Human colony-stimulating factor-1 (CSF-1) was purified from the serum-free conditioned medium of a human pancreatic carcinoma cell line. The four-step procedure included chromatography on DEAE Sepharose, Con A Sepharose and HPLC on phenyl column and reverse-phase C-3 column. The purity of human CSF-1 was demonstrated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS—PAGE) as a single diffuse band with a molecular weight (Mr) of 42,000-50,000 and was further confirmed by a single amino-terminal amino acid residue of glutamate. Under reducing conditions, purified CSF-1 appeared on SDS-PAGE as a single protein band with a Mr of 21,000-25,000 and concurrently lost its biological activity, indicating that human CSF-1 consists of two similar subunits and that the intact quaternary structure is essential for biological activity. When treated with neuraminidase and endo-8~D~N—acetylglucosaminidase D, the Mr of CSF-1 was reduced to 36,000-40,000 and to a Mr of 18,000-20,000 in the presence of mercaptoethanol.

Legumes are unique among plants for their ability to fix atmospheric nitrogen with the help of soil bacteria rhizobia. Medicago truncatula is used as a model legume to study different aspects of symbiotic nitrogen fixation. M. truncatula, in association with its symbiotic partner Sinorhizobium meliloti, fix atmospheric nitrogen into ammonia, which the plant uses for amino acid biosynthesis and the bacteria get reduced photosynthate in return. M. truncatula NPF1.7 previously called MtNIP/LATD is required for symbiotic nitrogen fixing root nodule development and for normal root architecture. Mutations in MtNPF1.7 have defects in these processes. MtNPF1.7 encodes a member of the NPF family of transporters. Experimental results showing that MtNPF1.7 functioning as a high-affinity nitrate transporter are its expression restoring chlorate susceptibility to the Arabidopsis chl1-5 mutant and high nitrate transport in Xenopus laevis oocyte system. However, the weakest Mtnip-3 mutant allele also displays high-affinity nitrate transport in X. laevis oocytes and chlorate susceptibility to the Atchl1-5 mutant, suggesting that MtNPF1.7 might have another biochemical function. Experimental evidence shows that MtNPF1.7 also functions in hormone signaling. Constitutive expression of MtNPF1.7 in several species including M. truncatula results in plants with a robust growth phenotype. Using a synthetic auxin reporter, the presence …

N-Acylethanolamines (NAEs) are endogenous lipid metabolites that occur in a variety of dry seeds, and their levels decline rapidly during the first few hours of imbibition (Chapman et al., 1999, Plant Physiol., 120:1157-1164). Biochemical studies supported the existence of an NAE amidohydrolase activity in seeds and seedlings, and efforts were directed toward identification of DNA sequences encoding this enzyme. Mammalian tissues metabolize NAEs via an amidase enzyme designated fatty acid amide hydrolase (FAAH). Based on the characteristic amidase signature sequence in mammalian FAAH, a candidate Arabidopsis cDNA was identified and isolated by reverse transcriptase-PCR. The Arabidopsis cDNA was expressed in E. coli and the recombinant protein indeed hydrolyzed a range of NAEs to free fatty acids and ethanolamine. Kinetic parameters for the recombinant protein were consistent with those properties of the rat FAAH, supporting identification of this Arabidopsis cDNA as a FAAH homologue. Two T-DNA insertional mutant lines with disruptions in the Arabidopsis NAE amidohydrolase gene (At5g64440) were identified. The homozygous mutant seedlings were more sensitive than the wild type to exogenously applied NAE 12:0. Transgenic seedlings overexpressing the NAE amidohydrolase enzyme showed noticeably greater tolerance to NAE 12:0 than wild type seedlings. These results together provide evidence in vitro …

To elaborate on the function(s) of the ENOD8 gene in the nodules of M. truncatula, several different experimental approaches were used. A census of the ENOD8 genes was first completed indicating that only ENOD8.1 (nt10554-12564 of GenBank AF463407) is highly expressed in nodule tissues. A maltose binding protein-ENOD8 fusion protein was made with an E. coli recombinant system. A variety of biochemical assays were undertaken with the MBP-ENOD8 recombinant protein expressed in E. coli, which did not yield the esterase activity observed for ENOD8 protein nodule fractions purified from M. sativa, tested on general esterase substrates, α-naphthyl acetate, and p-nitrophenylacetate. Attempts were also made to express ENOD8 in a Pichia pastoris system; no ENOD8 protein could be detected from Pichia pastoris strains which were transformed with the ENOD8 expression cassette. Additionally, it was shown that the ENOD8 protein can be recombinantly synthesized by Nicotiana benthamiana in a soluble form, which could be tested for activity toward esterase substrates, bearing resemblance to nodule compounds, such as the Nod factor. Transcription localization studies using an ENOD8 promoter gusA fusion indicated that ENOD8 is expressed in the bacteroid-invaded zone of the nodule. The ENOD8 protein was also detected in that same zone by …