Malignant transformation of early lymphoid progenitors in mice expressing an activated Blk tyrosine kinase

The intracellular signals governing cellular proliferation and developmental progression during lymphocyte development are incompletely understood. The tyrosine kinase Blk is expressed preferentially in the B lineage, but its function in B cell development has been largely unexplored. We have generated transgenic mice expressing constitutively active Blk [Blk(Y495F)] in the B and T lymphoid compartments. Expression of Blk(Y495F) in the B lineage at levels similar to that of endogenous Blk induced B lymphoid tumors of limited clonality, whose phenotypes are characteristic of B cell progenitors at the proB/preB-I to preB-II transition. Expression of constitutively active Blk in the T lineage resulted in the appearance of clonal, thymic lymphomas composed of intermediate single positive cells. Taken together, these results indicate that specific B and T cell progenitor subsets are preferentially susceptible to transformation by Blk(Y495F) and suggest a role for Blk in the control of proliferation during B cell development.

Estimating sequestered parasite population dynamics in cerebral malaria

Clinical investigation of malaria is hampered by the lack of a method for estimating the number of parasites that are sequestered in the tissues, for it is these parasites that are thought to be crucial to the pathogenesis of life-threatening complications such as cerebral malaria. We present a method of estimating this hidden population by using clinical observations of peripheral parasitemia combined with an age-structured mathematical model of the parasite erythrocyte cycle. Applying the model to data from 217 Gambian children undergoing treatment for cerebral malaria we conclude that although artemether clears parasitemia more rapidly than quinine, the clearance of sequestered parasites is similar for the two drugs. The estimated sequestered mass was found to be a more direct predictor of fatal outcome than clinically observed parasitemia. This method allows a sequential analysis of sequestered parasite population dynamics in children suffering from cerebral malaria, and the results offer a possible explanation for why artemether provides less advantage than might have been expected over quinine in reducing mortality despite its rapid effect on circulating parasites.

Molecular Cloning and Characterization of a Radial Spoke Head Protein of Sea Urchin Sperm Axonemes: Involvement of the Protein in the Regulation of Sperm Motility

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40°C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form α-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.

Corticotropin-releasing factor-binding protein ligand inhibitor blunts excessive weight gain in genetically obese Zucker rats and rats during nicotine withdrawal

Elevation of the neuropeptide corticotropin-releasing factor (CRF) in the brain is associated with a reduction of food intake and body weight gain in normal and obese animals. A protein that binds CRF and the related peptide, urocortin, with high affinity, CRF-binding protein (CRF-BP), may play a role in energy homeostasis by inactivating members of this peptide family in ingestive and metabolic regulatory brain regions. Intracerebroventricular administration in rats of the high-affinity CRF-BP ligand inhibitor, rat/human CRF (6-33), which dissociates CRF or urocortin from CRF-BP and increases endogenous brain levels of “free” CRF or urocortin significantly blunted exaggerated weight gain in Zucker obese subjects and in animals withdrawn from chronic nicotine. Chronic administration of CRF suppressed weight gain nonselectively by 60% in both Zucker obese and lean control rats, whereas CRF-BP ligand inhibitor treatment significantly reduced weight gain in obese subjects, without altering weight gain in lean control subjects. Nicotine abstinent subjects, but not nicotine-naive controls, experienced a 35% appetite suppression and a 25% weight gain reduction following acute and chronic administration, respectively, of CRF-BP ligand inhibitor. In marked contrast to the effects of a CRF-receptor agonist, the CRF-BP ligand inhibitor did not stimulate adrenocorticotropic hormone secretion or elevate heart rate and blood pressure. These results provide support for the hypothesis that the CRF-BP may function within the brain to limit selected actions of CRF and/or urocortin. Furthermore, CRF-BP may represent a novel and functionally selective target for the symptomatic treatment of excessive weight gain associated with obesity of multiple etiology.

Role of Fission Yeast Primase Catalytic Subunit in the Replication Checkpoint

To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe, spp1+ and spp2+ (S. pombe primase 1 and 2). spp1+ encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Polα to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1+ gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle. Spp1 is essential for cell viability, and thermosensitive mutants of spp1+ exhibit an allele-specific abnormal mitotic phenotype. Mutations of spp1+ reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Polα–primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Polα has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Polα. Together, these have led us to propose that suboptimal cellular levels of polα–primase complex due to the allele-specific mutations of Spp1 might not allow Polα to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response. Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.

Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis

Signal transducer and activator of transcription (STAT) proteins perform key roles in mediating signaling by cytokines and growth factors, including platelet-derived growth factor (PDGF). In addition, Src family kinases activate STAT signaling and are required for PDGF-induced mitogenesis in normal cells. One STAT family member, Stat3, has been shown to have an essential role in cell transformation by the Src oncoprotein. However, the mechanisms by which STAT-signaling pathways contribute to mitogenesis and transformation are not fully defined. We show here that disruption of Stat3 signaling by using dominant-negative Stat3β protein in NIH 3T3 fibroblasts suppresses c-Myc expression concomitant with inhibition of v-Src-induced transformation. Ectopic expression of c-Myc is able to partially reverse this inhibition, suggesting that c-Myc is a downstream effector of Stat3 signaling in v-Src transformation. Furthermore, c-myc gene knockout fibroblasts are refractory to transformation by v-Src, consistent with a requirement for c-Myc protein in v-Src transformation. In normal NIH 3T3 cells, disruption of Stat3 signaling with dominant-negative Stat3β protein inhibits PDGF-induced mitogenesis in a manner that is reversed by ectopic c-Myc expression. Moreover, inhibition of Src family kinases with the pharmacologic agent, SU6656, blocks Stat3 activation by PDGF. These findings, combined together, delineate the signaling pathway, PDGF → Src → Stat3 → Myc, that is important in normal PDGF-induced mitogenesis and subverted in Src transformation.

Expediting the transfer of evidence into practice: building clinical partnerships*

A librarian/clinician partnership was fostered in one hospital through the formation of the Evidence-based Practice Committee, with an ulterior goal of facilitating the transfer of evidence into practice. The paper will describe barriers to evidence-based practice and outline the committee's strategies for overcoming these barriers, including the development and promotion of a Web-based guide to evidence-based practice specifically designed for clinicians (health professionals). Educational strategies for use of the Web-based guide will also be addressed. Advantages of this partnership are that the skills of librarians in meeting the needs of clinicians are maximized. The evidence-based practice skills of clinicians are honed and librarians make a valuable contribution to the knowledgebase of the clinical staff. The knowledge acquired through the partnership by both clinicians and librarians will increase the sophistication of the dialogue between the two groups and in turn will expedite the transfer of evidence into practice.