The role of Lin28b in myeloid and mast cell differentiation and mast cell malignancy.

Mast cells (MCs) are critical components of the innate immune system and important for host defense, allergy, autoimmunity, tissue regeneration and tumor progression. Dysregulated MC development leads to systemic mastocytosis (SM), a clinically variable but often devastating family of hematologic disorders. Here we report that induced expression of Lin28, a heterochronic gene and pluripotency factor implicated in driving a fetal hematopoietic program, caused MC accumulation in adult mice in target organs such as the skin and peritoneal cavity. In vitro assays revealed a skewing of myeloid commitment in LIN28B-expressing hematopoietic progenitors, with increased levels of LIN28B in common myeloid and basophil-MC progenitors altering gene expression patterns to favor cell fate choices that enhanced MC specification. In addition, LIN28B-induced MCs appeared phenotypically and functionally immature, and in vitro assays suggested a slowing of MC terminal differentiation in the context of LIN28B upregulation. Finally, interrogation of human MC leukemia samples revealed upregulation of LIN28B in abnormal MCs from patients with SM. This work identifies Lin28 as a novel regulator of innate immune function and a new protein of interest in MC disease.

Kinetic resolution of a conformational transition and the ATP hydrolysis step using relaxation methods with a Dictyostelium myosin II mutant containing a single tryptophan residue.

The fluorescence emission intensity from a conserved tryptophan residue (W501) located in the relay loop (F466 to L516) of the Dicytostelium discoideum myosin II motor domain is sensitive to ATP binding and hydrolysis. The initial binding process is accompanied by a small quench in fluorescence, and this is followed by a large enhancement that appears coincident with the hydrolysis step. Using temperature and pressure jump methods, we show that the enhancement process is kinetically distinct from but coupled to the hydrolysis step. The fluorescence enhancement corresponds to the open-closed transition (k(obs) approximately 1000 s(-1) at 20 degrees C). From the overall steady-state fluorescence signal and the presence or absence of a relaxation transient, we conclude that the ADP state is largely in the open state, while the ADP.AlF(4) state is largely closed. At 20 degrees C the open-closed equilibria for the AMP.PNP and ADP.BeF(x) complexes are close to unity and are readily perturbed by temperature and pressure. In the case of ATP, the equilibrium of this step slightly favors the open state, but coupling to the subsequent hydrolysis step gives rise to a predominantly closed state in the steady state. Pressure jump during steady-state ATP turnover reveals the distinct transients for the rapid open-closed transition and the slower hydrolysis step.

Evaluation of stress-inducible hsp90 gene expression as a potential molecular biomarker in Xenopus laevis.

In this study we have evaluated stress-inducible hsp90 mRNA accumulation as a potential molecular biomarker in Xenopus laevis. In order to obtain a probe for Northern blot analysis we employed a PCR-based approach using degenerate primers for the amplification and cloning of an hsp90 gene sequence from Xenopus laevis. The deduced amino acid sequence is 102 amino acids in length and exhibited the highest degree of identity with zebrafish and human hsp90 beta genes. Furthermore, the putative intron and exon boundaries of this fragment are the same as hsp90 beta in chicken, mouse and human, indicating that the fragment represents a Xenopus hsp90 beta-like gene. Northern blot analyses revealed that this gene was constitutively expressed in cultured A6 cells. While heat shock and sodium arsenite exposure resulted in the increased accumulation of hsp90 mRNA in A6 cells, treatment with cadmium chloride and zinc chloride did not. Also, exposure of A6 cells to concurrent heat shock and sodium arsenite produced a mild synergistic response with respect to hsp90 mRNA levels in contrast to hsp70 mRNA levels which displayed a strong synergistic effect. Finally, hsp90 mRNA was detected constitutively throughout early embryogenesis but was heat-inducible only in late blastula and later stages of development. Given the normal abundance and limited stress-induced accumulation of hsp90 mRNA, it may not have a great deal of potential as a molecular biomarker compared to hsp70 and hsp30 mRNA. However, it may be useful in conjunction with other stress protein mRNAs to establish a set of biomarker profiles to characterize the cellular response to a stressful or toxic agent.

Cloning and characterization of a cDNA encoding the collagen-binding stress protein hsp47 in zebrafish.

Hsp47 is a major stress-inducible protein that is localized to the endoplasmic reticulum of avian and mammalian cells and is thought to act as a molecular chaperone specific for the processing of procollagen. Although hsp47 is coordinately expressed together with several collagen types, and vertebrate embryos are known to express collagen genes in complex spatial and temporal patterns, limited information is available regarding the function or regulation of hsp47 during early embryonic development. We have initiated an examination of hsp47 in the zebrafish, Danio rerio, which offers a number of features that make it attractive as a model developmental system with which to examine the expression and function of hsp47. A polymerase chain reaction (PCR)-based cloning strategy was used to isolate a hsp47 cDNA from an embryonic zebrafish cDNA library. The deduced translation product of the cDNA is a 404-amino-acid polypeptide that is 72% identical to chicken, 64% identical to mouse and rat, and 69% identical to human hsp47. The protein contains a typical hydrophobic signal sequence, an RDEL endoplasmic reticulum retention signal, and a serine protease inhibitor signature sequence, all of which are characteristic of hsp47 in higher vertebrates. Thus, it is likely that hsp47 in zebrafish is also localized to the endoplasmic reticulum and may play a similar role to its counterpart in higher vertebrates. Northern blot analysis revealed that the hsp47 gene is expressed at relatively low levels in embryos during normal development but is strongly induced following exposure to heat shock at the gastrula, midsomitogenesis, 2-day, and 3-day larval stages. The level of induction was much higher than has previously been reported in chicken and mouse cells.