Lipid phosphate phosphatases dimerise,but this interaction is not required for <Emphasis Type="Italic">in vivo</Emphasis> activity

Background  

Lipid phosphate phosphatases (LPPs) are integral membrane proteins believed to dephosphorylate bioactive lipid messengers, so modifying or attenuating their activities. Wunen, a Drosophila LPP homologue, has been shown to play a pivotal role in primordial germ cell (PGC) migration and survival during embryogenesis. It has been hypothesised that LPPs may form oligomeric complexes, and may even function as hexamers. We were interested in exploring this possibility, to confirm whether LPPs can oligomerise, and if they do, whether oligomerisation is required for either in vitroor in vivoactivity.     

Insights into the role of Val45 and Gln182 of Escherichia coli MutY in DNA substrate binding and specificity

Background  

Escherichia coli MutY (EcMutY) reduces mutagenesis by removing adenines paired with guanines or 7,8-dihydro-8-oxo-guanines (8-oxoG). V45 and Q182 of EcMutY are considered to be the key determinants of adenine specificity. Both residues are spatially close to each other in the active site and are conserved in MutY family proteins but not in Methanobacterium thermoautotrophicum Mig.MthI T/G mismatch DNA glycosylase (A50 and L187 at the corresponding respective positions).     

alpha-Sarcin catalytic activity is not required for cytotoxicity

Background  

α-Sarcin is a protein toxin produced by Aspergillus giganteus. It belongs to a family of cytotoxic ribonucleases that inactivate the ribosome and inhibit protein synthesis. α-Sarcin cleaves a single phosphodiester bond within the RNA backbone of the large ribosomal subunit, which makes the ribosome unrecognizable to elongation factors and, in turn, blocks protein synthesis. Although it is widely held that the protein synthesis inhibition caused by the toxin leads to cell death, it has not been directly shown that catalytically inactive mutants of α-sarcin are non-toxic when expressed directly within the cytoplasm of cells. This is important since recent studies have cast doubt on whether protein synthesis inhibition is sufficient to initiate apoptosis.     

Comparison of sterols and fatty acids in two species of <Emphasis Type="Italic">Ganoderma</Emphasis>

Background  

Two species of Ganoderma, G. sinense and G. lucidum, are used as Lingzhi in China. Howerver, the content of triterpenoids and polysaccharides, main actives compounds, are significant different, though the extracts of both G. lucidum and G. sinense have antitumoral proliferation effect. It is suspected that other compounds contribute to their antitumoral activity. Sterols and fatty acids have obvious bioactivity. Therefore, determination and comparison of sterols and fatty acids is helpful to elucidate the active components of Lingzhi.     

Studies of the intermediary metabolism in cultured cells of the insect <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis> using <Superscript>13</Superscript>C- or <Superscript>15</Superscript>N-labelled tracers

Background  

Insect cells can serve as host systems for the recombinant expression of eukaryotic proteins. Using this platform, the controlled expression of ¹⁵N/¹³C labelled proteins requires the analysis of incorporation paths and rates of isotope-labelled precursors present in the medium into amino acids. For this purpose, Spodoptera frugiperda cells were grown in a complex medium containing [U-¹³C₆]glucose. In a second experiment, cultures of S. frugiperda were grown in the presence of ¹⁵N-phenylalanine.     

Combinatorial diversity of fission yeast SCF ubiquitin ligases by homo- and heterooligomeric assemblies of the F-box proteins Pop1p and Pop2p

Background  

SCF ubiquitin ligases share the core subunits cullin 1, SKP1, and HRT1/RBX1/ROC1, which associate with different F-box proteins. F-box proteins bind substrates following their phosphorylation upon stimulation of various signaling pathways. Ubiquitin-mediated destruction of the fission yeast cyclin-dependent kinase inhibitor Rum1p depends on two heterooligomerizing F-box proteins, Pop1p and Pop2p. Both proteins interact with the cullin Pcu1p when overexpressed, but it is unknown whether this reflects their co-assembly into bona fide SCF complexes.     

Functional redundancy between <Emphasis Type="Italic">trans</Emphasis>-Golgi network SNARE family members in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background

Vesicle fusion is an essential process for maintaining the structure and function of the endomembrane system. Fusion is mediated by t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) fusion proteins on the target membrane and v-SNAREs on the vesicle membrane; v-and t-SNAREs interact with each other, driving vesicle fusion with the target membrane. The Arabidopsis thaliana trans-Golgi network resident SNAREs SYP41 and VTI12, along with YKT61/62, have been shown to function in vesicle fusion in vitro, consistent with immunoprecipitation results showing their interaction in Arabidopsis cell extracts. Conflicting published results have indicated that SYP4 family members are either functionally redundant or have distinct and essential functions; the reason for this discrepancy is unclear.

Results

Here we used a proteoliposome fusion assay to demonstrate that SYP42 and SYP43 can substitute for SYP41 in driving lipid mixing, providing support for functional overlap between family members. Previous reports have also suggested that VTI11 and VTI12 SNAREs show partial overlap in function, despite having mostly distinct localizations and binding partners. We show that VTI11 can substitute for VTI12 in in vitro lipid mixing reactions, providing molecular support for the genetic evidence for partial functional redundancy in vivo.

Conclusions

Our data provide biochemical evidence for functional overlap in membrane fusion between members of the SYP4 or VTI1 SNARE groups, supporting previous genetic data suggesting redundancy.

     

Evaluation of phenolic contents and antioxidant activity of various solvent extracts of <Emphasis Type="Italic">Sonchus asper</Emphasis> (L.) Hill

Background  

Sonchus asper (SA) is traditionally used for the treatment of various ailments associated with liver, lungs and kidneys. This study was aimed to investigate the therapeutic potential of nonpolar (hexane, SAHE; ethyl acetate, SAEE and chloroform, SACE) and polar (methanol, SAME) crude extracts of the whole plant.     

Biochemical characterization of the maltokinase from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG

Background  

Maltose-1-phosphate was detected in Mycobacterium bovis BCG extracts in the 1960's but a maltose-1-phosphate synthetase (maltokinase, Mak) was only much later purified from Actinoplanes missouriensis, allowing the identification of the mak gene. Recently, this metabolite was proposed to be the intermediate in a pathway linking trehalose with the synthesis of glycogen in M. smegmatis. Although the M. tuberculosis H37Rv mak gene (Rv0127) was considered essential for growth, no mycobacterial Mak has, to date, been characterized.     

Kinetics and mechanism of monomolecular heterolysis of commercial organohalogen compounds: XXXVIII. Correlation analysis of solvent effects on the activation parameters of heterolysis of <Emphasis Type="Italic">t</Emphasis>-BuBr and <Emphasis Type="Italic">t</Emphasis>-BuI

Heterolysis of t-BuBr and t-BuI in aprotic solvents involves a H - S compensation effect. The G of t-BuBr heterolysis in aprotic solvents decreases with increasing solvent polarity and cohesion, whereas the respective value for t-BuI heterolysis decreases with increasing solvent polarity, nucleophilicity, and polarizability. In protic solvents, a negative effect of nucleophilic solvation is observed.Translated from Zhurnal Obshchei Khimii, Vol. 74, No. 9, 2004, pp. 1476–1483.Original Russian Text Copyright © 2004 by Ponomarev, Zaliznyi, Dvorko.This revised version was published online in April 2005 with a corrected cover date.     

Thermophile-specific proteins: the gene product of <Emphasis Type="Italic">aq_1292</Emphasis> from <Emphasis Type="Italic">Aquifex aeolicus</Emphasis> is an NTPase

Background  

To identify thermophile-specific proteins, we performed phylogenetic patterns searches of 66 completely sequenced microbial genomes. This analysis revealed a cluster of orthologous groups (COG1618) which contains a protein from every thermophile and no sequence from 52 out of 53 mesophilic genomes. Thus, COG1618 proteins belong to the group of thermophile-specific proteins (THEPs) and therefore we here designate COG1618 proteins as THEP1s. Since no THEP1 had been analyzed biochemically thus far, we characterized the gene product of aq_1292 which is THEP1 from the hyperthermophilic bacterium Aquifex aeolicus (aaTHEP1).     

Effects of the deletion of the <Emphasis Type="Italic">Escherichia coli</Emphasis> frataxin homologue CyaY on the respiratory NADH:ubiquinone oxidoreductase

Background  

Frataxin is discussed as involved in the biogenesis of iron-sulfur clusters. Recently it was discovered that a frataxin homologue is a structural component of the respiratory NADH:ubiquinone oxidoreductase (complex I) in Thermus thermophilus. It was not clear whether frataxin is in general a component of complex I from bacteria. The Escherichia coli homologue of frataxin is coined CyaY.     

Bergamot (<Emphasis Type="Italic">Citrus bergamia</Emphasis> Risso) fruit extracts and identified components alter expression of interleukin 8 gene in cystic fibrosis bronchial epithelial cell lines

Background  

Cystic fibrosis (CF) airway pathology is a fatal, autosomal, recessive genetic disease characterized by extensive lung inflammation. After induction by TNF-α, elevated concentrations of several pro-inflammatory cytokines (i.e. IL-6, IL-1β) and chemokines (i.e. IL-8) are released from airway epithelial cells. In order to reduce the excessive inflammatory response in the airways of CF patients, new therapies have been developed and in this respect, medicinal plant extracts have been studied. In this article we have investigated the possible use of bergamot extracts (Citrus bergamia Risso) and their identified components to alter the expression of IL-8 associated with the cystic fibrosis airway pathology.     

Analysis of ketone‐based neurosteroids by reactive low‐temperature plasma mass spectrometry

Rationale

Neurosteroids are important signalling molecules that modulate neuronal activity. Their low concentrations and low volatility make neurosteroid detection and quantification by ambient mass spectrometry challenging. Here we develop a reactive low‐temperature plasma mass spectrometry (LTP‐MS) method and demonstrate its potential for fast screening and quantification of neurosteroids in mouse brain.

Methods

Ketone‐based neurosteroids were analysed with the LTP‐MS method. The plasma of the LTP was heated in order to improve the desorption efficiency of low‐volatility neurosteroids. Methylamine with a concentration of 500 ppbv was employed as the reactive reagent. Neurosteroids in mouse brain tissue extracts were detected in 70 s with mass errors less than ±3 ppm due to coupling of the ion source with a high‐performance mass spectrometer.

Results

Reaction between neurosteroids and methylamine, seeded into the LTP gas stream, resulted in the formation of protonated methylamine–neurosteroid adducts with 5‐ to 100‐fold abundances, compared to [M + H]⁺ ions detected in non‐reactive LTP‐MS. The lowest detectable concentrations of neurosteroid standards were in the range of ng/mL. Concentrations of neurosteroids in male and female mouse brain extracts as determined with reactive LTP‐MS were on the level of ng/g, comparable to results obtained with high‐performance liquid chromatography–tandem mass spectrometry.

Conclusions

The developed reactive LTP‐MS is capable of providing sensitive identification and quantification of ketone‐based neurosteroids in mouse brain extracts with minimal sample treatment, and showcases the potential of reactive LTP‐MS as a tool for fast screening of neurosteroid levels in brain.

     

Myosin-cross-reactive antigen (MCRA) protein from <Emphasis Type="Italic">Bifidobacterium breve</Emphasis> is a FAD-dependent fatty acid hydratase which has a function in stress protection

Background  

The aim of this study was to determine the catalytic activity and physiological role of myosin-cross-reactive antigen (MCRA) from Bifidobacterium breve NCIMB 702258. MCRA from B. breve NCIMB 702258 was cloned, sequenced and expressed in heterologous hosts (Lactococcus and Corynebacterium) and the recombinant proteins assessed for enzymatic activity against fatty acid substrates.     

Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation

Background  

Mycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host. In order to protect itself from the redox onslaughts of host immune system, M. tuberculosis appears to have developed accessory thioredoxin-like proteins which are represented by ORFs encoding WhiB-like proteins. We have earlier reported that WhiB1/Rv3219 is a thioredoxin like protein of M. tuberculosis and functions as a protein disulfide reductase. Generally thioredoxins have many substrate proteins. The current study aims to identify the substrate protein(s) of M. tuberculosis WhiB1.     

Identification of inhibitors of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> phosphoethanolamine methyltransferase using an enzyme-coupled transmethylation assay

Background  

The phosphoethanolamine methyltransferase, PfPMT, of the human malaria parasite Plasmodium falciparum, a member of a newly identified family of phosphoethanolamine methyltransferases (PMT) found solely in some protozoa, nematodes, frogs, and plants, is involved in the synthesis of the major membrane phospholipid, phosphatidylcholine. PMT enzymes catalyze a three-step S-adenosylmethionine-dependent methylation of the nitrogen atom of phosphoethanolamine to form phosphocholine. In P. falciparum, this activity is a limiting step in the pathway of synthesis of phosphatidylcholine from serine and plays an important role in the development, replication and survival of the parasite within human red blood cells.     

Comparison of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isocitrate dehydrogenases (ICD-1 and ICD-2) reveals differences in coenzyme affinity,oligomeric state,pH tolerance and phylogenetic affiliation

Background  

M.tb icd-1 and M.tb icd-2, have been identified in the Mycobacterium tuberculosis genome as probable isocitrate dehydrogenase (ICD) genes. Earlier we demonstrated that the two isoforms can elicit B cell response in TB patients and significantly differentiate TB infected population from healthy, BCG-vaccinated controls. Even though immunoassays suggest that these proteins are closely related in terms of antigenic determinants, we now show that M.tb icd-1 and M.tb icd-2 code for functional energy cycle enzymes and document the differences in their biochemical properties, oligomeric assembly and phylogenetic affiliation.     

Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation

Background  

Dictyostelium cells secrete the proteins AprA and CfaD. Cells lacking either AprA or CfaD proliferate faster than wild type, while AprA or CfaD overexpressor cells proliferate slowly, indicating that AprA and CfaD are autocrine factors that repress proliferation. CfaD interacts with AprA and requires the presence of AprA to slow proliferation. To determine if CfaD is necessary for the ability of AprA to slow proliferation, whether AprA binds to cells, and if so whether the binding requires the presence of CfaD, we examined the binding and effect on proliferation of recombinant AprA.     

Limited proteolysis of human histone deacetylase 1

Background  

Histone deacetylase (HDAC) proteins are associated with cell proliferation, differentiation, apoptosis, and cancer. Specifically, HDAC1 is linked with cell growth, a hallmark of cancer formation. HDAC1 is a phosphoprotein and phosphorylation at S421 and S423 promotes HDAC1 enzymatic activity and protein association. While single and double point mutants of HDAC1 at S421 and S423 appear functionally similar, the evidence suggests that HDAC1 is phosphorylated simultaneously at both S421 and S423 in vivo. Additional experiments are necessary to probe the role of double phosphorylation of HDAC1 at S421 and S423.