellular pH, ADP transport, and F(0)F(1)-ATPase.

Cytokines such as interleukin-3 (IL-3) promote the survival of hematopoietic cells through mechanisms that are not well characterized. Withdrawal of IL-3 from an IL-3-dependent pro-B cell line induced early stress-related events that preceded cell death by more than 40 h. Intracellular pH rose above pH 7.8, peaking 2-3 h post-IL-3 withdrawal, and induced a transient increase in mitochondrial membrane potential (Delta Psi(m)) detected using several different dyes. Similar events were observed following IL-7 withdrawal from a different dependent cell line. Bcl-2, Bax, and caspases were unrelated to these early events. Intracellular alkaline pH inhibited the mitochondrial import of ADP, which would limit ATP synthesis. Total cellular ATP sharply declined during this early period, presumably as a consequence of suppressed ADP import. This was followed by an increase in reduced pyridine nucleotides. The transient increase in Delta Psi(m) was blocked by oligomycin, an inhibitor of F(0)F(1-)ATPase that may have undergone reversal caused by the abnormal ADP-ATP balance within mitochondria. These findings suggest a novel sequence of early events following trophic factor withdrawal in which alkaline pH inhibits ADP import into mitochondria, reversing the F(0)F(1-)ATPase, which in turn consumes ATP and pumps out protons, raising Delta Psi(m).

Gastrointestinal cells of IL-7 receptor null mice exhibit increased sensitivity to irradiation.

IL-7 is a critical cytokine in the development of T and B cells but little is known about its activity on nonhematopoietic cells. An unexpected finding was noted in allogeneic bone marrow transplant studies using IL-7 receptor null (IL-7R alpha(-/-)) mice as recipients. These mice exhibited a significantly greater weight loss after total body irradiation compared with wild type, IL-7R alpha(+/+), mice. Pathological assessment indicated greater intestinal crypt damage in IL-7R alpha(-/-) recipients, suggesting these mice may be predisposed to gut destruction. Therefore, we determined the effect of the conditioning itself on the intestinal tract of these mice. IL-7R alpha(-/-) mice and IL-7R alpha(+/+) mice were irradiated and examined for lesions and apoptosis within the small intestine. In moribund animals, IL-7R alpha(-/-) mice had extensive damage in the small intestine, including marked ablation of the crypts and extreme shortening of villi following 1500 cGy total body irradiation. In contrast, by 8 days after irradiation, the small intestines of IL-7R alpha(+/+) mice had regenerated as distinguished by normal villus length and hyperplastic crypts. Following 750 cGy irradiation, IL-7R alpha(-/-) mice had a higher proportion of apoptotic cells in the crypts and an accompanying increase in the pro-apoptotic protein Bak was expressed in intestinal epithelial cells. These results demonstrate the increased radiosensitivity of intestinal stem cells within the crypts in IL-7R alpha(-/-) mice and a role for IL-7 in the protection of radiation-induced apoptosis in these same cells. This study describes a novel role of IL-7 in nonhematopoietic tissues.