Gibberellin (GA) 20‐oxidases are multifunctional enzymes that catalyse reactions at an important branch point in the GA biosynthetic pathway. These enzymes oxidise the C‐20 methyl group of a ...diterpene carboxylic acid precursor (e.g. GA12) to form an alcohol (in our case GA15‐open lactone) and an aldehyde (GA24). The aldehyde is either oxidised to a tricarboxylic acid (GA25) or, with loss of carbon‐20 and lactonisation, to a C19‐GA (GA9). This branching is interesting to study, because C19‐GA derivatives function as plant hormones in different tissues, whereas the C20‐GA tricarboxylic acids have no known function. We have constructed chimaeric proteins by combining a GA 20‐oxidase from immature seeds of Cucurbita maxima L., which produces mainly C‐20 carboxylic acids, with a 20‐oxidase from Marah macrocarpus immature seeds, which forms predominantly CC19‐GAs. The cDNAs encoding these two very similar 20‐oxidases were digested with restriction endonucleases Van 911. Bcl 1, and Bsa WI, and six chimaeric sequences were produced by recombination of the DNA fragments. The pCM1 ‐construct was obtained by exchanging nt 303–809 of the Cucurbita cDNA with the homologous DNA from the March 20‐oxidase. In pCM2, pCM3, pCM4, pCM5 and pCM6, nt 810–992, nt 993–end, nt 303–992, nt 810–end, and nt 311–end were exchanged, respectively. All constructs were cloned in a pUC18 vector and functionally expressed in E. coli NM522 cells. GA 20‐oxidase activity was detectable in cell‐lysates from the transformed E. coli, but the extent and kind of conversion depended on the construct. Highest conversion of GA12was found with pCM1 and pCM3, one‐tenth of this conversion was observed with pCM5 and pCM6, and one‐hundredth was obtained with the hybrid proteins from pCM2 and pCM4. With pCM2 and pCM4, neither the C19‐end product, GA9, nor the C20‐end product, GA25‐was formed. However, after transformation with constructs pCM1, pCM3, pCM5 or pCM6. GA9accounted for 30, 40, 60 and 90%, respectively, of the end products formed. Thus, the segments originating from M. macrocarpus conferred upon the chimaeric proteins an increasing ability to direct the biosynthetic flow into C19‐GAs in this order. Although GA24is the immediate precursor, much less end products were formed by using this substrate.
Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted ...antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP-related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP-inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.
Recent treatments of leukemias with T cells expressing chimeric antigen receptors (CARs) underline their impressive therapeutic potential but also their risk of severe side effects including cytokine ...release storms and tumor lysis syndrome. In case of cross-reactivities, CAR T cells may also attack healthy tissues. To overcome these limitations, we previously established a switchable CAR platform technology termed UniCAR. UniCARs are not directed against typical tumor-associated antigens (TAAs) but instead against a unique peptide epitope: Fusion of this peptide epitope to a recombinant antibody domain results in a target module (TM). TMs can cross-link UniCAR T cells with tumor cells and thereby lead to their destruction. So far, we constructed TMs with a short half-life. The fast turnover of such a TM allows to rapidly interrupt the treatment in case severe side effects occur. After elimination of most of the tumor cells, however, longer lasting TMs which have not to be applied via continous infusion would be more convenient for the patient. Here we describe and characterize a TM for retargeting UniCAR T cells to CD19 positive tumor cells. Moreover, we show that the TM can efficiently be produced
from producer cells housed in a sponge-like biomimetic cryogel and, thereby, serving as an
TM factory for an extended retargeting of UniCAR T cells to CD19 positive leukemic cells.
Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted ...antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP-related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP-inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.
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