Purification and Properties of Phospholipase D from Actinomadura sp. Strain No. 362

An extracellular phospholipase D from Actinomadura sp. Strain No. 362 was purified about 430-fold from the culture filtrate. The purified enzyme preparation was judged to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight and isoelectric point of the enzyme were estimated to be about 50,000—60,000 and 6.4, respectively. The enzyme was most active at pH 5.5 and 50°C in the presence of Triton X-100, but showed the highest activity at pH 7.0 and 60 — 70°C in its absence. The enzyme was stable up to 30°C at pH 7.2 and also stable in the pH range of 4.0 to 8.0 on 2 hr incubation at 25°C. With regard to substrate specificity, this enzyme hydrolysed lecithin best among the phospholipids tested. It was activated by Fe^{3 +}, Al³⁺, Mn^{2 +}, Ca^{2 +}, diethyl ether, sodium deoxycholate and Triton X-100, but was inhibited by cetyl pyridinium chloride and dodecylsulfate.     

Depression of Complement Regulatory Factors in Rat and Human Renal Grafts Is Associated with the Progress of Acute T-Cell Mediated Rejection

Background

The association of complement with the progression of acute T cell mediated rejection (ATCMR) is not well understood. We investigated the production of complement components and the expression of complement regulatory proteins (Cregs) in acute T-cell mediated rejection using rat and human renal allografts.

Methods

We prepared rat allograft and syngeneic graft models of renal transplantation. The expression of Complement components and Cregs was assessed in the rat grafts using quantitative real-time PCR (qRT-PCR) and immunofluorescent staining. We also administered anti-Crry and anti-CD59 antibodies to the rat allograft model. Further, we assessed the relationship between the expression of membrane cofactor protein (MCP) by immunohistochemical staining in human renal grafts and their clinical course.

Results

qRT-PCR results showed that the expression of Cregs, CD59 and rodent-specific complement regulator complement receptor 1-related gene/protein-y (Crry), was diminished in the rat allograft model especially on day 5 after transplantation in comparison with the syngeneic model. In contrast, the expression of complement components and receptors: C3, C3a receptor, C5a receptor, Factor B, C9, C1q, was increased, but not the expression of C4 and C5, indicating a possible activation of the alternative pathway. When anti-Crry and anti-CD59 mAbs were administered to the allograft, the survival period for each group was shortened. In the human ATCMR cases, the group with higher MCP expression in the grafts showed improved serum creatinine levels after the ATCMR treatment as well as a better 5-year graft survival rate.

Conclusions

We conclude that the expression of Cregs in allografts is connected with ATCMR. Our results suggest that controlling complement activation in renal grafts can be a new strategy for the treatment of ATCMR.     

Life cycles of 17 riffle-dwelling mayfly species (Baetidae,Heptageniidae, and Ephemerellidae) in central Japan

We described the life cycles of 17 riffle-dwelling mayfly species in a central Japanese stream. Both species of Baetidae (Alainites yoshinensis Gose and Baetis thermicus Uéno) and two of nine species of Heptageniidae (Ecdyonurus scalaris Kluge and Epeorus latifolium Uéno/l-nigrus Matsumura) in this stream were multivoltine (more than one generation per year). Seven other heptageniid species (two unidentified species of Cinygmula, Epeorus aesculus Imanishi, E. curvatulus Matsumura, E. ikanonis Takahashi, E. napaeus Imanishi, and Rhithrogena japonica Uéno) and all six species of Ephemerellidae [Cincticostella elongatula (McLachlan), C. nigra (Uéno), Drunella basalis (Imanishi), D. ishiyamana Matsumura, D. sachalinensis (Matsumura), and D. trispina (Uéno)] were essentially univoltine.     

Structural analysis of an acidic, fatty acid ester-bonded extracellular polysaccharide produced by a pristane-assimilating marine bacterium, Rhodococcus erythropolis PR4

Rhodococcus erythropolis PR4 is a marine bacterium that can degrade various alkanes including pristane, a C(19) branched alkane. This strain produces a large quantity of extracellular polysaccharides, which are assumed to play an important role in the hydrocarbon tolerance of this bacterium. The strain produced two acidic extracellular polysaccharides, FR1 and FR2, and the latter showed emulsifying activity toward clove oil, whereas the former did not. FR2 was composed of D-galactose, D-glucose, D-mannose, D-glucuronic acid, and pyruvic acid at a molar ratio of 1:1:1:1:1, and contained 2.9% (w/w) stearic acid and 4.3% (w/w) palmitic acid attached via ester bonds. Therefore, we designated FR2 as a PR4 fatty acid-containing extracellular polysaccharide or FACEPS. The chemical structure of the PR4 FACEPS polysaccharide chain was determined by 1D (1)H and (13)C NMR spectroscopies as well as by 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The sugar chain of PR4 FACEPS was shown to consist of tetrasaccharide repeating units having the following structure: [structure: see text].     

Screening procedure for the analysis of distigmine bromide in serum by high-performance liquid chromatography-electrospray ionization mass spectrometry

A screening procedure was developed for the identification and quantification of distigmine bromide in serum samples by using liquid chromatography (LC)-electrospray ionization (ESI)-mass spectrometry (MS). In this method, distigmine bromide was analyzed in 0.5 mL serum by using pancuronium bromide as the internal standard, and gradient elution was performed using a reversed-phase column and a mixture of 10 mM-ammonium formate and methanol as the mobile phase. A highly sensitive assay could be performed with simple solid phase extraction using a cation exchange cartridge column by carrying out selected ion monitoring analysis in the positive ion detection mode. The procedure was validated in terms of linearity (0.9973 at 2.5 ng/mL). The inter- and intra-day precisions (coefficient of variation; CV%) were <8.5% and < 9.7%, respectively. The analytes were evaluated for stability and were found to be stable in serum for 1 week at 4 degrees C and 4 weeks at -30 degrees C, and successfully applied to in the analysis of two overdose cases. This method is sensitive and useful for the detection, quantification, and confirmation of distigmine bromide in serum.     

Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage

Mitochondrial damage is a well known cause of mitochondria-related diseases. A major mechanism underlying the development of mitochondria-related diseases is thought to be an increase in intracellular oxidative stress produced by impairment of the mitochondrial electron transport chain (ETC). However, clear evidence of intracellular free radical generation has not been clearly provided for mitochondrial DNA (mtDNA)-damaged cells. In this study, using the novel fluorescence dye, 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF), which was designed to detect hydroxyl radicals (*OH), intracellular free radical formation was examined in 143B cells (parental cells), 143B-rho(0) cells (mtDNA-lacking cells), 87 wt (cybrid), and cybrids of 4977-bp mtDNA deletion (common deletion) cells containing the deletion with 0%, 5%, 50% and >99% frequency (HeLacot, BH5, BH50 and BH3.12, respectively), using a laser confocal microscope detection method. ETC inhibitors (rotenone, 3-nitropropionic acid, thenoyltrifluoroacetone, antimycin A and sodium cyanide) were also tested to determine whether inhibitor treatment increased intracellular reactive oxygen species (ROS) generation. A significant increase in ROS for 143B-rho(0) cells was observed compared with 143B cells. However, for the 87 wt cybrid, no increase was observed. An increase was also observed in the mtDNA-deleted cells BH50 and BH3.12. The ETC inhibitors increased intracellular ROS in both 143B and 143B-rho(0) cells. Furthermore, in every fluorescence image, the fluorescence dye appeared localized around the nuclei. To clarify the localization, we double-stained cells with the dye and MitoTracker Red. The resulting fluorescence was consistently located in mitochondria. Furthermore, manganese superoxide dismutase (MnSOD) cDNA-transfected cells had decreased ROS. These results suggest that more ROS are generated from mitochondria in ETC-inhibited and mtDNA-damaged cells, which have impaired ETC.     

Changes of protein profiles during pollen development in<Emphasis Type="Italic"> Lilium longiflorum</Emphasis>

Pollen proteins of Lilium longiflorum were examined at different developmental stages (young, mature and cultured) using two-dimensional differential gel electrophoresis. Quantitative changes of six proteins (MP1–MP6) during pollen development were observed in the acidic and low molecular weight region. After water absorption on the culture medium, the quantities of all six proteins were drastically changed. Mass spectrometric analysis revealed that MP2, MP3, MP4 and MP6 are late embryogenesis abundant (LEA) (D-7) protein, profilin 3, profilin 1 and enolase, respectively. The remaining two proteins (MP1 and MP5) could not be identified by mass spectrometric analysis. Immunogold electron microscopic examination showed the presence of these proteins in different regions: MP1 around lipid bodies, suggesting possible involvement in lipid metabolism, MP4 near actin in the cytoplasm, indicating the possibility of its interaction with actin in the regulatory pathways of pollen, and MP2 and MP6 in the cytoplasm.     

Six1 controls patterning of the mouse otic vesicle

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wild-type otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.     

Effects of chloramphenicol on photosynthesis, protein profiles and transketolase activity under extremely high CO2 concentration in an extremely-high-CO2-tolerant green microalga, Chlorococcum littorale

An extremely-high-CO2-tolerant alga, Chlorococcum littorale, showed high quantum efficiency of PSII (PhiII) in the light at 40% CO2, as well as at 5% CO2. However, PhiII decreased greatly when chloramphenicol (CAP) was added at 40% CO2, while no such decrease was observed at 5% CO2. Cycloheximide showed no effect on PhiII at either 5% or 40% CO2. The amount of a 76 kDa polypeptide (p76) on SDS-PAGE decreased markedly in the presence of CAP at 40% CO2 but not at 5% CO2. A partial amino acid sequence of p76 was 71-100% identical (10-14 identical residues out of 14 amino acids determined) to those of transketolases (TKLs) reported in higher plants and a cyanobacterium. In agreement with these observations, the TKL activity in C. littorale was decreased by CAP at 40% CO2, but not at 5% CO2. The transient decrease in TKL activity caused by CAP under 40% CO2 was well correlated with that in PhiII. These results indicate that the addition of CAP directly or indirectly influences the stability of TKL in C. littorale at 40% CO2, but not at 5% CO2, and that photosynthetic activity was reduced by a decrease in TKL activity.