Spirulan from Blue‐Green Algae Inhibits Fibrin and Blood Clots: Its Potent Antithrombotic Effects

We investigated in vitro and in vivo fibrinolytic and antithrombotic activity of spirulan and analyzed its partial biochemical properties. Spirulan, a sulfated polysaccharide from the blue‐green alga Arthrospira platensis, exhibits antithrombotic potency. Spirulan showed a strong fibrin zymogram lysis band corresponding to its molecular mass. It specifically cleaved Aα and Bβ, the major chains of fibrinogen. Spirulan directly decreased the activity of thrombin and factor X activated (FXa), procoagulant proteins. In vitro assays using human fibrin and mouse blood clots showed fibrinolytic and hemolytic activities of spirulan. Spirulan (2 mg/kg) showed antithrombotic effects in the ferric chloride (FeCl₃)‐induced carotid arterial thrombus model and collagen and epinephrine‐induced pulmonary thromboembolism mouse model. These results may be attributable to the prevention of thrombus formation and partial lysis of thrombus. Therefore, we suggest that spirulan may be a potential antithrombotic agent for thrombosis‐related diseases.     

A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium

An anaerobic microorganism termed AN-C16-KBRB was isolated from the bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The 16S rRNA gene of the strain was 98% similar to that of Clostridiaceae bacterium SK082 (AB298754) as the highest homology. A novel celEdx16 gene encoding a bifunctional endo-/exocellulase (CelEdx16) was cloned by the shotgun method from AN-C16-KBRB, and the enzyme was characterized. The celEdx16 gene had an open reading frame of 1,104-base pairs, which encoded 367 amino acids to yield a protein of molecular mass 40.4 kDa. The amino acid sequence was 53% identical to that of an endoglucanase from Clostridium thermocellum. CelEdx16 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific endocellulase and exocellulase activities of CelEdx16 were 15.9 and 3.6 × 10⁻² U mg⁻¹, respectively. The Michaelis–Menten constant (K _m values) and the maximal reaction velocities (V _max values) of CelEdx16 were 47.1 μM and 9.6 × 10⁻³ μmole min⁻¹ when endocellulase activity was measured and 106.3 μM and 2.1 × 10⁻⁵ μmole min⁻¹ when exocellulase activity was assessed. CelEdx16 was optimally active at pH 5.0 and 40°C.     

Reaction products of a new anti-cancer agent, Pt(IV)(cyclohexyldiamine)Cl4, with guanosine and 9-methylguanine: first crystal structures of Pt(cyclohexyldiamine) complexes with a nucleobase and a nucleoside

Pt(IV)(dach)Cl4 (dach = cyclohexyldiamine) was reacted with guanosine and 9-methylguanine and their reaction products were analyzed by single-crystal x-ray diffraction. In both cases the resulting complexes, [Pt(dach)(guanosine)2]2+ and [Pt(dach)(9-methylguanine)2]2+ respectively, corresponded to an unanticipated reduction of the octahedral Pt(IV) starting material to a square planar Pt(II) species. The nature of the reducing agent is presently unknown.     

The role of YKL-40 in the pathogenesis of autoimmune diseases: a comprehensive review

YKL-40, a chitinase-3-like protein 1 (CHI3L1) or human cartilage glycoprotein 39 (HC gp-39), is expressed and secreted by various cell-types including macrophages, chondrocytes, fibroblast-like synovial cells and vascular smooth muscle cells. Its biological function is not well elucidated, but it is speculated to have some connection with inflammatory reactions and autoimmune diseases. Although having important biological roles in autoimmunity, there were only attempts to elucidate relationships of YKL-40 with a single or couple of diseases in the literature. Therefore, in order to analyze the relationship between YKL-40 and the overall diseases, we reviewed 51 articles that discussed the association of YKL-40 with rheumatoid arthritis, psoriasis, systemic lupus erythematosus, Behçet disease and inflammatory bowel disease. Several studies showed that YKL-40 could be assumed as a marker for disease diagnosis, prognosis, disease activity and severity. It is also shown to be involved in response to disease treatment. However, other studies showed controversial results particularly in the case of Behçet disease activity. Therefore, further studies are needed to elucidate the exact role of YKL-40 in autoimmunity and to investigate its potential in therapeutics.     

Chromatofocusing of mammalian estrone sulfate sulfohydrolase activity

Estrone sulfate sulfohydrolase (estrogen sulfatase) activity was solubilized by treatment with Triton X-100 from 105,000 g pellets of guinea pig uterus, testis and brain, as well as from rat liver and human placenta. The solubilized forms were subjected to chromatofocusing in the fast protein liquid chromatography (FPLC) system and on conventional columns packed in our laboratory. The guinea pig tissue pattern was complex. Uterus showed peaks of activity with apparent pI's of 9.11 and 7.6; testis contained 3 peaks with pI's of 9.18, 8.7 and 7.5; brain possessed peaks with pI's of 9.28 and 8.6. In each case the major activity peak was that with pI greater than 9. Rat liver activity chromatofocused as a single peak of apparent pI = 6.87 and the human placental enzyme also showed a single, though broad, peak, of pI = 6.57. This suggests not only that the guinea pig enzyme(s) differs markedly from those of rat liver and human placenta, but that there may be qualitative differences between the forms in the three guinea pig tissues. Chromatofocusing behaviour was not independent of the specific exchange resins and ampholytes utilized. The recovered enzyme activity was fairly stable and it seems that chromatofocusing could be a useful step in purification of the guinea pig enzyme(s), particularly the main form possessing a pI greater than 9.     

Cloning and localization of MCdef, a defensin from Manila clams (Ruditapes philippinarum)

A defensin-like peptide was previously detected in hemocytes of Manila clams (Ruditapes philippinarum). In the current study, we cloned and characterized this defensin, designated MCdef. Cloning produced a full-length gene sequence of 201 bp predicted to encode a 66-amino-acid precursor protein maturing to a 44-amino-acid residue. Amino acid sequence analysis showed that MCdef is similar to defensins from marine mollusks and ticks. Phylogenetic analysis suggested that MCdef is closely related to defensins from Mytilus galloprovincialis (Mediterranean mussel) and Crassostrea gigas (Pacific cupped oyster). The three-dimensional structure of MCdef was modeled using the solution structure of C. gigas defensin as a template. With the exception of three variable loop areas, the modeled structure of MCdef was identical to that of C. gigas defensin. MCdef antiserum was raised against a synthetic MCdef peptide and verified by Western blotting using recombinant MCdef. RT-PCR analysis demonstrated high levels of MCdef mRNA in hemocytes and adductor, foot, gill, mantle, palp, and siphon tissues of Vibrio tapetis-infected Manila clams, whereas in V. tapetis-uninfected Manila clams, the level of MCdef mRNA was low in adductor, palp, and siphon tissues and even lower in the other tested tissues. Immunohistochemical analysis revealed high MCdef expression was detected in the gill, the mantle, and the digestive tubules of the diverticulum of V. tapetis-infected Manila clams. Minimum inhibitory concentration (MIC) of the purified rMCdef was determined. MCdef showed highest activity against Streptococcus iniae and Staphylococcus aureus.     

Inhibition of Taq DNA polymerase by seaweed extracts from British Columbia, Canada and Korea

Fifty-nine species of marine macrophytes from the coasts of British Columbia, Canada and Korea have been screened for the presence of PCR inhibitors, namely inhibitors of Taq DNA polymerase. Eleven of the species displayed some inhibitor activity. At the concentration of 5 μg of methanol extract in 25μL reaction mixture of PCR containing 1.5 unit of Taq DNA polymerase, one (Ulva sp.) of 8 Chlorophyta, eight (Colpomenia bullosa, Ecklonia cava, Endarachne binghamiae, Fucus distichus, Hizikia fusiformis, Sargassum confusum, Sargassum sagamianum, and Sargassum thunbergii) of 28 Phaeophyta, and one (Symphyocladia latiuscula) of 34 Rhodophyta showed inhibition in PCR amplification. In the case of the water extract, two (Cladophora columbiana, Ulva sp.) Chlorophyta, seven (Endarachne binghamiae, Fucus distichus, Hizikia fusiformis, Sargassum confusum, Sargassum sagamianum, Sargassum horneri, Scytosiphon dotyi) Phaeophyta, no Rhodophyta and one (Phyllospadix scouleri) seagrass showed inhibition in PCR amplification. the methanol fraction of Sargassum confusum and the water fraction of Fucus gardneri (mid–intertidal) have been found to inhibit PCR at level as low as 0.5 μg in 25μL of PCR reaction mixture. This revised version was published online in August 2006 with corrections to the Cover Date.     

Metabolic discrimination of Catharanthus roseus leaves infected by phytoplasma using 1H-NMR spectroscopy and multivariate data analysis

A comprehensive metabolomic profiling of Catharanthus roseus L. G. Don infected by 10 types of phytoplasmas was carried out using one-dimensional and two-dimensional NMR spectroscopy followed by principal component analysis (PCA), an unsupervised clustering method requiring no knowledge of the data set and used to reduce the dimensionality of multivariate data while preserving most of the variance within it. With a combination of these techniques, we were able to identify those metabolites that were present in different levels in phytoplasma-infected C. roseus leaves than in healthy ones. The infection by phytoplasma in C. roseus leaves causes an increase of metabolites related to the biosynthetic pathways of phenylpropanoids or terpenoid indole alkaloids: chlorogenic acid, loganic acid, secologanin, and vindoline. Furthermore, higher abundance of Glc, Glu, polyphenols, succinic acid, and Suc were detected in the phytoplasma-infected leaves. The PCA of the (1)H-NMR signals of healthy and phytoplasma-infected C. roseus leaves shows that these metabolites are major discriminating factors to characterize the phytoplasma-infected C. roseus leaves from healthy ones. Based on the NMR and PCA analysis, it might be suggested that the biosynthetic pathway of terpenoid indole alkaloids, together with that of phenylpropanoids, is stimulated by the infection of phytoplasma.     

Combined effect of organic acids and supercritical carbon dioxide treatments against nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 in fresh pork

Aims:  To evaluate the effectiveness of organic acids and supercritical carbon dioxide (SC-CO₂) treatments as well as their combined effect for the reduction of nonpathogenic Escherichia coli and three pathogenic bacteria in fresh pork.
Methods and Results:  The different treatment conditions were as follows: (i) treatment with acetic (1%, 2% or 3%) or lactic acid (1%, 2% or 3%) only, (ii) treatment with SC-CO₂ at 12 MPa and 35°C for 30 min only and (iii) treatment with 3% acetic or lactic acid followed by treatment with SC-CO₂. Within the same organic acid concentration, the lactic and acetic acid treatments had similar reductions. For the combined treatment of lactic acid and SC-CO₂, micro-organism levels were maximally reduced, ranging from 2·10 to 2·60 log CFU cm⁻² ( E. coli , 2·58 log CFU cm⁻²; Listeria monocytogenes , 2·60 log CFU cm⁻²; Salmonella typhimurium , 2·33 log CFU cm⁻²; E. coli O157:H7, 2·10 log CFU cm⁻²).
Conclusions:  The results of this study indicate that the combined treatments of SC-CO₂ and organic acids were more effective at destroying foodborne pathogens than the treatments of SC-CO₂ or organic acids alone.
Significance and Impact of the Study:  The combination treatment of SC-CO₂ and organic acids may be useful in the meat industry to help increase microbial safety.