Pore-forming colicins: synthesis, extracellular release, mode of action, immunity

Colicins are bacterial toxins encoded by plasmids which also confer immunity to producing cells. In a first stage, colicins are synthesized in the cytoplasm of colicinogenic cells. Subsequently they are released into the extracellular medium following the action of a small protein synthesized coordinately with the colicins. This protein is a lipoprotein and causes a non-specific increase in the envelope permeability, in particular, through the activation of an outer membrane phospholipase. After release into the medium, colicins kill sensitive cells in 3 defined steps: adsorption onto a specific receptor at the surface of the bacterium, translocation across the outer membrane and action. A specific domain of the colicin molecule is responsible for each of these steps. The most common colicins are those which kill by depolarizing the cytoplasmic membrane with the formation of voltage-dependent ionic channels. Immunity is conferred to producing cells by a membrane protein which interacts with the colicin and prevents formation or functioning of these ionic channels formed by its C-terminal domain.     

"Unknown genome" proteomics: a new NADP-dependent epimerase/dehydratase revealed by N-terminal sequencing, inverted PCR, and high resolution mass spectrometry

We present here a new approach that enabled the identification of a new protein from a bacterial strain with unknown genomic background using a combination of inverted PCR with degenerate primers derived from N-terminal protein sequences and high resolution peptide mass determination of proteolytic digests from two-dimensional electrophoretic separation. Proteins of the sulfate-reducing bacterium Desulfotignum phosphitoxidans specifically induced in the presence of phosphite were separated by two-dimensional gel electrophoresis as a series of apparent soluble and membrane-bound isoforms with molecular masses of approximately 35 kDa. Inverted PCR based on N-terminal sequences and high resolution peptide mass fingerprinting by Fourier transform-ion cyclotron resonance mass spectrometry provided the identification of a new NAD(P) epimerase/dehydratase by specific assignment of peptide masses to a single ORF, excluding other possible ORF candidates. The protein identification was ascertained by chromatographic separation and sequencing of internal proteolytic peptides. Metal ion affinity isolation of tryptic peptides and high resolution mass spectrometry provided the identification of five phosphorylations identified in the domains 23-47 and 91-118 of the protein. In agreement with the phosphorylations identified, direct molecular weight determination of the soluble protein eluted from the two-dimensional gels by mass spectrometry provided a molecular mass of 35,400 Da, which is consistent with an average degree of three phosphorylations.     

Novel Ru(II) oximato complexes with silent oxygen atom: Synthesis,chemistry and biological activities

A series of octahedral complexes, [Ru(CO)(EPh₃)₂(bhmh)] (E = P or As; H₂bhmh = benzoic acid (2-hydroxyimino-1-methyl-propylidene)-hydrazide), [Ru(CO)(EPh₃)₂(ihmh)] (H₂ihmh = isonicotinic acid (2-hydroxyimino-1-methyl-propylidene)-hydrazide), [Ru(CO)(EPh₃)₂(hhmh)] (H₂hhmh = 2-hydroxy-benzoic acid (2-hydroxyimino-1-methyl-propylidene)-hydrazide) have been prepared by a facile procedure. X-ray structure determination of three of the complexes revealed that the hydrazone ligand coordinates through the imine and the oxime nitrogen and the amide oxygen atoms. In all the complexes, the N–OH moiety of the oxime is deprotonated to give an N–O^? species and this oxygen atom did not coordinate to the central metal atom. The oxidation–reduction processes for each of these complexes have been determined in CH₃CN by cyclic voltammetry. The complexes displayed two oxidation couples and one irreversible reduction response between +1.6 and ?1.6 V. The trend in the half wave potentials reflects the electronic nature of the hydrazone ligand. Antibacterial activity of the ligands and the complexes has been evaluated against five pathogenic bacteria. The binding of the complexes with herring sperm DNA has also been investigated by UV–Vis spectroscopy and cyclic voltammetry.     

Prebiotic chemistry: a new modus operandi

A variety of macromolecules and small molecules-(oligo)nucleotides, proteins, lipids and metabolites-are collectively considered essential to early life. However, previous schemes for the origin of life-e.g. the 'RNA world' hypothesis-have tended to assume the initial emergence of life based on one such molecular class followed by the sequential addition of the others, rather than the emergence of life based on a mixture of all the classes of molecules. This view is in part due to the perceived implausibility of multi-component reaction chemistry producing such a mixture. The concept of systems chemistry challenges such preconceptions by suggesting the possibility of molecular synergism in complex mixtures. If a systems chemistry method to make mixtures of all the classes of molecules considered essential for early life were to be discovered, the significant conceptual difficulties associated with pure RNA, protein, lipid or metabolism 'worlds' would be alleviated. Knowledge of the geochemical conditions conducive to the chemical origins of life is crucial, but cannot be inferred from a planetary sciences approach alone. Instead, insights from the organic reactivity of analytically accessible chemical subsystems can inform the search for the relevant geochemical conditions. If the common set of conditions under which these subsystems work productively, and compatibly, matches plausible geochemistry, an origins of life scenario can be inferred. Using chemical clues from multiple subsystems in this way is akin to triangulation, and constitutes a novel approach to discover the circumstances surrounding the transition from chemistry to biology. Here, we exemplify this strategy by finding common conditions under which chemical subsystems generate nucleotides and lipids in a compatible and potentially synergistic way. The conditions hint at a post-meteoritic impact origin of life scenario.     

Activation of "silent" antibodies and their interaction with antigens

Animal and human blood serum contains great amount of blocked (or "silent") immunoglobulins which, being activated by heating to 60 degrees C, pH decrease to 2.0-2.5 or treatment with 5M KSCN acquire a capacity to interact with different antigens. This interaction may be equally prevented or weakened by both identical and serologically non-related antigen, i.e. activated immunoglobulins are polyspecific. Polyspecific immunoglobulins show less affinity in comparison with monospecific antibodies, their interaction with antigens depends considerably on temperature.     

Radionuclide-conjugated monoclonal antibodies: a synthesis of immunology,inorganic chemistry and nuclear science

Radionuclide-conjugated monoclonal antibodies are new modalities for tumor diagnosis/imaging and therapy. The availability and common usage of these ‘magic bullets’ depends on the biotechnological development and synthesis of three fields of science.     

Amide analogues of TSA: synthesis,binding mode analysis and HDAC inhibition

The synthesis of new amide type histone deacetylase inhibitors is described, having an (R)-methyl substituent and a diene or saturated structure of the chain linking the hydroxamic acid and dimethylaminobenzoyl groups. The saturated compound shows stronger HDAC inhibition than the unsaturated analogue. Molecular modeling suggests that the flexibility of the linker chain is important for an optimal orientation of the dimethylaminobenzoyl group in the enzyme.     

Humicola insolens cellobiose dehydrogenase: cloning, redox chemistry, and "logic gate"-like dual functionality

1Cellobiose dehydrogenase is a hemoflavoenzyme that catalyzes the sequential electron-transfer from an electron-donating substrate (e.g. cellobiose) to a flavin center, then to an electron-accepting substrate (e.g. quinone) either directly or via a heme center after an internal electron-transfer from the flavin to heme. We cloned the dehydrogenase from Humicola insolens, which encodes a protein of 761 amino acid residues containing an N-terminal heme domain and a C-terminal flavin domain, and studied how the catalyzed electron transfers are regulated. Based on the correlation between the rate and redox potential, we demonstrated that with a reduced flavin center, the enzyme, as a reductase, could export electron from its heme center by a "outer-sphere" mechanism. With the "resting" flavin center, however, the enzyme could have a peroxidase-like function and import electron to its heme center after a peroxidative activation. The dual functionality of its heme center makes the enzyme a molecular "logic gate", in which the electron flow through the heme center can be switched in direction by the redox state of the coupled flavin center.     

Triketones active against antibiotic-resistant bacteria: synthesis, structure-activity relationships, and mode of action

A series of acylated phloroglucinols and triketones was synthesized and tested for activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE) and multi-drug-resistant Mycobacterium tuberculosis (MDR-TB). A tetra-methylated triketone with a C₁₂ side chain was the most active compound (MIC of around 1.0 μg/ml against MRSA) and was shown to stimulate oxygen consumption by resting cell suspensions, suggesting that the primary target was the cytoplasmic membrane.     

Fragrance chemistry,nocturnal rhythms and pollination "syndromes" in Nicotiana

GC-MS analyses of nocturnal and diurnal floral volatiles from nine tobacco species (Nicotiana; Solanaceae) resulted in the identification of 125 volatiles, including mono- and sesquiterpenoids, benzenoid and aliphatic alcohols, aldehydes and esters. Fragrance chemistry was species-specific during nocturnal emissions, whereas odors emitted diurnally were less distinct. All species emitted greater amounts of fragrance at night, regardless of pollinator affinity. However, these species differed markedly in odor complexity and emission rates, even among close relatives. Species-specific differences in emission rates per flower and per unit fresh or dry flower mass were significantly correlated; fragrance differences between species were not greatly affected by different forms of standardization. Flowers of hawkmoth-pollinated species emitted nitrogenous aldoximes and benzenoid esters on nocturnal rhythms. Four Nicotiana species in section Alatae sensu strictu have flowers that emit large amounts of 1,8 cineole, with smaller amounts of monoterpene hydrocarbons and alpha-terpineol on a nocturnal rhythm. This pattern suggests the activity of a single biosynthetic enzyme (1,8 cineole synthase) with major and minor products; however, several terpene synthase enzymes could contribute to total monoterpene emissions. Our analyses, combined with other studies of tobacco volatiles, suggest that phenotypic fragrance variation in Nicotiana is shaped by pollinator- and herbivore-mediated selection, biosynthetic pathway dynamics and shared evolutionary history.     

Design, synthesis and coordination chemistry of sidearm substituted bisoxazoline ligands

We describe herein the design, synthesis and coordination chemistry of a novel ligand motif that combines a bisoxazoline with a third flexible chelating substituent (“tail”). Four such ligands (6-9) were synthesized from phenylglycine in 4-5 steps each in ca. 20% overall yield. Coordination of 6 and 7 to [Re(CO)₄Cl]₂ yielded crystals suitable for X-ray analysis. Likewise, crystals were obtained from coordination of 6 to FeCl₂. The solid state structures of the resulting complexes (10-12) reveal κ² binding of the bisoxazoline motif; however, the structures of complexes 10 and 12 show that the “tail” is poised above the metal center, demonstrating the potential for alternate binding modes in solution. The complexes exhibit a relatively planar 6-membered chelate structure, in contrast to the boat conformation typical of trispyrazolylborate complexes.     

"Drink Milk for Fitness": The Cultural Politics of Human Biological Variation and Milk Consumption in the United States

Cow's milk is accorded a high cultural value in the contemporary United States. Its white color, association with the maternal and the pastoral, and repeated mention in the Bible add positive symbolic weight to this major national agricultural commodity. Thus, it comes as no surprise that influential policy-making institutions in the United States recommend milk consumption for all U.S. groups. This is despite variation in adult populations' abilities to digest milk, which has been documented by biological anthropologists. This article assesses various U.S. "stories" about milk consumption and its relationship to biological variation against the biological anthropological explanation of variation in lactase activity/lactose tolerance. Many of these serve as normalizing discourses that ultimately pathologize biological difference and may undermine the dietary traditions of some ethnic groups. In particular, the close relationship between government and the dairy industry leads to policies that fail to seriously consider variation in digestive physiology among the diverse U.S. populations.     

Gibberellin metabolism: new insights revealed by the genes

The identification of most of the genes involved in the metabolic pathways for gibberellin hormones has helped us to understand these pathways and their regulation. Many of these enzymes are multifunctional and therefore fewer enzymes than might be expected are required to synthesize the various gibberellin structures. However, several of the enzymes are encoded by multiple genes that are regulated differently, adding unexpected genetic complexity. Several endogenous and environmental factors modify the expression of gibberellin biosynthesis genes, including developmental stage, hormonal status and light. A future challenge will be to dissect the complex, interacting pathways that mediate the regulation of gibberellin metabolism.     

Total syntheses of iso-, neuro- and phytoprostanes: new insight in lipid chemistry

Isoprostanes (IsoPs) are a complex family of compounds produced, in vivo, from peroxidation of polyunsaturated fatty acids (AA, DHA, EPA, alpha-linolenic) via a free-radical-catalyzed mechanism. Carbocyclic annulations are extremely important reactions and the stereocontrolled intramolecular free-radical cyclization has emerged as a powerful tool for carbon-carbon bond formation in synthetic chemistry. The hex-5-enyl radical cyclization is the most well-known for the synthesis of cyclopentane rings. After a short review of the literature, concerning the total synthesis of isoprostanes and intermediates, we will present our own contributions on the preparation of chiral cyclopentane rings from glucose leading to new isoprostanes. This study allowed us to control the cyclization outcome to yield the all-syn and/or syn-anti-syn precursors which permit us to the total synthesis of a large set of iso-, neuro-, and phytoprostanes.