Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs.

OBJECTIVES: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. METHODS: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. RESULTS: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. CONCLUSIONS: TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter

Species-specific Typing of DNA Based on Palindrome Frequency Patterns

DNA in its natural, double-stranded form may contain palindromes, sequences which read the same from either side because they are identical to their reverse complement on the sister strand. Short palindromes are underrepresented in all kinds of genomes. The frequency distribution of short palindromes exhibits more than twice the inter-species variance of non-palindromic sequences, which renders palindromes optimally suited for the typing of DNA. Here, we show that based on palindrome frequency, DNA sequences can be discriminated to the level of species of origin. By plotting the ratios of actual occurrence to expectancy, we generate palindrome frequency patterns that allow to cluster different sequences of the same genome and to assign plasmids, and in some cases even viruses to their respective host genomes. This finding will be of use in the growing field of metagenomics

Renal Arterial Calibre after Acute Unilateral Ureteric Occlusion

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72406/1/j.1440-1673.1970.tb02908.x.pd

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs

Objectives Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. Methods The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. Results All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. Conclusions TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporte

Ctp1 and the MRN-Complex Are Required for Endonucleolytic Rec12 Removal with Release of a Single Class of Oligonucleotides in Fission Yeast

DNA double-strand breaks (DSBs) are formed during meiosis by the action of the topoisomerase-like Spo11/Rec12 protein, which remains covalently bound to the 5′ ends of the broken DNA. Spo11/Rec12 removal is required for resection and initiation of strand invasion for DSB repair. It was previously shown that budding yeast Spo11, the homolog of fission yeast Rec12, is removed from DNA by endonucleolytic cleavage. The release of two Spo11 bound oligonucleotide classes, heterogeneous in length, led to the conjecture of asymmetric cleavage. In fission yeast, we found only one class of oligonucleotides bound to Rec12 ranging in length from 17 to 27 nucleotides. Ctp1, Rad50, and the nuclease activity of Rad32, the fission yeast homolog of Mre11, are required for endonucleolytic Rec12 removal. Further, we detected no Rec12 removal in a rad50S mutant. However, strains with additional loss of components localizing to the linear elements, Hop1 or Mek1, showed some Rec12 removal, a restoration depending on Ctp1 and Rad32 nuclease activity. But, deletion of hop1 or mek1 did not suppress the phenotypes of ctp1Δ and the nuclease dead mutant (rad32-D65N). We discuss what consequences for subsequent repair a single class of Rec12-oligonucleotides may have during meiotic recombination in fission yeast in comparison to two classes of Spo11-oligonucleotides in budding yeast. Furthermore, we hypothesize on the participation of Hop1 and Mek1 in Rec12 removal

Indistinguishable Landscapes of Meiotic DNA Breaks in rad50+ and rad50S Strains of Fission Yeast Revealed by a Novel rad50+ Recombination Intermediate

The fission yeast Schizosaccharomyces pombe Rec12 protein, the homolog of Spo11 in other organisms, initiates meiotic recombination by creating DNA double-strand breaks (DSBs) and becoming covalently linked to the DNA ends of the break. This protein–DNA linkage has previously been detected only in mutants such as rad50S in which break repair is impeded and DSBs accumulate. In the budding yeast Saccharomyces cerevisiae, the DSB distribution in a rad50S mutant is markedly different from that in wild-type (RAD50) meiosis, and it was suggested that this might also be true for other organisms. Here, we show that we can detect Rec12-DNA linkages in Sc. pombe rad50+ cells, which are proficient for DSB repair. In contrast to the results from Sa. cerevisiae, genome-wide microarray analysis of Rec12-DNA reveals indistinguishable meiotic DSB distributions in rad50+ and rad50S strains of Sc. pombe. These results confirm our earlier findings describing the occurrence of widely spaced DSBs primarily in large intergenic regions of DNA and demonstrate the relevance and usefulness of fission yeast studies employing rad50S. We propose that the differential behavior of rad50S strains reflects a major difference in DSB regulation between the two species—specifically, the requirement for the Rad50-containing complex for DSB formation in budding yeast but not in fission yeast. Use of rad50S and related mutations may be a useful method for DSB analysis in other species

Iron: a target for the management of Kaposi's sarcoma?

BACKGROUND: Kaposi's sarcoma (KS) is a mesenchymal tumour associated with human herpesvirus-8 infection. However, the incidence of human herpesvirus-8 infection is far higher than the prevalence of KS, suggesting that viral infection per se is not sufficient for the development of malignancy and that one or more additional cofactors are required. DISCUSSION: Epidemiological data suggest that iron may be one of the cofactors involved in the pathogenesis of KS. Iron is a well-known carcinogen and may favour KS growth through several pathways. Based on the apoptotic and antiproliferative effect of iron chelation on KS cells, it is suggested that iron withdrawal strategies could be developed for the management of KS. Studies using potent iron chelators in suitable KS animal models are critical to evaluate whether iron deprivation may be a useful anti-KS strategy. SUMMARY: It is suggested that iron may be one of non-viral co-factors involved of KS pathogenesis and that iron withdrawal strategies might interfere with tumour growth in patients with KS

Molecular Blocking of CD23 Supports Its Role in the Pathogenesis of Arthritis

BACKGROUND: CD23 is a differentiation/activation antigen expressed by a variety of hematopoietic and epithelial cells. It can also be detected in soluble forms in biological fluids. Initially known as the low-affinity receptor for immunoglobulin E (Fc epsilonRII), CD23 displays various other physiologic ligands such as CD21, CD11b/c, CD47-vitronectin, and mannose-containing proteins. CD23 mediates numerous immune responses by enhancing IgE-specific antigen presentation, regulating IgE synthesis, influencing cell differentiation and growth of both B- and T-cells. CD23-crosslinking promotes the secretion of pro-inflammatory mediators from human monocytes/macrophages, eosinophils and epithelial cells. Increased CD23 expression is found in patients during allergic reactions and rheumatoid arthritis while its physiopathologic role in these diseases remains to be clarified. METHODOLOGY/PRINCIPAL FINDINGS: We previously generated heptapeptidic countrestructures of human CD23. Based on in vitro studies on healthy and arthritic patients' cells, we showed that CD23-specific peptide addition to human macrophages greatly diminished the transcription of genes encoding inflammatory cytokines. This was also confirmed by significant reduction of mediator levels in cell supernatants. We also show that CD23 peptide decreased IgE-mediated activation of both human and rat CD23(+) macrophages. In vivo studies in rat model of arthritis showed that CD23-blocking peptide ameliorates clinical scores and prevent bone destruction in a dose dependent manner. Ex-vivo analysis of rat macrophages further confirmed the inhibitory effect of peptides on their activation. Taken together our results support the role of CD23 activation and subsequent inflammatory response in arthritis. CONCLUSION: CD23-blocking peptide (p30A) prevents the activation of monocytes/macrophages without cell toxicity. Thus, targeting CD23 by antagonistic peptide decreases inflammatory markers and may have clinical value in the treatment of human arthritis and allergic reactions involving CD23

Comparative genomics of drug resistance in <i>Trypanosoma brucei rhodesiense</i>

Trypanosoma brucei rhodesiense is one of the causative agents of human sleeping sickness, a fatal disease that is transmitted by tsetse flies and restricted to Sub-Saharan Africa. Here we investigate two independent lines of T. b. rhodesiense that have been selected with the drugs melarsoprol and pentamidine over the course of 2 years, until they exhibited stable cross-resistance to an unprecedented degree. We apply comparative genomics and transcriptomics to identify the underlying mutations. Only few mutations have become fixed during selection. Three genes were affected by mutations in both lines: the aminopurine transporter AT1, the aquaporin AQP2, and the RNA-binding protein UBP1. The melarsoprol-selected line carried a large deletion including the adenosine transporter gene AT1, whereas the pentamidine-selected line carried a heterozygous point mutation in AT1, G430R, which rendered the transporter non-functional. Both resistant lines had lost AQP2, and both lines carried the same point mutation, R131L, in the RNA-binding motif of UBP1. The finding that concomitant deletion of the known resistance genes AT1 and AQP2 in T. b. brucei failed to phenocopy the high levels of resistance of the T. b. rhodesiense mutants indicated a possible role of UBP1 in melarsoprol-pentamidine cross-resistance. However, homozygous in situ expression of UBP1-Leu(131) in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine