Medical Research Council

About: Medical Research Council is a government organization based out in London, United Kingdom. It is known for research contribution in the topics: Population & Malaria. The organization has 16430 authors who have published 19150 publications receiving 1475494 citations.

Bioorthogonal Reactions for Labeling Proteins

Abstract: O the past 15 years a great deal of progress has been made on the discovery, rediscovery, and invention of bioorthogonal reactions between functional groups that do not react with biological entities under physiological conditions but selectively react with each other. Strategies for labeling different classes of biomolecules have been developed by coopting the biosynthetic machinery of cells to introduce molecules containing bioorthogonal functional groups. Tagging approaches have allowed some additional functional groups to be attached to proteins, and genetic code expansion and reprogramming have facilitated the site-specific incorporation of unnatural amino acids bearing bioorthogonal functional groups into proteins in bacteria, mammalian cells, and animals via the discovery and synthetic evolution of orthogonal aminoacyl-tRNA synthetase/tRNA pairs and orthogonal ribosomes. In addition, selective pressure incorporation and its derivatives have allowed the statistical labeling of proteins and proteomes with analogues of natural amino acids. The incorporation of unnatural amino acids bearing bioorthogonal functional groups and their chemoselective labeling has great potential for imaging and controlling individual proteins and labeling proteomes, but the ability of investigators to leverage these approaches for biological discovery will be crucially dependent on the properties of the chemical reactions used. The reactants in a bioorthogonal reaction should be kinetically, thermodynamically, and metabolically stable before the reaction takes place and not toxic to living systems. The reaction should yield stable covalent linkages with no or innocuous byproducts. Moreover, the two bioorthogonal moieties have to react selectively with each other under physiological conditions (ambient temperature and pressure, neutral pH, aqueous conditions), without either of them crossreacting with the plethora of chemical functionalities found in living cells. Despite the challenges of meeting these criteria, a number of reactions have been developed that show good biocompatibility and selectivity in living systems (see Figure 1). Some of these reactions are chemoselective with respect to many but not all biological functionalities and have been used to label proteins in vitro and on the cell surface, while other reactions have additionally been used for the more challenging task of labeling proteins inside cells or living animals. Most bioorthogonal reactions follow second-order kinetics, and their rates depend directly on the concentrations of both reaction partners as well as on the intrinsic second-order rate constant k2 [M −1 s−1] of the reaction. Rapid reactions with high second-order rate constants are therefore advantageous for labeling during biological processes that occur on a very short time scale or for the labeling of low abundance proteins. Lower abundance proteins can sometimes be labeled with a large excess of labeling reagent, but this strategy may be practically limited by solubility, off target reactions and toxicity. Bioorthogonal reactions for which one partner can be installed into proteins are summarized in Figure 1. Their second-order rate constants span 9 orders of magnitude with the fastest bioorthogonal labeling reactions reaching rates up to 10 M−1 s−1, which approaches the rate constants for many enzymatic labeling approaches. Here we briefly introduce the bioorthogonal chemistries used for labeling proteins and comment on their utility for protein labeling before providing a perspective on future directions. Amongst the first functionalities to be explored as bioorthogonal reporters were ketones and aldehydes. Under acidic conditions (pH 4−6) their carbonyl groups react with strong α-effect nucleophiles such as hydrazines and alkoxyamines. Ketone/aldehyde condensations show rather slow kinetics with second-order rate constants in the range of 10−4 to 10−3 M−1 s−1, necessitating high concentrations of labeling reagent in order to achieve good labeling, which might be problematic in terms of toxicity and background signal. In general ketone/aldehyde condensations are best suited for in vitro or cell-surface labeling, because the reaction requires an acidic pH, which is difficult to obtain inside most cellular compartments. Furthermore, inside living cells, α-effect nucleophiles may undergo side-reactions with carbonyl-bearing metabolites. A functionality that is essentially absent from biological systems and truly orthogonal in its reactivity to the majority of biological functionalities is the azide group. Azide-bearing unnatural amino acids have been incorporated into proteins and used in a variety of chemical reactions. One potential limitation of the use of azides for protein labeling is that some unnatural amino acids bearing azides appear to be reduced in some proteins examined. Azide-modified proteins have been reacted with phosphines in Staudinger ligations. This reaction has been used to label biomolecules in living cells and animals. The Staudinger ligation, however, has slow kinetics: the reaction proceeds with second-order rate constants in the low 10−3 M−1 s−1 range. In addition many of the phosphine reagents are oxidized by air or metabolic enzymes. Azides can also react with terminal alkynes in [3 + 2] cycloadditions, catalyzed by Cu salts. The CuAAC (Cucatalyzed alkyne−azide cycloaddition) reaction proceeds considerably faster than the Staudinger ligation in physiological settings. However, its reliance on the Cu catalyst is not without problems, since Cu may be toxic to living systems, and decreasing the copper concentration is generally accompanied by a large decrease in reaction rate. The development of tailored water-soluble Cu ligands and/or

Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria

Abstract: The catalytic domain of the F-ATPase in mitochondria protrudes into the matrix of the organelle, and is attached to the membrane domain by central and peripheral stalks. Energy for the synthesis of ATP from ADP and phosphate is provided by the transmembrane proton-motive-force across the inner membrane, generated by respiration. The proton-motive force is coupled mechanically to ATP synthesis by the rotation at about 100 times per second of the central stalk and an attached ring of c-subunits in the membrane domain. Each c-subunit carries a glutamate exposed around the midpoint of the membrane on the external surface of the ring. The rotation is generated by protonation and deprotonation successively of each glutamate. Each 360° rotation produces three ATP molecules, and requires the translocation of one proton per glutamate by each c-subunit in the ring. In fungi, eubacteria, and plant chloroplasts, ring sizes of c10–c15 subunits have been observed, implying that these enzymes need 3.3–5 protons to make each ATP, but until now no higher eukaryote has been examined. As shown here in the structure of the bovine F1-c-ring complex, the c-ring has eight c-subunits. As the sequences of c-subunits are identical throughout almost all vertebrates and are highly conserved in invertebrates, their F-ATPases probably contain c8-rings also. Therefore, in about 50,000 vertebrate species, and probably in many or all of the two million invertebrate species, 2.7 protons are required by the F-ATPase to make each ATP molecule.

The follicular dendritic cell: its role in antigen presentation in the generation of immunological memory

Abstract: There is an ever-growing tendency for cellular immunologists to work with suspension cultures of lymphocytes in a variety of in vitro systems, in the hopes that these will render the analysis of immune phenomena more amenable to detailed analysis. The advantages of these systems are obvious. However, anyone who has studied histological sections of lymphoid tissues will surely have reached the conviction that ultimately our understanding of the complexities of the immune system must take into account the fact that in vivo lymphocytes live in highly organised tissues: that there are T cell areas and B cell areas, and in addition a variety of (as yet largely uncharacterised) nonlymphoid cells which may present antigens and/or provide appropriate microenvironments for different lymphocyte populations to interact, and to differentiate into appropriate effector cells (for a review of lymphoid tissue structure, see Nossal & Ada 1971). This chapter is concerned with the function of one of the most prominent features of peripheral lymphoid tissues, which has fascinated histologists for many years, namely the germinal center. In an unstimulated animal primary lymphoid follicles consist largely of densely packed aggregates of small, resting B lymphocytes, and very few T lymphocytes (see Weissman 1975). Indeed in conventional sections of the spleen, it is difficult to distinguish these B areas from the closely adjacent T areas, the periarteri-

Antibodies to blood stage antigens of Plasmodium falciparum in rural Gambians and their relation to protection against infection.

Abstract: Cross-sectional and longitudinal studies were performed in a rural population living in The Gambia to examine the relationship between several in vitro assays of the host immune response to asexual stages of Plasmodium falciparum and protection from malaria in vivo. Assays included an enzyme-linked immunosorbent assay for antibodies to schizont antigens; an indirect immunofluorescence assay for total antiblood-stage antibodies; an immunofluorescence assay on glutaraldehyde-fixed parasites to detect antibodies to antigen Pf 155; an assay for serum inhibition of red blood cell invasion; a micro-agglutination assay to detect antibodies to neo-antigens on the surface of infected red blood cells; and an assay using polymorphonuclear leucocytes to detect antibodies capable of opsonizing schizont infected red blood cells. There were marked differences in the age-related pattern of response for different assays performed on sera obtained at a cross-sectional survey of 280 individuals. Examination of the correlation between the various immune responses and malariometric indices at the population level and at the individual level provided no evidence that any of the in vitro assays were related to protective immunity. The relationship between in vitro measurements of the anti-malarial immune response and protection from clinical episodes of malaria was examined in a group of 134 children aged 11 years and under who were monitored weekly throughout an entire malaria transmission season. The only immune factor to show a consistent protective effect against clinical malaria was the titre of antibodies to neo-antigens on the infected erythrocyte surface (P = 0.01). The same longitudinal techniques were used to examine the effect of two non-immunological factors, sickle cell trait and mosquito net usage, both of which showed significant protection against clinical episodes and malaria.

Worldwide Survey of Fumonisin Contamination of Corn and Corn-Based Products

Abstract: As part of a comprehensive risk assessment study for fumonisins, reliable data on exposure of populations to these dietary toxins must be obtained. To assess the extent of worldwide exposure, the published literature on the contamination of food and feed supplies has been reviewed and supplemented with unpublished material from various international sources. Fumonisin contamination of corn and corn-based products occurs in many countries. Animal mycotoxicoses such as equine leukoencephalomalacia and porcine pulmonary edema are caused by heavily contaminated animal feeds. For example, as much as 330 micrograms/g fumonisin B1 (FB1) has been found in swine feed. Although commercially available refined corn products for human consumption are generally contaminated at levels below 1 microgram/g FB1, individual products in certain countries can reach far higher levels. Health risks associated with consumption of these products depend on the extent to which they are consumed in a varied diet. Home-grown corn in certain rural areas, where it also constitutes the staple diet, can be contaminated at > 100 micrograms/g. Consumption of corn contaminated at these high levels has been associated with a high incidence of esophageal cancer in these areas.