In vivo bioassay to detect irinotecan-stabilized DNA/topoisomerase I complexes in rats

Irinotecan is an anticancer agent that stabilizes topoisomerase I/DNA complexes. So far, no test system has been reported for directly determining irinotecan-induced stabilization of topoisomerase I/DNA complexes in organs in vivo. We adapted an ‘in vivo complexes of enzyme to DNA’ (ICE) bioassay to assess irinotecan activity in the stomach, duodenum, colon and liver of male Wistar rats after a single treatment with irinotecan (100 mg/kg body weight, intraperitoneally). This was compared to the control group receiving 0.9% sodium chloride intraperitoneally. In addition, the DNA strand breaking properties of irinotecan were measured in mucosal cells from the distal colon by single-cell gel electrophoresis (comet assay) to investigate the association of topoisomerase poisoning and DNA damage in vivo. A single dose of irinotecan significantly increased amounts of topoisomerase I covalently bound to DNA in stomach, duodenum, colon and liver. Concomitantly, the irinotecan-treated group showed significantly higher amounts of DNA strand breaks in colon mucosa cells compared to the control group. The ICE bioassay and the comet assay represent two test systems for investigating the impact of topoisomerase I poisons on DNA integrity in colon tissues of Wistar rats.     

Dynamics of HIV-1 Assembly and Release

Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1–2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of ∼5×10⁻³ s⁻¹, corresponding to 8–9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at ∼1,500±700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics.     

Anti-Apoptotic Genes in the Survival of Monocytic Cells During Infection

Macrophages are cells of the immune system that protect organisms against invading pathogens by fulfilling critical roles in innate and adaptive immunity and inflammation. They originate from circulating monocytes and show a high degree of heterogeneity, which reflects the specialization of function given by different anatomical locations. Differentiation of monocytes towards a macrophage phenotype is also accompanied by an increase of resistance against various apoptotic stimuli, a required characteristic that allows macrophages to accomplish their function in a stressful environment.Apoptosis, a form of programmed cell death, is a tightly regulated process, needed to maintain homeostasis by balancing proliferation with cellular demise. Caspases, a family of cysteine proteases that are highly conserved in multicellular organisms, function as central regulators of apoptosis. FLIP (FLICE-inhibitory protein), anti-apoptotic members of the Bcl2 family and inhibitors of apoptosis (IAP) are the main three groups of anti-apoptotic genes that counteract caspase activation through both the extrinsic and intrinsic apoptotic pathways.Modulation of the apoptotic machinery during viral and bacterial infections, as well as in various malignancies, is a wellestablished mechanism that promotes the survival of affected cells. The involvement of anti-apoptotic genes in the survival of monocytes/macrophages, either physiological or pathological, will be described in this review. How viral and bacterial infections that target cells of the monocytic lineage affect the expression of anti-apoptotic genes is important in understanding the pathological mechanisms that lead to manifested disease. The latest therapeutic approaches that target anti-apoptotic genes will also be discussed.Key Words: Apoptosis, monocytes/macrophages, HIV, anti-apoptotic genes, tuberculosis.     

ELMOD2 is a candidate gene for familial idiopathic pulmonary fibrosis

We performed a genomewide scan in six multiplex families with familial idiopathic pulmonary fibrosis (IPF) who originated from southeastern Finland. The majority of the Finnish multiplex families were clustered in the region, and the population history suggested that the clustering might be explained by an ancestor shared among the patients. The genomewide scan identified five loci of interest. The hierarchical fine mapping in an extended data set with 24 families originating from the same geographic region revealed a shared 110 kb to 13 Mb haplotype on chromosome 4q31, which was significantly more frequent among the patients than in population-based controls (odds ratio 6.3; 95% CI 2.5-15.9; P = .0001). The shared haplotype harbored two functionally uncharacterized genes, ELMOD2 and LOC152586, of which only ELMOD2 was expressed in lung and showed significantly decreased messenger-RNA expression in IPF lung (n = 6) when compared with that of healthy lung (n = 7; P = .05). Our results suggest ELMOD2 as a novel candidate gene for susceptibility in familial IPF.     

Celastrol: Molecular targets of Thunder God Vine

Celastrol, a quinone methide triterpene, is a pharmacologically active compound present in Thunder God Vine root extracts used as a remedy of inflammatory and autoimmune diseases, e.g. rheumatoid arthritis. Celastrol is one of the most promising medicinal molecules isolated from the plant extracts of traditional medicines. Molecular studies have identified several molecular targets which are mostly centered on the inhibition of IKK-NF-κB signaling. Celastrol (i) inhibits directly the IKKα and β kinases, (ii) inactivates the Cdc37 and p23 proteins which are co-chaperones of HSP90, (iii) inhibits the function of proteasomes, and (iv) activates the HSF1 and subsequently triggers the heat shock response. It seems that the quinone methide structure present in celastrol can react with the thiol groups of cysteine residues, forming covalent protein adducts. In laboratory experiments, celastrol has proved to be a potent inhibitor of inflammatory responses and cancer formation as well as alleviating diseases of proteostasis deficiency. Celastrol needs still to pass several hurdles, e.g. ADMET assays, before it can enter the armoury of western drugs.     

Genetic manipulation of polyamine catabolism in rodents

Activation of polyamine catabolism through the overexpression of spermidine/spermine N1-acetyltransferase (SSAT) in transgenic rodents does not only lead to distorted tissue polyamine homeostasis, manifested as striking accumulation of putrescine, appearance N1-acetylspermidine and reduction of tissue spermidine and/or spermine pools, but likewise creates striking phenotypic changes. The latter include loss of hair, lipoatrophy and female infertility. Forced expression of SSAT modulates skin, prostate and intestinal carcinogenesis, induces acute pancreatitis and blocks early liver regeneration. Although many of these features are directly attributable to altered tissue polyamine pools, some of them are more likely related to the greatly accelerated flux of the polyamines caused by activated catabolism and compensatorily enhanced biosynthesis.