Isolation and structural analysis of efficient autonomously replicating sequences (ARSs) of the yeast Candida utilis

The industrially important yeast Candida utilis is widely used in production of food and medical materials, but its host-vector system has not been well developed. We screened for compact and efficient ARSs to construct practically useful vectors. The C. utilis strain AHU3053 was found to be efficiently transformed by the conventional lithium acetate method and was used as the host. The C. utilis IAM4264 genomic library was constructed by inserting the partial Sau3AI digests in pRI51, which has a kanMX gene expressible in C. utilis. By examining 98 C. utilis G418-resistant transformants, five plasmids had the highest ARS activity. By trimming of the inserts, the 1490 and 552 bp fragments with transformation activity of over 10(3)/microg DNA were obtained from ARS3 and ARS4, respectively. Although several sequences identical to S. cerevisiae ARS consensus sequences (ACSs) were found in ARS3 and ARS4, our deletion analysis indicated that these were not essential for the activity. Because the minimal functional ARS fragment was also several-fold larger than that of S. cerevisiae, the C. utilis ARSs have some unique characteristics resembling the Sz. pombe ARSs. These ARSs were functional in other C. utilis strains tested and useful for constructing practical vectors.     

In Situ Observation of Phase Separation of a Barium Borate Melt in a Stable Immiscibility Region under Microgravity

The precipitation of droplets was directly observed on a BaO–B₂O₃ melt in a drop shaft experiment. This is the first time that precipitation of droplets has been observed in a 4.5 s drop test. The melt film of 4BaOz96B₂O₃ (mol%) held on a platinum wire loop was heated above the critical tem-perature to produce uniformity and was cooled down to the phase separation temperature range. Phase separation of the melt was observed directly with a video camera. The IR image of the melt was simultaneously detected with a CCD array and was converted into a two-dimensional thermograph.     

Terpendole E and its Derivative Inhibit STLC‐ and GSK‐1‐Resistant Eg5

Terpendole E is first natural product found to inhibit mitotic kinesin Eg5, but its inhibitory mechanism remains to be revealed. Here, we report the effects of terpendole E and 11ketopaspaline (a new natural terpendole E analogue) on the Eg5–microtubule interaction and in several Eg5 mutants. 11‐Ketopaspaline is a shunt product from terpendole E, and it shows potent inhibitory activity against the microtubule‐stimulated ATPase activity of Eg5. Unlike other Eg5 inhibitors, such as S‐trityl‐L ‐cysteine (STLC) and GSK‐1, both terpendole E and 11‐ketopaspaline only partially inhibited Eg5–microtubule interaction. Furthermore, terpendole E and 11‐ketopaspaline inhibited several Eg5 mutants that are resistant to STLC (Eg5^D130A, Eg5^L214A) or GSK‐1 (Eg5^I299F, Eg5^A356T), but with the same extent of inhibition against wild‐type Eg5. Because Eg5^D130A and Eg5^L214A show cross‐resistance to most known Eg5 inhibitors, which bind the L5 loop, these results suggest that terpendole E and its analogues have a different binding site and/or inhibitory mechanism to those for L5 loop‐binding type Eg5 inhibitors.     

An absolute requirement of fructose 1,6-bisphosphate for the Lactobacillus caseiL-lactate dehydrogenase activity induced by a single amino acid substitution

Lactobacillus casei allosteric L-lactate dehydrogenase (L-LDH)absolutely requires fructose 1,6-bisphosphate [Fru(1,6)P2] forits catalytic activity under neutral conditions, but exhibitsmarked catalytic activity in the absence of Fru(1,6)P₂ underacidic conditions through the homotropic activation effect ofsubstrate pyruvate. In this enzyme, a single amino acid replacement,i.e. that of His205 conserved in the Fru(1,6)P₂-binding siteof certain allosteric L-LDHs of lactic acid bacteria with Thr,did not induce a marked loss of the activation effect of Fru(1,6)P₂or divalent metal ions, which are potent activators that improvethe activation function of Fru(1,6)P₂ under neutral conditions.However, this replacement induced a great loss of the Fru(1,6)P₂-independentactivation effect of pyruvate or pyruvate analogs under acidicconditions, consequently indicating an absolute Fru(1,6)P₂ requirementfor the enzyme activity. The replacement also induced a significantreduction in the pH-dependent sensitivity of the enzyme to Fru(1,6)P₂,through a slight decrease and increase of the Fru(1,6)P₂ sensitivityunder acidic and neutral conditions, respectively, indicatingthat His205 is also largely involved in the pH-dependent sensitivityof L.casei L-LDH to Fru(1,6)P₂. The role of His205 in the allostericregulation of the enzyme is discussed on the basis of the knowncrystal structures of L-LDHs.     

Reaction of Syringylglycerol-β-Syringyl Ether Type of Lignin Model Compounds in Alkaline Medium

Reactions of three kind of syringylglycerol- β-syringyl ether type model compounds under alkaline medium were investigated. Sinapyl alcohol and β-hydroxypropiosyringone were formed as phenyl propanoid moieties from syringylglycerol- β-(methyl-syringyl) ether 1 by the β-aryl ether cleavage under soda treatment, while only sinapyl alcohol was formed from syringylglycerol- β-syringyl ether 2. The formation of both two degradation products are quite interesting because there is no nucleophilic additives in soda liquor. A possible reaction mechanisms for the β-aryl cleavage of syringylglycerol-β-syringyl ether type is homolytical cleavage via quinone methide.     

ADDUCT FORMATION OF β-DIKETONATO LANTHANOIDS(III) WITH CARBOXYLIC ACIDS HAVING HYDROXYL AND METHOXYL GROUPS

Adduct formation of 2-thenoyltrifluoroacetonato(tta) lantha-num(III), europium(III), and ytterbium(I II) with α-pheny 1 1 actic, α-methoxy phenyl acetic, salicylic, and o-methoxy benzoic acids in chloroform has been studied by solvent extraction technique. It has been found that 1anthanoid(111) chelates form more stable adducts with these carboxylic acids in the sequence: Yb(tta)₃ < Eu(tta) ₃ < La(tta) ₃. The adduct formation constants as well as the separation factors obtained as the ratio of the constants between a pair of metals are the largest for α-phenyl lactic acid among these acids. The characteristics with α-phenyl 1actic acid would be caused by the action as bidentate unlike benzoic acid derivatives which work as monodentates owing to the internal complexa-tion through hydrogen bonding.     

Asymmetric Multidimensional Scaling of Brand Switching Among Margarine Brands

Behaviormetrika - Brand switching data among 12 margarine brands were analyzed by the asymmetric multidimensional scaling based on the singular value decomposition. A two-dimensional result was...     

Te concentration dependent photoemission and inverse-photoemission study of FeSe1?xTex

We have characterized the electronic structure of FeSe_1−xTe_x for various x values using soft x-ray photoemission spectroscopy (SXPES), high-resolution photoemission spectroscopy (HRPES) and inverse photoemission spectroscopy (IPES). The SXPES valence band spectral shape shows that the 2 eV feature in FeSe, which was ascribed to the lower Hubbard band in previous theoretical studies, becomes less prominent with increasing x. HRPES exhibits systematic x dependence of the structure near the Fermi level (E_F): its splitting near E_F and filling of the pseudogap in FeSe. IPES shows two features, near E_F and approximately 6 eV above E_F; the former may be related to the Fe 3d states hybridized with chalcogenide p states, while the latter may consist of plane-wave-like and Se d components. In the incident electron energy dependence of IPES, the density of states near E_F for FeSe and FeTe has the Fano lineshape characteristic of resonant behavior. These compounds exhibit different resonance profiles, which may reflect the differences in their electronic structures. By combining the PES and IPES data the on-site Coulomb energy was estimated at 3.5 eV for FeSe.