Chromosome catastrophes involve replication mechanisms generating complex genomic rearrangements

Complex genomic rearrangements (CGRs) consisting of two or more breakpoint junctions have been observed in genomic disorders. Recently, a chromosome catastrophe phenomenon termed chromothripsis, in which numerous genomic rearrangements are apparently acquired in one single catastrophic event, was described in multiple cancers. Here, we show that constitutionally acquired CGRs share similarities with cancer chromothripsis. In the 17 CGR cases investigated, we observed localization and multiple copy number changes including deletions, duplications, and/or triplications, as well as extensive translocations and inversions. Genomic rearrangements involved varied in size and complexities; in one case, array comparative genomic hybridization revealed 18 copy number changes. Breakpoint sequencing identified characteristic features, including small templated insertions at breakpoints and microhomology at breakpoint junctions, which have been attributed to replicative processes. The resemblance between CGR and chromothripsis suggests similar mechanistic underpinnings. Such chromosome catastrophic events appear to reflect basic DNA metabolism operative throughout an organism's life cycle.     

Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II

Bacterial asparaginases (EC 3.5.1.1) have attracted considerable attention because enzymes of this group are used in the therapy of certain forms of leukemia. Class II asparaginase from Escherichia coli (EcA), a homotetramer with a mass of 138 kDa, is especially effective in cancer therapy. However, the therapeutic potential of EcA is impaired by the limited stability of the enzyme in vivo and by the induction of antibodies in the patients. In an attempt to modify the properties of EcA, several variants with amino acid replacements at subunit interfaces were constructed and characterized. Chemical and thermal denaturation analysis monitored by activity, fluorescence, circular dichroism, and differential scanning calorimetry showed that certain variants with exchanges that weaken dimer–dimer interactions exhibited complex denaturation profiles with active dimeric and/or inactive monomeric intermediates appearing at low denaturant concentrations. By contrast, other EcA variants showed considerably enhanced activity and stability as compared to the wild-type enzyme. Thus, even small changes at a subunit interface may markedly affect EcA stability without impairing its catalytic properties. Variants of this type may have a potential for use in the asparaginase therapy of leukemia.     

Role of salicylic acid and oxidative burst in expression of resistance to Alternaria blight

The accumulation of salicylic acid and H₂O₂ during pathogenic infection of mustard plants with Alternaria brassicae spores was investigated to understand the role of these two defense compounds in the expression of resistance. Comparisons were made between a susceptible Brassica juncea variety RH30 and a Brassica carinata variety HC1, which is known to be resistant. An oxidative burst was detected as in situ accumulation of H₂O₂, in both the Brassica spp. after pathogen application. However, H₂O₂ generation was extracellular in the resistant variety and both extra- and intracellular in the susceptible variety. Endogenous levels of SA increased over 2.5-fold in the resistant variety HC1 in response to pathogen application and this increase was observed only in conjugated SA levels. Pathogen application also led to an increase in the antioxidant enzymes, guaiacol-dependent peroxidase (GDP) and superoxide dismutase (SOD) in HC1. Exogenous SA application to leaves led to over threefold increase in the free and conjugated SA levels in both varieties. Pathogen application to the SA pretreated plants led to over 10-fold increase in endogenous SA levels in both varieties as compared to the levels in controls and this correlated with a decrease in disease symptoms in both species. SA appeared to regulate defense responses in Brassica spp. in a concentration-dependent manner. While 2.7-fold increase in endogenous SA levels (as seen in HC1) led to an induction of antioxidant enzymes, over 10-fold increases in endogenous SA levels (as seen after exogenous SA application in both varieties) brought about no induction of the antioxidant enzymes, probably because SA itself served as an antioxidant.     

Synthesis and functional characterization of a fluorescent peptide probe for non invasive imaging of collagen in live tissues

Targeted molecular imaging to detect changes in the structural and functional organization of tissues, at the molecular level, is a promising approach for effective and early diagnosis of diseases. Quantitative and qualitative changes in type I collagen, which is a major component in the extra cellular matrix (ECM) of skin and other vital organs like lung, liver, heart and kidneys, are often associated with the pathophysiology of these organs. We have synthesized a fluorescent probe that comprises collagelin, a specific collagen binding peptide, coupled to fluorescent porphyrin that can effectively detect abnormal deposition of collagen in live tissues by emitting fluorescence in the near infra red (NIR) region. In this report we have presented the methodology for coupling of 5-(4-carboxy phenyl)-10, 15, 20-triphenyl porphyrin (C-TPP) to the N-terminal of collagelin or to another mutant peptide (used as a control). We have evaluated the efficacy of these fluorescent peptides to detect collagen deposition in live normal and abnormal tissues. Our results strongly suggest that porphyrin-tagged collagelin can be used as an effective probe for the non invasive in vivo detection of tissue fibrosis, especially in the liver.     

Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ

RlmJ catalyzes the m⁶A2030 methylation of 23S rRNA during ribosome biogenesis in Escherichia coli. Here, we present crystal structures of RlmJ in apo form, in complex with the cofactor S-adenosyl-methionine and in complex with S-adenosyl-homocysteine plus the substrate analogue adenosine monophosphate (AMP). RlmJ displays a variant of the Rossmann-like methyltransferase (MTase) fold with an inserted helical subdomain. Binding of cofactor and substrate induces a large shift of the N-terminal motif X tail to make it cover the cofactor binding site and trigger active-site changes in motifs IV and VIII. Adenosine monophosphate binds in a partly accommodated state with the target N6 atom 7 Å away from the sulphur of AdoHcy. The active site of RlmJ with motif IV sequence ₁₆₄DPPY₁₆₇ is more similar to DNA m⁶A MTases than to RNA m⁶₂A MTases, and structural comparison suggests that RlmJ binds its substrate base similarly to DNA MTases T4Dam and M.TaqI. RlmJ methylates in vitro transcribed 23S rRNA, as well as a minimal substrate corresponding to helix 72, demonstrating independence of previous modifications and tertiary interactions in the RNA substrate. RlmJ displays specificity for adenosine, and mutagenesis experiments demonstrate the critical roles of residues Y4, H6, K18 and D164 in methyl transfer.     

Studies on the Exopolysaccharide from Sphingomonas paucimobilis-GS1: Nutritional requirements and precursor-forming enzymes

Studies on the nutritional requirements for optimal exopolysaccharide (EPS) production by Sphingomonas paucimobilis-GS1, in a synthetic medium revealed sucrose (40 g/L) and glutamate (0.5 g/L) or KNO₃ (1 g/L) to be the most suitable carbon and nitrogen sources, respectively. Ammonium salts were unfavorable to EPS accumulation, and inorganic phosphate above 10 mM affected the polymer quality. Specific activities of the EPS precursor-forming enzymes, UDP glucose pyrophosphorylase (UDPGPP) and phosphoglucomutase (PGluM), were four to five times lower, whereas that of UDPglucose dehydrogenase (UDPGDH) was 15–20 times lower under media conditions not favoring EPS production than under conditions favoring EPS accumulation. The activity of hexokinase (HK), however, remained constant. Considerably lower specific activities of PGluM and UDPGPP were also detected in some of the non-mucoid mutants.     

Chemotherapy of leishmaniasis. Part XI: Synthesis and bioevaluation of novel isoxazole containing heteroretinoid and its amide derivatives

Novel isoxazole containing heteroretinoid (4) and its amide derivatives (5a–j) have been synthesized and evaluated for their in vivo antileishmanial activity against Leishmania donovani in hamsters. Compounds 3, 5a, 5d, 5k and 5l inhibited 70–76% parasite growth at 50 mg kg^?1 × 5 days. The present study has helped us in identifying a new lead that could be exploited as a potential antileishmanial agent.     

Chemotherapy of leishmaniasis. Part IX: Synthesis and bioevaluation of aryl substituted ketene dithioacetals as antileishmanial agents

A new series of aryl substituted ketene dithioacetals 6a–h was synthesized and evaluated for their in vitro and in vivo antileishmanial activity against Leishmania donovani. Two compounds exhibited significant in vitro activity against intracellular amastigotes of L. donovani with IC₅₀ values 3.56 and 5.12 μM and were found promising as compared with reference drug, miltefosine. On the basis of good Selectivity Indices (S.I.), they were further tested for their in vivo response against L. donovani/hamster model and showed significant inhibition of parasite multiplication 78% and 83%, respectively. These compounds were better than the existing antileishmanials in respect to IC₅₀ and SI values, but were less active than miltefosine in vivo.