The universal ancestor

A genetic annealing model for the universal ancestor of all extant life is presented; the name of the model derives from its resemblance to physical annealing. The scenario pictured starts when “genetic temperatures” were very high, cellular entities (progenotes) were very simple, and information processing systems were inaccurate. Initially, both mutation rate and lateral gene transfer levels were elevated. The latter was pandemic and pervasive to the extent that it, not vertical inheritance, defined the evolutionary dynamic. As increasingly complex and precise biological structures and processes evolved, both the mutation rate and the scope and level of lateral gene transfer, i.e., evolutionary temperature, dropped, and the evolutionary dynamic gradually became that characteristic of modern cells. The various subsystems of the cell “crystallized,” i.e., became refractory to lateral gene transfer, at different stages of “cooling,” with the translation apparatus probably crystallizing first. Organismal lineages, and so organisms as we know them, did not exist at these early stages. The universal phylogenetic tree, therefore, is not an organismal tree at its base but gradually becomes one as its peripheral branchings emerge. The universal ancestor is not a discrete entity. It is, rather, a diverse community of cells that survives and evolves as a biological unit. This communal ancestor has a physical history but not a genealogical one. Over time, this ancestor refined into a smaller number of increasingly complex cell types with the ancestors of the three primary groupings of organisms arising as a result.

Growth and viability of macrophages continuously stimulated to produce nitric oxide

Deregulated production of nitric oxide (NO) has been implicated in the development of certain human diseases, including cancer. We sought to assess the damaging potential of NO produced under long-term conditions through the development of a suitable model cell culture system. In this study, we report that when murine macrophage-like RAW264.7 cells were exposed continuously to bacterial lipopolysaccharide (LPS) or mouse recombinant interferon-γ (IFN-γ) over periods of 21–23 days, they continued to grow, but with doubling times 2 to 4 times, respectively, longer than the doubling time of unstimulated cells. Stimulated cells produced NO at rates of 30 to 70 nmol per million cells per day throughout the stimulation period. Within 24 hr after removal of stimulant, cells resumed exponential growth. Simultaneous exposure to LPS and IFN-γ resulted in decreased cell number, which persisted for 2 days after removal of the stimulants. Exponential growth was attained only after an additional 4 days. Addition of N-methyl-l-arginine (NMA), an NO synthase inhibitor, to the medium inhibited NO production by 90% of all stimulated cells, partially reduced doubling time of cells stimulated with LPS or IFN-γ, and partially increased viability and growth rates in those exposed to both LPS and IFN-γ. However, when incubated with LPS and IFN-γ at low densities both in the presence and in the absence of NMA, cells grew at a rate slower than that of unstimulated cells, with no cell death, and they resumed exponential growth 24 hr after removal of stimulants. Results from cell density experiments suggest that macrophages are protected from intracellularly generated NO; much of the NO damaging activity occurred outside of the producer cells. Collectively, results presented in this study suggest that the type of cellular toxicity observed in macrophages is markedly influenced by rate of exposure to NO: at low rates of exposure, cells exhibit slower growth; at higher rates, cells begin to die; at even higher rates, cells undergo growth arrest or die. The ability of RAW264.7 cells to produce NO over many cell generations makes the cell line a useful system for the study of other aspects of cellular damage, including genotoxicity, resulting from exposure to NO under long-term conditions.

A human myeloma cell line suitable for the generation of human monoclonal antibodies

Ever since monoclonal antibodies were produced in 1975 with mouse myeloma cells there has been interest in developing human myeloma cultures for the production of monoclonal antibodies. However, despite multiple attempts, no human myeloma line suitable for hybridoma production has been described. Here we report the derivation of a hypoxanthine–aminopterin–thymidine-sensitive and ouabain-resistant human myeloma cell line (Karpas 707H) that contains unique genetic markers. We show that this line is useful for the generation of stable human hybridomas. It can easily be fused with ouabain-sensitive Epstein–Barr virus-transformed cells as well as with fresh tonsil and blood lymphocytes, giving rise to stable hybrids that continuously secrete very large quantities of human immunoglobulins. The derived hybrids do not lose immunoglobulin secretion over many months of continuous growth. The availability of this cell line should enable the in vitro immortalization of human antibody-producing B cells that are formed in vivo. The monoclonal antibodies produced may have advantages in immunotherapy.

Mutagenesis of the peptidyltransferase center of 23S rRNA: the invariant U2449 is dispensable

U2449 is one of many invariant residues in the central loop of domain V of 23S rRNA, a region that constitutes part of the peptidyltransferase center of the ribosome. In Escherichia coli, this U is post-transcriptionally modified to dihydrouridine (D) and is the only D modification found in E.coli rRNAs. To analyze the role of this base and its modification in ribosomal function, all three base substitutions were constructed on a plasmid copy of the rrnB operon and assayed for their ability to support cell growth in a strain of E.coli lacking chromosomal rrn operons. Both purine substitution mutations were not viable. However, growth and antibiotic sensitivity of cells expressing only the mutant D2449C rRNA was indistinguishable from wild type. We conclude that while a pyrimidine is required at position 2449 for proper ribosomal function, the D modification is dispensable.

Implications of immune-to-brain communication for sickness and pain

This review presents a view of hyperalgesia and allodynia not typical of the field as a whole. That is, exaggerated pain is presented as one of many natural consequences of peripheral infection and injury. The constellation of changes that results from such immune challenges is called the sickness response. This sickness response results from immune-to-brain communication initiated by proinflammatory cytokines released by activated immune cells. In response to signals it receives from the immune system, the brain orchestrates the broad array of physiological, behavioral, and hormonal changes that comprise the sickness response. The neurocircuitry and neurochemistry of sickness-induced hyperalgesia are described. One focus of this discussion is on the evidence that spinal cord microglia and astrocytes are key mediators of sickness-induced hyperalgesia. Last, evidence is presented that hyperalgesia and allodynia also result from direct immune activation, rather than neural activation, of these same spinal cord glia. Such glial activation is induced by viruses such as HIV-1 that are known to invade the central nervous system. Implications of exaggerated pain states created by peripheral and central immune activation are discussed.

α-Catenin-independent Recruitment of ZO-1 to Nectin-based Cell-Cell Adhesion Sites through Afadin

ZO-1 is an actin filament (F-actin)–binding protein that localizes to tight junctions and connects claudin to the actin cytoskeleton in epithelial cells. In nonepithelial cells that have no tight junctions, ZO-1 localizes to adherens junctions (AJs) and may connect cadherin to the actin cytoskeleton indirectly through β- and α-catenins as one of many F-actin–binding proteins. Nectin is an immunoglobulin-like adhesion molecule that localizes to AJs and is associated with the actin cytoskeleton through afadin, an F-actin–binding protein. Ponsin is an afadin- and vinculin-binding protein that also localizes to AJs. The nectin-afadin complex has a potency to recruit the E-cadherin–β-catenin complex through α-catenin in a manner independent of ponsin. By the use of cadherin-deficient L cell lines stably expressing various components of the cadherin-catenin and nectin-afadin systems, and α-catenin–deficient F9 cell lines, we examined here whether nectin recruits ZO-1 to nectin-based cell-cell adhesion sites. Nectin showed a potency to recruit not only α-catenin but also ZO-1 to nectin-based cell-cell adhesion sites. This recruitment of ZO-1 was dependent on afadin but independent of α-catenin and ponsin. These results indicate that ZO-1 localizes to cadherin-based AJs through interactions not only with α-catenin but also with the nectin-afadin system.

Nuclear protein import: Ran-GTP dissociates the karyopherin alphabeta heterodimer by displacing alpha from an overlapping binding site on beta.

The alpha subunit of the karyopherin heterodimer functions in recognition of the protein import substrate and the beta subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p10, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus. Ran-GTP dissociates the karyopherin heterodimer and forms a stoichiometric complex with Ran-GTP. Here we report the mapping of karyopherin beta's binding sites both for Ran-GTP and for karyopherin alpha. We discovered that karyopherin beta's binding site for Ran-GTP shows a striking sequence similarity to the cytoplasmic Ran-GTP binding protein, RanBP1. Moreover, we found that Ran-GTP and karyopherin alpha bind to overlapping sites on karyopherin beta. Having a higher affinity to the overlapping site, Ran-GTP displaces karyopherin alpha and binds to karyopherin beta. Competition for overlapping binding sites may be the mechanism by which GTP bound forms of other small GTPases function in corresponding dissociation-association reactions. We also mapped Ran's binding site for karyopherin beta to a cluster of basic residues analogous to those previously shown to constitute karyopherin alpha's binding site to karyopherin beta.

Reconstitution of p53-ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP).

The E6 protein of the high-risk human papillomaviruses inactivates the tumor suppressor protein p53 by stimulating its ubiquitinylation and subsequent degradation. Ubiquitinylation is a multistep process involving a ubiquitin-activating enzyme, one of many distinct ubiquitin-conjugating enzymes, and in certain cases, a ubiquitin ligase. In human papillomavirus-infected cells, E6 and the E6-associated protein are thought to act as a ubiquitin-protein ligase in the ubiquitinylation of p53. Here we describe the cloning of a human ubiquitin-conjugating enzyme that specifically ubiquitinylates E6-associated protein. Furthermore, we define the biochemical pathway of p53 ubiquitinylation and demonstrate that in vivo inhibition of various components in the pathway leads to an inhibition of E6-stimulated p53 degradation.

Ribosomes can slide over and beyond “hungry” codons, resuming protein chain elongation many nucleotides downstream

In cells subjected to moderate aminoacyl-tRNA limitation, the peptidyl-tRNA–ribosome complex stalled at the “hungry” codon can slide well beyond it on the messenger RNA and resume translation further downstream. This behavior is proved by unequivocal amino acid sequence data, showing a protein that lacks the bypassed sequence encoded between the hungry codon and specific landing sites. The landing sites are codons cognate to the anticodon of the peptidyl-tRNA. The efficiency of this behavior can be as high as 10–20% but declines with the length of the slide. Interposition of “trap” sites (nonproductive landing sites) in the bypassed region reduces the frequency of successful slides, confirming that the ribosome–peptidyl-tRNA complex passes through the untranslated region of the message. This behavior appears to be quite general: it can occur at the two kinds of hungry codons tested, AUA and AAG; the sliding peptidyl-tRNA can be any of three species tested, phenylalanine, tyrosine, or leucine tRNA; the peptidyl component can be either of two very different peptide sequences; and translation can resume at any of the three codons tested.

Single neuron control over a complex motor program.

While there are many instances of single neurons that can drive rhythmic stimulus-elicited motor programs, such neurons have seldom been found to be necessary for motor program function. In the isolated central nervous system of the marine mollusc Tritonia diomedea, brief stimulation (1 sec) of a peripheral nerve activates an interneuronal central pattern generator that produces the long-lasting (approximately 30-60 sec) motor program underlying the animal's rhythmic escape swim. Here, we identify a single interneuron, DRI (for dorsal ramp interneuron), that (i) conveys the sensory information from this stimulus to the swim central pattern generator, (ii) elicits the swim motor program when driven with intracellular stimulation, and (iii) blocks the depolarizing "ramp" input to the central pattern generator, and consequently the motor program itself, when hyperpolarized during the nerve stimulus. Because most of the sensory information appears to be funneled through this one neuron as it enters the pattern generator, DRI presents a striking example of single neuron control over a complex motor circuit.