Consequences of placing an intramolecular crosslink in myosin S1

This paper describes the placement of a crosslinking agent (dibromobimane) between two thiols (Cys-522 and Cys-707) of a fragment, “S1,” of the motor protein, myosin. It turns out that fastening the first anchor of the crosslinker is easy and rapid, but fastening the second anchor (Cys-522) is very temperature dependent, taking 30 min at room temperature but about a week on ice. Moreover, crystallography taken at 4°C would seem to predict that the linkage is impossible, because the span of the crosslinking agent is much less than the interthiol distance. The simplest resolution of this seeming paradox is that structural fluctuations of the protein render the linkage increasingly likely as the temperature increases. Also, measurements of the affinity of MgADP for the protein, as well as the magnetic resonance of the P-atoms of the ADP once emplaced, suggest that binding the first reagent anchor to Cys-707 initiates an influence that travels to the rather distant ADP-binding site, and it is speculated what this “path of influence” might be.

Mycobacterial infection of macrophages results in membrane-permeable phagosomes

Cell-mediated immunity is critical for host resistance to tuberculosis. T lymphocytes recognizing antigens presented by the major histocompatibility complex (MHC) class I and class II molecules have been found to be necessary for control of mycobacterial infection. Mice genetically deficient in the generation of MHC class I and class Ia responses are susceptible to mycobacterial infection. Although soluble protein antigens are generally presented by macrophages to T cells through MHC class II molecules, macrophages infected with Mycobacterium tuberculosis or bacille Calmette-Guerin have been shown to facilitate presentation of ovalbumin through the MHC class I presentation pathway via a TAP-dependent mechanism. How mycobacteria, thought to reside within membrane-bound vacuoles, facilitate communication with the cytoplasm and enable MHC class I presentation presents a paradox. By using confocal microscopy to study the localization of fluorescent-tagged dextrans of varying size microinjected intracytoplasmically into macrophages infected with bacille Calmette-Guerin expressing the green fluorescent protein, molecules as large as 70 kilodaltons were shown to gain access to the mycobacterial phagosome. Possible biological consequences of the permeabilization of vacuolar membranes by mycobacteria would be pathogen access to host cell nutrients within the cytoplasm, perhaps contributing to bacterial pathogenesis, and access of microbial antigens to the MHC class I presentation pathway, contributing to host protective immune responses.

Complementary mutations in an antigenic peptide allow for crossreactivity of autoreactive T-cell clones

T cells recognize antigen by formation of a trimolecular complex in which the T-cell receptor (TCR) recognizes a specific peptide antigen within the groove of a major histocompatibility complex (MHC) molecule. It has generally been assumed that T-cell recognition of two distinct MHC–antigen complexes is due to similarities in the three-dimensional structure of the complexes. Here we report results of experiments examining the crossreactivity of TCRs recognizing the myelin basic protein peptide MBPp85–99 and several of its analogs in the context of MHC. We demonstrate that single conservative amino acid substitutions of the antigenic peptide at the predominant TCR contact residues at positions 91 and 93 totally abrogate reactivity of specific T-cell clones. Yet, when a conservative substitution is made at position 91 concomitant with a substitution at position 93, the T-cell clones regain reactivity equivalent with that of the original stimulating peptide. Thus, the exact nature of the amino acid side chains engaging one TCR functional pocket may change the apparent selectivity of the other predominant TCR functional pocket, thus suggesting a remarkable degree of receptor plasticity. This ability of the TCR–MHC–peptide complex to undergo conformational changes provides a conceptual framework for reconciling the apparent paradox of the extreme selectivity of the TCR and its remarkable crossreactivity with different MHC–peptide complexes.

Inhibition of advanced glycation endproduct formation by acetaldehyde: Role in the cardioprotective effect of ethanol

Epidemiological studies suggest that there is a beneficial effect of moderate ethanol consumption on the incidence of cardiovascular disease. Ethanol is metabolized to acetaldehyde, a two-carbon carbonyl compound that can react with nucleophiles to form covalent addition products. We have identified a biochemical modification produced by the reaction of acetaldehyde with protein-bound Amadori products. Amadori products typically arise from the nonenzymatic addition of reducing sugars (such as glucose) to protein amino groups and are the precursors to irreversibly bound, crosslinking moieties called advanced glycation endproducts, or AGEs. AGEs accumulate over time on plasma lipoproteins and vascular wall components and play an important role in the development of diabetes- and age-related cardiovascular disease. The attachment of acetaldehyde to a model Amadori product produces a chemically stabilized complex that cannot rearrange and progress to AGE formation. We tested the role of this reaction in preventing AGE formation in vivo by administering ethanol to diabetic rats, which normally exhibit increased AGE formation and high circulating levels of the hemoglobin Amadori product, HbA1c, and the hemoglobin AGE product, Hb-AGE. In this model study, diabetic rats fed an ethanol diet for 4 weeks showed a 52% decrease in Hb-AGE when compared with diabetic controls (P < 0.001). Circulating levels of HbA1c were unaffected by ethanol, pointing to the specificity of the acetaldehyde reaction for the post-Amadori, advanced glycation process. These data suggest a possible mechanism for the so-called “French paradox,” (the cardioprotection conferred by moderate ethanol ingestion) and may offer new strategies for inhibiting advanced glycation.

The paradox of alloreactivity and self MHC restriction: Quantitative analysis and statistics

Although 1–24% of T cells are alloreactive, i.e., respond to MHC molecules encoded by a foreign haplotype, it is generally believed that T cells cannot recognize foreign peptides binding foreign MHC molecules. We show using a quantitative model that, if T cell selection and activation are affinity-driven, then an alloreactivity of 1–24% is incompatible with the textbook notion that self MHC restriction is absolute. If an average of 1% of clones are alloreactive, then according to our model, at most 20-fold more clones should, on average, be activated by antigens presented on self MHC than by antigens presented on foreign MHC. This ratio is at best 5 if alloreactivity is 5%. These results describe average properties of the murine immune system, but not the outcome of individual experiments. Using supercomputer technology, we simulated 100,000 MHC restriction experiments. Although the average restriction ratio was 7.1, restriction was absolute in 10% of the simulated experiments, greater than 100, although not absolute, in 29%, and below 6 in 24%. This extreme variability agrees with experimental estimates. Our analysis suggests that alloreactivity and average self MHC restriction both cannot be high, but that a low average restriction level is compatible with high levels in a significant number of experiments.

Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens

This review summarizes recent evidence from knock-out mice on the role of reactive oxygen intermediates and reactive nitrogen intermediates (RNI) in mammalian immunity. Reflections on redundancy in immunity help explain an apparent paradox: the phagocyte oxidase and inducible nitric oxide synthase are each nonredundant, and yet also mutually redundant, in host defense. In combination, the contribution of these two enzymes appears to be greater than previously appreciated. The remainder of this review focuses on a relatively new field, the basis of microbial resistance to RNI. Experimental tuberculosis provides an important example of an extended, dynamic balance between host and pathogen in which RNI play a major role. In diseases such as tuberculosis, a molecular understanding of host–pathogen interactions requires characterization of the defenses used by microbes against RNI, analogous to our understanding of defenses against reactive oxygen intermediates. Genetic and biochemical approaches have identified candidates for RNI-resistance genes in Mycobacterium tuberculosis and other pathogens.

Multineuronal codes in retinal signaling.

The visual world is presented to the brain through patterns of action potentials in the population of optic nerve fibers. Single-neuron recordings show that each retinal ganglion cell has a spatially restricted receptive field, a limited integration time, and a characteristic spectral sensitivity. Collectively, these response properties define the visual message conveyed by that neuron's action potentials. Since the size of the optic nerve is strictly constrained, one expects the retina to generate a highly efficient representation of the visual scene. By contrast, the receptive fields of nearby ganglion cells often overlap, suggesting great redundancy among the retinal output signals. Recent multineuron recordings may help resolve this paradox. They reveal concerted firing patterns among ganglion cells, in which small groups of nearby neurons fire synchronously with delays of only a few milliseconds. As there are many more such firing patterns than ganglion cells, such a distributed code might allow the retina to compress a large number of distinct visual messages into a small number of optic nerve fibers. This paper will review the evidence for a distributed coding scheme in the retinal output. The performance limits of such codes are analyzed with simple examples, illustrating that they allow a powerful trade-off between spatial and temporal resolution.