Liposome-encapsulated superoxide dismutase suppresses liposome-mediated augmentation of TNF-alpha production from peripheral blood leucocytes

Liposome-encapsulated hemoglobin (LEH), a candidate for a red cell substitute, has been reported to be cleared from circulation primarily by the phagocytic system and modulate the production of inflammatory cytokines, such as TNF-alpha and IL-6, both in vivo and in vitro. In this study, we investigated the effects of liposome vesicles on the LPS-induced TNF-alpha production using a whole blood culture system. We also studied the effects of superoxide dismutase (SOD) encapsulated in liposome on the cytokine production. The pre-treatment of whole blood with liposome vesicles potentiated the LPS-induced TNF-alpha production. The encapsulation of SOD in the liposome vesicles suppressed the liposome-mediated augmentation of TNF-alpha production in a dose-dependent manner. These results suggest that encapsulation of SOD in LEH decreases the production of inflammatory cytokines from the phagocytic system which may be caused or augmented by LEH infusion in vivo.     

Constructing a local district telepathology network in Japan. Diagnosis of intraoperative frozen sections via telepathology over an integrated service digital network and the National Television Standard Committee System

OBJECTIVE: To construct a local telepathology network between the Department of Pathology, Tohoku University Hospital, and Koritu Kesennuma Hospital, about 150 km away. STUDY DESIGN: Tohoku University Hospital is connected with Koritu Kesennuma Hospital by an integrated service digital network for telepathology using the National Television Standard Committee system. The cases submitted for telepathology were limited to those in which a rapid intraoperative diagnosis was made on frozen sections. RESULTS: At this writing, more than 200 cases were diagnosed during a period of 2.5 years. The cases submitted increased with time, amounting to 150 in 1996. In some cases the use of telepathology proved to be fairly advantageous. For example, in one case a radical operation was avoided because of a diagnosis on intraoperative frozen sections. DISCUSSION: There are problems to be solved before telepathology becomes available for practical use: (1) misdiagnosis due to poor quality of instruments, including the transmission cable and pictures; (2) cost-benefit ratio, (3) protection of patients' privacy, and (4) overwork for pathologists. The Japanese government will officially accept telepathology as a means of medical examination in the future. Despite some problems left, telepathology is a promising technology.     

Inactivation of mitogen-activated protein kinases by a mammalian tyrosine-specific phosphatase, PTPBR7

Mitogen-activated protein kinase (MAPK) is inactivated through dephosphorylation of tyrosyl and threonyl regulatory sites. In yeast, both dual-specificity and tyrosine-specific phosphatases are involved in dephosphorylation. In mammals, however, no tyrosine-specific phosphatase has been identified molecularly to dephosphorylate MAPK in vivo. Recently, we and others have cloned a murine tyrosine-specific phosphatase, PTPBR7/PTP-SL, which is expressed predominantly in the brain. Here we report inactivation of the extracellular signal-regulated kinase (ERK) family MAPK by PTPBR7. PTPBR7 made complexes with ERK1/ERK2 in vivo and dephosphorylated ERK1 in vitro. When overexpressed in mammalian cells, wild-type PTPBR7 suppressed the phosphorylation and activation of ERK by epidermal growth factor (EGF), nerve growth factor (NGF), and constitutively active MEK1, a mutant MAPK kinase. In contrast, catalytically inactive and ERK-binding-deficient mutants revealed little inhibition on the ERK cascade. These results indicate that PTPBR7 suppresses MAPK directly in vivo.     

Binding kinetics of engineered mutants provide insight about the pathway for entering and exiting the intestinal fatty acid binding protein

To better understand the mechanism by which fatty acids bind to and dissociate from the binding cavities of fatty acid binding proteins (FABPs), we constructed 31 single amino acid mutants of the intestinal FABP (I-FABP) and determined the rate constants for binding and dissociation, primarily for long-chain fatty acids (FA). FA dissociation from these proteins was measured both by the ADIFAB method and by the change in tryptophan fluorescence of the FABPs. Rate constants for binding (kon) were calculated from the rate constants for dissociation (koff) and the equilibrium binding affinities. Amino acid substitutions were made at locations within the binding cavity, in the region of the gap between the betaD- and betaE-strands, and within the "portal" region of the protein. The koff values for the mutant proteins ranged from about 20-fold slower to 4-fold faster than the wild-type (WT) protein. Values for kon were as much as 20-fold slower than the WT protein, but in no case was kon significantly faster than the WT. Mutants with slower and faster koff values were generally those involving sites within the binding cavity and, relative to the WT protein, revealed higher and lower affinities, respectively. Reduced rates of binding were generally, but not exclusively, associated with sites within the portal region. For example, for F68A which is located closer to the opposite end of the protein from the portal region, the kon is more than 10-fold slower than WT. Even for these distal sites, however, the evidence is consistent with reductions in kon being due to alterations of the portal region. Binding affinities and rate constants measured as a function of ionic strength also suggest that the FA initially binds, through an electrostatic interaction, to Arg-56 on the surface of the protein, before inserting into the binding cavity. Thus, the results of this study are consistent with FA binding to I-FABP involving an initial interaction with Arg-56 followed by insertion of the FA, through the portal region, into the binding cavity and with a reversal of these steps for the dissociation reaction.     

Absorption of acylated anthocyanins in rats and humans after ingesting an extract of Ipomoea batatas purple sweet potato tuber

We evaluated the absorbability of anthocyanins in humans and rats administered with a beverage prepared from an extract of the tuber of purple sweet potato (Ipomoea batatas Cultivar Ayamurasaki), or with an anthocyanin concentrate. Two major anthocyanin components, cyanidin 3-O-(2-O-(6-O-(E)-caffeoyl-beta-D-glucopyranosyl)-beta-D-glucopyranoside)-5-O-beta-D-glucopyranoside) and peonidin 3-O-(2-O-(6-O-(E)-caffeoyl-beta-D-glucopyranosyl)-beta-D-glucopyranoside)-5-O-beta-D-glucopyranoside), were detected in the plasma and urine of both rats and humans by HPLC or liquid chromatography/mass spectrometry (LC/MS). The plasma concentration of anthocyanins in humans reached a maximum 90 minutes after ingestion, and the recovery of anthocyanins in the urine was estimated as 0.01-0.03%. These results indicate that acylated anthocyanins could be selectively absorbed after ingesting food.     

Crystallization and MAD data collection of high-molecular weight cytochrome c from Desulfovibrio vulgaris Miyazaki F

Hexadecaheme high molecular weight cytochrome c from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F has been successfully purified and crystallized. X-ray diffraction data have been collected by the multiple wavelength anomalous dispersion method. The crystal belongs to the space group P2(1)2(1)2(1) with unit-cell parameters a=60.42, b=84.29 and c=144.16 A and contains one molecule per asymmetric unit.     

Evidence that exogenous substances can be phagocytized by alveolar epithelial cells and transported into blood capillaries

Since the ability of alveolar epithelial cells to ingest inhaled fine particles has not been characterized in detail, the present study seeks to evaluate this physiological activity. We used a 0.2% suspension of intact or lecithin-coated polystyrene latex beads (240 nm in diameter). A 5-ml suspension of intact or lecithin-coated latex beads was intratracheally administered to rats using a compressor nebulizer. Thereafter, the lungs were perfused intratracheally with glutaraldehyde solution and cut into small pieces. The samples were postfixed with osmium tetroxide, embedded in epoxy resin and examined under an electron microscope. Both lecithin-coated and uncoated beads were incorporated into alveolar macrophages. Some of the ingested beads in the alveolar macrophages were sequestered within lysosomes. Types I and II alveolar epithelial cells selectively incorporated only lecithin-coated beads, which were also observed within the cytoplasm of monocytes in the capillary lumen. These findings suggest that alveolar epithelial cells can incorporate exogenous particles, which are then transferred from the alveoli to intravascular spaces by transcytosis.     

Influence of interleukin-12 receptor β1 polymorphisms on tuberculosis

Host genetic factors may be important determinants of susceptibility to tuberculosis, and several candidate gene polymorphisms have been shown to date. A series of recent reports concerning rare human deficiencies in the type-1 cytokine pathway suggest that more subtle variants of relevant genes may also contribute to susceptibility to tuberculosis at the general population level. To investigate whether polymorphisms in the interleukin-12 receptor (IL-12R) gene predispose individuals to tuberculosis, we studied these genes by single-strand conformational polymorphism analysis and direct sequencing. Although no common polymorphisms could be identified in the IL-12R beta 2 gene ( IL-12RB2), we confirmed four single nucleotide polymorphisms (SNPs; 641A-->G, 684C-->T, 1094T-->C, and 1132G-->C) causing three missense variants (Q214R, M365T, G378R) and one synonymous substitution in the extracellular domain of the IL-12R beta 1 gene ( IL12RB1). All SNPs were in almost perfect linkage disequilibrium (D'=0.98), and two common haplotypes of IL12RB1(allele 1: Q214-M365-G378; allele 2: R214-T365-R378) were revealed. Polymerase chain reaction/restriction fragment length polymorphism and sequence analyses were used to type IL12RB1polymorphisms in 98 patients with tuberculosis and 197 healthy controls in Japanese populations. In our case-control association study of tuberculosis, the R214-T365-R378 allele (allele 2) was over-represented in patients with tuberculosis, and homozygosity for R214-T365-R378 (the 2/2 genotype) was significantly associated with tuberculosis (odds ratio: 2.45; 95% CI: 1.20-4.99; P=0.013). In healthy subjects, homozygotes for R214-T365-R378 had lower levels of IL-12-induced signaling, according to differences in cellular responses to IL-12 between two haplotypes. These data suggest that the R214-T365-R378 allele, i.e., variation in IL12RB1, contribute to tuberculosis susceptibility in the Japanese population. This genetic variation may predispose individuals to tuberculosis infection by diminishing receptor responsiveness to IL-12 and to IL-23, leading to partial dysfunction of interferon-gamma-mediated immunity.     

Expression and characterization of the C345C/NTR domains of complement components C3 and C5

Complement components C3, C4, and C5 are members of the thioester-containing alpha-macroglobulin protein superfamily. Within this superfamily, a unique feature of the complement proteins is a 150-residue-long C-terminal extension of their alpha-subunits that harbors three internal disulfide bonds. Previous reports have suggested that this is an independent structural module, homologous to modules found in other proteins, including netrins and tissue inhibitors of metalloproteinases. Because of its distribution, this putative module has been named both C345C and NTR. To assess the structures of these segments of the complement proteins, their relationships with other domains, and activities as independent structures, we expressed C345C from C3 and C5 in a bacterial strain that permits cytoplasmic disulfide bond formation. Affinity purification directly from cell lysates yielded recombinant C3- and C5-C345C with properties consistent with multiple intramolecular disulfide bonds and high beta-sheet contents. rC5-, but not rC3-C345C inhibited complement hemolytic activity, and surface plasmon resonance studies revealed that rC5-C345C binds to complement components C6 and C7 with dissociation constants of 10 and 3 nM, respectively. Our results provide strong evidence that this binding corresponds to the previously described reversible binding of C5 to C6 and C7, and taken together with earlier work, indicate that the C5-C345C module interacts directly with the factor I modules in C6 and C7. The high binding affinities suggest that complexes composed of C5 bound to C6 or C7 exist in plasma before activation and may facilitate assembly of the complement membrane attack complex.