Molecular Cloning and Characterization of a Novel Retinoblastoma-Binding Protein

We describe the isolation and characterization of a novel cDNA encoding a polypeptide that interacts in a yeast two-hybrid system as well as in mammalian cells with the retinoblastoma (RB) protein. This new protein, which we call Rim, consists of 897 amino acids, has two leucine zipper motifs, and has a LECEE sequence previously identified as an RB-binding domain. Rim also has an E1A/CtBP-binding motif and four putative nuclear localization signals.RimmRNA is expressed ubiquitously at low levels in all human adult tissues tested and at much higher levels in several tumor cell lines. TheRimgene (HGMW-approved symbol RBBP8) is localized on human chromosome 18q11.2.     

Industry involvement and baseline assumptions of cost-effectiveness analyses: diagnostic accuracy of the Papanicolaou test

Background

Industry involvement has been associated with more favourable cost-effectiveness ratios in cost-effectiveness analyses, but the mechanisms for this association are unclear. We evaluated whether the assumed accuracy of the Papanicolaou (Pap) test was correlated with the features of cost-effectiveness analysis studies.

Methods

We searched PubMed (last updated April 2010) for cost-effectiveness analysis studies in which at least one strategy involved the Pap test for cervical cancer. We assessed the baseline assumed diagnostic sensitivity and specificity of the Pap test in each study and the association of these values with three levels of manufacturer involvement in the study.

Results

Among 88 analyzed cost-effectiveness analysis studies, the assumed sensitivity of the Pap test was lower in studies with manufacturer-affiliated authors, manufacturer funding or manufacturer-related competing interests versus studies without (mean sensitivity 60% v. 70%, p < 0.001). The assumed specificity of the Pap test was lower in cost-effectiveness analyses involving new screening tests (mean 93% v. 96%, p = 0.016). The assumed specificity did not differ between trials with manufacturer involvement versus those without (mean 95% v. 95%, p = 0.755).

Interpretation

The results of cost-effectiveness analyses may be affected by a downgrading of the assumed diagnostic accuracy of the standard Pap test against which newer tests or interventions are compared. New technology then seems to have more favourable results against a straw-man comparator.Given the substantial impact of cost-effectiveness analyses on public health policies, it is important to safeguard their robustness. However, most published cost-effectiveness analyses report favourable incremental cost-effectiveness ratios, and industry-funded analyses are more likely to report desirable ratios.¹ Some journals discourage cost-effectiveness analysis studies that have been conducted or funded by sponsors of the examined interventions.² Examples exist in which cost-effectiveness analysis studies on the same topic have reached different conclusions depending on who funded them.³ Yet the mechanism for such discrepancies in seemingly rigorous quantitative analyses is unclear.The process of cost-effectiveness analysis involves making assumptions about the main parameters of interest that enter calculations. Baseline values and plausible ranges are specified. When baseline assumptions vary across different cost-effectiveness analyses, results may diverge considerably. We evaluated the baseline assumptions of the diagnostic accuracy of a widely used and studied screening test,⁴ the Papanicolaou (Pap) test (i.e., conventional cervical cytology) in cost-effectiveness analysis studies. The diagnostic accuracy of the Pap test has been studied extensively,^5–7 and assumptions about its accuracy are pivotal for any cost-effectiveness analysis related to screening for and prevention of cervical cancer. Moreover, alternative approaches, such as DNA testing for human papilloma virus (HPV) or HPV vaccines, are quite expensive. New tests may get favourable cost-effectiveness ratios against the Pap test if the performance of the Pap test is assumed to be suboptimal.We therefore examined whether cost-effectiveness analysis studies involving screening for and prevention of cervical cancer assumed different diagnostic performance estimates for the Pap test and whether differences reflected sponsor-related biases.     

Role of guanylate binding protein‐1 in vascular defects associated with chronic inflammatory diseases

Rheumatic autoimmune disorders are characterized by a sustained pro‐inflammatory microenvironment associated with impaired function of endothelial progenitor cells (EPC) and concomitant vascular defects. Guanylate binding protein‐1 (GBP‐1) is a marker and intracellular regulator of the inhibition of proliferation, migration and invasion of endothelial cells induced by several pro‐inflammatory cytokines. In addition, GBP‐1 is actively secreted by endothelial cells. In this study, significantly increased levels of GBP‐1 were detected in the sera of patients with chronic inflammatory disorders. Accordingly we investigated the function of GBP‐1 in EPC. Interestingly, stable expression of GBP‐1 in T17b EPC induced premature differentiation of these cells, as indicated by a robust up‐regulation of both Flk‐1 and von Willebrand factor expression. In addition, GBP‐1 inhibited the proliferation and migration of EPC in vitro. We confirmed that GBP‐1 inhibited vessel‐directed migration of EPC at the tissue level using the rat arterio‐venous loop model as a novel quantitative in vivo migration assay. Overall, our findings indicate that GBP‐1 contributes to vascular dysfunction in chronic inflammatory diseases by inhibiting EPC angiogenic activity via the induction of premature EPC differentiation.     

The Evolution of Fungal Metabolic Pathways

Fungi contain a remarkable range of metabolic pathways, sometimes encoded by gene clusters, enabling them to digest most organic matter and synthesize an array of potent small molecules. Although metabolism is fundamental to the fungal lifestyle, we still know little about how major evolutionary processes, such as gene duplication (GD) and horizontal gene transfer (HGT), have interacted with clustered and non-clustered fungal metabolic pathways to give rise to this metabolic versatility. We examined the synteny and evolutionary history of 247,202 fungal genes encoding enzymes that catalyze 875 distinct metabolic reactions from 130 pathways in 208 diverse genomes. We found that gene clustering varied greatly with respect to metabolic category and lineage; for example, clustered genes in Saccharomycotina yeasts were overrepresented in nucleotide metabolism, whereas clustered genes in Pezizomycotina were more common in lipid and amino acid metabolism. The effects of both GD and HGT were more pronounced in clustered genes than in their non-clustered counterparts and were differentially distributed across fungal lineages; specifically, GD, which was an order of magnitude more abundant than HGT, was most frequently observed in Agaricomycetes, whereas HGT was much more prevalent in Pezizomycotina. The effect of HGT in some Pezizomycotina was particularly strong; for example, we identified 111 HGT events associated with the 15 Aspergillus genomes, which sharply contrasts with the 60 HGT events detected for the 48 genomes from the entire Saccharomycotina subphylum. Finally, the impact of GD within a metabolic category was typically consistent across all fungal lineages, whereas the impact of HGT was variable. These results indicate that GD is the dominant process underlying fungal metabolic diversity, whereas HGT is episodic and acts in a category- or lineage-specific manner. Both processes have a greater impact on clustered genes, suggesting that metabolic gene clusters represent hotspots for the generation of fungal metabolic diversity.     

Antibodies to the envelope glycoprotein of human T cell leukemia virus type 1 robustly activate cell-mediated cytotoxic responses and directly neutralize viral infectivity at multiple steps of the entry process

Infection of human cells by human T cell leukemia virus type 1 (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to cell surface receptors, including heparan sulfate proteoglycans, neuropilin 1, and glucose transporter 1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognized by neutralizing Abs and CTL following a protective immune response, and therefore, represent attractive components for a HTLV-1 vaccine. To begin to explore the immunological properties of potential envelope-based subunit vaccine candidates, we have used a soluble recombinant surface glycoprotein (gp46, SU) fused to the Fc region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice. The recombinant SU protein is highly immunogenic and induces high titer Ab responses, facilitating selection of hybridomas that secrete mAbs targeting SU. Many of these mAbs recognize envelope displayed on the surface of HTLV-1-infected cells and virions and several of the mAbs robustly antagonize envelope-mediated membrane fusion and neutralize pseudovirus infectivity. The most potently neutralizing mAbs recognize the N-terminal receptor-binding domain of SU, though there is considerable variation in neutralizing proficiency of the receptor-binding domain-targeted mAbs. By contrast, Abs targeting the C-terminal domain of SU tend to lack robust neutralizing activity. Importantly, we find that both neutralizing and poorly neutralizing Abs strongly stimulate neutrophil-mediated cytotoxic responses to HTLV-1-infected cells. Our data demonstrate that recombinant forms of SU possess immunological features that are of significant utility to subunit vaccine design.     

Transfer of the AQP1 cDNA for the correction of radiation-induced salivary hypofunction

The treatment of most patients with head and neck cancer includes ionizing radiation (IR). Salivary glands in the IR field suffer significant and irreversible damage, leading to considerable morbidity. Previously, we reported that adenoviral (Ad)-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to rat [C. Delporte, B.C. O'Connell, X. He, H.E. Lancaster, A.C. O'Connell, P. Agre, B.J. Baum, Increased fluid secretion after adenoviral-mediated transfer of the aquaporin-1 cDNA to irradiated rat salivary glands. Proc. Natl. Acad. Sci. U S A. 94 (1997) 3268-3273] and miniature pig [Z. Shan, J. Li, C. Zheng, X. Liu, Z. Fan, C. Zhang, C.M. Goldsmith, R.B. Wellner, B.J Baum, S. Wang. Increased fluid secretion after adenoviral-mediated transfer of the human aquaporin-1 cDNA to irradiated miniature pig parotid glands. Mol. Ther. 11 (2005) 444-451] salivary glands approximately 16 weeks following IR resulted in a dose-dependent increase in salivary flow to > or =80% control levels on day 3. A control Ad vector was without any significant effect on salivary flow. Additionally, after administration of Ad vectors to salivary glands, no significant lasting effects were observed in multiple measured clinical chemistry and hematology values. Taken together, the findings show that localized delivery of AdhAQP1 to IR-damaged salivary glands is useful in transiently increasing salivary secretion in both small and large animal models, without significant general adverse events. Based on these results, we are developing a clinical trial to test if the hAQP1 cDNA transfer strategy will be clinically effective in restoring salivary flow in patients with IR-induced parotid hypofunction.     

The PERK-eIF2α phosphorylation arm is a pro-survival pathway of BCR-ABL signaling and confers resistance to imatinib treatment in chronic myeloid leukemia cells

Activation of adaptive mechanisms plays a crucial role in cancer progression and drug resistance by allowing cell survival under stressful conditions. Therefore, inhibition of the adaptive response is considered as a prospective therapeutic strategy. The PERK-eIF2α phosphorylation pathway is an important arm of the unfolded protein response (UPR), which is induced under conditions of endoplasmic reticulum (ER) stress. Our previous work showed that ER stress is induced in chronic myeloid leukemia (CML) cells. Herein, we demonstrate that the PERK-eIF2α phosphorylation pathway is upregulated in CML cell lines and CD34⁺ cells from CML patients and is associated with CML progression and imatinib resistance. We also show that induction of apoptosis by imatinib results in the downregulation of the PERK-eIF2α phosphorylation arm. Furthermore, we demonstrate that inactivation of the PERK-eIF2α phosphorylation arm decreases the clonogenic and proliferative capacities of CML cells and sensitizes them to death by imatinib. These findings provide evidence for a pro-survival role of PERK-eIF2α phosphorylation arm that contributes to CML progression and development of imatinib resistance. Thus, the PERK-eIF2α phosphorylation arm may represent a suitable target for therapeutic intervention for CML disease.