Leptin and its receptor, obese receptor (OB-R), comprise an important signaling system for the regulation of body weight. Splice variants of OB-R mRNA encode proteins that differ in the length of their cytoplasmic domains. We cloned a long isoform of the wild-type leptin receptor that is preferentially expressed in the hypothalamus and show that it can activate signal transducers and activators of transcription (STAT)-3, and . A point mutation within the OB-R gene of diabetic (db) mice generates a new splice donor site that dramatically reduces expression of this long isoform in homozygous db/db mice. In contrast, an OB-R protein with a shorter cytoplasmic domain is present in both db/db and wild-type mice. We show that this short isoform is unable to activate the STAT pathway. These data provide further evidence that the mutation in OB-R causes the db/db phenotype and identify three STAT proteins as potential mediators of the anti-obesity effects of leptin.Leptin, the product of the obese (ob) gene, is a 16-kDa secreted protein primarily produced by adipocytes (1). There is a good correlation between the percentage of body fat and serum leptin levels suggesting that leptin production is regulated by the mass of adipocytes (2, 3). Leptin levels were normal or elevated in obese individuals (2, 4) arguing against a simple leptin deficiency as the cause of obesity in the majority of humans (5). Serum leptin concentrations increased under a fatty diet but failed to prevent weight gain (3). Therefore, insensitivity to the action of leptin appears to be a common mechanism in obese individuals and in several rodent models. This suggests that dysregulation at the level of the leptin receptor, the downstream signaling pathway, or an unknown modifying mechanism may constitute the basis for weight disorders. The crucial role of leptin and its receptor in obesity is well illustrated by two phenotypically very similar mutants obese (ob) and diabetes (db) (6). Mice homozygous for a loss of function mutation of ob display obesity, hyperglycemia, and insulin resistance resembling type II diabetes. Administration of recombinant leptin to ob mice corrected these abnormalities (7-9). Based on early parabiosis experiments it was expected that db would be caused by a mutation in the ob receptor (OB-R) (6).OB-R was cloned by virtue of its high affinity to leptin through an expression cloning strategy (10). The OB-R gene was mapped to the same 5-centimorgan interval on mouse chromosome 4 to which db had been localized (10). Surprisingly, no mutation in the coding region of OB-R cDNA of db/db mice was found and leptin binding sites were unaltered in db/db mice (10). However, the cloned mouse OB-R cDNA encoded a protein with a much shorter cytoplasmic domain than the human homologue, suggesting that a longer mouse isoform exists. We cloned this longer form of OB-R from wild-type mice and found that the mRNA for this isoform is dramatically reduced in db/db mice. A G to T mutation in db mice generates a new splice donor and su...

Malarial parasites growing inside erythrocytes digest up to 80% of the host cell's haemoglobin within a lysosomal organelle, the digestive vacuole. They sequester the potentially toxic haem (Fe (II) protohaematoporphyrin) that is released during this process into an insoluble pigment called haemozoin, which consists of polymerized Fe (III) protohaematoporphyrin subunits. We have studied this process of haem polymerization, which was previously reported to be enzyme-mediated and the target of the quinoline antimalarial drugs chloroquine and quinine. Here we show that, rather than being enzyme-mediated, haem polymerization is actually a chemical process, dependent only on the presence of haem-derived material associated with haemozoin and not on protein. This discovery does not invalidate haem polymerization as a target for drug intervention and the mechanism by which haemozoin formation is initiated is still not understood, but our view of this process and of the action of choroquine must be reconsidered.

Thrombopoietin (TPO), originally described as an activity in the serum of thrombocytopenic animals that leads to increased production of platelets, has recently been isolated and cloned. Its closest relative in the cytokine superfamily, erythropoietin (EPO), is transcriptionally regulated during anemia, and it was expected that TPO would similarly be regulated during thrombocytopenia. We induced thrombocytopenia in mice and confirmed that TPO activity was upregulated, as determined by a bioassay. Liver and kidney were found to be the major sources of TPO mRNA. Surprisingly, TPO mRNA in these tissues was not upregulated in thrombocytopenic mice. Using a sensitive RNase protection assay that can distinguish between TPO isoforms, we found no change in the profile of mRNA for these isoforms. A semiquantitative reverse transcription- polymerase chain reaction assay also did not demonstrate upregulation of TPO mRNA in the spleen. Thus, the increase of TPO activity during thrombocytopenia is not caused by regulation at the level of TPO mRNA. Furthermore, isolated mouse platelets absorbed high amounts of bioactive TPO out of TPO-conditioned medium in a dose-dependent fashion. Our results are consistent with TPO protein being regulated at a posttranscriptional level and/or directly through absorption and metabolism by platelets.

The question of whether extracellular signals inf luence hematopoiesis by instructing stem cells to commit to a specific hematopoietic lineage (instructive model) or solely by permitting the survival and proliferation of predetermined progenitors (permissive model) has been controversial since the discovery of lineage-dominant hematopoietic cytokines. To study the potential role of cytokines and their receptors in hematopoietic cell fate decisions, we used homologous recombination to replace the thrombopoietin receptor gene (mpl) with a chimeric construct encoding the extracellular domain of mpl and the cytoplasmic domain of the granulocyte colonystimulating factor receptor (G-CSFR). This chimeric receptor binds thrombopoietin but signals through the G-CSFR intracellular domain. We found that, despite the absence of a functional mpl signaling domain, homozygous knock-in mice had a normal platelet count, indicating that in vivo the cytoplasmic domain of G-CSFR can functionally replace mpl signaling to support normal megakaryopoiesis and platelet formation. This finding is compatible with the permissive model, according to which cytokine receptors provide a nonspecific survival or proliferation signal, and argues against an instructive role of mpl or G-CSFR in hematopoietic cell fate decisions.