Purification and characterization of a cytosolic, 42-kDa and Ca2+-dependent phospholipase A2 from bovine red blood cells: its involvement in Ca2+-dependent release of arachidonic acid from mammalian red blood cells

It has become evident that a Ca(2+)-dependent release of arachidonic acid (AA) and subsequent formation of bioactive lipid mediators such as prostaglandins and leukotrienes in red blood cells (RBCs) can modify physiological functions of neighboring RBCs and platelets. Here we identified a novel type of cytosolic PLA(2) in bovine and human RBCs and purified it to apparent homogeneity with a 14,000-fold purification. The purified enzyme, termed rPLA(2), has a molecular mass of 42 kDa and reveals biochemical properties similar to group IV cPLA(2), but shows different profiles from cPLA(2) in several column chromatographies. Moreover, rPLA(2) did not react with any of anti-cPLA(2) and anti-sPLA(2) antibodies and was identified as an unknown protein in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Divalent metal ions tested exhibited similar effects between rPLA(2) and cPLA(2), whereas mercurials inhibited cPLA(2) but had no effect on rPLA(2). Antibody against the 42-kDa protein not only precipitated the rPLA(2) activity, but also reacted with the 42-kDa protein from bovine and human RBCs in immunoblot analysis. The 42-kDa protein band was selectively detected in murine fetal liver cells known as a type of progenitor cells of RBCs. It was found that EA4, a derivative of quinone newly developed as an inhibitor for rPLA(2), inhibited a Ca(2+) ionophore-induced AA release from human and bovine RBCs, indicating that this enzyme is responsible for the Ca(2+)-dependent AA release from mammalian RBCs. Finally, erythroid progenitor cell assay utilizing diaminobenzidine staining of hemoglobinized fetal liver cells showed that rPLA(2) detectable in erythroid cells was down-regulated when differentiated to non-erythroid cells. Together, our results suggest that the 42-kDa rPLA(2) identified as a novel form of Ca(2+)-dependent PLA(2) may play an important role in hemostasis, thrombosis, and/or erythropoiesis through the Ca(2+)-dependent release of AA.     

Allosteric effects potentiating the release of the second fibrinopeptide A from fibrinogen by thrombin

Fibrin formation depends on the release of the two N-terminal fibrinopeptides A (FPA) from fibrinogen, and its formation is accompanied by an intermediate, alpha-profibrin, which lacks only one of the FPA. In this study, we confirm that the maximal levels of alpha-profibrin found over the course of thrombin reactions with human fibrinogen are only half of what would be expected if the first and second FPA were being released independently with equal rate constants. The rapidity of release of the fibrinopeptides by thrombin had been shown to depend on an allosteric transformation that is induced when Na(+) binds to a site defined by the 215-227 residues of thrombin, a transformation that results in the exposure of its fibrinogen-binding exosites transforming the thrombin from a slow to a fast acting form toward fibrinogen. When choline was substituted for sodium to transform thrombin to its slow form, the maximal levels of alpha-profibrin rose to those expected for independent release of the two FPA. Thus, it is only the fast thrombin that releases the second FPA fast, and that fast release only occurs when both FPA are present because of a partial coupling of its release with that of the first FPA. The release of the FPA from purified alpha-profibrin with the first FPA already missing is no faster than the release of any FPA. Surprisingly, we also found that slow thrombin became increasingly transformed to a fast form in the absence of sodium when the fibrinogen was elevated to high concentrations. This potentiation by concentrated fibrinogen also occurs with the recombinant mutant thrombin (Y225P), which is otherwise slow in both the presence and absence of Na(+). The potentiation of thrombin by fibrinogen must be short-lived so that the thrombin reverts to its slow acting form in the interim among encounters with other fibrinogen molecules in dilute fibrinogen solutions lacking Na(+), whereas at high fibrinogen concentrations the thrombin encounters other molecules before it reverts back to the slow form.     

Characterization of a highly pathogenic H5N1 avian influenza A virus isolated from duck meat

Since the 1997 H5N1 influenza virus outbreak in humans and poultry in Hong Kong, the emergence of closely related viruses in poultry has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur. In May 2001, an avian H5N1 influenza A virus was isolated from duck meat that had been imported to South Korea from China. Phylogenetic analysis of the hemagglutinin (HA) gene of A/Duck/Anyang/AVL-1/01 showed that the virus clustered with the H5 Goose/Guandong/1/96 lineage and 1997 Hong Kong human isolates and possessed an HA cleavage site sequence identical to these isolates. Following intravenous or intranasal inoculation, this virus was highly pathogenic and replicated to high titers in chickens. The pathogenesis of DK/Anyang/AVL-1/01 virus in Pekin ducks was further characterized and compared with a recent H5N1 isolate, A/Chicken/Hong Kong/317.5/01, and an H5N1 1997 chicken isolate, A/Chicken/Hong Kong/220/97. Although no clinical signs of disease were observed in H5N1 virus-inoculated ducks, infectious virus could be detected in lung tissue, cloacal, and oropharyngeal swabs. The DK/Anyang/AVL-1/01 virus was unique among the H5N1 isolates in that infectious virus and viral antigen could also be detected in muscle and brain tissue of ducks. The pathogenesis of DK/Anyang/AVL-1/01 virus was characterized in BALB/c mice and compared with the other H5N1 isolates. All viruses replicated in mice, but in contrast to the highly lethal CK/HK/220/97 virus, DK/Anyang/AVL-1/01 and CK/HK/317.5/01 viruses remained localized to the respiratory tract. DK/Anyang/AVL-1/01 virus caused weight loss and resulted in 22 to 33% mortality, whereas CK/HK/317.5/01-infected mice exhibited no morbidity or mortality. The isolation of a highly pathogenic H5N1 influenza virus from poultry indicates that such viruses are still circulating in China and may present a risk for transmission of the virus to humans.     

ProNGF induces p75-mediated death of oligodendrocytes following spinal cord injury

The neurotrophin receptor p75 is induced by various injuries to the nervous system, but its role after injury has remained unclear. Here, we report that p75 is required for the death of oligodendrocytes following spinal cord injury, and its action is mediated mainly by proNGF. Oligodendrocytes undergoing apoptosis expressed p75, and the absence of p75 resulted in a decrease in the number of apoptotic oligodendrocytes and increased survival of oligodendrocytes. ProNGF is likely responsible for activating p75 in vivo, since the proNGF from the injured spinal cord induced apoptosis among p75(+/+), but not among p75(-/-), oligodendrocytes in culture, and its action was blocked by proNGF-specific antibody. Together, these data suggest that the role of proNGF is to eliminate damaged cells by activating the apoptotic machinery of p75 after injury.     

Endophytic bacillus sp. isolated from the interior of balloon flower root

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C15:0 (37%), C17:0 (5.1%), and iso-C15:0 (27.7%). DNA G+C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.     

Proinflammatory cytokine profile in Vibrio vulnificus septicemic patients' sera

Vibrio vulnificus causes a fulminant and frequently fatal septicemia in susceptible hosts. The present study was designed to evaluate the proinflammatory cytokine profile in V. vulnificus septicemia patients' sera and the effect of doxycycline therapy on the levels of proinflammatory cytokines. Levels of proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, were measured in the sera of V. vulnificus septicemic patients and normal healthy volunteers using colorimetric sandwich ELISA. The mean values of TNF-alpha, IL-1beta and IL-6 in the sera of V. vulnificus patients (n=33) increased by 210-, 232- and 40-fold in comparison with those of normal healthy volunteers (n=5), but only the IL-6 level showed a statistically significant difference (P<0.05) between the two groups. Sera from the cases for which doxycycline treatment histories were obvious were designated 'before-treatment' (TX). All the others were included in the after-TX group. In the before-TX group (n=5), the levels of TNF-alpha and IL-1beta significantly increased (P<0.05) in comparison with the after-TX group (n=5). IL-6 levels in the two groups showed no difference. In conclusion, the levels of the well known proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 increased in the V. vulnificus septicemic patients' sera, and the levels of TNF-alpha and IL-1beta decreased significantly after doxycycline treatment. These data indicate that proinflammatory cytokines might play a critical role in V. vulnificus septicemia like in other endotoxemic shocks. The use of doxycycline as an effective bactericidal agent and as an effective modulator of proinflammatory cytokines is supported.     

Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery

A novel water-soluble lipopolymer was synthesized by linking cholesteryl chloroformate to the secondary amino groups of branched poly(ethylenimine) (PEI) of 1,800 and 10,000 Da. Conjugation through PEI secondary amines gives this newly synthesized lipopolymer (abbreviated as PEI-Chol) special advantage over our previously synthesized lipopolymers, which utilized the primary amino groups for conjugation, as the primary amino groups have a significant role in DNA condensation. Also, significantly, only one cholesterol molecule was grafted onto each PEI molecule (confirmed by (1)H NMR and MALDI-TOF mass spectrometry), leaving enough space for the steric interactions of the PEI's primary amines with the DNA. The PEI-Chol lipopolymer was characterized for the critical micellar concentration (cmc), buffer capacity, DNA condensation (by band retardation and circular dichroism), in vitro transfection efficiency, and cell viability. The cmcs of PEI-Chol 1,800 and PEI-Chol 10,000 were 496.6 and 1,330.5 microg/mL, respectively. The acid-base titration indicated high buffering capacity of the polymers around the pH range of 5-7, which indicated their potential for buffering in the acidic pH environment of the endosomes. The band retardation studies indicated that efficient condensation of the plasmid DNA could be achieved using these lipopolymers. The circular dichroism spectra indicated a change in DNA conformation and adoption of lower energy state upon condensation with these lipopolymers when an N/P ratio of 2.5/1 or above was formulated. The mean particle size of these complexes was in the range 110-205 nm, except for the complexes prepared using PEI of 1,800 Da, which had a mean particle size of 384 +/- 300 nm. The zeta potential of DNA complexes prepared using PEI-Chol 1,800, PEI-Chol 10,000 and PEI of 1,800, 10,000, and 25,000 Da at an N/P ratio of 15/1 was in the range 23-30 mV and was dependent on the N/P ratios. The in vitro transfection of PEI-Chol/pCMS-EGFP complexes in Jurkat cells showed high levels of expressed Green Fluorescent Protein (GFP) with little toxicity as determined by flow cytometry. These novel water-soluble lipopolymers provided good transfection efficiency with other desirable characteristics such as water solubility, free primary amino groups for efficient DNA condensation and high buffer capacity that indicated the possibility of efficient endosomal release.     

A functional proteomic analysis of secreted fibrinolytic enzymes from Bacillus subtilis 168 using a combined method of two-dimensional gel electrophoresis and zymography

Here we describe a proteomic approach to detect fibrinolytic enzymes from the culture supernatant of Bacillus subtilis 168. Following isoelectric focusing without dithiothreitol, two gels, one for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the other for zymography, were run in parallel. After silver staining of SDS-PAGE and activity staining of zymography gel, the two gels were superimposed to detect protein spots that coincided with clear zones on the zymography gel. We identified four protein spots and characterized them with matrix-assisted laser desorption/ionization mass spectrometry. Database search revealed that four spots contained at least one of the extracellular serine proteases such as WprA and Vpr. This combined method of two-dimensional gel and zymography can be used as a powerful tool to detect proteases from various organisms.     

Reaction equilibrium of penicillin G with amberlite LA-2 in a nonpolar organic solvent

Studies have been made of the reactive extraction of penicillin G by Amberlite LA-2, a secondary amine, dissolved in kerosene. On the basis of the previous works about extraction equilibria of monocarboxylic acids by some secondary amines in low polar organic solvents, four equilibrium models were suggested to describe the reaction equilibrium of penicillin G in the liquid-liquid extraction system. The calculated results from the models were compared with the experimental data of 96 runs, and only two equilibrium models seemed to be probable. Ultimately, the most reasonable extraction equilibrium model was chosen through spectroscopic studies on organic solutions obtained by five specific extraction equilibrium experiments.     

Medicinal herb extracts inhibit growth of<Emphasis Type="Italic">Rhabditis elongate</Emphasis> (Nematoda:<Emphasis Type="Italic">Rhabditidae</Emphasis>)

We tested the influence of extracts from three medicinal herbs —Salvia miltiorrhiza, Schizandra chinensis, andEugenia caryophyllata — on activity of the nematodeRhabditis elongate. Treatment with f.caryophyllata was most useful, causing the greatest decrease in populations and mobility, but did not have any detrimental effect on the initial growth of the host microorganism,Escherichia coli. For example, when 0.5 g/L of the extract was added to an inoculated liquid culture, we counted 710 nematodes/mL, with a multiplication rate 5 times greater than the initial population. This was in contrast to the control sample, which had a count of 1100 nematodes/mL and a growth ratio of 11. For our field test, nematode mobility in the presence of the extract also decreased, to 6.8 mm/day, compared with 20 mm/day for the control. Likewise, when 1.0 g/L of the extract was added to the soil, the total number of nematodes was reduced to only 30- to 40% of the control population.