Additional box B of RNA polymerase III promoter in SINE B1 can be functional

Many genes of small RNAs and short interspersed elements (SINEs) are transcribed by RNA polymerase III due to an internal promoter that is composed of two boxes (A and B) spaced by 30-45 bp. Rodent SINE B1 originated from 7SL RNA, and a 29-bp tandem duplication took place in B1 at an early stage of its evolution. As a result of this duplication, an additional box B (named B') located at a distance of 79-82 bp from box A arose in SINE B1. Here we have shown that despite the unusually large distance between boxes A and B', they can form an active promoter. In chinchillas, guinea pigs, and other rodents belonging to clade Ctenohystrica, structure of the B' box was well preserved and closely resembles the canonical B box. One may suggest therefore, that box B' can functionally replace box B in those copies of B1 where the latter has lost activity due to mutations.     

Leptin in human physiology and pathophysiology

Leptin, discovered through positional cloning 15 years ago, is an adipocyte-secreted hormone with pleiotropic effects in the physiology and pathophysiology of energy homeostasis, endocrinology, and metabolism. Studies in vitro and in animal models highlight the potential for leptin to regulate a number of physiological functions. Available evidence from human studies indicates that leptin has a mainly permissive role, with leptin administration being effective in states of leptin deficiency, less effective in states of leptin adequacy, and largely ineffective in states of leptin excess. Results from interventional studies in humans demonstrate that leptin administration in subjects with congenital complete leptin deficiency or subjects with partial leptin deficiency (subjects with lipoatrophy, congenital or related to HIV infection, and women with hypothalamic amenorrhea) reverses the energy homeostasis and neuroendocrine and metabolic abnormalities associated with these conditions. More specifically, in women with hypothalamic amenorrhea, leptin helps restore abnormalities in hypothalamic-pituitary-peripheral axes including the gonadal, thyroid, growth hormone, and to a lesser extent adrenal axes. Furthermore, leptin results in resumption of menses in the majority of these subjects and, in the long term, may increase bone mineral content and density, especially at the lumbar spine. In patients with congenital or HIV-related lipoatrophy, leptin treatment is also associated with improvements in insulin sensitivity and lipid profile, concomitant with reduced visceral and ectopic fat deposition. In contrast, leptin's effects are largely absent in the obese hyperleptinemic state, probably due to leptin resistance or tolerance. Hence, another emerging area of research pertains to the discovery and/or usefulness of leptin sensitizers. Results from ongoing studies are expected to further increase our understanding of the role of leptin and the potential clinical applications of leptin or its analogs in human therapeutics.     

Tumor necrosis factor alpha and inflammation disrupt the polarity complex in intestinal epithelial cells by a posttranslational mechanism

Inflammatory processes disrupt the barrier function in epithelia. Increased permeability often leads to chronic of inflammation. Important among other cytokines, tumor necrosis factor alpha (TNF-α) initiates an NF-κB-mediated response that leads to upregulation of myosin light chain kinase (MLCK), a hallmark of the pathogenesis of inflammatory bowel disease. Here, we found that two components of the evolutionarily conserved organizer of tight junctions and polarity, the polarity complex (atypical protein kinase C [aPKC]-PAR6-PAR3) were downregulated by TNF-α signaling in intestinal epithelial cells and also in vivo during intestinal inflammation. Decreases in aPKC levels were due to decreased chaperoning activity of Hsp70 proteins, with failure of the aPKC rescue machinery, and these effects were rescued by NF-κB inhibition. Comparable downregulation of aPKC shRNA phenocopied effects of TNF-α signaling, including apical nonmuscle myosin II accumulation and myosin light chain phosphorylation. These effects, including ZO-1 downregulation, were rescued by overexpression of constitutively active aPKC. We conclude that this novel mechanism is a complementary effector pathway for TNF-α signaling.     

Whole-genome molecular haplotyping of single cells

Conventional experimental methods of studying the human genome are limited by the inability to independently study the combination of alleles, or haplotype, on each of the homologous copies of the chromosomes. We developed a microfluidic device capable of separating and amplifying homologous copies of each chromosome from a single human metaphase cell. Single-nucleotide polymorphism (SNP) array analysis of amplified DNA enabled us to achieve completely deterministic, whole-genome, personal haplotypes of four individuals, including a HapMap trio with European ancestry (CEU) and an unrelated European individual. The phases of alleles were determined at ～99.8% accuracy for up to ～96% of all assayed SNPs. We demonstrate several practical applications, including direct observation of recombination events in a family trio, deterministic phasing of deletions in individuals and direct measurement of the human leukocyte antigen haplotypes of an individual. Our approach has potential applications in personal genomics, single-cell genomics and statistical genetics.     

Dosage suppression genetic interaction networks enhance functional wiring diagrams of the cell

Dosage suppression is a genetic interaction in which overproduction of one gene rescues a mutant phenotype of another gene. Although dosage suppression is known to map functional connections among genes, the extent to which it might illuminate global cellular functions is unclear. Here we analyze a network of interactions linking dosage suppressors to 437 essential genes in yeast. For 424 genes, we curated interactions from the literature. Analyses revealed that many dosage suppression interactions occur between functionally related genes and that the majority do not overlap with other types of genetic or physical interactions. To confirm the generality of these network properties, we experimentally identified dosage suppressors for 29 genes from pooled populations of temperature-sensitive mutant cells transformed with a high-copy molecular-barcoded open reading frame library, MoBY-ORF 2.0. We classified 87% of the 1,640 total interactions into four general types of suppression mechanisms, which provided insight into their relative frequencies. This work suggests that integrating the results of dosage suppression studies with other interaction networks could generate insights into the functional wiring diagram of a cell.     

Identification of potentially involved proteins in levofloxacin resistance mechanisms in Coxiella burnetii

The etiological agent of Q fever, Coxiella burnetii, is an obligate intracellular bacterium that multiplies within a phagosome-like parasitophorous vacuole. Fluoroquinolones have been used as an alternative therapy for Q fever. Resistance to fluoroquinolones can arise via several mechanisms utilized by pathogens to avoid killing. Until today, genome-based studies have shown that the main mechanism of C. burnetii to resist inhibition by fluoroquinolones is based on mutations in quinolone-resistance-determining region (QRDR). In this study, in a broader search at the protein level for C. burnetii mechanisms that confer resistance to fluoroquinolones, the proteomes of in vitro developed fluoroquinolone resistant bacteria and susceptible bacteria were compared using the MS-driven combined fractional diagonal chromatography (COFRADIC) proteomics technique. Quantitative comparison of the 381 proteins identified in both strains indicated the different expression of 15 bacterial proteins. These proteins are involved in different cellular processes indicating that the antibiotic resistance mechanism of the bacterium is a multifaceted process.     

Synthesis and biological activity of bimorpholine and its carbanucleoside

A new enantiomerically pure carbacyclic nucleoside analogue with bimorpholine as a nonaromatic nucleobase was synthesized. The nucleoside analogue and bimorpholine were tested for cytotoxicity using an MTT assay and the xCELLigence System. Both assays revealed that compound 3 was highly cytotoxic at a 50 μM concentration while the cytotoxic effect of compound 1 was much less prominent. No antiretroviral activity was detected for this compound. In contrast, it acted as a potent inhibitor of hepatitis C virus (HCV) replication. Most likely this effect originates largely from the cytotoxicity of the compound; however, it is possible that a specific mechanism of HCV inhibition also exists.     

Oxidation of Lanthionines Renders the Lantibiotic Nisin Inactive

The peptide antibiotic nisin A belongs to the group of antibiotics called lantibiotics. They are classified as lantibiotics because they contain the structural group lanthionine. Lanthionines are composed of a single sulfur atom that is linked to the β-carbons of two alanine moieties. These sulfur atoms are vulnerable to environmental oxidation. A mild oxidation reaction was performed on nisin A to determine the relative effects it would have on bioactivity. High-mass-accuracy Fourier transform ion cyclotron resonance mass spectrometry data revealed the addition of seven, eight, and nine oxygens. These additions correspond to the five lanthionines, two methionines, and two histidines that would be susceptible to oxidation. Subsequent bioassays revealed that the oxidized form of nisin A had a complete loss of bactericidal activity. In a competition study, the oxidized nisin did not appear to have an antagonistic affect on the bioactivity of nisin A, since the addition of an equal molar concentration of the oxidized variant did not have an influence on the bactericidal activity of the native antibiotic. Electron microscopy data revealed that the oxidized forms were still capable of assembling into large circular complexes, demonstrating that oxidation does not disrupt the lateral assembly mechanism of the antibiotic. Affinity thin-layer chromatography and fluorescence microscopy experiments suggested that the loss of activity is due to the inability of the oxidized form of nisin to bind to the cell wall precursor lipid II. Given the loss of bioactivity following oxidation, oxidation should be an important factor to consider in future production, purification, pharmacokinetic, and pharmacodynamic studies.Lantibiotics are ribosomally synthesized peptide bacteriocins that undergo extensive posttranslational modifications to yield unusual amino acids, like lanthionine, methyllanthionine, 2,3-didehydroalanine, 2,3-didehydrobutyrine, and S-[aminovinyl] cysteine (8). The name lantibiotic is derived from the presence of the posttranslationally modified lanthionine residues. Nisin A (3,351.5 Da), produced by Lactococcus lactis, belongs to this class of antibiotics and is further subclassified as a type A(I) lantibiotic. Type A(I) lantibiotics are cationic and have a rigid ring conformation separated by areas of flexibility. Another well-studied lantibiotic, gallidermin, also belongs to this class of lantibiotics and has significant homology to nisin A in the first two lanthionine rings, A and B (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Schematic of the covalent structures of nisin A and gallidermin. The N-terminal rings A and B are believed to be responsible for binding to lipid II.The antibiotics in this class have drawn considerable attention for their bactericidal potential as preservatives and for their potential for treating Staphylococcus and Streptococcus infections. Nisin A has been used for over 40 years in Europe as a preservative in the food industry and was approved for use in the United States by the FDA in 1988. Its uses include controlling the growth of various bacteria in pasteurized cheese and liquid egg ingredients, as well as preserving salad dressings (12), canned foods (10, 32), and, most recently, ground beef (24). Other lantibiotics, including gallidermin and epidermin, have been shown to be useful as treatments for acne and in the maintenance of oral health (19, 21). Recent literature shows that both nisin A and gallidermin can be used to treat and/or prevent mastitis in bovines (3, 25a), and they are currently marketed as wipes.Lantibiotics have multiple modes of bactericidal activity (2, 16). In the case of nisin A, the sensitivity of the host bacterium has been shown to be dependent on the charge states of its cell wall and membrane (1, 6, 25). More importantly, the bactericidal activity is attributed to lipid II abduction (4, 5, 7). A novel mechanism of antimicrobial activity for nisin A has been described, in which it binds to lipid II and sequesters it into large complexes. These complexes aid in the abduction of lipid II from the growth zones of bacteria, where lipid II is required for new cell wall formation (16). A novel lipid II binding motif for nisin A has been characterized by nuclear magnetic resonance (NMR) (18) in which the N-terminal portion of nisin A, lanthionine rings A and B, interacts with the pyrophosphate, the peptidoglycan MurNAc, and the first isoprene of lipid II.An oxidized form of nisin A was characterized using bactericidal assays, high-mass-accuracy Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), chromatography methods, and electron and fluorescence microscopy techniques. The objectives of this study were to determine changes in the biophysical properties of oxidized nisin A as they relate to its bactericidal activity, its ability to interact with bacterial membranes, its capacity for lateral assembly, and its ability to bind to lipid II.