Membrane thickness design of implantable bio-MEMS sensors for the in-situ monitoring of blood flow.

This paper presents some ideas for the membrane thickness design of of implantable bio-micro-electro-mechanical systems (bio-MEMS) for the in situ monitoring of blood flow. The objective is to develop a smart wireless sensing unit for non-invasive early stenosis detection in heart bypass grafts. The design includes considerations of nonlinear material models, multiscale blood flows, and appropriate analyical models for data interpretation, as well as preliminary studies of the pressure and flow sensing concepts. The paper also examines the use of surface coatings for the design on biocompatibility and non-adhesion of blood platelets and constituents. The implications of the results are discussed for in vivo deployment of such sensor systems.

Anaerobic treatment of pre-hydrolysate liquor (PHL) from a rayon grade pulp mill: pilot and full-scale experience with UASB reactors.

Pilot studies were carried out for the treatment of pre-hydrolysate liquor (PHL), a high strength effluent (COD: 70,000-80,000 mg/l) emanating from a rayon grade pulp mill using up-flow anaerobic sludge blanket reactor (UASB). Substrate inhibition was avoided with optimum COD feed of around 25,000 mg/l. This was achieved by diluting the PHL with a low strength effluent stream known as alkali back wash (ABW) available in the plant and also by partially recycling the reactor liquid outlet. An optimum organic loading rate (OLR) of 10 could be achieved with a COD reduction of 70-75%, a BOD reduction of 85-90% and a methane yield of 0.31-0.33 m3/kg of COD reduced. The pilot scale studies also revealed that addition of milk of lime (MOL) was essential for neutralization and buffering and DAP and urea to supplement the nutrients in the PHL. Based on the pilot studies, a full-scale high rate biomethanation plant was designed and erected for treating the PHL, which after some modification showed similar performance for COD, BOD reduction and methane yield.

Interleukin-10 induces macrophage apoptosis and expression of CD16 (FcgammaRIII) whose engagement blocks the cell death programme and facilitates differentiation.

The development of monocytes into macrophages is regulated by helper T cells (Th) cells and, vice versa, the differentiation of Th cells into Th1 and Th2 is regulated by macrophages. Herein we examined the role of the Th2 cytokine, interleukin-10 (IL-10), on the development of macrophages. IL-10 is known to block the expression of antigen-presenting major histocompatibility complex (MHC) II and of costimulatory B7 molecules but it induces the expression of FcRs, especially the FcgammaRIII (CD16). The expression of CD16 enables the macrophage to carry out antibody-dependent cell-mediated cytotoxicity (ADCC) functions. However, this differentiation step is largely undercut by the capacity of IL-10 to induce macrophage apoptosis before the process of differentiation ensues. We found that the negative effect of IL-10 on the survival of macrophages is reversed in an environment that contains immunoglobulin G (IgG). IgG, especially when immune complexed with antigen, stimulates CD16 to transmit survival signals in macrophages which enable them to complete the differentiation process into CD16+ cells. Thus, IL-10 suppresses macrophage accumulation in healthy tissues where IgG is absent, and facilitates macrophage accumulation and differentiation in tissues that contain IgG, for example inflamed tissues or tissues that present autoreactive antibodies.

Biochemical characterization of osteo-testicular protein tyrosine phosphatase and its functional significance in rat primary osteoblasts.

Rat osteo-testicular protein tyrosine phosphatase (OST-PTP), expressed in osteoblasts and testis, is a receptor-like transmembrane protein with two tandemly repeated phosphatase domains in the cytoplasmic region. In this report, we show that the first domain (CD1) is enzymatically active and appears to be influenced by the catalytically inactive second domain (CD2). The activity of CD1 is specific to phosphorylated tyrosine. Full-length OST-PTP protein expressed in COS cells has a molecular mass of approximately 185 kDa, and immunoprecipitates of this protein using OST-PTP-specific antisera show strong tyrosine phosphatase activity. Expression of OST-PTP mRNA in primary rat calvarial osteoblasts is temporally regulated, and peak expression is found at approximately day 15, which correlated well with the appearance of OST-PTP protein and its associated tyrosine phosphatase activity. Treatment of osteoblasts in culture with antisense oligonucleotides directed against the 5' untranslated region of OST-PTP results in abrogation of differentiation, confirming the functional importance of OST-PTP expression in osteoblast development.

Polyspecific self-reactive antibodies in individuals infected with human immunodeficiency virus facilitate T cell deletion and inhibit costimulatory accessory cell function.

Self-reactive polyspecific IgG antibodies (PSAs) arise in human immunodeficiency virus (HIV)-seropositive subjects before they develop AIDS. Self-reactive PSA levels correlate with the destruction of CD8 T cells in HIV-infected individuals and mediate the antibody-dependent cellular toxicity-based destruction of human T cells in tissue culture. PSAs react across the species barrier and bind to T cell antigens in mice. Such reactivity with mouse lymphocytes was not detected in normal human serum. Injection of human PSA IgG causes massive T cell depletion in the spleen, lymph nodes, and thymus in mice: evidence that PSA IgG facilitates T cell destruction in vivo. In addition to facilitating macrophage cytotoxicity, self-reactive PSA IgG inhibits the macrophage-mediated activation of T cells with antigen receptor-specific monoclonal antibody or with antigen. Exogenous costimulatory stimuli or interleukin (IL)-12 can reverse the inhibition. In contrast, exogenous IL-10 mimics this inhibition. These data implicate PSA IgG as a pathogenic factor in the development of HIV disease.

Expression of estrogen receptor-beta protein in rodent ovary.

Estrogen is an essential hormone for the LH surge and ovulation. The primary source of estrogen is from ovarian granulosa cells and in rats, estrogen, in turn, increases granulosa cell number and enhances FSH-stimulated gene expression in these cells. Thus, rat granulosa cells both respond to and synthesize estrogen. To further elucidate the mechanisms mediating the actions of estrogen in granulosa cells, we have identified and characterized the estrogen receptor-beta (ER-beta) subtype in rodent granulosa cells. ER-beta protein was localized to the nuclei of rat granulosa cells in preantral and antral follicles by immunocytochemistry, coincident with the location of ER-beta messenger RNA (mRNA). Immunoprecipitation and Western blot analysis using ER-beta specific antisera demonstrated a protein of approximately 60 kDa in granulosa cells prepared from PMSG-primed immature mice and estrogen-treated immature rats. Extracts from granulosa cells specifically bound an estrogen response element and the complex was recognized by antisera to ER-beta. A synthetic steroid estrogen radioligand, [125I]-17alpha-iodovinyl-11beta-methoxyestradiol ([125I]-VME2), bound to cytosolic granulosa cell preparations with high affinity (estimated K(D) value of 401 +/- 83 pM, and Bmax value of 102 +/- 9 fmol/mg protein). ER-beta protein levels rapidly declined following hCG treatment consistent with the reported decrease in binding activity and ER-beta mRNA levels by high levels of gonadotropins. Overall, we have demonstrated that 1) ER-beta protein is the dominant estrogen receptor subtype present in rodent granulosa cells, 2) this receptor is functional, and 3) it is regulated by ovulatory doses of gonadotropins. Thus, ER-beta is likely to be a mediator of estrogen action in rodent granulosa cells during follicular development.

MHC class I molecules on CD4 T cells regulate receptor-mediated activation signals.

Three T cell populations can be distinguished based on their response to antigen receptor engagement. A sizable fraction dies within hours of TCR ligation, a smaller fraction enters the mitotic cycle, and the remaining T cells merely upregulate the expression of certain cell surface markers. An MHC-I-controlled regulatory mechanism has been identified. MHC I MAbs, or Fab fragments, prevent T cells from mounting a proliferative mitogen response but do not inhibit the mitogen-induced deletion of T cells. IFN-gamma enlarges the fraction of T cells which proliferate in response to mitogen stimulation but, in the presence of MHC I MAb, these cells fail to clonally expand and enter the deletion pathway. Phenotypically, MHC I MAb Fab fragments induce T cells to upregulate the expression of the apoptosis marker CD95, even in the absence of TCR ligand, and prevent the upregulation of costimulatory CD28 molecule expression.

Formulation of a generalized experimental model for a manually driven flywheel motor and its optimization.

A manually driven brick-making machine has recently been developed without the benefit of any design data. The machine consists of three main units: a pedal-driven flywheel motor; the transmission between the flywheel shaft and the input shaft of the process machine; and the process unit, consisting of auger, cone and die. The machine was essentially developed on the basis of general mechanical design experience and intuition. In spite of this, it proved to be functional and economically viable. However, it was felt essential to develop it scientifically. This paper reports on the full development of the pedal-driven flywheel motor. As this is a human-machine system it is highly unlikely that a logic-based model can be established. Therefore an experimental method is adopted to evolve a generalized experimental model, which is further optimized to satisfy several objective functions.

Pentostatin increases the acute toxicity of high dose cyclophosphamide.

One dose of pentostatin was added to a standard cyclophosphamide (CY) based transplant regimen in two patients in an attempt to decrease the rate of non-engraftment in haploidentical allogeneic BMT. Despite a normal cardiac history and evaluation prior to transplant, both patients suffered fatal cardiac toxicity within 48 h of receiving the chemotherapy. This phenomenon was further investigated in an animal model. Laboratory rats were treated with progressive doses of CY in a range that produces acute cardiac toxicity. Successive groups of rats were treated with either pentostatin or fludarabine and CY at 400 mg/kg. Neither pentostatin nor fludarabine alone produced early mortality. However, a marked increase in early mortality was noted in those animals treated with pentostatin and high-dose CY. The addition of fludarabine did not increase the early toxicity of CY. Autopsy revealed no gross or microscopic abnormalities in the animals. The implications of adding agents that interfere with adenosine metabolism to CY based transplant regimens is discussed.

Identification and some properties of a unique DNA polymerase from cells infected with human B-lymphotropic virus.

A new DNA polymerase and DNase activity were identified from cells infected with human B-lymphotropic herpesvirus (HBLV). DNA polymerase associated with HBLV infection was similar in its sensitivity to inhibition by ppi analogs as other herpesvirus-specific DNA polymerases but was dissimilar in its inhibition by certain nucleoside triphosphates.

Interaction of 2-halogenated dATP analogs (F, Cl, and Br) with human DNA polymerases, DNA primase, and ribonucleotide reductase.

Recently, 2-halogenated deoxyadenosine analogs (F, Cl, and Br) have been shown to have antitumor activity. These analogs are phosphorylated by cells and are believed to exert their cytotoxic action at the nucleoside triphosphate level. In this work the interaction of these nucleoside triphosphate analogs with potential targets, such as DNA polymerase alpha, beta, and gamma, DNA primase, and ribonucleotide reductase was examined in detail. All of these compounds competitively inhibited the incorporation of dAMP into DNA by DNA polymerase alpha, beta, or gamma. F-dATP was able to completely substitute for dATP using DNA polymerase alpha and gamma, but not with DNA polymerase beta. Cl-dATP and Br-dATP substituted poorly for dATP using DNA polymerase alpha and beta. Extension of a 32P-labeled primer by DNA polymerase alpha, beta, or gamma on a single-stranded M13 template showed that these compounds were incorporated into the 3' end of the growing DNA chain and that elongation beyond the incorporated analogs was significantly retarded for Cl-dATP and Br-dATP using either DNA polymerase alpha or beta. DNA primase using poly(dC) as template was inhibited by these compounds at a concentration 4 to 5 times greater than that required for 2-F-araATP. The 2-halogenated dATP analogs were potent inhibitors of ADP reduction by ribonucleotide reductase. In conclusion, the cytotoxic action of 2-Cl-deoxyadenosine and 2-Br-deoxyadenosine may partially be mediated through the mechanism of "self-potentiation," by depression of the deoxynucleoside triphosphate pools due to inhibition of ribonucleotide reductase, which would facilitate their incorporation into DNA and result in the inhibition of DNA synthesis.

Synthesis and interaction with uridine phosphorylase of 5'-deoxy-4',5-difluorouridine, a new prodrug of 5-fluorouracil.

5'-Deoxy-4',5-difluorouridine (4'-F-5'-dFUrd) (10) has been synthesized on the basis of the rationale that the labilization of the glycosidic linkage caused by the 4'-fluoro substituent might allow this compound to be a better prodrug form of the anticancer drug 5-fluorouracil (FUra) than is the widely studied fluoropyrimidine 5'-deoxy-5-fluorouridine (5'-dFUrd). The rate of solvolytic hydrolysis of the glycosidic linkage of 4'-F-5'-dFUrd at pH 1 was about 500-fold faster than that of 5'-dFUrd. Since uridine phosphorylase is thought to be the enzyme that causes degradation of 5'-dFUrd in vivo to generate FUra, we compared the substrate interactions of 5'-dFUrd and 4'-F-5'-dUrd with this enzyme. The Vmax for hydrolysis of 4'-F-5'-dFUrd to FUra by uridine phosphorylase was about 5-fold greater than that of 5'-dFUrd, whereas the Km value of 4'-F-5'-dFUrd was 10-fold lower. The combination of these two factors results in 4'-F-5'-dFUrd having a 50-fold higher value of V/K than does 5'-dFUrd. Against L1210 cells in culture, the IC50 value for growth inhibition by 4'-F-5'-dFUrd was 3 X 10(-7) compared to 3 X 10(-6) for 5'-dFUrd.

Study of ribonucleotide reductase in cells infected with six clinical isolates of herpes simplex virus type 2 (HSV-2) with mutations in its larger subunit.

Herpes simplex virus type 2 (HSV-2) induces a novel ribonucleotide reductase (RR) composed of two subunits (140 and 38 kDa) in infected cells. Other investigators have developed a monoclonal antibody, A6, against the 140-kDa subunit of RR and have found, in about 1% of the cases, an inability to detect this protein in cells infected with clinical isolates of HSV-2. We therefore investigated whether in such cases the clinical isolates were capable of inducing viral RR activity and whether the lack of detection of the 140-kDa protein by the monoclonal antibody was due to an alteration in the antigenic site of this protein. Six such isolates were examined and were found to induce RR activity, similar to HSV-2 (strain 333) RR, which did not require ATP for CDP reduction. Western blot analyses using A6 failed to detect the protein. However, R1, a polyclonal antibody raised against viral RR was capable of detecting this subunit. In addition, R1 was also capable of neutralizing RR activity induced by all the isolates and HSV-2 (strain 333). In conclusion, the lack of detection of the large subunit of RR was not due to the lack of induction but was due to an alteration in the antigenic site recognized by A6; this alteration did not appear to affect the properties of the induced RR activity.

Studies on DNA topoisomerases I and II in herpes simplex virus type 2-infected cells.

It has been suggested that herpes simplex virus (HSV) type 1 may induce a virus-specific DNA topoisomerase activity which copurifies with virus-induced DNA polymerase. We have examined DNA topoisomerase (TOPO) I and II activities in HSV-2-infected HeLa S3 cells. Both activities were partially purified using DEAE-cellulose, phosphocellulose and double-stranded DNA cellulose column chromatography. It was found that both activities could be separated from HSV-2-specific DNA polymerase. Throughout the purification TOPO I could be immunologically detected with a monoclonal antibody developed against human TOPO I. Regardless of the source, mock- or HSV-2-infected human cells, both types of topoisomerase were equally tolerant of 200 mM-KCl. There appeared to be no apparent heterogeneity of TOPO I in HeLa S3 cells through the course of the HSV-2 infection. We conclude that host cell topoisomerases are quite stable in HSV-2-infected HeLa S3 cells and that there is no evidence that HSV-2 is capable of inducing HSV-2-specific TOPO I and TOPO II activities.

Synthesis and biological activity of 5-fluoro-2',3'-dideoxy-3'-fluorouridine and its 5'-phosphate.

5-Fluoro-2',3'-dideoxy-3'-fluorouridine (3'-FFdUrd) and 5-fluoro-2',3'-dideoxy-3'-fluorouridine 5'-phosphate (3'-FFdUMP) have been synthesized, and their interactions with thymidine (dThd) phosphorylase and thymidylate (dTMP) synthetase, respectively, have been examined. 3'-FFdUrd is not a substrate for dThd phosphorylase, but is a weak, noncompetitive inhibitor (Ki = 1.7 mM). 3'-FFdUMP inhibits dTMP synthetase competitively with deoxyuridylate (Ki = 0.13 mM) when both the substrate and inhibitor are present simultaneously. However, in the presence of 5,10-methylenetetrahydrofolate, the inhibition increases with time in a first-order manner (konobsd = 0.029 s-1). A complex is formed between [6-3H]3'-FFdUMP and dTMP synthetase, which is isolable on nitrocellulose filters, and has a dissociation rate (koffobsd = 1.4 X 10(-2) min-1) similar to that of the potent inhibitor 5-fluoro-2'-deoxyuridylate (koffobsd = 1.3 X 10(-2) min-1) from its ternary complex with dTMP synthetase. These results are explained in terms of a two-stage model involving the initial formation of a reversible adsorption complex, followed by a slow conversion to a tight-binding catalytic complex.

Human leukemic cells resistant to 5-fluoro-2'-deoxyuridine contain a thymidylate synthetase with lower affinity for nucleotides.

A line of human lymphocytic leukemia cells (CCRF-CEM) has been obtained which is 140-fold resistant to the potent cell growth inhibitor 5-fluoro-2'-deoxyuridine (FdUrd). The cells were also 11-fold cross-resistant to 5-fluorouracil. In contrast to several previous studies involving FdUrd-resistant mouse cells, thymidylate synthetase levels were not substantially elevated in these FdUrd-resistant human leukemic cells. Thymidine kinase activity was also unchanged in the resistant cells, although the levels of 5-fluoro-2'-deoxyuridylate (FdUMP), the potent inhibitor of thymidylate synthetase, generated at equimolar doses of FdUrd were about 40% lower than in the sensitive cells. Studies of the kinetics of FdUMP binding to thymidylate synthetase isolated from the FdUrd-resistant cells disclosed a considerably higher dissociation constant (Kd = 1.0 X 10(-9) M) for the ternary covalent enzyme . FdUMP . 5,10-methylene tetrahydrofolate complex compared to the value obtained with enzyme from sensitive cells (Kd = 4.4 X 10(-11) M). The thymidylate synthetase from the FdUrd-resistant cells also showed 17-fold weaker binding of 2'-deoxyuridylate, even though the Km value for 2'-deoxyuridylate was 3-fold lower compared to the enzyme from FdUrd-sensitive cells. The turnover number of the altered enzyme was 1.8-fold higher than that for the normal enzyme but the rate constants for the release of FdUMP from the ternary complex, which is also an enzyme-catalyzed reaction, were identical for both enzymes. Electrophoresis of the radiolabeled ternary complexes on nondenaturing gels showed small but reproducible differences in migration rates. These results demonstrate that the mechanism of resistance to FdUrd in this cell line involves an alteration in the target enzyme, thymidylate synthetase, which causes it have a lower affinity for nucleotides.