Intestinal tumor suppression in ApcMin/+ mice by prostaglandin D2 receptor PTGDR.

Our earlier work showed that knockout of hematopoietic prostaglandin D synthase (HPGDS, an enzyme that produces prostaglandin D2) caused more adenomas in Apc(Min/+) mice. Conversely, highly expressed transgenic HPGDS allowed fewer tumors. Prostaglandin D2 (PGD2) binds to the prostaglandin D2 receptor known as PTGDR (or DP1).

Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of α-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts.

Enzyme replacement therapy for MPS IIIB (mucopolysaccharidosis type IIIB; also known as Sanfilippo B syndrome) has been hindered by inadequate mannose 6 phosphorylation and cellular uptake of rhNAGLU (recombinant human α-N-acetylglucosaminidase). We expressed and characterized a modified rhNAGLU fused to the receptor-binding motif of IGF-II (insulin-like growth factor 2) (rhNAGLU-IGF-II) to enhance its ability to enter cells using the cation-independent mannose 6-phosphate receptor, which is also the receptor for IGF-II (at a different binding site). RhNAGLU-IGF-II was stably expressed in CHO (Chinese-hamster ovary) cells, secreted and purified to apparent homogeneity.

Mannose 6-phosphate conjugation is not sufficient to allow induction of immune tolerance to phenylalanine ammonia-lyase in dogs.

The immune response to exogenous protein has been shown to reduce therapeutic efficacy in animal models of enzyme replacement therapy. A previously published study demonstrated an immunosuppressive regimen which successfully induced immune tolerance to α-L-iduronidase in canines with mucopolysaccharidosis I. The two key requirements for success were high-affinity receptor-mediated enzyme uptake, conferred by mannose 6-phosphate conjugation, and immunosuppression with low-dose antigen exposure.

Biochemical characterization of fluorescent-labeled recombinant human alpha-L-iduronidase in vitro.

In vivo tracking of the delivery of therapeutic proteins is a useful tool for preclinical studies. However, many labels are too large to use without disrupting the normal uptake, function, or other properties of the protein. Low-molecular-weight fluorescent labels allow in vivo and ex vivo tracking of the distribution of therapeutic proteins, and should not alter the protein's characteristics.

Hematopoietic prostaglandin D synthase (HPGDS): a high stability, Val187Ile isoenzyme common among African Americans and its relationship to risk for colorectal cancer.

Intestinal tumors in Apc(Min/+) mice are suppressed by over-production of HPGDS, which is a glutathione transferase that forms prostaglandin D(2) (PGD(2)). We characterized naturally occurring HPGDS isoenzymes, to see if HPGDS variation is associated with human colorectal cancer risk. We used DNA heteroduplex analysis and sequencing to identify HPGDS variants among healthy individuals.

High dietary niacin may increase prostaglandin formation but does not increase tumor formation in ApcMin/+ mice.

High doses of niacin (nicotinic acid) used to treat dyslipidemias cause flushing, due to high levels of prostaglandin D(2) (PGD(2)). GPR109A, a G-protein coupled receptor, triggers the flushing in the skin. In addition to boosting PGD(2), niacin binding to GPR109A activates the entire prostanoid cascade.

Early versus late treatment of spinal cord compression with long-term intrathecal enzyme replacement therapy in canine mucopolysaccharidosis type I.

Enzyme replacement therapy (ERT) with intravenous recombinant human alpha-l-iduronidase (IV rhIDU) is a treatment for patients with mucopolysaccharidosis I (MPS I). Spinal cord compression develops in MPS I patients due in part to dural and leptomeningeal thickening from accumulated glycosaminoglycans (GAG). We tested long-term and every 3-month intrathecal (IT) and weekly IV rhIDU in MPS I dogs age 12-15months (Adult) and MPS I pups age 2-23days (Early) to determine whether spinal cord compression could be reversed, stabilized, or prevented.

XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps.

XRCC4 and DNA ligase IV form a complex that is essential for the repair of all double-strand DNA breaks by the nonhomologous DNA end joining pathway in eukaryotes. We find here that human XRCC4:DNA ligase IV can ligate two double-strand DNA ends that have fully incompatible short 3' overhang configurations with no potential for base pairing. Moreover, at DNA ends that share 1-4 annealed base pairs, XRCC4:DNA ligase IV can ligate across gaps of 1 nt.

Hematopoietic prostaglandin D synthase suppresses intestinal adenomas in ApcMin/+ mice.

Aspirin and other nonsteroidal anti-inflammatory drugs prevent some cases of colon cancer by inhibiting prostaglandin (PG) synthesis. PGE(2) promotes colon neoplasia, as shown by knockout mouse studies on enzymes and receptors in the PG cascade. A few experiments 20 to 30 years ago suggested that PGD(2) may suppress tumors, but a role for biosynthetic enzymes for PGD(2) in tumor development has not been studied.

A biochemically defined system for mammalian nonhomologous DNA end joining.

Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice.

To slip or skip, visualizing frameshift mutation dynamics for error-prone DNA polymerases.

Three models describing frameshift mutations are "classical" Streisinger slippage, proposed for repetitive DNA, and "misincorporatation misalignment" and "dNTP-stabilized misalignment," proposed for non-repetitive DNA. We distinguish between models using pre-steady state fluorescence kinetics to visualize transiently misaligned DNA intermediates and nucleotide incorporation products formed by DNA polymerases adept at making small frameshift mutations in vivo. Human polymerase (pol) mu catalyzes Streisinger slippage exclusively in repetitive DNA, requiring as little as a dinucleotide repeat.

Error-prone replication for better or worse.

Precise genome duplication requires accurate copying by DNA polymerases and the elimination of occasional mistakes by proofreading exonucleases and mismatch repair enzymes. The commonly held belief that 'if something is worth doing, then it's worth doing well' normally applies to DNA replication and repair, however, there are exceptions. This review describes elements that are crucial to cell fitness, evolution and survival in the recently discovered error-prone DNA polymerases.