Combination of hTERT and bmi-1, E6, or E7 induces prolongation of the life span of bone marrow stromal cells from an elderly donor without affecting their neurogenic potential

Murine bone marrow stromal cells differentiate not only into mesodermal derivatives, such as osteocytes, chondrocytes, adipocytes, skeletal myocytes, and cardiomyocytes, but also into neuroectodermal cells in vitro. Human bone marrow stromal cells are easy to isolate but difficult to study because of their limited life span. To overcome this problem, we attempted to prolong the life span of bone marrow stromal cells and investigated whether bone marrow stromal cells modified with bmi-1, hTERT, E6, and E7 retained their differentiated capability, or multipotency. In this study, we demonstrated that the life span of bone marrow stromal cells derived from a 91-year-old donor could be extended and that the stromal cells with an extended life span differentiated into neuronal cells in vitro. We examined the neuronally differentiated cells morphologically, physiologically, and biologically and compared the gene profiles of undifferentiated and differentiated cells. The neuronally differentiated cells exhibited characteristics similar to those of midbrain neuronal progenitors. Thus, the results of this study support the possible use of autologous-cell graft systems to treat central nervous system diseases in geriatric patients.     

Purification and characterization of enantioselective N-acetyl-β-Phe acylases from Burkholderia sp. AJ110349

For the production of enantiopure β-amino acids, enantioselective resolution of N-acyl β-amino acids using acylases, especially those recognizing N-acetyl-β-amino acids, is one of the most attractive methods. Burkholderia sp. AJ110349 had been reported to exhibit either (R)- or (S)-enantiomer selective N-acetyl-β-Phe amidohydrolyzing activity, and in this study, both (R)- and (S)-enantioselective N-acetyl-β-Phe acylases were purified to be electrophoretically pure and determined the sequences, respectively. They were quite different in terms of enantioselectivities and in their amino acids sequences and molecular weights. Although both the purified acylases were confirmed to catalyze N-acetyl hydrolyzing activities, neither of them show sequence similarities to the N-acetyl-α-amino acid acylases reported thus far. Both (R)- and (S)-enantioselective N-acetyl-β-Phe acylase were expressed in Escherichia coli. Using these recombinant strains, enantiomerically pure (R)-β-Phe (>99% ee) and (S)-β-Phe (>99% ee) were obtained from the racemic substrate.     

Identification of 5-Hydroxy Eicosatetraenoic Acid in Cerebrospinal Fluid After Subarachnoid Hemorrhage

Abstract: To detect and identify lipid peroxides in the CFS following subarachnoid hemorrhage (SAH), CSF samples were obtained sequentially from 10 patients who developed typical vasospasm and were analyzed by HPLC and gas chromatography-mass spectrometry. One of the peaks appearing on the 7th day after SAH was identified as 5-hydroxy eicosatetraenoic acid. On HPLC, an identical peak was detected in samples from other SAH patients. The results gave unequivocal evidence that peroxides of arachidonic acid are present in the CSF following SAH, and a correlation between them and the occurrence of vasospasm seemed likely. The hypothesis that lipid peroxides are involved in the genesis of vasospasm deserves further investigation.     

Cholesterol esterase accelerates intestinal cholesterol absorption

Lipid bilayers of dimyristoyl phosphatidylcholine (DMPC) containing opioid peptide dynorphin A(1-17) are found to be spontaneously aligned to the applied magnetic field near at the phase transition temperature between the gel and liquid crystalline states (T(m)=24 degrees C), as examined by 31P NMR spectroscopy. The specific interaction between the peptide and lipid bilayer leading to this property was also examined by optical microscopy, light scattering, and potassium ion-selective electrode, together with a comparative study on dynorphin A(1-13). A substantial change in the light scattering intensity was noted for DMPC containing dynorphin A(1-17) near at T(m) but not for the system containing A(1-13). Besides, reversible change in morphology of bilayer, from small lipid particles to large vesicles, was observed by optical microscope at T(m). These results indicate that lysis and fusion of the lipid bilayers are induced by the presence of dynorphin A(1-17). It turned out that the bilayers are spontaneously aligned to the magnetic field above T(m) in parallel with the bilayer surface, because a single 31P NMR signal appeared at the perpendicular position of the 31P chemical shift tensor. In contrast, no such magnetic ordering was noted for DMPC bilayers containing dynorphin A(1-13). It was proved that DMPC bilayer in the presence of dynorphin A(1-17) forms vesicles above T(m), because leakage of potassium ion from the lipid bilayers was observed by potassium ion-selective electrode after adding Triton X-100. It is concluded that DMPC bilayer consists of elongated vesicles with the long axis parallel to the magnetic field, together with the data of microscopic observation of cylindrical shape of the vesicles. Further, the long axis is found to be at least five times longer than the short axis of the elongated vesicles in view of simulated 31P NMR lineshape.     

Species composition and assemblage structure of chironomid larvae (Diptera: Chironomidae) attaching to the artificial substrates in a Japanese temperate basin,in relation to the longitudinal gradient

The relative importance of natural and anthropogenic factors, especially topographic type, riparian canopy, altitude, temperature and bank protection, on larval chironomid assemblage was investigated in a Japanese basin. To focus on the macro-scale factors, a concrete block, as an artificial substrate, was used for chironomid collection so that sampling regime may be identical among the sites. Partial CCA using sampling month as a covariable revealed that topographic type, riparian canopy coverage, water temperature and altitude were the main factors influencing species distribution. Stempellinella tamaseptima, Polypedilum tamanigrum and five Rheotanytarsus species showed positive, whereas five Cricotopus species showed negative associations with canopy coverage. Some traditional longitudinal zonations of species were still shown. Chironomus flaviplumus and Chironomus yoshimatsui were merely associated with lower reaches. Stepwise multiple regressions of the assemblage indices on the environmental variables were applied. Bank protection and depth showed negative correlations with Shannon diversity H′. Both topographic type and depth showed negative correlations with Pielou equitability J. Topographic type (lower reach) and specific conductance showed positive, while bank protection showed a negative correlation with abundance. Species richness was not explained by any variables. As a whole, topographic type was the most directly related factor to chironomid assemblages.     

Dried-up zone as a temporal stock of chironomid larvae: survival periods and density in a reservoir bank

The survival period, community composition and density of chironomid larvae were investigated at the reservoir bank in Yachiyoko Lake (Haji Dam), Hiroshima, Japan to verify their survivability in a dried-up zone. We define “dried-up zone” as the one that had been exposed to drought due to drawdown by evaporation and/or drainage. The larvae were collected from sediments at different altitudes along a transectional line. After larval rearing, 189 indiv. m⁻² of adults emerged even from the sediment of a 7-day drought (17.1% sedimental water content). The proportion of Tanytarsus oyamai Sasa in the community tended to be larger with dry periods, indicating that this species has a higher drought tolerance than other species. The mean density in the dried-up zone was estimated as 886.4 indiv. m⁻². Biomass is generally estimated by using the samples of only underwater sediment. These results show that some species have the potential to survive for at least one week in a dried-up zone and suggest that the biomass is likely to be highly underestimated for the entire ecosystem. A pathway of matter flow from aquatic to terrestrial ecosystems via the larvae in a dried-up zone should be taken into consideration.     

Substrate-free structure of a monomeric NADP isocitrate dehydrogenase: an open conformation phylogenetic relationship of isocitrate dehydrogenase

Both monomeric and dimeric NADP+-dependent isocitrate dehydrogenase (IDH) belong to the metal-dependent beta-decarboxylating dehydrogenase family and catalyze the oxidative decarboxylation from 2R,3S-isocitrate to yield 2-oxoglutarate, CO2, and NADPH. It is important to solve the structures of IDHs from various species to correlate with its function and evolutionary significance. So far, only two crystal structures of substrate/cofactor-bound (isocitrate/NADP) NADP+-dependent monomeric IDH from Azotobacter vinelandii (AvIDH) have been solved. Herein, we report for the first time the substrate/cofactor-free structure of a monomeric NADP+-dependent IDH from Corynebacterium glutamicum (CgIDH) in the presence of Mg2+. The 1.75 A structure of CgIDH-Mg2+ showed a distinct open conformation in contrast to the closed conformation of AvIDH-isocitrate/NADP+ complexes. Fluorescence studies on CgIDH in the presence of isocitrate/or NADP+ suggest the presence of low energy barrier conformers. In CgIDH, the amino acid residues corresponding to the Escherichia coli IDH phosphorylation-loop are alpha-helical compared with the more flexible random-coil region in the E. coli protein where IDH activation is controlled by phosphorylation. This more structured region supports the idea that activation of CgIDH is not controlled by phosphorylation. Monomeric NADP+-specific IDHs have been identified from about 50 different bacterial species, such as proteobacteria, actinobacteria, and planctomycetes, whereas, dimeric NADP+-dependent IDHs are diversified in both prokaryotes and eukaryotes. We have constructed a phylogenetic tree based on amino acid sequences of all bacterial monomeric NADP+-dependent IDHs and also another one with specifically chosen species which either contains both monomeric and dimeric NADP+-dependent IDHs or have monomeric NADP+-dependent, as well as NAD+-dependent IDHs. This is done to examine evolutionary relationships.     

A practical device for pinpoint delivery of molecules into multiple neurons in culture

We have developed a device for pinpoint delivery of chemicals, proteins, and nucleic acids into cultured cells. The principle underlying the technique is the flow of molecules from the culture medium into cells through a rupture in the plasma membrane made by a needle puncture. DNA transfection is achieved by stabbing the needle tip into the nucleus. The CellBee device can be attached to any inverted microscope, and molecular delivery can be coupled with conventional live cell imaging. Because the position of the needle relative to the targeted cultured cells is computer-controlled, efficient delivery of molecules such as rhodamine into as many as 100 HeLa cells can be completed in 10 min. Moreover, specific target cells within a single dish can be transfected with multiple DNA constructs by simple changes of culture medium containing different plasmids. In addition, the nano-sized needle tip enables gentle molecular delivery, minimizing cell damage. This method permits DNA transfection into specific hippocampal neurons without disturbing neuronal circuitry established in culture.     

A comparison of desiccation tolerance among 12 species of chironomid larvae

Tolerance to desiccation was compared among 12 Japanese species of chironomid larvae under the condition of 60% in relative humidity at 25.5?°C. Three parameters were assessed: time to 50% survival (T ₅₀), water loss at 50% survival (WL₅₀) and water loss rate (WLR). T ₅₀, WL₅₀ and WLR were determined as measures of desiccation tolerance, dehydration tolerance, and dehydration resistance, respectively. T ₅₀ was 64.4–142 min for most species, except Propsilocerus akamusi (Tokunaga) which took 872 min. WL₅₀ was 60.6–82.4% for all species. WLR was only 0.0664% per minute for Pr. akamusi, while it was 0.629–1.50% for the other species. These results showed that Pr. akamusi had a high desiccation tolerance due to a high preventive ability of evaporation from body surface. T ₅₀ showed no significant relationships to WL₅₀ or WLR among the 12 species, while there was a significant positive relationship between WL₅₀ and WLR. These results suggest that chironomid species have a trade-off tendency that a species has a high tolerance – low resistance or a high resistance – low tolerance for dehydration.     

Expression, purification, and characterization of a bacterial GTP-dependent PEP carboxykinase

The Corynebacterium glutamicum (C. glutamicum) phosphoenolpyruvate carboxykinase (PCK) gene (pckA) was cloned into an Escherichia coli expression vector with a glutathione S-transferase (GST) tag. This recombinant DNA can produce highly overexpressed tagged protein in soluble form. This is the first report of the production of C. glutamicum PCK overexpressed in E. coli. The GST-fused PCK was purified using the glutathione-Sepharose 4B affinity column and the GST tag was removed in one-step. This one-step, easy purification method would be very useful for future mutational and structural studies. The molecular mass of the purified protein is approximately 68 kDa as confirmed by mass spectrometry and it is a monomeric enzyme. Also, the enzyme assays revealed that C. glutamicum PCK has a GTP-specific activity and that its activity is maximal in the presence of both Mn2+ and Mg2+.