Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the spinosyn gene cluster. Duplication of the spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis — the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased spinosyn yield significantly. Journal of Industrial Microbiology & Biotechnology (2001) 27, 399–402. Received 31 May 2001/ Accepted in revised form 09 July 2001     

Fibrinolysis by urokinase endowed with magnetic property

The activated magnetic modifier was synthesized from magnetite, alpha, omega-dicarboxymethylpoly(oxyethylene) and N-hydroxysuccinimide (Biochem. Biophys. Res. Commun., 145, 908-914, 1987). Urokinase was directly coupled with the activated magnetic modifier to obtain magnetic urokinase. The magnetic urokinase dispersed in saline and exerted high fibrinolytic activity (13.8 X 10(4) IU/mg protein), and was readily recovered from saline by magnetic force of 250 Oe. By applying magnetic force, the urokinase was attracted at our will and local fibrinolysis was achieved on fibrin gel in a petri dish.     

The activity of alkaline phosphatase in the sera of DBA/2NCrj and CBA/BrA mice with calcified tongue lesions

Spontaneously occurring calcified lesions were found in the tongues of DBA/2NCrj and CBA/BrA mice. In the DBA/2NCrj strain, the frequency of the lesion was 80% (males) and 88% (females). The youngest age of a mouse with this lesion was 18 days after birth, and 3-4 lesions were found in the tongue of 6- to 8-week-old mice. In CBA/BrA mice, 20% of females and 48% of males had the lesions. No significant differences were found in the serum calcium concentrations in high and low lesion-developing strains, but the alkaline phosphatase activities in the high-developing DBA/2NCrj, DBA/LiA, and CBA/BrA strains were higher than in strains with no calcified lesions.     

Production of monocyte chemotactic and activating factor (MCAF) by human dermal fibroblasts in response to interleukin 1 or tumor necrosis factor

Normal human dermal fibroblasts rapidly expressed (less than 30 min.) considerable mRNA for monocyte chemotactic and activating factor (MCAF) and released high levels of biological activity in response to interleukin 1 (IL 1) or tumor necrosis factor (TNF). In contrast, cultured normal human keratinocytes did not express MCAF mRNA when stimulated with IL 1 or TNF. These results suggest the important role of dermal fibroblasts, the predominant cells in dermal connective tissue, in the recruitment of monocytes during inflammation. This is the first report of the induction of MCAF by IL 1 or TNF in any cell type.     

Cloning and sequencing of the cDNA for human monocyte chemotactic and activating factor (MCAF)

cDNA clones having a nucleotide sequence encoding a human monocyte chemotactic and activating factor (MCAF) were isolated and sequenced. The amino acid sequence deduced from the nucleotide sequence reveals the primary structure of the MCAF precursor to be composed of a putative signal peptide sequence of 23 amino acid residues and a mature MCAF sequence of 76 amino acid residues. The amino acid sequence of MCAF showed 25-55% homology with other members of an inducible cytokine family, including macrophage inflammatory protein and some putative polypeptide mediators known as JE, LD78, RANTES and TCA-3. This suggests that MCAF is a member of family of factors involved in immune and inflammatory responses.     

Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA.

Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment.     

Phosphorylation of intracellular precursors of human IL-1

The human IL-1 molecules (IL-1 alpha and IL-1 beta) are post-translationally cleaved from 31-kDa precursor to 18-kDa biologically active molecules. During the course of studies of post-translational modifications of human IL-1, we have observed that although LPS induced the production of both intracellular IL-1 alpha and IL-1 beta in human monocytes, [32P]orthophosphate labeling of these cells revealed that intracellular precursor of IL-1 alpha (pre-IL-1 alpha) to be phosphorylated at least 10-fold more than intracellular pre-IL-1 beta. However, no 32P-incorporation could be detected in the 18-kDa processed IL-1 alpha and IL-1 beta. Analysis by TLC revealed that the major phosphorylation site occurred at serine residue(s). The 32P was incorporated into multiply cleaved precursors of IL-1 alpha, which appeared in the absence of protease inhibitors. Since the smallest Mr pre-IL-1 alpha that was labeled with 32P was 22 kDa, the phosphorylated serine residue is presumably located adjacent to a sequence of four basic amino acids located in the 4-kDa region at the amino terminus of the 22-kDa precursor of IL-1 alpha. This serine residue might also be a major phosphorylation site for a cAMP-dependent protein kinase. This hypothesis was substantiated by the demonstration that a synthetic peptide analogue of this region (residue 84 to 112) could be similarly phosphorylated in vitro by a cAMP-dependent protein kinase. Furthermore, a truncated pre-IL-1 alpha (residue 64 to 271) and a "fusion" protein containing staphylococcal protein A and an amino-terminal half-portion of pre-IL-1 alpha (residue 1 to 112), but not mature IL-1 alpha (residue 113 to 271), could also be phosphorylated by cAMP-dependent protein kinase. There is no comparable amino acid sequence in IL-1 beta which could be expected to be phosphorylated by a cAMP-dependent protein kinase. The physiologic relevance of phosphorylation of pre-IL-1 alpha was investigated. The data showed that phosphorylation of truncated pre-IL-1 alpha greatly enhanced its susceptibility to digestion by trypsin and promoted the conversion of pre-IL-1 alpha to the more biologically active IL-1. Although the precise role of the rather selective phosphorylation of pre-IL-1 alpha is not known, our findings do suggest that the phosphorylation of serine close to dibasic/tetrabasic amino acid sequence functions to facilitate the processing and/or release of IL-1 alpha.     

Purification and characterization of human papillomavirus type 16 E7 protein with preferential binding capacity to the underphosphorylated form of retinoblastoma gene product.

Human papillomavirus type 16 E7 is considered to be a major viral oncoprotein playing an important role(s) in cervical cancers. E7 protein was shown to bind to the protein product of the retinoblastoma gene (RB), while simian virus 40 large T and adenovirus E1A were also shown to possess binding activity to RB protein. The RB protein is a cell cycle regulator that is highly phosphorylated specifically in S, G2, and M, whereas it is underphosphorylated in G0 and G1. Recently, large T was demonstrated to bind preferentially to the underphosphorylated RB protein, which is considered to be an active form restricting cell proliferation. However, it is not known whether E7 can bind to phosphorylated RB protein. We successfully purified large quantities of unfused human papillomavirus type 16 E7 protein expressed in Escherichia coli by using a T7 promoter-T7 RNA polymerase system. The purified E7 protein was demonstrated to bind preferentially to the underphosphorylated RB protein.