The UniMarker (UM) method for synteny mapping of large genomes

MOTIVATION: Synteny mapping, or detecting regions that are orthologous between two genomes, is a key step in studies of comparative genomics. For completely sequenced genomes, this is increasingly accomplished by whole-genome sequence alignment. However, such methods are computationally expensive, especially for large genomes, and require rather complicated post-processing procedures to filter out non-orthologous sequence matches. RESULTS: We have developed a novel method that does not require sequence alignment for synteny mapping of two large genomes, such as the human and mouse. In this method, the occurrence spectra of genome-wide unique 16mer sequences present in both the human and mouse genome are used to directly detect orthologous genomic segments. Being sequence alignment-free, the method is very fast and able to map the two mammalian genomes in one day of computing time on a single Pentium IV personal computer. The resulting human-mouse synteny map was shown to be in excellent agreement with those produced by the Mouse Genome Sequencing Consortium (MGSC) and by the Ensembl team; furthermore, the syntenic relationship of segments found only by our method was supported by BLASTZ sequence alignment.     

Enhancing effect of low culture temperature on specific antibody productivity of recombinant Chinese hamster ovary cells: clonal variation

To understand the different responses of recombinant Chinese hamster ovary (rCHO) cells to low culture temperature regarding specific productivity (q), 12 parental clones and their corresponding amplified clones producing a humanized antibody were cultivated at 32 and 37 degrees C. The specific growth rate of all clones, including both parental and amplified clones, decreased by 30-63% at 32 degrees C, compared to rates at 37 degrees C. In contrast, their specific antibody productivity (qAb) was significantly enhanced at 32 degrees C. Furthermore, the degree of qAb enhancement at 32 degrees C varied a lot from 4- to 25-fold among the parental clones. At 32 degrees C, most of the amplified clones, regardless of methotrexate (MTX) levels, also showed enhanced qAb but to a lesser extent than their parental clones. However, clone 14 amplified at 0.32 microM MTX (clone 14-0.32) and clone 20 amplified at 1 microM MTX (clone 20-1.00), unlike their parental clones, did not show enhanced qAb at 32 degrees C. Thus, it was found that the enhancing effect of low culture temperature on q of rCHO cells depends on clones. Taken together, the results obtained here emphasize the importance of clonal selection for the successful application of low culture temperature to the enhanced foreign protein production in rCHO cells.     

Arginine vasopressin produces inhibition upon respiration without pressor effect in the rat

The purpose of the current study was to examine where arginine vasopressin (AVP) inhibits respiration by direct action on the areas of the ventrolateral medulla (VLM) in the rat. The animal was anesthetized by urethane (1.2 g/kg, i.p.), paralyzed with gallamine triethiodide, and artificially ventilated. Catheterization of the femoral artery and vein, and bilateral vagotomy were performed. The rat was then placed upon a stereotaxic instrument in a prone position. The phrenic nerve was separated and cut peripherally. Phrenic nerve activity (PNA) was monitored at normocapnia and hypercapnia in hyperoxia. Microinjection of AVP into various subregions of the VLM was then performed. In response to AVP microinjection, a transient period of apnea and then a significant decrease in PNA amplitude were observed. Arterial blood pressure was unchanged. This inhibition of PNA with AVP treatment was site-specific, attenuated by raising CO2 concentration, and totally abolished by pretreatment with AVP V1A receptor antagonist. Data of the present study indicate that endogenous resource of AVP may produce an inhibitory effect upon respiration via AVP receptors presented on neurons within the VLM.     

Differential expression of the tetracycline-controlled transactivator-driven human CYP1B1 gene in double-transgenic mice is due to androgens: application for detecting androgens and antiandrogens

Differential expression of the tetracycline-controlled transactivator (tTA)-driven human cytochrome p450 (CYP) 1B1 gene was found in the livers of male mice, at high levels in neonates, but at low levels in adults. The goals of this study were to determine whether the differential expression of the tTA-driven human CYP1B1 (hCYP1B1) gene in neonates and adults was testosterone dependent and whether flutamide, a representative potent antiandrogen, led to the induction of hCYP1B1. This was tested by treating castrated transgenic mice with testosterone propionate and musk extracts. It was concluded that: (i). the levels of expression of both tTA and hCYP1B1 gradually declined, with clear changes being apparent between 2 and 4 weeks of age, (ii). castration of adult males resulted in the increased expressions of both tTA and hCYP1B1 to levels similar to those found in adult females, (iii). treatment of castrated male and adult female mice with testosterone propionate and musk extracts led to the restoration of the levels of expression of hCYP1B1 in the adult males, and (iv). treatment of adult males with flutamide caused an increase in the levels of expression of hCYP1B1 in the adult females, as indicated by the antiandrogenic activity. Thus, the differential expression of the tTA-driven hCYP1B1 gene in the transgenic mice was caused by androgen, and it is possible that castrated male and adult female mice expressing the tTA-controlled hCYP1B1 could be used as the basis for a strategy for the detection of androgens and antiandrogens.     

Distortion of the three-dimensional structure of the vnd/NK-2 homeodomain bound to DNA induced by an embryonically lethal A35T point mutation

The three-dimensional solution structure obtained by NMR of the A35T mutant vnd/NK-2 homeodomain bound to the vnd/NK-2 consensus 16 bp DNA sequence was determined. This mutation to threonine from alanine in position 35 in helix II of the vnd/NK-2 homeodomain is associated with early embryonic lethality in Drosophila melanogaster. Although the unbound mutant protein is not structured, in the DNA-bound state it adopts the three-helix fold characteristic of all known homeodomains, but with alterations relative to the structure of the wild-type analogue. These structural modifications occur, and are accompanied by a 50-fold reduction in the DNA binding affinity, even though most of the protein-DNA interactions originally seen for the wild-type homeodomain are found likewise in the threonine analogue. Alterations include torsional angle changes in the loop between helix I and helix II, and in the turn between helix II and helix III, as well as in a distortion of the usual antiparallel orientation of helix I with respect to helix II. The alteration of the position of leucine 40 in the A35T mutant is proposed to explain the observed 1.27 ppm upfield shift of the corresponding amide proton resonance relative to the value observed for the wild-type analogue. A detailed comparison of the structures of the mutant A35T and wild-type vnd/NK-2 homeodomains bound to the cognate DNA is presented. The consequences of the structural alteration of the DNA-bound A35T mutant vnd/NK-2 protein may constitute the basis of the observed early embryonic lethality.     

Effects of structure of beta-cyclodextrin-containing polymers on gene delivery

Linear cationic beta-cyclodextrin-based polymers (betaCDPs) are capable of forming polyplexes with nucleic acids and transfecting cultured cells. The betaCDPs are synthesized by the condensation of a diamino-cyclodextrin monomer A with a diimidate comonomer B. In this paper, the effects of polymer structure on polyplex formation, in vitro transfection efficiency and toxicity are elucidated. By comparison of the betaCDPs to polyamidines lacking cyclodextrins, the inclusion of a cyclodextrin moiety in the comonomer A units reduces the IC50s of the polymer by up to 3 orders of magnitude. The spacing between the cationic amidine groups is also important. Different polymers with 4, 5, 6, 7, 8, and 10 methylene units (betaCDP4, 5, 6, 7, 8, and 10) in the comonomer B molecule are synthesized. Transfection efficiency is dependent on comonomer B length with up to 20-fold difference between polymers. Optimum transfection is achieved with the betaCDP6 polymer. In vitro toxicity varied by 1 order of magnitude and the lowest toxicity is observed with betaCDP8. The LD40 of the betaCDP6 to mice is 200 mg/kg, making this polymer a promising agent for in vivo gene delivery applications.     

Structural basis for the NAD-dependent deacetylase mechanism of Sir2

The NAD-dependent histone/protein deacetylase activity of Sir2 (silent information regulator 2) accounts for its diverse biological roles including gene silencing, DNA damage repair, cell cycle regulation, and life span extension. We provide crystallographic evidence that 2'-O-acetyl ADP-ribose is the reaction product that is formed at the active site of Sir2 from the 2.6-A co-crystal structure of 2'-O-acetyl-ADP-ribose and Sir2 from Archaeoglobus fulgidus. In addition, we show that His-116 and Phe-159 play critical roles in the catalysis and substrate recognition. The conserved Ser-24 and Asp-101 contribute to the stability for NAD binding rather than being directly involved in the catalysis. The crystal structures of wild type and mutant derivatives of Sir2, in conjunction with biochemical analyses of the mutants, provide novel insights into the reaction mechanism of Sir2-mediated deacetylation.     

Antiphospholipid antibodies induce monocyte chemoattractant protein-1 in endothelial cells

The presence of antiphospholipid Ab is associated with increased risk of thrombosis. The monocyte-endothelial cell interaction has been suggested to play a key role at the site of vascular injury during thrombosis. Therefore, we tested the effect of anticardiolipin Abs (aCL) on the production of monocyte chemoattractant protein-1 (MCP-1) in HUVEC. We found that monoclonal aCL as well as IgG fractions from patients with antiphospholipid syndrome (APS-IgG) could induce the production of MCP-1 in HUVEC. The ability of IgG aCL to induce MCP-1 production could be abrogated by preabsorption with cardiolipin liposomes. Simultaneous addition of either monoclonal aCL or APS-IgG with IL-1beta resulted in synergistic increase in MCP-1 production, whereas the addition of control IgG lacking aCL activity did not alter IL-1beta-induced levels of MCP-1. MCP-1 mRNA expression was also up-regulated when HUVEC were incubated with either APS-IgG or monoclonal aCL, and down-regulated by the treatment of dexamethasone. In addition, we found that serum levels of MCP-1 in 76 systemic lupus erythematosus patients correlated well with the titers of IgG aCL. Collectively, these results indicate that aCL could promote endothelial cell-monocyte cross-talk by enhancing the endothelial production of MCP-1, thereby shifting the hemostatic balance toward the prothrombotic state of APS.