Production of high-purity isomalto-oligosaccharides syrup by the enzymatic conversion of transglucosidase and fermentation of yeast cells

A method for the production of high-purity isomalto-oligosaccharides (IMO) involving the transglucosylation by transglucosidase and yeast fermentation was proposed. The starch of rice crumbs was enzymatically liquefied and saccharified, and then converted to low-purity IMO syrup by transglucosylation. The low-purity IMO produced either from rice crumbs or tapioca flour as the starch source could be effectively converted to high-purity IMO by yeast fermentation to remove the digestible sugars including glucose, maltose, and maltotriose. Both Saccharomyces carlsbergensis and Saccharomyces cerevisiae were able to ferment glucose in the IMO syrup. Cells of S. carlsbergensis harvested from the medium of malt juice were also able to ferment maltose and maltotriose. A combination of these two yeasts or S. carlsbergensis alone could be used to totally remove the digestible sugars in the IMO, coupled with the production of ethanol. The resultant high-purity IMO, including mainly isomaltose, panose, and isomaltotriose made up more than 98% w/w of the total sugars after a 3-day fermentation. When the low-purity IMO was produced from the starch of tapioca flour, 3-day fermentation under the same conditions resulted in IMO with purity lower than that from rice crumbs. For low-purity IMO from rice crumbs, fermentation with washed S. carlsbergensis cells harvested at log phase was the most effective. However, for the low-purity IMO from tapioca flour, incubation with S. cerevisiae for the first 24 h and then supplementing with an equal amount of S. carlsbergensis cells for further fermentation was the most effective approach for producing high-purity IMO.     

Role of transmembrane segment 5 of the plant vacuolar H+-pyrophosphatase

Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase consisting of a single type of polypeptide with a molecular mass of approximately 81 kDa. Topological analysis tentatively predicts that mung bean V-PPase contains 14 transmembrane domains. Alignment analysis of V-PPase demonstrated that the transmembrane domain 5 (TM5) of the enzyme is highly conserved in plants and located at the N-terminal side of the putative substrate-binding loop. The hydropathic analysis of V-PPase showed a relatively lower degree of hydrophobicity in the TM5 region as compared to other domains. Accordingly, it appears that TM5 is probably involved in the proton translocation of V-PPase. In this study, we used site-directed mutagenesis to examine the functional role of amino acid residues in TM5 of V-PPase. A series of mutants singly replaced by alanine residues along TM5 were constructed and over-expressed in Saccharomyces cerevisiae; they were then used to determine their enzymatic activities and proton translocations. Our results indicate that several mutants displayed minor variations in enzymatic properties, while others including those mutated at E225, a GYG motif (residues from 229 to 231), A238, and R242, showed a serious decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase. Moreover, the mutation at Y230 relieved several cation effects on the V-PPase. The GYG motif presumably plays a significant role in maintaining structure and function of V-PPase.     

PD98059 and U0126 activate AMP-activated protein kinase by increasing the cellular AMP:ATP ratio and not via inhibition of the MAP kinase pathway

The MAP kinase pathway inhibitor U0126 caused phosphorylation and activation of AMP-activated protein kinase (AMPK) and increased phosphorylation of its downstream target acetyl-CoA carboxylase, in HEK293 cells. This effect only occurred in cells expressing the upstream kinase, LKB1. Of two other widely used MAP kinase pathway inhibitors not closely related in structure to U0126, PD98059 also activated AMPK but PD184352 did not. U0126 and PD98059, but not PD184352, also increased the cellular ADP:ATP and AMP:ATP ratios, accounting for their ability to activate AMPK. These results suggest the need for caution in interpreting experiments conducted using U0126 and PD98059.     

The phosphatase subunit tap42 functions independently of target of rapamycin to regulate cell division and survival in Drosophila

The protein phosphatase 2A (PP2A) regulatory subunit Tap42 is essential for target of rapamycin (TOR)-mediated signaling in yeast, but its role in higher eukaryotes has not been established. Here we show that Tap42 does not contribute significantly to TOR signaling in Drosophila, as disruption of the Tap42 gene does not cause defects in cell growth, metabolism, or S6-kinase activity characteristic of TOR inactivation. In addition, Tap42 is not required for increased cell growth in response to activation of TOR signaling. Instead, we find that Tap42 mutations cause disorganization of spindle microtubules in larval neuroblasts, leading to a preanaphase mitotic arrest in these cells. Loss of Tap42 ultimately results in increased JNK signaling, caspase activation, and cell death. These phenotypes are associated with increased accumulation and nuclear localization of PP2A in Tap42 mutant cells. Our results demonstrate that the role of Tap42 in TOR signaling has not been conserved in higher eukaryotes, indicating fundamental differences in the mechanisms of TOR signaling between yeast and higher eukaryotes.     

Kinetic and structural properties of disulfide engineered phospholipase A2: insight into the role of disulfide bonding patterns

The family of secreted 14 kDa phospholipase A(2) (PLA2) enzymes have a common motif for the catalytic site but differ in their disulfide architecture. The functional significance of such structural changes has been analyzed by comparing the kinetic and spectroscopic properties of a series of disulfide mutants engineered into the sequence of pig pancreatic IB PLA2 to resemble the mammalian paralogues of the PLA2 family [Janssen et al. (1999) Eur. J. Biochem. 261, 197-207, 1999]. We report a detailed comparison of the functional parameters of pig iso-PLA2, as well as several of the human homologues, with these disulfide engineered mutants of pig IB PLA2. The crystal structure of the ligand free and the active site inhibitor-MJ33 bound forms of PLA2 engineered to have the disulfide bonding pattern of group-X (eng-X) are also reported and compared with the structure of group-IB and human group-X PLA2. The engineered mutants show noticeable functional differences that are rationalized in terms of spectroscopic properties and the differences detected in the crystal structure of eng-X. A major difference between the eng-mutants is in the calcium binding to the enzyme in the aqueous phase, which also influences the binding of the active site directed ligands. We suggest that the disulfide architecture of the PLA2 paralogues has a marginal influence on interface binding. In this comparison, the modest differences observed in the interfacial kinetics are attributed to the changes in the side chain residues. This in turn influences the coupling of the catalytic cycle to the calcium binding and the interfacial binding event.     

Highly specific expression of luciferase gene in lungs of naïve nude mice directed by prostate-specific antigen promoter

PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10(9)PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases.     

Cephalosporin C production by immobilized Cephalosporium acremonium cells in a repeated batch tower bioreactor

The industrial production of antibiotics with filamentous fungi is usually carried out in conventional aerated and agitated tank fermentors. Highly viscous non-Newtonian broths are produced and a compromise must be found between convenient shear stress and adequate oxygen transfer. In this work, cephalosporin C production by bioparticles of immobilized cells of Cephalosporium acremonium ATCC 48272 was studied in a repeated batch tower bioreactor as an alternative to the conventional process. Also, gas-liquid oxygen transfer volumetric coefficients, k(L)a, were determined at various air flow-rates and alumina contents in the bioparticle. The bioparticles were composed of calcium alginate (2.0% w/w), alumina ( < 44 micra), cells, and water. A model describing the cell growth, cephalosporin C production, oxygen, glucose, and sucrose consumption was proposed. To describe the radial variation of oxygen concentration within the pellet, the reaction-diffusion model forecasting a dead core bioparticle was adopted. The k(L)a measurements with gel beads prepared with 0.0, 1.0, 1.5, and 2.0% alumina showed that a higher k(L)a value is attained with 1.5 and 2.0%. An expression relating this coefficient to particle density, liquid density, and air velocity was obtained and further utilized in the simulation of the proposed model. Batch, followed by repeated batch experiments, were accomplished by draining the spent medium, washing with saline solution, and pouring fresh medium into the bioreactor. Results showed that glucose is consumed very quickly, within 24 h, followed by sucrose consumption and cephalosporin C production. Higher productivities were attained during the second batch, as cell concentration was already high, resulting in rapid glucose consumption and an early derepression of cephalosporin C synthesizing enzymes. The model incorporated this improvement predicting higher cephalosporin C productivity.     

Craniodental variation in the African macaque, with reference to various Asian species

Based on twenty-seven craniodental measurements and ratios derived from them, the relationship between the African macaque (M. sylvanus) and the others in Asia were examined with principal components analyses (PCA) and Euclidean distance analysis based upon prior discriminant function analyses (DFA). Results based on analyses of raw measurements indicate that the variation between species lies in the first axis of PCA; the species are dispersed according to their differences in size. The variation between sexes (sexual dimorphism) lies in the second axis. In the analyses of ratio variables, though these two patterns of separation remain orthogonal, they lie at approximately forty-five degrees to each axis. Variables relating to anterior teeth were found to play an important role in variation analysis, and this may be related to the special food preferences of these monkeys: more frequent usage of the incisor teeth for processing frugivorous diets than in other primates that are mainly folivorous. The results from Euclidean distance analyses indicate that the average distance of species within the Asian group is shorter than that between Asian and African groups regardless of sex and variable type. In addition the variation between African and Asian groups is larger than that within Asian group. Thus, it is reasonable to suggest that the African macaque has a range of measurements and ratios quite distinct from the species found in Asia (though the greatest separations result from the analyses of ratio data). These results therefore support the view that M. sylvanus may be regarded as an independent species group in the genus Macaca as proposed by Delson [1980].     

Histidine 167 is the phosphate acceptor in glucose-6-phosphatase-beta forming a phosphohistidine enzyme intermediate during catalysis

The glucose-6-phosphatase (Glc-6-Pase) family comprises two active endoplasmic reticulum (ER)-associated isozymes: the liver/kidney/intestine Glc-6-Pase-alpha and the ubiquitous Glc-6-Pase-beta. Both share similar kinetic properties. Sequence alignments predict the two proteins are structurally similar. During glucose 6-phosphate (Glc-6-P) hydrolysis, Glc-6-Pase-alpha, a nine-transmembrane domain protein, forms a covalently bound phosphoryl enzyme intermediate through His(176), which lies on the lumenal side of the ER membrane. We showed that Glc-6-Pase-beta is also a nine-transmembrane domain protein that forms a covalently bound phosphoryl enzyme intermediate during Glc-6-P hydrolysis. However, the intermediate was not detectable in Glc-6-Pase-beta active site mutants R79A, H114A, and H167A. Using [(32)P]Glc-6-P coupled with cyanogen bromide mapping, we demonstrated that the phosphate acceptor in Glc-6-Pase-beta is His(167) and that it lies inside the ER lumen with the active site residues, Arg(79) and His(114). Therefore Glc-6-Pase-alpha and Glc-6-Pase-beta share a similar active site structure, topology, and mechanism of action.