Berberine-DNA complexation: new insights into the cooperative binding and energetic aspects

The equilibrium binding of the cytotoxic plant alkaloid berberine to various DNAs and energetics of the interaction have been studied. At low ratios of bound alkaloid to base pair, the binding exhibited cooperativity to natural DNAs having almost equal proportions of AT and GC sequences. In contrast, the binding was non-cooperative to DNAs with predominantly high AT or GC sequences. Among the synthetic DNAs, cooperative binding was observed with poly(dA).poly(dT) and poly(dG).poly(dC) while non-cooperative binding was seen with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Both cooperative and non-cooperative bindings were remarkably dependent on the salt concentration of the media. Linear plots of ln K(a) versus [Na(+)] for poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) showed the release of 0.56 and 0.75 sodium ions respectively per bound alkaloid. Isothermal titration calorimetry results revealed the binding to be exothermic and favoured by both enthalpy and entropy changes in all DNAs except the two AT polymers and AT rich DNA, where the same was predominantly entropy driven. Heat capacity values (DeltaCp(o)) of berberine binding to poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), Clostridium perfringens and calf thymus DNA were -98, -140, -120 and -110 cal/mol K respectively. This study presents new insights into the binding dependent base pair heterogeneity in DNA conformation and the first complete thermodynamic profile of berberine binding to DNAs.     

Biochemical and genetic characterization of mundticin KS,an antilisterial peptide produced by Enterococcus mundtii NFRI 7393

Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.     

Molecular aspects of atherogenesis: new insights and unsolved questions

Summary The development of atherosclerotic disease results from the interaction between environment and genetic make up. A key factor in atherogenesis is the oxidative modification of lipids, which is involved in the recruitment of mononuclear leukocytes to the arterial intima – a process regulated by several groups of adhesion molecules and cytokines. Activated leukocytes, as well as endothelial mitochondria, can produce reactive oxygen species (ROS) that are associated with endothelial dysfunction, a cause of reduced nitric oxide (NO) bioactivity and further ROS production. Peroxisome proliferator-activated receptors (PPAR) and liver X receptors (LXR) are nuclear receptors significantly involved in the control of lipid metabolism, inflammation and insulin sensitivity. Also, an emerging role has been suggested for G protein coupled receptors and for the small Ras and Rho GTPases in the regulation of the expression of endothelial NO synthase (eNOS) and of tissue factor, which are involved in thrombus formation and modulation of vascular tone. Further, the interactions among eNOS, cholesterol, oxidated LDL and caveola membranes are probably involved in some molecular changes observed in vascular diseases. Despite the relevance of oxidative processes in atherogenesis, anti-oxidants have failed to significantly improve atherosclerosis (ATS) prevention, while statins have proved to be the most successful drugs.     

Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity

Mouse telokin and SM22 promoters have previously been shown to direct smooth muscle cell-specific expression of transgenes in vivo in adult mice. However, the activity of these promoters is highly dependent on the integration site of the transgene. In the current study, we found that the ectopic expression of telokin promoter transgenes could be abolished by flanking the transgene with insulator elements from the H19 gene. However, the insulator elements did not increase the proportion of mouse lines that exhibited consistent, detectable levels of transgene expression. In contrast, when transgenes were targeted to the hprt locus, both telokin and SM22 promoters resulted in reproducible patterns and levels of transgene expression in all lines of mice examined. Telokin promoter transgene expression was restricted to smooth muscle tissues in adult and embryonic mice. As reported previously, SM22 transgenes were expressed at high levels specifically in arterial smooth muscle cells; however, in contrast to randomly integrated transgenes, the hprt-targeted SM22 transgenes were also expressed at high levels in smooth muscle cells in veins, bladder, and gallbladder. Using hprt-targeted transgenes, we further analyzed elements within the telokin promoter required for tissue specific activity in vivo. Analysis of these transgenes revealed that the CArG element in the telokin promoter is required for promoter activity in all tissues and that the CArG element and adjacent AT-rich region are sufficient to drive transgene expression in bladder but not intestinal smooth muscle cells. visceral smooth muscle; development; myosin light chain kinase; embryos; CArG element     

Phosphoproteomics: new insights into cellular signaling

Developments in the field of phosphoproteomics have been fueled by the need simultaneously to monitor many different phosphoproteins within the signaling networks that coordinate responses to changes in the cellular environment. This article presents a brief review of phosphoproteomics with an emphasis on the biological insights that have been derived so far.     

Phylogenomics of Enterococcus faecalis from wild birds: new insights into host-associated differences in core and accessory genomes of the species

Wild birds have been suggested to be reservoirs of antimicrobial resistant and/or pathogenic Enterococcus faecalis (Efs) strains, but the scarcity of studies and available sequences limit our understanding of the population structure of the species in these hosts. Here, we analysed the clonal and plasmid diversity of 97 Efs isolates from wild migratory birds. We found a high diversity, with most sequence types (STs) being firstly described here, while others were found in other hosts including some predominant in poultry. We found that pheromone-responsive plasmids predominate in wild bird Efs while 35% of the isolates entirely lack plasmids. Then, to better understand the ecology of the species, the whole genome of fivestrains with known STs (ST82, ST170, ST16 and ST55) were sequenced and compared with all the Efs genomes available in public databases. Using several methods to analyse core and accessory genomes (AccNET, PLACNET, hierBAPS and PANINI), we detected differences in the accessory genome of some lineages (e.g. ST82) demonstrating specific associations with birds. Conversely, the genomes of other Efs lineages exhibited divergence in core and accessory genomes, reflecting different adaptive trajectories in various hosts. This pangenome divergence, horizontal gene transfer events and occasional epidemic peaks could explain the population structure of the species.     

Impact of genetic insights into calpain biology

Calpain has long been an enigmatic enzyme, although it is involved in a variety of biological phenomena. Recent progress in calpain genetics has highlighted numerous physiological contexts in which the functions of calpain are of great significance. This review focuses on recent findings in the field of calpain genetics and the importance of calpain function. Calpain is an intracellular Ca(2+)-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02) found in almost all eukaryotes. It is also present in a few bacteria, but not in archaebacteria. Calpain has limited proteolytic activity; rather, it transforms or modulates the structure and/or activity of its substrates. It is, therefore, referred to as a 'modulator protease'. Within the human genome, 15 genes (CAPN1-3, CAPN5-16) encode a calpain-like protease (CysPc) domain along with several different functional domains. Thus, calpains can be regarded as a distinct family of versatile enzymes that fulfil numerous tasks in vivo. Genetic studies show that a variety of defects in many different organisms, including lethality, muscular dystrophies and gastropathy, actually stem from calpain deficiencies. The cause-effect relationships identified by these studies form the basis for ongoing and future studies regarding the physiological role of calpains.     

Neonatal diabetes: new insights into aetiology and implications

Neonatal diabetes mellitus (NDM) is defined as hyperglycaemia occurring in the first few weeks of life. It can be either transient (TNDM) or permanent (PNDM), and until recently, little was known about the condition. A cohort of 30 infants with a history of TNDM has been studied, and findings have suggested that NDM does not have the same aetiology as classical type 1 childhood diabetes. Uniparental isodisomy of chromosome 6 and an unbalanced duplication of paternal chromosome 6 have both been described as a genetic basis for TNDM in over 75% of the cases. In addition, cerebellar hypoplasia and Walcott-Rallison syndrome have been associated with PNDM, suggesting an autosomal recessive inheritance pattern; furthermore, a mutation in the gene insulin promoter factor 1 has been identified as a cause of pancreatic agenesis in PNDM. In the long term, TNDM may reduce beta cell functional capacity and present a predisposition to type 2 diabetes mellitus.     

Orotidylate decarboxylase: insights into the catalytic mechanism from substrate specificity studies.

Pyrimidine nucleotides were tested as substrates for pure yeast orotidylate decarboxylase in an attempt to gain insight into the nature of the catalytic mechanism of the enzyme. Substitutions of the 5-position in the pyrimidine ring of the orotidylate substrate resulted in compounds that are either excellent inhibitors or substrates of the enzyme. The 5-bromo- and 5-chloroorotidylates are potent inhibitors while the 5-fluoro derivative is a good substrate with a turnover number 30 times that observed with orotidylate. When carbon 5 of the pyrimidine ring is replaced by nitrogen in 5-azaorotidylate, the resulting compound is unstable in solution with a half-life of 25 min at pH 6. However, studies with freshly generated 5-azaorotidylate show that an enzyme-dependent reaction occurs, presumably decarboxylation. This enzyme reaction follows simple Michaelis-Menten kinetics. Because the 5-aza group is not electrophilic, an enzyme mechanism utilizing a nucleophilic addition of the enzyme at the 5-position is ruled out. We also present studies that are not compatible with a mechanism requiring the formation of a Schiff's base prior to decarboxylation. The enzyme is tolerant of modest substitution at the 4-position, for the 4-keto group can be replaced with a thioketone. However, no catalysis is observed when the same substitution is made at the 2-position. Similarities in the substrate specificity of orotate phosphoribosyltransferase and orotidylate decarboxylase led us to compare the amino acid sequences of the two enzymes; significant (20%) sequence homology was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial UCPs: new insights into regulation and impact

Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins sustaining an inducible proton conductance. They weaken the proton electrochemical gradient built up by the mitochondrial respiratory chain. Brown fat UCP1 sustains a free fatty acid (FA)-induced purine nucleotide (PN)-inhibited proton conductance. Inhibition of the proton conductance by PN has been considered as a diagnostic of UCP activity. However, conflicting results have been obtained in isolated mitochondria for UCP homologues (i.e., UCP2, UCP3, plant UCP, and protist UCP) where the FFA-activated proton conductance is poorly sensitive to PN under resting respiration conditions. Our recent work clearly indicates that the membranous coenzyme Q, through its redox state, represents a regulator of the inhibition by PN of FFA-activated UCP1 homologues under phosphorylating respiration conditions. Several physiological roles of UCPs have been suggested, including a control of the cellular energy balance as well as the preventive action against oxidative stress. In this paper, we discuss new information emerging from comparative proteomics about the impact of UCPs on mitochondrial physiology, when recombinant UCP1 is expressed in yeast and when UCP2 is over-expressed in hepatic mitochondria during steatosis.     

Capping protein: new insights into mechanism and regulation

Temporal and spatial control of the actin cytoskeleton are crucial for a range of eukaryotic cellular processes. Capping protein (CP), a ubiquitous highly conserved heterodimer, tightly caps the barbed (fast-growing) end of the actin filament and is an important component in the assembly of various actin structures, including the dynamic branched filament network at the leading edge of motile cells. New research into the molecular mechanism of how CP interacts with the actin filament in vitro and the function of CP in vivo, including discoveries of novel interactions of CP with other proteins, has greatly enhanced our understanding of the role of CP in regulating the actin cytoskeleton.     

Adenosylcobalamin-dependent isomerases: new insights into structure and mechanism

Adenosylcobalamin-dependent isomerases catalyze a variety of chemically difficult 1,2-rearrangements that proceed through a mechanism involving free radical intermediates. These radicals are initially generated by homolysis of the cobalt-carbon bond of the coenzyme. Recently, the crystal structures of several of these enzymes have been solved, revealing two modes of coenzyme binding and highlighting the role of the protein in controlling the rearrangement of reactive substrate radical intermediates. Complementary data from kinetic, spectroscopic and theoretical studies have produced insights into the mechanism by which substrate radicals are generated at the active site, and the pathways by which they rearrange.     

Mitochondria and dopamine: new insights into recessive parkinsonism

Recessively inherited mutations in parkin, DJ-1, and PINK1 have recently been linked to familial forms of parkinsonism. These syndromes are often clinically indistinguishable from Parkinson's disease, as similar neuronal groups, notably dopaminergic neurons, are selectively affected. Studies of the functions of these gene products may provide insights into the pathogenic mechanisms underlying the selective degeneration of dopaminergic neurons. Emerging evidence that one or several of these genes play important roles in mitochondrial function and the dopaminergic system suggests that these events may be early steps of the pathophysiological changes of the disease. This review will summarize recent advances in our understanding of these gene products, with emphasis on the surprising convergence of their functions.     

Subnuclear organelles: new insights into form and function

The cell nucleus is a complex and highly dynamic environment with many functionally specialized regions of substructure that form and maintain themselves in the absence of membranes. Relatively little is known about the basic physical properties of the nuclear interior or how domains within the nucleus are structurally and functionally organized and interrelated. Here, we summarize recent data that shed light on the structural and functional properties of three prominent subnuclear organelles--nucleoli, Cajal bodies (CBs) and speckles. We discuss how these findings impact our understanding of the guiding principles of nuclear organization and various types of human disease.     

Nuocytes and beyond: new insights into helminth expulsion

T helper 2 (Th2) responses, characterized by the expression of the type-2 cytokines interleukin (IL)-4, IL-5, IL-9 and IL-13, are essential for the control of parasitic helminth infections and also drive the pathogenesis of allergy and asthma. Such responses are initiated, maintained and regulated, in part, by an array of innate effector cells and cytokines. However, relatively little is known about how the initiation of type-2 immune responses occurs in vivo. The recent discovery, using helminth models, of several novel innate immune cells capable of shaping type-2 immune responses allows us to reflect on the progress made in this area. It also affords us the opportunity to highlight the diversity of immune responses that can be driven by innate cells responding rapidly to early cytokine cues.     

Carboxylesterase 2 production and characterization in human cells: new insights into enzyme oligomerization and activity

Carboxylesterase 2 (CES2), the main carboxylesterase expressed in human intestine, is an increasingly important enzyme in anti-cancer combined therapies for the treatment of different pathologies like colon adenocarcinoma and malignant glioma. The production of human recombinant CES2, in human embryonic kidney cells (HEK-293T cells) using serum-free media, is herein described. CES2 secretion to the media was achieved by the simple addition of an in-frame C-terminal 10× histidine tag (CES2-10xHis) without the need of addition of extra N-terminal signalling sequences or the mutation or deletion of the C-terminal HTEL motif responsible for retaining the protein in the lumen of endoplasmic reticulum. This secretion allowed a fourfold increase in CES2 production. The characterization of human recombinant CES2 showed that this protein exists in other active and inactive forms than the described 60 kDa monomer.