Structural analysis of the N-glycans of the major cysteine proteinase of Trypanosoma cruzi. Identification of sulfated high-mannose type oligosaccharides

Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine proteinase, cruzipain. This lysosomal enzyme bears an unusual C-terminal extension that contains a number of post-translational modifications, and most antibodies in natural and experimental infections are directed against it. In this report we took advantage of UV-MALDI-TOF mass spectrometry in conjunction with peptide N-glycosidase F deglycosylation and high performance anion exchange chromatography analysis to address the structure of the N-linked oligosaccharides present in this domain. The UV-MALDI-TOF MS analysis in the negative-ion mode, using nor-harmane as matrix, allowed us to determine a new striking feature in cruzipain: sulfated high-mannose type oligosaccharides. Sulfated GlcNAc2Man3 to GlcNAc2Man9 species were identified. In accordance, after chemical or enzymatic desulfation, the corresponding signals disappeared. In addition, by UV-MALDI-TOF MS analysis (a) a main population of high-mannose type oligosaccharides was shown in the positive-ion mode, (b) lactosaminic glycans were also identified, among them, structures corresponding to monosialylated species were detected, and (c) as an interesting fact a fucosylated oligosaccharide was also detected. The presence of the deoxy sugar was further confirmed by high performance anion exchange chromatography. In conclusion, the total number of oligosaccharides occurring in cruzipain was shown to be much higher than previous estimates. This constitutes the first report on the presence of sulfated glycoproteins in Trypanosomatids.     

Viral genome methylation as an epigenetic defense against geminiviruses

Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through double-stranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2- mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.     

Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.     

Germline stem cells: the first guards of heredity

The genes involved in the cellular response to DNA damage are crucial for ensuring DNA integrity during spermatogenesis. In this issue of Cell Stem Cell, Takubo et al. (2008) show that ATM, a key kinase of the DNA damage response, is also involved in maintaining the stem cell potential of undifferentiated spermatogonia.     

Serotonin activation of the ERK pathway in Hermissenda: contribution of calcium-dependent protein kinase C

The mitogen‐activated protein kinase (MAPK) cascade is an important contributor to synaptic plasticity and learning in both vertebrates and invertebrates. In the nudibranch mollusk Hermissenda, phosphorylation and activation of the extracellular signal‐regulated protein kinase (ERK), a key member of a MAPK cascade, is produced by one‐trial and multitrial Pavlovian conditioning. Several signal transduction pathways that are activated by 5‐hydroxytryptamine (5‐HT) and may contribute to conditioning have been identified in type B photoreceptors. However, the regulation of ERK activity by ‘upstream’ signaling molecules has not been previously investigated in Hermissenda. In the present study we examined the role of protein kinase C (PKC) in the serotonin (5‐HT) activation of the ERK pathway. The phorbol ester TPA produced an increase in ERK phosphorylation that was blocked by the PKC inhibitors GF109203X or Gö6976. TPA‐dependent ERK phosphorylation was also blocked by the MEK1 inhibitors PD098059 or U0126. The increased phosphorylation of ERK by 5‐HT was reduced but not blocked by pretreatment with the calcium chelator BAPTA‐AM or pretreatment with Gö6976 or GF109203X. These results indicate that Ca²⁺‐dependent PKC activation contributes to ERK phosphorylation, although a PKC‐independent pathway is also involved in 5‐HT‐dependent ERK phosphorylation and activation.     

Chemical Composition of Chlamydospores of Candida albicans

A variety of analytical techniques was employed to study the composition of the chlamydospore of Candida albicans. The outer, thin, electron-transparent layer was found to be composed of glucan, together with a small amount of chitin. The inner, thick, electron-dense layer is proteinaceous. The central structure is composed largely of ribonucleic acid and lipid globules. In addition to being acid-fast, the chlamydospore was found to contain glycolipids and to lose the property of acid-fastness on extraction with ethanol-ether.     

Towards unifying racial and ethnic paradigms

Bridging the divide between the ethnic/assimilation and the race literatures has long vexed sociologists. Valdez and Golash-Boza are to be commended for the intellectual effort they display in this article to engage this messy and contentious relationship. The distinction between the ethnic paradigm which produces assimilation projects and the racial paradigm which produces structural racism projects is important. I further this by noting that the process involved in the racial paradigm is racialization. I engage three examples: Mexican Americans, African-Americans, and white Americans. Together, these provide clarity to the discussion of how to pursue a unified approach.     

5-Aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles as inhibitors of Hsp90 chaperone

A series of 5-aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles were synthesized and their binding to several constructs of human Hsp90 chaperone measured by isothermal titration calorimetry (ITC). The most potent compound bound Hsp90 with the dissociation constant of about 5 nM.