Molecular cloning of levan fructotransferase gene from Arthrobacter ureafaciens K2032 and its expression in Escherichia coli for the production of difructose dianhydride IV

AIMS: To clone and overexpress a novel levan fructotransferase gene lftA from Arthrobacter ureafaciens K2032. METHODS AND RESULTS: The lftA gene, encoding a levan fructotransferase (LFTase) of 521 amino acids (aa) residues, was cloned from the genomic DNA of A. ureafaciens K2032, and overexpressed in Escherichia coli. The recombinant LFTase overexpressed in E. coli was then used to produce a difructose dianhydride (DFA IV) from levan. DFA IV crystals with 97% purity could be obtained from the reaction mixture in 83.7% yield by using a natural crystallization method. CONCLUSIONS: The lftA gene cloned from A. ureafaciens K2032 encode a novel levan fructotransferase which produces difructose dianhydride (DFA IV) from levan. SIGNIFICANCE AND IMPACT OF THE STUDY: Levan fructotransferase is a useful enzyme with great promise in the production of DFA IV and various fructosides.     

C/EBPalpha is required for proteolytic cleavage of cyclin A by calpain 3 in myeloid precursor cells

In this report, we present novel findings that implicate CCAAT/enhancer-binding protein (C/EBPalpha) in regulating the expression and activity of calpain 3 in vivo and data showing a new physiological substrate for calpain 3, cyclin A. Our results demonstrate that cleavage of cyclin A by calpain 3 occurs in mouse and human myeloid precursor cells. Calpain 3 cleaves cyclin A in vitro and in vivo, resulting in the production of a truncated product that lacks the N-terminal destruction box required for its degradation at the end of mitosis. The cleaved form of cyclin A retains the cyclin-dependent kinase (cdk) binding domain and forms active complexes with cdk2. Calpain 3-mediated cleavage of cyclin A is lacking in C/EBPalpha-/- mice, which are not able to produce mature granulocytes. Our data support a model in which calpain 3-mediated cleavage of cyclin A in dividing myeloid progenitor cells is important for the onset of differentiation. Deficits in this pathway in C/EBPalpha-/- mice might contribute to the failure of these mice to produce mature granulocytes. These data reveal a new pathway involving tightly controlled post-translational processing of cyclin A during differentiation of granulocytes.     

Procedures for improving maternal behavior in captive chimpanzees

Captive female chimpanzees who have had no opportunity to observe mothers with infants or to interact with infants often show inappropriate maternal behavior, particularly with their first-born infant, and this usually results in the removal of the infant to be human-reared. The present study used two techniques to encourage appropriate maternal behavior in ten pregnant female chimpanzees. These females were housed together with unrelated infant chimpanzees to adopt, or with lactating female chimpanzees and infants to observe. In five cases both techniques were used, in two cases only the first technique was used, and in three cases only the second technique was used. All ten female chimpanzees showed appropriate maternal behavior when their infants were born, in contrast to a group of eight female chimpanzees who had no such experience whose infants had to be removed for human-rearing. It is suggested that these techniques, or adaptations of them, could be applied to many other captive female chimpanzees with similar results.     

High production of D-β-hydroxyisobutyric acid from methacrylic acid by Candida rugosa and its mutant

Production of D-β-hydroxyisobutyric acid (D-HIBA) from methacrylic acid (MA) was investigated using Candida rugosa IFO 0750 and its mutant. Cell growth decreased as the MA concentration increased and was inhibited at D-HIBA concentrations higher than 30 g/l. Optimal MA concentration for D-HIBA production was in the range of 10–20 g/l. It was also noted that cell growth and D-HIBA production were inhibited by higher concen-trations of Na⁺, K⁺, and NH₄ ⁺, which were required for pH control during cultivation. With a suitably designed feeding mode of MA, the parent strain produced 65 g/l of D-HIBA after 120?h of fed-batch cultivation, but molar conversion yield of D-HIBA was less than 40%. The mutant, unable to assimilate propionic acid, produced as high as 70 g/l of D-HIBA in the same culture period with a molar conversion yield of more than 70%.     

Activation of phospholipase C by the alpha subunits of the Gq and G11 proteins in transfected Cos-7 cells.

High efficiency transient transfection was used to introduce cDNA corresponding to various G protein alpha subunits into Cos-7 cells. The proteins that were subsequently synthesized were detected with specific G protein alpha subunit antipeptide antiserum and were localized in the membrane fraction of the cell. Cells that were prelabeled with the [3H]inositol and transfected with G alpha q and G alpha 11 cDNA showed marked increases in formation of [3H]inositol phosphates after stimulation with aluminum fluoride. Co-transfection with cDNAs corresponding to phosphoinositide specific phospholipase C beta 1 (PI-PLC beta 1) and to G alpha q or G alpha 11 resulted in even higher levels of inositol phosphate formation. The introduction of mutations that convert residue glutamine 209 to leucine in G alpha q and G alpha 11 resulted in persistent activation of PI-PLC and high steady state levels of inositol phosphates. On the other hand, transfection with a variety of other G alpha subunit cDNAs, i.e. G alpha Z, G alpha OA, G alpha OB, transducin, and the glutamine 205 to leucine mutants of G alpha Z and of G alpha OA did not increase inositol phosphate formation. To further test the specificity of G protein activation of PI-PLC, a cell-free system was prepared by using washed membranes of transiently transfected cells and purified PI-PLC beta 1. Membranes derived from G alpha q and G alpha 11, but not G alpha OA transfected cells, showed guanosine 5-O-thiotriphosphate (GTP gamma S)-stimulated PIP2 hydrolysis. The activity seen in the system reconstituted with membranes derived from G alpha 11-transfected cells was blocked by preincubation with specific G alpha 11 antipeptide antibodies. All of these results are consistent with the conclusion that G alpha q and G alpha 11 cDNA encode proteins that in the presence of GTP gamma S specifically activate PI-PLC.     

Ectodomain cleavage and shedding of the type III transforming growth factor-beta receptor in lung membranes effect of temperature, ligand binding and membrane solubilization.

Previous studies from our laboratory [Philip, A. & O'Connor-McCourt, M. D. (1991) J. Biol. Chem. 266, 22290--22296] have shown that the lung exhibited the highest uptake of circulating [125I]-transforming growth factor-beta1 (TGF-beta1) on a per gram basis. This observation, together with the lack of information on TGF-beta receptor expression in the lung, prompted us to attempt to characterize TGF-beta receptors in this tissue. In the present report we show that the type III TGF-beta receptor is the most abundant TGF-beta binding protein in rat lung membranes and that it exhibits a 10-fold higher affinity for TGF-beta2 than for TGF-beta1. We observed that the majority of the type III receptor population in lung membranes is cleaved at a site in the central portion of the ectodomain, the resulting two fragments (95 kDa and 58 kDa) being held together by disulfide bonds. Furthermore, we demonstrate that a soluble form of the ectodomain of the type III receptor is shed from rat lung membranes in an efficient manner, with protease cleavage occurring at a site close to the transmembrane domain. This shedding is controllable by temperature, thus providing a system to study the mechanism of ectodomain release. Using this system, we show that the shedding is inhibited by prior ligand binding and by membrane solubilization. The identification of a membrane preparation which exhibits controllable and quantitative release of the type III receptor ectodomain provides a unique cell-free system for further studies of the mechanism of shedding of the type III TGF-beta receptor ectodomain.