12S,20-dihydroxyicosatetraenoic acid: a new icosanoid synthesized by neutrophils from 12S-hydroxyicosatetraenoic acid produced by thrombin- or collagen-stimulated platelets.

A new metabolite of arachidonic acid, formed during interaction between thrombin- or collagen-stimulated platelets and unstimulated neutrophils, has been demonstrated by both thin-layer radiochromatography and high-performance liquid chromatography. Production of the 3H-labeled metabolite in combined suspensions containing [3H]arachidonate-labeled platelets and unlabeled neutrophils from aspirin-treated donors suggested that platelet 3H-labeled 12S-hydroxy-5,8-cis,10-trans,14-cis-icosatetraenoic acid (12-HETE) was the precursor. This was confirmed by identification of the same product when purified 12-[3H]HETE was added directly to unstimulated neutrophils. Hydrogenation and oxidation of the isolated product, followed by gas chromatography-mass spectrometry showed the structure to be 12S,20-dihydroxyicosatetraenoic acid. These experiments further show that platelet stimuli known to occur in vivo may initiate metabolic interactions between different cell types via the arachidonic acid pathway.     

Expression cloning of a cDNA encoding the murine interleukin 4 receptor based on ligand binding.

Interleukin 4 (IL-4) is a potent mediator of growth and differentiation for various lymphoid and myeloid cells. To isolate a cDNA encoding the murine IL-4 receptor, we have developed an expression cloning method that uses biotinylated ligand as a probe and that may be generally applicable to cloning of receptor genes. COS-7 cells transiently transfected with the cloned full-length cDNA bind murine IL-4 specifically with a Kd = 165 pM. Crosslinking of 125I-labeled IL-4 to COS-7 cells transfected with the cDNA reveals binding to proteins of 120-140 kDa. IL-4-responsive cells also express IL-4-binding proteins of 120-140 kDa but show additional bands at 60-70 kDa; the relationship of the smaller proteins to the larger ones is unclear. The nucleotide sequence indicates that the full-length cDNA encodes 810 amino acids including the signal sequence. While no consensus sequence for protein kinases is present in the cytoplasmic domain, a sequence comparison with the erythropoietin receptor, the IL-6 receptor, and the beta chain of the IL-2 receptor reveals a significant homology in the extracellular domain, indicating that the IL-4 receptor is a member of a cytokine receptor family.     

Use of spleen organ cultures to monitor hemopoietic progenitor cell regeneration following irradiation and marrow transplantation

After lethal irradiation of C57BL mice followed by the injection of 10(7) marrow cells, total cellularity and progenitor cell levels exceeded pretreatment levels within 12 days in the spleen, but regeneration remained incomplete in the marrow. The exceptional regenerative capacity of progenitor populations in the spleen was observed in organ cultures of spleen slices prepared 24 hr after irradiation and transplantation, excluding continuous repopulation from the marrow as a significant factor in splenic regeneration.     

SMAD4 mutations in colorectal cancer probably occur before chromosomal instability, but after divergence of the microsatellite instability pathway

Loss of chromosome 18q21 is well documented in colorectal cancer, and it has been suggested that this loss targets the DCC, DPC4/SMAD4, and SMAD2 genes. Recently, the importance of SMAD4, a downstream regulator in the TGF-beta signaling pathway, in colorectal cancer has been highlighted, although the frequency of SMAD4 mutations appears much lower than that of 18q21 loss. We set out to investigate allele loss, mutations, protein expression, and cytogenetics of chromosome 18 copy number in a collection of 44 colorectal cancer cell lines of known status with respect to microsatellite instability (MSI). Fourteen of thirty-two MSI(-) lines showed loss of SMAD4 protein expression; usually, one allele was lost and the other was mutated in one of a number of ways, including deletions of various sizes, splice site changes, and missense and nonsense point mutations (although no frameshifts). Of the 18 MSI(-) cancers with retained SMAD4 expression, four harbored missense mutations in the 3' part of the gene and showed allele loss. The remaining 14 MSI(-) lines had no detectable SMAD4 mutation, but all showed allele loss at SMAD4 and/or DCC. SMAD4 mutations can therefore account for about 50-60% of the 18q21 allele loss in colorectal cancer. No MSI(+) cancer showed loss of SMAD4 protein or SMAD4 mutation, and very few had allelic loss at SMAD4 or DCC, although many of these MSI(+) lines did carry TGFBIIR changes. Although SMAD4 mutations have been associated with late-stage or metastatic disease, our combined molecular and cytogenetic data best fit a model in which SMAD4 mutations occur before colorectal cancers become aneuploid/polyploid, but after the MSI(+) and MSI(-) pathways diverge. Thus, MSI(+) cancers may diverge first, followed by CIN(+) (chromosomal instability) cancers, leaving other cancers to follow a CIN(-)MSI(-) pathway.     

Effect of high-fat diet on glucose homeostasis and gene expression in glucokinase knockout mice

Aim: We have generated a heterozygous glucokinase knockout mouse (gk^del/wt), upon which we investigated the effect of high‐fat diet (HFD) with respect to metabolic control and both hepatic and β‐cell gene expression. We also investigated the in vitro efficacy of a glucokinase activator (GKA) on glucose‐stimulated insulin secretion (GSIS) in gk^del/wtmouse islets. Methods: Male gk^del/wtand gk^wt/wtmice were grouped (n = 8–10) at 10 weeks of age and fed HFD or chow diet (CD) for 10 weeks. Multiple parameters including blood glucose, plasma insulin and glucose tolerance were assessed. Further animal groups were used for in vitro GSIS and islet and liver gene expression analysis. Results and Conclusions: gk^del/wtmice showed early‐onset persistent hyperglycaemia, raised glycated haemoglobin levels, impaired GSIS and glucose tolerance but no change in plasma cholesterol, non‐esterified fatty acids or triglyceride levels. After HFD feeding, insulin levels of gk^del/wtmice were less than half that of gk^wt/wtmice, although they were equivalent to gk^wt/wtmice on CD. While gk^wt/wtmice maintained moderate hyperglycaemia, gk^del/wtmice became overtly diabetic, with worsened glucose tolerance. A GKA (GKA50) increased GSIS, at 10 mM glucose, in gk^del/wtmice to an extent at least as great as that seen in gk^wt/wtmice on both CD and HFD. gk^del/wtmice showed only a small number of changes in gene expression compared with gk^wt/wtmice. We propose the high fat–fed gk^del/wtmouse as a model of type 2 diabetes and report retained efficacy of a GKA on in vitro GSIS.