A denitrifying bacterium from the deep sea at 11 000-m depth

The denitrifying bacterium strain MT-1 was isolated from the mud of the Mariana Trench. The optimal temperature and pressure for growth of this bacterium were found to be 30°C and 0.1 MPa, respectively. However, it showed greater tolerance to low temperature (4°C) and high hydrostatic pressure (50 MPa) as compared with denitrifiers obtained from land. From the results, it can be said that this organism is adapted to the environment of the deep sea. Strain MT-1 was shown to belong to the genus Pseudomonas by analysis of its 16S rDNA. The cytochrome contents of the bacterium were similar to those of Ps. stutzeri in spectrophotometric studies. Received: June 2, 1997 / Accepted: August 9, 1997     

Studies on γ Globulin of Rice Embryo

All globulin components hitherto found in many species of seeds, α, β, γ and δ globulins, were identified in rice grain by ultracentrifugal experiments and gel-filtration chromatography. Among them, γ globulin was found to occur in high concentration in embryo and bran which were the most active parts in biological functions of rice grain. Then γ globulin was isolated from embryo by gel-filtration chromatography on a Sephadex G-200 column. Purified γ globulin was homogeneous in ultracentrifugal analysis and it was found to be insoluble in cold saline solution. On the other hand, α and β globulins were found to be more concentrated in endosperm with considerable heterogeneity.     

Concentrated polymer brushes do not induce the expression of inflammatory and angiogeneic genes in human umbilical vein endothelial cells

Objective

When polymer brushes are applied as the inner coating for artificial blood vessels, they may induce unwanted responses in vascular endothelial cells continuously exposed to the polymer surface. Accordingly, we have examined the in vitro effect of non-biofouling concentrated polymer brushes (CPBs) on pro-inflammatory and angiogenic responses of human umbilical vein endothelial cells (HUVECs).

Results

Micro-patterned CPBs were prepared on silicon wafers using biocompatible polymers, poly(poly(ethylene glycol)methyl ether methacrylate) (PPEGMA) and poly(2-hydroxyethyl methacrylate) (PHEMA). HUVECs were cultured on PPEGMA-CPBs and PHEMA-CPBs with different channel widths (20, 50, and 80 µm) and analyzed for mRNA expression of the pro-inflammatory cytokines IL-6 and IL-8 and angiogeneic vascular endothelial growth factor (VEGF). Irrespective of channel width, PHEMA-CPBs reduced the expression of all target genes, whereas PPEGMA-CPBs reduced VEGF and did not affect IL-6 and IL-8 levels.

Conclusion

Micro-patterned CPBs, irrespective of chemical structure or adhesion area, do not induce the expression of important pro-inflammatory and angiogenic mediators in endothelial cells.

     

Intramolecular and intermolecular perturbation on electronic state of FAD free in solution and bound to flavoproteins: FTIR spectroscopic study by using the C = O stretching vibrations as probes

The intramolecular and intermolecular perturbation on the electronic state of FAD was investigated by FTIR spectroscopy by using the C=O stretching vibrations as probes in D(2)O solution. Natural and artificial FADs, i.e. 8-CN-, 8-Cl-, 8-H-, 8-OCH(3)-, and 8-NH(2)-FAD labelled by 2-(13)C, (18)O=C(2), or 4,10a-(13)C(2) were used for band assignments. The C(2)=O and C(4)=O stretching vibrations of oxidized FAD were shifted systematically by the substitution at the 8-position, i.e. the stronger the electron-donating ability (NH(2) > OCH(3) > CH(3) > H > Cl > CN) of the substituent, the lower the wavenumber region where both the C(2)=O and C(4)=O bands appear. In contrast, the C(4)=O band of anionic reduced FAD scarcely shifted. The 1,645-cm(-1) band containing C(2)=O stretching vibration shifted to 1,630 cm(-1) in the medium-chain acyl-CoA dehydrogenase (MCAD)-bound state, which can be explained by hydrogen bonds at C(2)=O of the flavin ring. The band was observed at 1,607 cm(-1) in the complex of MCAD with 3-thiaoctanoyl-CoA. The 23 cm(-1) shift was explained by the charge-transfer interaction between oxidized flavin and the anionic acyl-CoA. In the case of electron-transferring flavoprotein, two bands associated with the C(4)=O stretching vibration were obtained at 1,712 and 1,686 cm(-1), providing evidence for the multiple conformations of the protein.     

Chemical and genetic differentiation of Ligularia tsangchanensis in Yunnan and Sichuan provinces of China

The intraspecific diversity in L. tsangchanensis collected in the Chinese Provinces Yunnan and southwestern Sichuan was studied by chemical and genetic approaches. The samples collected in Yunnan were found to contain cacalol (1) as the sole major component, while samples from Sichuan contained 7alpha- and 7beta-eremophila-9,11-dien-8-one (5 and 6) as well as the 3alpha-angeloyloxy derivative 7 as major components. In addition, the sequences of the internal transcribed spacers (ITSs) of the ribosomal RNA gene indicated that the Yunnan and the Sichuan samples constitute separate clades. These results demonstrate that L. tsangchanensis in Yunnan and Sichuan are distinct.     

Promotive effect of macrophage colony-stimulating factor on long-term engraftment of murine hematopoietic stem cells

Large ex vivo expansion of hematopoietic stem cells (HSCs) sufficient for use in clinical applications has not been achieved, although the influence of some cytokines including SCF, IL-11, Flt3-L, and TPO for this purpose has been reported. We present evidence for an indirect effect of macrophage colony-stimulating factor (M-CSF) on expansion of murine HSCs. Fresh Lin(-/low) cells were isolated from Ly5.1 mouse bone marrow and cultured with or without M-CSF in the presence of SCF + IL-11 + Flt3-L or SCF + IL-11 + TPO for 6 days. The expanded cells were harvested and transplanted into lethally irradiated Ly5.2 recipients with competitor cells. Culture of Lin(-/low) cells with M-CSF significantly enhanced long-term engraftment. When the more enriched HSC populations of Lin(-/low) c-Kit(+) Sca-1(+) cells were used as a source of HSCs, such a promotive effect was not observed, in agreement with negative expression of the M-CSF receptor (c-Fms). However, co-culture with Lin(-/low) c-Fms(+) resulted in a significant increase of long-term engraftment. These results suggested that M-CSF is an indirect stimulator for ex vivo expansion of HSCs in the presence of SCF, IL-11, Flt3-L, and TPO. These observations provide new directions for ex vivo expansion and insight into new engraftment regulation through M-CSF signaling.     

Purification and properties of extracellularβ-fructofuranosidases fromAureobasidium sp. ATCC 20524

Summary Two extracellular -fructofuranosidases (E-1 andE-2) fromAureobasidium sp. ATCC 20524, producing 1-kestose (1^F--fructofuranosyl-sucrose) from sucrose, were purified to homogeneity. Molecular weights of the enzymes were estimated to be about 304000 (E-1) and 315000 (E-2) Da by gel filtration. The enzymes contained 33% (w/w) (E-1) and 27% (w/w) (E-2) carbohydrate. TheK _m values for sucrose ofE-1 andE-2 andE-2 were 0.34 and 0.28 M, respectively. were 0.34 and 0.28 M, respectively. The enzymatic profiles of these enzymes were almost identical to intracellular enzymesP-1 andP-2 except for the differences in carbohydrate content andK _m values ofE-2 andP-2.     

Design and synthesis of novel DFG-out RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors: 2. Synthesis and characterization of a novel imide-type prodrug for improving oral absorption

As an alternative to the previously reported solid dispersion formulation for enhancing the oral absorption of thiazolo[5,4-b]pyridine 1, we investigated novel N-acyl imide prodrugs of 1 as RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors. Introducing N-acyl promoieties at the benzanilide position gave chemically stable imides. N-tert-Butoxycarbonyl (Boc) introduced imide 6 was a promising prodrug, which was converted to the active compound 1 after its oral administration in mice. Cocrystals of 6 with AcOH (6b) possessed good physicochemical properties with moderate thermodynamic solubility (19μg/mL). This crystalline prodrug 6b was rapidly and enzymatically converted into 1 after its oral absorption in mice, rats, dogs, and monkeys. Prodrug 6b showed in vivo antitumor regressive efficacy (T/C=-6.4%) in an A375 melanoma xenograft model in rats. Hence, we selected 6b as a promising candidate and are performing further studies. Herein, we report the design, synthesis, and characterization of novel imide-type prodrugs.     

Eicosapentaenoic acid attenuates statin-induced ER stress and toxicity in myoblast

T cell activation requires both antigen specific and co-stimulatory signals that include the interaction of CD28 with its ligands CD80 and CD86. These signals are delivered by antigen presenting cells (APC) in the context of the immunological synapse (IS). Reorganization of the cytoskeleton is required for the formation and maintenance of the IS. Our results show that a highly conserved polylysine motif in CD86 cytoplasmic tail, herein referred to as the K4 motif, is responsible for the constitutive association of CD86 to the cytoskeleton in primary human APC as well as in a murine APC model. This motif is not involved in initial APC:T cell conjugate formation but mutation of the K4 motif affects CD86 reorientation at the IS. Importantly, APCs expressing CD86 with mutated K4 motif are severely compromised in their capacity to trigger complete T cell activation upon peptide presentation as measured by IL-2 secretion. Altogether, our results reveal the critical importance of the cytoskeleton-dependent CD86 polarization to the IS and more specifically the K4 motif for effective co-signaling.