Dual benefit of reduced Cx43 on atherosclerosis in LDL receptor-deficient mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR(-/-)) mice were intercrossed with mice heterozygous for Cx43 (Cx43(+/-) mice). Male mice with normal (Cx43(+/+)LDLR(-/-)) or reduced (Cx43(+/-)LDLR(-/-)) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43(+/-)LDLR(-/-) mice compared with Cx43(+/+)LDLR(-/-) littermate controls. In addition, atheroma in Cx43(+/-)LDLR(-/-) mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

Microplate screening for apoptosis with antibody to single-stranded DNA distinguishes anticancer drugs from toxic chemicals

The effect of anticancer drugs and toxic compounds on cultures of human leukemic cells was evaluated by an enzyme-linked immunosorbent assay (Apoptosis ELISA) that uses a monoclonal antibody against single-stranded DNA to quantitate the apoptotic cells. The concentrations of 13 anticancer drugs, which increased Apoptosis ELISA absorbance, were close to the cytotoxic concentrations determined by the long-term cell survival assay. Short-term tetrazolium-based microculture tetrazolium (MTT) assay was significantly less sensitive than the Apoptosis ELISA and the cell survival assay for all anticancer drugs. For 6 drugs, cytotoxic concentrations measured by the MTT assay were at least 1 log higher than the concentrations inducing apoptosis. Importantly, in contrast to the anticancer drugs, 14 toxic chemicals did not increase the Apoptosis ELISA absorbance at cytotoxic concentrations. The difference in apoptosis induction by the anticancer drugs and the toxic chemicals was especially large in cultures treated with drug concentrations 2-fold higher than the IC(50) dose. Although all of the anticancer drugs tested induced intense ELISA reaction (mean absorbance 2.0), all toxic chemicals tested did not induce apoptosis. The Apoptosis ELISA assay could have useful applications in drug development as it can distinguish between clinically useful anticancer drugs and toxic compounds, has sensitivity similar to that of the long-term cell survival assay, and provides insight into the mechanism of drug cytotoxicity by differentiating between compounds killing cells by apoptosis and necrosis.     

Current regulatory issues in cell and tissue therapy

Cell-based therapies have grown dramatically in power and scope in recent years. Once limited to blood and BM transplantation, these therapies now encompass tissue repair and regeneration, metabolic support, gene replacement, and immune effector functions, with established and investigational clinical applications in disorders affecting nearly every tissue and organ system. The complexity and novel applications of human cells, tissues, and cellular and tissue-based products (HCT/Ps), however, present potential risks for adverse events. The US Food and Drug Administration, responding to these concerns, has established a tiered, risk-based regulatory structure, in which more rigorous controls and safeguards are required for products thought to pose increased risk. The proposed good tissue practices (GTP) rule and existing good manufacturing practices (GMP) requirements form the principal elements of this regulatory framework. The proposed GTPs are intended to prevent HCT/P contamination with infectious disease agents, and to ensure that these cells and tissues maintain their integrity and function. GMPs focus on production of safe, pure, and potent products, and entail a higher level of process control and product characterization. All HCT/Ps will be required to comply with GTPs. HCT/Ps considered to present greater risks of adverse events, however, will be subject to both GTPs and GMPs, and must obtain premarket approval using the Investigational New Drug (IND) mechanism established for biologics. Although these requirements will present significant challenges for clinician- investigators and laboratories producing HCT/Ps, the regulations fundamentally support good clinical care by increasing safety and control, and enable good science by improving the quality and reliability of data.     

Mutual influences of Pseudomonas aeruginosa and Desulfovibrio desulfuricans on their adhesion to stainless steel

The mutual influences of Pseudomonas aeruginosa PAO1 and Desulfovibrio desulfuricans subsp. desulfuricans (ATCC 29577) on their adhesion to stainless steel were investigated in batch and column experiments. It was found that P. aeruginosa promoted the adhesion of D. desulfuricans under conditions of turbulence, but not under quiescent conditions. The enhancement involved the alignment of most D. desulfuricans along P. aeruginosa cells and was attributed to the additional interaction surface area provided by adhered P. aeruginosa to aligning D. desulfuricans cells. A slightly positive effect of preadhered D. desulfuricans on the adhesion of P. aeruginosa was found. Under condition of laminar flow, substantially better adhesion of D. desulfuricans to confluent P. aeruginosa biofilms than to steel was observed. The mutual influences are discussed in terms of more favorable adhesion energies and the influence of changed hydraulic conditions due to the roughness of P. aeruginosa biofilms.     

Palaeogenetic analysis of (pre)historic artifacts and its significance for anthropology

The possibility of isolating ancient DNA (aDNA) from all kinds of (pre)historic anthropogenetic artifacts opens new perspectives. This study applies palaeogenetic techniques to three anthropological issues: 1. Palaeodiet. DNA sequences from organic residues in vessels identify Precolumbian Aztec diet. 2. (Pre)historic husbandry and economic structures. aDNA data can reveal the species and the genetic evolutionary stage of animals and plants and show the manner and the extent of their growth, cultivation, or domestication. 3. Production techniques, use, and functionality. Identification of the plant or animal source of an archaeological find can reveal the use or the function of the find. Examples from a Celtic "sausage-end" and an Aztec "eye salve" are given.     

Selective oestrogen receptor modulators

Selective oestrogen receptor modulators (SERMs) are compounds which act like oestrogens in some target tissues but which antagonise their effects in others. The first example of a SERM (referred to as a first-generation compound) was tamoxifen, for which oestrogen-like agonist activity on bone was seen to occur simultaneously with oestrogen antagonist activity on the breast. An unwanted effect of tamoxifen was its oestrogen-like action on the endometrium. Second-generation compounds have since been developed, most notably raloxifene, which has oestrogen-like actions on bone, lipids and the coagulation system, and oestrogen antagonist effects on the breast and uterus. Raloxifene has undergone very extensive, prospective, placebo-controlled, randomised trial evaluation, in which anti-fracture efficacy (to date only for vertebral fracture) has been accompanied by a major reduction in the incidence of new breast cancer. The compound is similar to placebo in its uterine effects, and similar to oestrogen in causing a two- to threefold increase in the risk of venous thromboembolism. Its lipid effects are similar to those of oestrogen, except for a relatively small effect on high-density lipoprotein cholesterol, and no significant effect on triglycerides. Data on cardiovascular event rates are not yet available; data on cognitive function are preliminary and, to date, reassuring. The mechanisms by which the same compound can exert oestrogen agonist effects on one target and antagonist effects on another are still being clarified. Important aspects include the fact that the oestrogen receptor undergoes different conformational changes according to the ligand. Thus the crystal structure of oestradiol bound to the oestrogen receptor differs from that of raloxifene bound to the same receptor. The existence of two oestrogen receptor subtypes may also be relevant. Mechanisms include differing interactions with various domains of the oestrogen receptor, and tissue-specific recruitment of steroid receptor co-activators and co-repressors may underlie some of the tissue-specific effects. The SERMs may be the prototype for other selective steroid receptor modulators, for example the androgen and progesterone receptors. The development of tissue target-specific agents is an exciting advance in endocrine pharmacology and can be extended to agents, such as tibolone, which exert some of their tissue specificity through their metabolites.     

The mitochondrial genome of the stramenopile alga Chrysodidymus synuroideus. Complete sequence, gene content and genome organization

This is the first report of a complete mitochondrial genome sequence from a photosynthetic member of the stramenopiles, the chrysophyte alga Chrysodidymus synuroideus. The circular-mapping mitochondrial DNA (mtDNA) of 34 119 bp contains 58 densely packed genes (all without introns) and five unique open reading frames (ORFs). Protein genes code for components of respiratory chain complexes, ATP synthase and the mitoribosome, as well as one product of unknown function, encoded in many other protist mtDNAs (YMF16). In addition to small and large subunit ribosomal RNAs, 23 tRNAs are mtDNA-encoded, permitting translation of all codons present in protein-coding genes except ACN (Thr) and CGN (Arg). The missing tRNAs are assumed to be imported from the cytosol. Comparison of the C.synuroideus mtDNA with that of other stramenopiles allowed us to draw conclusions about mitochondrial genome organization, expression and evolution. First, we provide evidence that mitochondrial ORFs code for highly derived, unrecognizable versions of ribosomal or respiratory genes otherwise ‘missing’ in a particular mtDNA. Secondly, the observed constraints in mitochondrial genome rearrangements suggest operon-based, co-ordinated expression of genes functioning in common biological processes. Finally, stramenopile mtDNAs reveal an unexpectedly low variability in genome size and gene complement, testifying to substantial differences in the tempo of mtDNA evolution between major eukaryotic lineages.     

Mechanical stress induces COX-2 mRNA expression in bone cells from elderly women

Mechanical loading-induced fluid flow in the lacuno-canalicular network is a possible signal for bone cell adaptive responses. In an earlier study we found that pulsating fluid flow (PFF, 0.7+/-0.02 Pa, 5 Hz, 0.4 Pa/s) stimulates the production of prostaglandins by neonatal mouse calvarial cells. In addition, mRNA expression of the inducible form of cyclooxygenase (COX-2), but not the constitutive form (COX-1), the major enzymes in prostaglandin production, was increased by PFF. The present study was performed to determine whether human primary bone cells from the iliac crest, respond to mechanical stress in a similar way as neonatal mouse calvarial cells. We subjected bone cells originating from the iliac crest of nine elderly women, between 56 and 80 yr of age, for 1 h to PFF and measured prostaglandin production and COX-1 and COX-2 mRNA expression. One hour PFF treatment stimulated the release of PGE2 by 3.5 fold and PGI2 by 2.2 fold. PFF also increased the expression of COX-2 mRNA by 2.9 fold, but did not change COX-1 mRNA. No correlation was found between donor age and PFF effect, neither on prostaglandin production nor on COX-2 mRNA expression. This study shows that bone cells from the iliac crest of elderly women react to PFF treatment in a similar way as neonatal mouse calvarial cells, namely with increased production of prostaglandins and upregulation of COX-2 mRNA expression. These results suggest that human bone cells from the iliac crest and neonatal mouse calvarial cells share a similar mechanotransduction pathway.     

A genome-wide screening in Saccharomyces cerevisiae for genes that confer resistance to the anticancer agent cisplatin

Cisplatin is a potent DNA-damaging agent that has demonstrated anticancer activities against several tumors. However, manifestation of cellular resistance is a major obstacle in anticancer therapy that severely limits the curative potential of cisplatin. Therefore, understanding the molecular basis of cisplatin resistance could significantly improve the clinical efficacy of this anticancer agent. Here, we employed Saccharomyces cerevisiae as a model organism to study cisplatin resistance mechanisms and describe a one-step cisplatin selection to identify and characterize novel cisplatin resistance genes. Screening a multicopy yeast genomic library enabled us to isolate several yeast clones for which we could confirm that the cisplatin resistance phenotype was linked to the introduced fragment. In a first attempt, a number of open reading frames could be identified. Among these genes, PDE2 and ZDS2 were repeatedly identified as genes whose overexpression confers cellular resistance to cisplatin. PDE2, encoding cAMP-phosphodiesterase 2, is of particular interest because the overexpression of this yeast gene is known to induce cisplatin resistance in mammalian cells as well, providing proof of the principle of our experimental approach. In addition, the identification of PDE2 shows that our yeast screening system can directly be informative for drug resistance in mammalian cells.     

Upregulation of hepatic glucose 6-phosphatase gene expression in rats treated with an inhibitor of glucose-6-phosphate translocase

The multicomponent hepatic glucose 6-phosphatase (Glc-6-Pase) system catalyzes the terminal step of hepatic glucose production and plays a key role in the regulation of blood glucose. We used the chlorogenic acid derivative S 3483, a reversible inhibitor of the glucose-6-phosphate (Glc-6-P) translocase component, to demonstrate for the first time upregulation of Glc-6-Pase expression in rat liver in vivo after inhibition of Glc-6-P translocase. In accordance with its mode of action, S 3483-treatment of overnight-fasted rats induced hypoglycemia and increased blood lactate, hepatic Glc-6-P, and glycogen. The metabolic changes were accompanied by rapid and marked increases in Glc-6-Pase mRNA (above 35-fold), protein (about 2-fold), and enzymatic activity (about 2-fold). Maximal mRNA levels were reached after 4 h of treatment. Glycemia, blood lactate, and Glc-6-Pase mRNA levels returned to control values, whereas Glc-6-P and glycogen levels decreased but were still elevated 2 h after S 3483 withdrawal. The capacity for Glc-6-P influx was only marginally increased after 8.5 h of treatment. Prevention of hypoglycemia by euglycemic clamp did not abolish the increase in Glc-6-Pase mRNA induced by S 3483 treatment. A similar pattern of hypoglycemia and possibly of associated counterregulatory responses elicited by treatment with the phosphoenolpyruvate carboxykinase inhibitor 3-mercaptopicolinic acid could account for only a 2-fold induction of Glc-6-Pase mRNA. These findings suggest that the significant upregulation of Glc-6-Pase gene expression observed after treatment of rats in vivo with an inhibitor of Glc-6-P translocase is caused predominantly either by S 3483 per se or by the compound-induced changes of intracellular carbohydrate metabolism.