SCFFbxw15 Mediates Histone Acetyltransferase Binding to Origin Recognition Complex (HBO1) Ubiquitin-Proteasomal Degradation to Regulate Cell Proliferation

Histone acetyltransferase binding to origin recognition complex (HBO1) plays a crucial role in DNA replication licensing and cell proliferation, yet its molecular regulation in cells is relatively unknown. Here an uncharacterized protein, Fbxw15, directly interacts with HBO1, a labile protein (t_½ = ∼3 h), to mediate its ubiquitination (Lys³³⁸) and degradation in the cytoplasm. Fbxw15-mediated HBO1 depletion required mitogen-activated protein kinase 1 (Mek1), which was sufficient to trigger HBO1 phosphorylation and degradation in cells. Mek1 ability to produce HBO1 degradation was blocked by Fbxw15 silencing. Lipopolysaccharide induced HBO1 degradation, an effect abrogated by Fbxw15 or Mek1 cellular depletion. Modulation of Fbxw15 levels was able to differentially regulate histone H3K14 acetylation and cellular proliferation by altering HBO1 levels. These studies authenticate Fbxw15 as a ubiquitin E3 ligase subunit that mediates endotoxin-induced HBO1 depletion in cells, thereby controlling cell replicative capacity.     

Streptomycin affinity depends on 13 amino acids forming a loop in homology modelled ribosomal S12 protein (rpsL gene) of Lysinibacillus sphaericus DSLS5 associated with marine sponge (Tedania anhelans)

Streptomycin, an antibiotic used against microbial infections, inhibits the protein synthesis by binding to ribosomal protein S12, encoded by rpsL12 gene, and associated mutations cause streptomycin resistance. A streptomycin resistant, Lysinibacillus sphaericus DSLS5 (MIC >300 µg/mL for streptomycin), was isolated from a marine sponge (Tedania anhelans). The characterisation of rpsL12 gene showed a region having similarity to long terminal repeat sequences of murine lukemia virus which added 13 amino acids for loop formation in RpsL12; in addition, a K56R mutation which corresponds to K43R mutation present in streptomycin-resistant Escherichia coli is also present. The RpsL12 protein was modelled and compared with that of Lysinibacillus boronitolerans, Escherichia coli and Mycobacterium tuberculosis. The modelled proteins docked with streptomycin indicate compound had less affinity. The effect of loop on streptomycin resistance was analysed by constructing three different models of RpsL12 by, (i) removing both loop and mutation, (ii) removing the loop alone while retaining the mutation and (iii) without mutation having loop. The results showed that the presence of loop causes streptomycin resistance (decreases the affinity), and it further enhanced in the presence of mutation at 56th codon. Further study will help in understanding the evolution of streptomycin resistance in organisms.     

Induction of IL-10 and TGFβ from CD4+CD25+FoxP3+ T Cells Correlates with Parasite Load in Indian Kala-azar Patients Infected with Leishmania donovani

Background

Visceral leishmaniasis (VL) is distinguished by a complex interplay of immune response and parasite multiplication inside host cells. However, the direct association between different immunological correlates and parasite numbers remains largely unknown.

Methodology/Principal Findings

We examined the plasma levels of different disease promoting/protective as well as Th17 cytokines and found IL-10, TGFβ and IL-17 to be significantly correlated with parasite load in VL patients (r = 0.52, 0.53 and 0.51 for IL-10, TGFβ and IL-17, respectively). We then extended our investigation to a more antigen-specific response and found leishmanial antigen stimulated levels of both IL-10 and TGFβ to be significantly associated with parasite load (r = 0.71 and 0.72 for IL-10 and TGFβ respectively). In addition to cytokines we also looked for different cellular subtypes that could contribute to cytokine secretion and parasite persistence. Our observations manifested an association between different Treg cell markers and disease progression as absolute numbers of CD4⁺CD25⁺ (r = 0.55), CD4⁺CD25^hi (r = 0.61) as well as percentages of CD4⁺CD25⁺FoxP3⁺ T cells (r = 0.68) all correlated with parasite load. Encouraged by these results, we investigated a link between these immunological components and interestingly found both CD4⁺CD25⁺ and CD4⁺CD25⁺FoxP3⁺ Treg cells to secrete significantly (p<0.05) higher amounts of not only IL-10 but also TGFβ in comparison to corresponding CD25^- T cells.

Conclusions/Significance

Our findings shed some light on source(s) of TGFβ and suggest an association between these disease promoting cytokines and Treg cells with parasite load during active disease. Moreover, the direct evidence of CD4⁺CD25⁺FoxP3⁺ Treg cells as a source of IL-10 and TGFβ during active VL could open new avenues for immunotherapy towards cure of this potentially fatal disease.     

Production of thermostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation: optimization of nutrients levels using response surface methodology

The optimization of nutrient levels for the production of thermostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation (SSF) was carried out using response surface methodology based on the central composite rotatable design. The design contains a total of 54 experimental trials with the first 32 organized in a fractional factorial design and experimental trials from 33-40 and 51-54 involving the replication of the central points. The design was employed by selecting potato starch, magnesium chloride, ferrous sulfate, corn steep liquor and pearl millet flour as model factors. Among the five independent variables studied, except magnesium chloride, all the nutrients were found significant. 16.5% potato starch, 2.5% corn steep, 0.015% ferrous sulfate and 14% pearl millet flour have been found optimal for the production of thermostable pullulanase. The strain SV2 produced 10% more pullulanase in the nutritionally optimized solid-state fermentation medium containing only four nutrients.     

Structure-function correlation during the etioplast-chloroplast transition in cucumber cotyledonary leaves

We studied the development of chloroplasts from etioplasts in the cotyledonary leaves of 4-d-old dark-grown cucumber (Cucumis sativus) seedlings after irradiation (20 μmol m^-2 s^-1). Upon irradiation, the triggering of chlorophyll (Chl) synthesis and accumulation showed a relatively short lag phase. The irradiation of etiolated seedlings initiated the synthesis of apoproteins of pigment-protein complexes. While Chl-protein 2 (CP2) was detected at 6 h after irradiation, CP1 only after 29 h. The appearance and accumulation of some of the apoproteins were monitored by Western-blotting. LHC2 apoprotein was detected after a 6 h-irradiation. The amounts of D1 protein of photosystem (PS) 2 and PsaA/B protein of PS1 were quantitated by ELISA. Further, the thylakoid membrane function during this time period in terms of PS1- and PS2-mediated electron transfer activity and intersystem electron pool size were analyzed. While PS1 activity was detected after 4 h, PS2-mediated O₂ evolution was detected only after a 17 h-irradiation. F_v/F_m value of Chl a fluorescence measurements indicated that the photochemical efficiency of these leaves reached its maximum after 29 h of irradiation. The intersystem pool size of cotyledonary leaves was equivalent to that of the control cotyledonary leaves grown for 25 h under continuous irradiation. Thus etioplasts develop into fully functional chloroplasts after approximately 25 h when 4 d-dark grown cucumber seedlings are continuously moderately irradiated. The development of photosynthetic electron transport chain seems to be limited in time at the level of PS2, possibly at the donor side. This revised version was published online in September 2006 with corrections to the Cover Date.     

The institute for genomic research Osa1 rice genome annotation database

We have developed a rice (Oryza sativa) genome annotation database (Osa1) that provides structural and functional annotation for this emerging model species. Using the sequence of O. sativa subsp. japonica cv Nipponbare from the International Rice Genome Sequencing Project, pseudomolecules, or virtual contigs, of the 12 rice chromosomes were constructed. Our most recent release, version 3, represents our third build of the pseudomolecules and is composed of 98% finished sequence. Genes were identified using a series of computational methods developed for Arabidopsis (Arabidopsis thaliana) that were modified for use with the rice genome. In release 3 of our annotation, we identified 57,915 genes, of which 14,196 are related to transposable elements. Of these 43,719 non-transposable element-related genes, 18,545 (42.4%) were annotated with a putative function, 5,777 (13.2%) were annotated as encoding an expressed protein with no known function, and the remaining 19,397 (44.4%) were annotated as encoding a hypothetical protein. Multiple splice forms (5,873) were detected for 2,538 genes, resulting in a total of 61,250 gene models in the rice genome. We incorporated experimental evidence into 18,252 gene models to improve the quality of the structural annotation. A series of functional data types has been annotated for the rice genome that includes alignment with genetic markers, assignment of gene ontologies, identification of flanking sequence tags, alignment with homologs from related species, and syntenic mapping with other cereal species. All structural and functional annotation data are available through interactive search and display windows as well as through download of flat files. To integrate the data with other genome projects, the annotation data are available through a Distributed Annotation System and a Genome Browser. All data can be obtained through the project Web pages at http://rice.tigr.org.     

Curcumin stably interacts with DNA hairpin through minor groove binding and demonstrates enhanced cytotoxicity in combination with FdU nucleotides

We report, based on biophysical studies and molecular mechanical calculations that curcumin binds DNA hairpin in the minor groove adjacent to the loop region forming a stable complex. UV–Vis and fluorescence spectroscopy indicated interaction of curcumin with DNA hairpin. In this novel binding motif, two ? H of curcumin heptadiene chain are closely positioned to the A₁₆-H8 and A₁₇-H8, while G₁₂-H8 is located in the close proximity of curcumin α H. Molecular dynamics (MD) simulations suggest, the complex is stabilized by noncovalent forces including; π-π stacking, H-bonding and hydrophobic interactions. Nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics simulations indicated curcumin is bound in the minor groove, while circular dichroism (CD) spectra suggested minute enhancement in base stacking and a little change in DNA helicity, without significant conformational change of DNA hairpin structure. The DNA:curcumin complex formed with FdU nucleotides rather than Thymidine, demonstrated enhanced cytotoxicity towards oral cancer cells relative to the only FdU substituted hairpin. Fluorescence co-localization demonstrated stability of the complex in biologically relevant conditions, including its cellular uptake. Acridine orange/EtBr staining further confirmed the enhanced cytotoxic effects of the complex, suggesting apoptosis as mode of cell death. Thus, curcumin can be noncovalently complexed to small DNA hairpin for cellular delivery and the complex showed increased cytotoxicity in combination with FdU nucleotides, demonstrating its potential for advanced cancer therapy.     

Skeletal troponin I as a marker of exercise-induced muscle damage

Sorichter, Stephan, Johannes Mair, Arnold Koller, WalterGebert, Daniel Rama, Charles Calzolari, Erika Artner-Dworzak, and BerndPuschendorf. Skeletal troponin I as a marker of exercise-inducedmuscle damage. J. Appl. Physiol.83(4): 1076-1082, 1997.The utility of skeletal troponin I (sTnI)as a plasma marker of skeletal muscle damage after exercise wascompared against creatine kinase (CK), myoglobin (Mb), and myosin heavychain (MHC) fragments. These markers were serially measured in normalphysical education teacher trainees after four different exerciseregimens: 20 min of level or downhill (16% decline) running(intensity: 70% maximal O₂uptake), high-force eccentric contractions (70 repetitions), orhigh-force isokinetic concentric contractions of the quadriceps group(40 repetitions). Eccentrically biased exercise (downhill running andeccentric contractions) promoted greater increases in all parameters.The highest plasma concentration were found after downhill running{median peaks: 309 U/l CK concentration ([CK])}, 466 µg/l Mb concentration([Mb]), 1,021 µU/l MHC concentration ([MHC]),and 27.3 µg/l sTnI concentration ([sTnI]). Level running produced a moderate response (median peaks: 178 U/l [CK],98 µg/l [Mb], 501 µU/l [MHC], and 6.6 µg/l [sTnI]), whereas the concentric contraction protocoldid not elicit significant changes in any of the markers assayed. sTnIincreased and peaked in parallel to CK and stayed elevated (>2.2µg/l) for at least 1-2 days after exercise. In contrast to MHC,sTnI is an initial, specific marker of exercise-induced muscle injury,which may be partly explained by their different intracellularcompartmentation with essentially no (MHC <0.1%) or a small solublepool (sTnI: median 3.4%).

     

Uncovering genomic causes of co-morbidity in epilepsy: gene-driven phenotypic characterization of rare microdeletions

Background

Patients with epilepsy often suffer from other important conditions. The existence of such co-morbidities is frequently not recognized and their relationship with epilepsy usually remains unexplained.

Methodology/Principal Findings

We describe three patients with common, sporadic, non-syndromic epilepsies in whom large genomic microdeletions were found during a study of genetic susceptibility to epilepsy. We performed detailed gene-driven clinical investigations in each patient. Disruption of the function of genes in the deleted regions can explain co-morbidities in these patients.

Conclusions/Significance

Co-morbidities in patients with epilepsy can be part of a genomic abnormality even in the absence of (known) congenital malformations or intellectual disabilities. Gene-driven phenotype examination can also reveal clinically significant unsuspected condition.