Showing papers by "Paul G. Richardson published in 2004"

Community structure and metabolism through reconstruction of microbial genomes from the environment

Abstract: Microbial communities are vital in the functioning of all ecosystems; however, most microorganisms are uncultivated, and their roles in natural systems are unclear. Here, using random shotgun sequencing of DNA from a natural acidophilic biofilm, we report reconstruction of near-complete genomes of Leptospirillum group II and Ferroplasma type II, and partial recovery of three other genomes. This was possible because the biofilm was dominated by a small number of species populations and the frequency of genomic rearrangements and gene insertions or deletions was relatively low. Because each sequence read came from a different individual, we could determine that single-nucleotide polymorphisms are the predominant form of heterogeneity at the strain level. The Leptospirillum group II genome had remarkably few nucleotide polymorphisms, despite the existence of low-abundance variants. The Ferroplasma type II genome seems to be a composite from three ancestral strains that have undergone homologous recombination to form a large population of mosaic genomes. Analysis of the gene complement for each organism revealed the pathways for carbon and nitrogen fixation and energy generation, and provided insights into survival strategies in an extreme environment.

The Genome of the Diatom Thalassiosira Pseudonana: Ecology, Evolution, and Metabolism

Abstract: Diatoms are unicellular algae with plastids acquired by secondary endosymbiosis. They are responsible for approximately 20% of global carbon fixation. We report the 34 million-base pair draft nuclear genome of the marine diatom Thalassiosira pseudonana and its 129 thousand-base pair plastid and 44 thousand-base pair mitochondrial genomes. Sequence and optical restriction mapping revealed 24 diploid nuclear chromosomes. We identified novel genes for silicic acid transport and formation of silica-based cell walls, high-affinity iron uptake, biosynthetic enzymes for several types of polyunsaturated fatty acids, use of a range of nitrogenous compounds, and a complete urea cycle, all attributes that allow diatoms to prosper in aquatic environments.

A phase 2 study of two doses of bortezomib in relapsed or refractory myeloma

Abstract: In a phase 2 open-label study of the novel proteasome inhibitor bortezomib, 54 patients with multiple myeloma who had relapsed after or were refractory to frontline therapy were randomized to receive intravenous 1.0 or 1.3 mg/m(2) bortezomib twice weekly for 2 weeks, every 3 weeks for a maximum of eight cycles. Dexamethasone was permitted in patients with progressive or stable disease after two or four cycles respectively. Responses were determined using modified European Group for Blood and Marrow Transplantation criteria. The complete response (CR) + partial response (PR) rate for bortezomib alone was 30% [90% confidence interval (CI), 15.7-47.1] and 38% (90% CI, 22.6-56.4) in the 1.0 mg/m(2) (8 of 27 patients) and 1.3 mg/m(2) (10 of 26 patients) groups respectively. The CR + PR rate for patients who received bortezomib alone or in combination with dexamethasone was 37% and 50% for the 1.0 and 1.3 mg/m(2) cohorts respectively. The most common grade 3 adverse events were thrombocytopenia (24%), neutropenia (17%), lymphopenia (11%) and peripheral neuropathy (9%). Grade 4 events were observed in 9% (five of 54 patients). Bortezomib alone or in combination with dexamethasone demonstrated therapeutic activity in patients with multiple myeloma who relapsed after frontline therapy.

Reverse methanogenesis: testing the hypothesis with environmental genomics.

Abstract: Microbial methane consumption in anoxic sediments significantly impacts the global environment by reducing the flux of greenhouse gases from ocean to atmosphere. Despite its significance, the biological mechanisms controlling anaerobic methane oxidation are not well characterized. One current model suggests that relatives of methane-producing Archaea developed the capacity to reverse methanogenesis and thereby to consume methane to produce cellular carbon and energy. We report here a test of the “reverse-methanogenesis” hypothesis by genomic analyses of methane-oxidizing Archaea from deep-sea sediments. Our results show that nearly all genes typically associated with methane production are present in one specific group of archaeal methanotrophs. These genome-based observations support previous hypotheses and provide an informed foundation for metabolic modeling of anaerobic methane oxidation.

The DNA sequence and biology of human chromosome 19

Abstract: Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G + C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

Mechanisms by which SGN-40, a Humanized Anti-CD40 Antibody, Induces Cytotoxicity in Human Multiple Myeloma Cells: Clinical Implications

Abstract: CD40 is expressed on B-cell malignancies, including human multiple myeloma (MM) and a variety of carcinomas. We examined the potential therapeutic utility of SGN-40, the humanized anti-CD40 monoclonal antibody, for treating human MM using MM cell lines and patient MM cells (CD138(++), CD40(+)). SGN-40 (0.01-100 micro g/ml) induces modest cytotoxicity in MM cell lines and patient MM cells. In the presence of de novo protein synthesis inhibitor cycloheximide, SGN-40 significantly induced apoptosis in Dexamethasone (Dex)-sensitive MM.1S and Dex-resistant MM.1R cells and in patient MM cells. SGN-40-mediated cytotoxicity is associated with up-regulation of cytotoxic ligands of the tumor necrosis factor family (Fas/FasL, tumor necrosis factor-related apoptosis-inducing ligand, and tumor necrosis factor alpha). SGN-40 treatment also induces a down-regulation of CD40 dependent on an endocytic pathway. Consequently, pretreatment of MM cells with SGN-40 blocked sCD40L-mediated phosphatidylinositol 3'-kinase/AKT and nuclear factor kappaB activation. Importantly, pretreatment of MM.1S and MM.1R cells with SGN-40 inhibited proliferation triggered by interleukin 6 (IL-6) but not by insulin-like growth factor-I. In addition, SGN-40 pretreatment of MM.1S cells blocked the ability of IL-6 to protect against Dex-induced inhibition of DNA synthesis. This was associated with a 2-4-fold reduction of IL-6 receptor at protein and mRNA levels in SGN-40-treated MM.1S cells and patient MM cells. Taken together, these results provide the preclinical rationale for the evaluation of SGN-40 as a potential new therapy to improve patient outcome in MM.

The sequence and analysis of duplication-rich human chromosome 16

Abstract: Human chromosome 16 features one of the highest levels of segmentally duplicated sequence among the human autosomes. We report here the 78,884,754 base pairs of finished chromosome 16 sequence, representing over 99.9% of its euchromatin. Manual annotation revealed 880 protein-coding genes confirmed by 1,670 aligned transcripts, 19 transfer RNA genes, 341 pseudogenes and three RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukaemia. Several large-scale structural polymorphisms spanning hundreds of kilobase pairs were identified and result in gene content differences among humans. Whereas the segmental duplications of chromosome 16 are enriched in the relatively gene-poor pericentromere of the p arm, some are involved in recent gene duplication and conversion events that are likely to have had an impact on the evolution of primates and human disease susceptibility.

The sequence and analysis of duplication rich human chromosome 16

Abstract: We report here the 78,884,754 base pairs of finished human chromosome 16 sequence, representing over 99.9 percent of its euchromatin. Manual annotation revealed 880 protein coding genes confirmed by 1,637 aligned transcripts, 19 tRNA genes, 341 pseudogenes and 3 RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukemia. Several large-scale structural polymorphisms spanning hundreds of kilobasepairs were identified and result in gene content differences across humans. One of the unique features of chromosome 16 is its high level of segmental duplication, ranked among the highest of the human autosomes. While the segmental duplications are enriched in the relatively gene poor pericentromere of the p-arm, some are involved in recent gene duplication and conversion events which are likely to have had an impact on the evolution of primates and human disease susceptibility.

p38 MAPK inhibition enhances PS-341 (bortezomib)-induced cytotoxicity against multiple myeloma cells

Abstract: Although PS-341 (bortezomib) is a promising agent to improve multiple myeloma (MM) patient outcome, 65% of patients with relapsed and refractory disease do not respond. We have previously shown that heat shock protein (Hsp)27 is upregulated after PS-341 treatment, that overexpression of Hsp27 confers PS-341 resistance, and that inhibition of Hsp27 overcomes PS-341 resistance. Since Hsp27 is a downstream target of p38 mitogen-activated protein kinase (MAPK)/MAPK-mitogen-activated protein kinase-2 (MAPKAPK2), we hypothesized that inhibition of p38 MAPK activity could augment PS-341 cytotoxicity by downregulating Hsp27. Although p38 MAPK inhibitor SCIO-469 (Scios Inc, CA, USA) alone did not induce significant growth inhibition, it blocked baseline and PS-341-triggered phosphorylation of p38 MAPK as well as upregulation of Hsp27, associated with enhanced cytotoxicity in MM.1S cells. Importantly, SCIO-469 enhanced phosphorylation of c-Jun NH2-terminal kinase (JNK) and augmented cleavage of caspase-8 and poly(ADP)-ribose polymerase. Moreover, SCIO-469 downregulated PS-341-induced increases in G2/M-phase cells, associated with downregulation of p21Cip1 expression. Importantly, SCIO-469 treatment augmented cytotoxicity of PS-341 even against PS-341-resistant cell lines and patient MM cells. These studies therefore provide the framework for clinical trials of SCIO-469 to enhance sensitivity and overcome resistance to PS-341, thereby improving patient outcome in MM.

Thalidomide for patients with relapsed multiple myeloma after high-dose chemotherapy and stem cell transplantation: results of an open-label multicenter phase 2 study of efficacy, toxicity, and biological activity.

Abstract: OBJECTIVE To determine the progression-free survival at 12 weeks, to evaluate the toxic effects, and to analyze the biological activity of thalidomide in patients with relapsed multiple myeloma (MM) after high-dose chemotherapy and stem cell transplantation. PATIENTS AND METHODS From 1999 to 2001, we performed a multicenter prospective phase 2 study in patients with MM that relapsed after high-dose chemotherapy and stem cell transplantation to evaluate the efficacy of oral thalidomide, with dose escalation from 200 to 600 mg/d over 12 weeks and a subsequent maintenance phase of 200 mg/d for up to 1 year. Outcome was correlated with serum and plasma levels of vascular endothelial growth factor and serum levels of tumor necrosis factor α, soluble intercellular adhesion molecule 1, interferon γ, interleukin (IL) 2, and IL-6 during treatment. RESULTS Thirty patients were treated (19 men and 11 women; median age, 58 years). The median number of prior therapies was 5, and the median duration from diagnosis of MM to study enrollment was 4.3 years. The 12-week progression-free survival rate was 67% (95% confidence interval [CI], 48%-86%). The observed response rate (partial response plus minor response) was 43% (95% CI, 28%-60%) with a median duration of 6 months. Attributable toxicities included constipation, fatigue, rash, and neuropathy, which was dose limiting in 8 patients (27%). Dose escalation from 200 to 600 mg/d was achieved in 50% of patients. Although responses were observed with lower doses, possibly eliminating the need to escalate the dose, responses were also seen in patients who completed the dose escalation. Some patients had disease progression while receiving the maintenance dose of 200 mg/d. Analysis of biomarker assays did not identify any biomarker associated with greater response, but a significant increase in levels of soluble intercellular adhesion molecule 1, IL-2, and interferon γ was seen with thalidomide therapy. CONCLUSION The optimal thalidomide dose varies, and adverse effects can be dose limiting. The dose of thalidomide therapy should be based on the individual patient to ensure that it is well tolerated and that a response is achieved.

Transforming growth factor β receptor I kinase inhibitor down-regulates Cytokine secretion and multiple myeloma cell growth in the bone marrow microenvironment

Abstract: Purpose: Transforming growth factors (TGFs) have pleiotropic biological effects on tumor cells and their environment. In multiple myeloma (MM), we have reported that bone marrow stromal cells (BMSCs) from MM patients produce more TGF-β1 than BMSCs from healthy donors, which in turn induces interleukin (IL)-6 secretion. We show here that the TGF-β receptor I kinase inhibitor SD-208 significantly decreases secretion of both IL-6 and vascular endothelial growth factor (VEGF) from BMSCs, as well as tumor cell growth triggered by MM cell adhesion to BMSCs. Experimental Design: Cytokine production and MM cell proliferation triggered by TGF-β1 or adhesion to BMSCs were examined in the presence or absence of SD-208. Effects of SD-208 on TGF-β1–induced signaling pathways triggering IL-6 and VEGF transcription in BMSCs were also delineated. Results: SD-208 significantly inhibits not only transcription but also secretion of both IL-6 and VEGF from BMSCs triggered by either TGF-β1 or adhesion of MM cells to BMSCs. Moreover, SD-208 decreased tumor cell growth triggered by MM cell adhesion to BMSCs. SD-208 works, at least in part, by blocking TGF-β1–triggered nuclear accumulation of Smad2/3 and hypoxia-inducible factor 1α, as well as related production of IL-6 and VEGF, respectively. Conclusions: These studies indicate that SD-208 inhibits production of cytokines mediating MM cell growth, survival, drug resistance, and migration in the BM milieu, thereby providing the preclinical rationale for clinical evaluation of SD-208 to improve patient outcome in MM.

Immunomodulatory Analogs of Thalidomide: An Emerging New Therapy in Myeloma

Abstract: In this issue of the Journal of Clinical Oncology (JCO), Schey et al present the results of a phase I study of the immunomodulatory thalidomide analog CC-4047 in relapsed, and relapsed refractory multiple myeloma (MM). This important work represents a next step in a series of studies for the development of new agents in the treatment of relapsed MM, which remains incurable, despite advances such as highdose therapy and stem-cell transplantation (SCT). Since the encouraging results of the thalidomide trial led by Singhal et al in advanced relapsed and refractory MM, thalidomide has been established as an effective therapy for the treatment of MM in the relapsed and upfront settings. However, the efficacy of thalidomide has been significantly limited by adverse effects, which include sedation, neuropathy, constipation, and deep vein thrombosis. This toxicity profile seems both dose-related and duration-related, especially post-SCT, spurring the development of thalidomide-derived immunomodulatory analogs known as immunomodulatory drugs (IMiDs), which have the potential of improved potency and reduced toxicity. CC-4047 is one of two small-molecule 4-amino derivatives currently under study in patients with MM. Studies demonstrate that both thalidomide and IMiDs not only act to inhibit angiogenesis, but also act directly to induce both apoptosis and growth arrest in resistant myeloma cells. They also block both the adhesion of myeloma cells to bone marrow stromal cells and the related protection against apoptosis, and block the increased secretion of myeloma cell growth, survival, and migratory factors such as interleukin-6, tumor necrosis factor alpha (TNF), and vascular endothelial growth factor, triggered by the binding of myeloma cells to bone marrow stromal cells. In addition, they expand natural killer cell and T-cell numbers, and improve function against human myeloma cells and enhance their susceptibility to antibody-dependent cellmediated cytotoxicity in vivo (Fig 1). By modifying thalidomide structure through the addition of an amino group at the 4 position of the phthaloyl ring (to generate CC-4047) and with the further removal of a carbonyl on the ring to form CC-5013, such analogs are up to 50,000 times more potent at inhibiting TNFthan the thalidomide parent compound in vitro and are markedly more stable. On the basis of this promising preclinical data, the first phase I study of the thalidomide analog CC-5013 was carried out in 2001, when a dose range of 5 to 50 mg/d was tested in 25 patients with relapsed and refractory MM. Patients in this study had received a median of three prior regimens, including autologous SCT and prior thalidomide in approximately twothirds of the patients. Importantly, no dose-limiting toxicity was observed at any dose level within the first 28 days, but grade 3 myelosuppression developed after 28 days in all 13 patients treated at the highest dose level of 50 mg/d. In 12 of these patients, dose reduction to 25 mg/d was well tolerated and was considered the maximum-tolerated dose. No significant constipation or neuropathy was seen in any cohort, and encouragingly, responses were seen in 17 (71%) of 24 assessable patients, including 11 patients (46%) who had received prior thalidomide. A second study by Zangari et al in 15 patients with advanced MM confirmed a maximum-tolerated dose of 25 mg/d. These studies provided the basis for the further evaluation of CC-5013, either alone or in combination with dexamethasone, to treat patients with MM at first relapse, or for relapsed refractory MM. Results of these studies have been encouraging, with impressive response rates and acceptable tolerability. A series of phase II trials with CC-5013 have been completed, and a multicenter, parallel group, controlled phase III randomized double-blind study of CC-5013 plus dexamethasone compared with dexamethasone alone in previously treated patients with MM has just completed accrual. JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 22 NUMBER 16 AUGUST 15 2004

The complete sequence of human chromosome 5

Abstract: Chromosome 5 is one of the largest human chromosomes yet has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding and syntenic conservation with non-mammalian vertebrates, suggesting they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-encoding genes including the protocadherin and interleukin gene families and the first complete versions of each of the large chromosome 5 specific internal duplications. These duplications are very recent evolutionary events and play a likely mechanistic role, since deletions of these regions are the cause of debilitating disorders including spinal muscular atrophy (SMA).

Caveolin-1 is required for vascular endothelial growth factor-triggered multiple myeloma cell migration and is targeted by bortezomib.

Abstract: We recently demonstrated that caveolae, vesicular flask-shaped invaginations of the plasma membrane, represent novel therapeutic targets in multiple myeloma. In the present study, we demonstrate that vascular endothelial growth factor (VEGF) triggers Src-dependent phosphorylation of caveolin-1, which is required for p130 Cas phosphorylation and multiple myeloma cell migration. Conversely, depletion of caveolin-1 by antisense methodology abrogates p130 Cas phosphorylation and VEGF-triggered multiple myeloma cell migration. The proteasome inhibitor bortezomib both inhibited VEGF-triggered caveolin-1 phosphorylation and markedly decreased caveolin-1 expression. Consequently, bortezomib inhibited VEGF-induced multiple myeloma cell migration. Bortezomib also decreased VEGF secretion in the bone marrow microenvironment and inhibited VEGF-triggered tyrosine phosphorylation of caveolin-1, migration, and survival in human umbilical vascular endothelial cells. Taken together, these studies demonstrate the requirement of caveolae for VEGF-triggered multiple myeloma cell migration and identify caveolin-1 in multiple myeloma cells and human umbilical vascular endothelial cells as a molecular target of bortezomib.

Tumour cell/dendritic cell fusions as a vaccination strategy for multiple myeloma

Abstract: Summary Multiple myeloma (MM) cells express certain tumour-associated antigens (TAAs) that could serve as targets for active-specific immunotherapy The aim of the present study was to test the MM/dendritic cell (DC) fusion as a vaccination strategy We fused MM cells with DC to generate fusion cells (FCs) and tested their antigen presenting cell (APC) function in mixed lymphocyte reactions and cytotoxicity assays First, the HS Sultan and SK0007 HAT sensitive human MM cell lines and DCs generated from peripheral blood of normal donors were fused in the presence of 50% polyethylene glycol to form FCs Next, tumour cells freshly isolated from patients were similarly fused with autologous DCs to generate FCs The FCs demonstrated a biphenotypic profile, confirmed both by flow-cytometry and dual immunofluorescence microscopy These FCs induced MM-specific cytotoxicity FCs, but not MM cells or DCs alone, were potent stimulators of autologous patient T cells More importantly, FC-primed autologous peripheral blood mononuclear cells demonstrated major histocompatibility complex-restricted MM-specific cytolysis These studies therefore demonstrated that MM/DC FC can trigger an autologous immune response to MM cells and formed the framework for a clinical trial currently underway

Proteasome inhibition in hematologic malignancies.

Abstract: Hematologic malignancies, including multiple myeloma (MM), will account for more than 100,000 new cases of cancer and over 57,000 deaths in the United States in 2003 Treatment of MM is a serious challenge, because despite a variety of available therapies, median survival is short A new therapeutic area focuses on inhibiting the activity of the proteasome, a 26S protease complex involved in cell cycle regulation, cell adhesion, inflammation, and protein turnover The novel proteasome inhibitor, bortezomib (Velcade), was recently approved for use in patients with refractory and relapsed MM and to date is the only proteasome inhibitor to have entered clinical trials Bortezomib has demonstrated activity with manageable toxicity in a variety of hematologic malignancies in addition to MM, including leukemia and non-Hodgkin's lymphoma This article reviews clinical information on bortezomib in hematologic malignancies both as monotherapy and in combination with dexamethasone Preliminary reports of bortezomib in combination with Doxil (pegylated liposomal doxorubicin), melphalan, and thalidomide are discussed, and current trials are described Available data suggest that bortezomib will be useful in the treatment of a variety of hematologic malignancies

Proteasome Inhibition: A Novel Approach to Anticancer Therapy

Abstract: Proteasome inhibition is a new approach under investigation for cancer therapy. Protein degradation by the proteasome is a fundamental metabolic process. Inhibition of the proteasome has been shown to result in cell-cycle arrest and programmed cell death.Bortezomib (Velcade®; formerly PS-341, Millennium Pharmaceuticals, Inc, Cambridge, Massachusetts) is the first proteasome inhibitor to enter clinical trials and is approved for the treatment of multiple myeloma in patients who have received at least two prior therapies and progressed on their last therapy. It is also being investigated for the treatment of a range of other cancers, both solid and hematologic. In preclinical studies, bortezomib causes apoptosis in cancer cells and seems to enhance the cytotoxicity of other conventional tumoricidal agents in human tumor cell lines, murine tumor models, and human xenograft models. In addition to targeting cancer cells directly, there is growing evidence that proteasome inhibition interferes with protective interactions between cancer cells and the bone marrow and may also act to prevent tumor-associated angiogenesis.Preliminary data suggest that bortezomib inhibits proteasome activity in a dose-dependent and reversible manner, and has manageable toxicities. A phase II trial in relapsed and refractory multiple myeloma patients indicated that patients experienced substantial anticancer activity when treated with bortezomib monotherapy.

Pseudosubstrate Peptides Inhibit Akt and Induce Cell Growth Inhibition

Abstract: We have designed peptide inhibitors that potently inhibit Akt both in vitro and inside cells. These peptide inhibitors are selective for Akt versus other closely related kinases. The peptides inhibit the in vitro phosphorylation of a biotinylated Bad peptide by Akt with potency up to 100 nM. We have shown that the binding between Akt1 and these peptide inhibitors requires MgATP. Mutating the two putative Akt phosphorylation sites to Ala (nonsubstrate) in these peptides increases the inhibitory potency while mutating the sites to aspartic acid (phosphorylation mimetic) reduces the potency. When delivered into cells, these peptide inhibitors can inhibit cellular Akt activity and cell growth. Thus, these Akt-specific peptide inhibitors provide prototypes for peptide mimetic drugs as well as very useful tools to dissect cellular functions of Akt.

A review of the proteasome inhibitor bortezomib in multiple myeloma.

Abstract: Multiple myeloma (MM) represented 14% of new haematological malignancies in the US in 2003 and almost 19% of anticipated deaths. Treatment with standard chemotherapy has resulted in a median survival of about 3 years and despite the improvements in survival seen with the use of intensive therapy supported by autologous stem cell transplantation, MM remains incurable; hence, new therapeutic strategies are urgently needed. One novel approach to the treatment of MM is the use of proteasome inhibitors. Proteasomes are ubiquitous protease complexes involved in diverse aspects of cell biology, such as protein homeostasis, cell cycle progression, apoptosis and inflammation, as well as resistance to antineoplastic therapy. The first-in-class proteasome inhibitor, bortezomib was recently approved in the US for the treatment of patients with MM who have received at least two prior therapies and are progressing on their last therapy. Its use in earlier-stage MM, other haematological malignancies and in solid tumours as monotherapy and in combination therapy is currently under investigation.

Closing the Gaps on Human Chromosome 19 Revealed Genes With a High Density of Repetitive Tandemly Arrayed Elements

Abstract: The reported human genome sequence includes about 400 gaps of unknown sequence that were not found in the bacterial artificial chromosome (BAC) and cosmid libraries used for sequencing of the genome. These missing sequences correspond to approximately 1% of euchromatic regions of the human genome. Gap filling is a laborious process because it relies on analysis of random clones of numerous genomic BAC or cosmid libraries. In this work we demonstrate that closing the gaps can be accelerated by a selective recombinational capture of missing chromosomal segments in yeast. The use of both methodologies allowed us to close the four remaining gaps on the human chromosome 19. Analysis of the gap sequences revealed that they contain several abnormalities that could result in instability of the sequences in microbe hosts, including large blocks of micro- and minisatellites and a high density of Alu repeats. Sequencing of the gap regions, in both BAC and YAC forms, allowed us to generate a complete sequence of four genes, including the neuronal cell signaling gene SCK1/SLI. The SCK1/SLI gene contains a record number of minisatellites, most of which are polymorphic and transmitted through meiosis following a Mendelian inheritance. In conclusion, the use of the alternative recombinational cloning system in yeast may greatly accelerate work on closing the remaining gaps in the human genome (as well as in other complex genomes) to achieve the goal of annotation of all human genes.

Efficacy, safety and tolerability of lumiracoxib in patients with rheumatoid arthritis.

Abstract: A randomised, double-blind study was performed to assess the efficacy and tolerability of lumiracoxib in patients with rheumatoid arthritis (RA). Patients received lumiracoxib 200mg once daily (o.d.) (n= 280), lumiracoxib 400mg o.d. (n= 281), naproxen 500 mg twice daily (n= 279) or placebo (n= 284) for 26 weeks. The primary efficacy variable was response to treatment according to ACR20 criteria (adjusted for prohibited concomitant or excessive rescue medication use and discontinuations due to unsatisfactory therapeutic response) at week 13. Safety and tolerability was also assessed. Significantly more patients receiving lumiracoxib than placebo were responders according to ACR20 criteria at week 13 (41.1 and 42.7% for lumiracoxib 200 and 400 mg o.d., respectively; 32.4% for placebo; both p < 0.05). The proportion responding to naproxen (39.1%) was not significantly different from placebo. Prespecified gastrointestinal adverse events were more frequent with naproxen than with either lumiracoxib dose or placebo. Lumiracoxib is therefore an effective and well-tolerated therapy for RA.